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c4n, anthanarayan
and
^^
Textbook
miker ' s
Microbiology
Edited by
C K J Paniker
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to
..
Dr R Afunthanarayin
17.6/1913-&2.1998
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way hudvwlHl enura.
C no nthana ra yan
anci
Moniker's
Textbook
of
Microbiology
This OUB
C'^Dnanthanarayon
and m
-A
«
.
niker
]
,
s
Textbook
of
Microbiology
(Late) ft Ananthanarayon
.
SA , MBBS I Madrail . 06. PhD \ ionion )
Formerly . FWrfeiuar of Micrptwlogy .
Medmol Colleges, Cd»cut, Koftnyom . Triwondhuiii and Gulbarga
raid Adviser sig tbe DHS, Kerala. on Laboratory Service
*
CK Jayaram Poniker
MD
Formerly , PirfdQrFrEifiHor of Microbiology and Principal,
Medmdl College, Calicut, Kerala
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Copyrighted material
Preface to the Seventh Edition
Only fnuryrars have pawed since the sixth edition of TbriboakafMkmtoiakjgjim published , bur rapid devetopirienrs
LEI die subject have made a new edition necessary. During this short period, new infectious diseases have emerged
-
nr rc cmcrged in new forms. For example* Severe Acu.ce Respiratory Syndrome (SARS) virus appeared suddenly
causing death and panic in many countries, and the Bird Flu virus posed repeated pandemic threats.
,
Microbiology has become an increasingly important discipline, set to (face new challenges- Exciting advances in
diagnostic microbiology using sophisticated techniques can help in the rapid identification of new pathogens and
serve to contain them.This was shown by the idendfLcation. of the new SA RS vims within weeks by concerted multi ”
discipl i nary international effons. While such scienri fie progress is a boon., some of it can be potentially dangerous, as
lor instance in the recent case of chemical synthesis of a complete pathogenic poliovirus in the laboratory.
In rhU edition., [he seventh, relevant new information has been added and all chapters revised and updated,
,
maintaining the general format of the book. The Textbook nfMicmhiofogy has been in use now for more than a
q mirier of a century. It has bent fired greatly from the comments and suggestions from students, teachers and other
reader?.
Their help is gratefully acknowledged.
C . K. Jayaram Paniker
ShantUi * 1/SSM, Eui Ml Rcrad ,
Cjflkifl, Kcndu 63? OOfr.
'
are important to us though only of less or academic interest to the developed countries. The Increasing importance
of the newer knowledge in immunology to health and disease is not adequately stressed in most of the extant
textbooks. Vi ms- disease* whic h are responsible for nearly 60 per cent of human illness require wider coverage. The
general approach_ to the tnLfun ofmkrobioloj - Inmraiuntryhas also been rather static. All these factors cjJlcd for
^ ^
a icxrhook of mu:mbi.o]ogy nrirore ir d cci L^uuinriv like lodil.
^^ ^
We therefore undertook th is endeavour based on mar experience of reaching undergraduates and postgraduates for
over two decades. We omitted the discipline of paroritolagy from our book since we already have an excellent
,
Th is book has taken us over three years co wrire and over a year in publication . N aturally we would be out of date to
a certain and inevitable extent. We do not claim any perfection. On the contrary, we have reqtbCSted medical
students and teachers all over the country to write tn us about any shortcomings and give us suggestions as to how
to improve- the hook. We shall spare no pains in seeing that their valuable suggestions are given effect to in our
-
second edition.
R. Aiwthanarayan
C. K. JayaLram Paniker
Copyrighted material
Acknowledgements
For kind perm i 5- FLon to UBC iilusf rations , supply of photographs, help lid suggestions and advice, the author is
indebted to the following civilisations and persons:
Wirld Health OipniiilMm; Indian Council of Medical Research; National Institute of Viralogy; Pune; Dr. G.
Baikrith Nsur, Du A.N. Ghosh, Dr. TN. Nsik and Dr Triverti Krishnan of National Institute of Cholera and
Enteric diseases, Calcutta, Prof. M. Maihui and Piol. T. Jacob John i >t Christian Medical College , Wllore, Prof ,
Anin Ch i tale,JailoJt Hospital Mumbai; Dr SJM.Sirsat, Canter Research 1 nstitute, Mumbai; Prof M.D, Mathur,
Maulana Azad Medical College, New Delhi, Ptof. J. Shanmugham, SCT Medical Centre, Trivandrum;
Dr. Girija G - Rao, Medical College, CaJicur; Dr. G .M. Warfce, TIiMedia Laboratories, Mumbai.
u opy
y righted materia
Contents
Purl I
1 lisitorical Introduction 1
2 . Morphology and Physiology of Bacteria 7
3 St ^iril isariun and 13is.infectii0iH 24
nlnire Media
"
4 (' 34
Culture Methods 39
6 Identification of Batttria 44
7, Racrenial Taxonomy 48
fi , Bacterial GcilCtifl 51
[]
9, Infection 64
10. Immunity 71
11 , Antigens SO
12. Antibodies-ImmunoglobuJ ins 94
13 , Antigen-Antibody Reactions 92
14. Complement System 110
15 , Structure and Functions of the Immune System 117
16, Immune Response 133
17. Immunodeficiency Diseases 152
IS. Hypersensitivity 159
19. Autoimmunity 169
20. Immunology of Transplantation and Malignancy 176
21 , Immunohematology 184
I'ilTl HI
22 , Staphylococcus 192
23 . Streptococcus 202
24. Pneumococcus 216
2L_ Neisseria 222
opyrigmea nr na
26. Corynebacterium 231
27 Bacillus 241
?R -
O rEftfri Hli LI rtii 24fi
29. Ncnsporing Anaerobes 266
in RntnmliafferiaiiMr TJ Cotifrtfms-Prrtteiis 271
31 . Enterobacteriaceae 11: Shigella 285
32. Enterobacteriaceae III: Salmonella 290
31 . Vihfift ins
34
35 .
^
Pwn nmnnas
Yersinia. Pasteurelk. Frandsella 324
36 . Haemophilus 333
37 Hoidetella n <j
38 Brucella
39. Mycobacterium 1: Tuberculosis 351
40. Mycobacterium II: Atypical Mycobacteria 36S
41 . Mycobacterium Ill: M, Leprae 370
42 . Spirochetes 377
43 . Mycoplasma 395
44, Actmomyoetes 400
45. Miscellaneous Bacteria 403
46, Ricketirdaceae 412
47 , Chkmydiae 422
Part IV
4S, General Properties of Viruses 430
49
50.
—
VirtiS HoAt I nTeraLtinns : Viral Tnfectinns
Bacteriophage
444
461
51 . Poxviruses 460
52. Herpesviruses 474
53. Adenoviruses 4fl £
54. Picomaviruses 490
55 . Orthomyxovirus 501
56 , Ikramyxoviruses 512
57. Arboviruses 521
5$, Rhabdovi ruses 535
.1 , i
J nanie ate nai
59, Hepatitis Viruses 547
fin Miscellaneous Viruses 563
61, Oncogenic Viruses 574
62, Human Immunodeficiency Virus: AIDS 582
T V
^63
rt
, Norma] Microbial Flora of the Human Body 599
64, Bacteriology of Water, Milk and Air 603
65, Medical Mycology 610
66 . Laboratory Control of Antimicrobial Therapy 628
67. Immunoprophylaxis 631
68, Hospital Infection 634
1 ndex 639
Copyrighted material
Copyrighted material
Historical Introduction
McdLuad microbiology is the study cst microbes that observations was not realised then * n d t(J
infect humans , the diseases they cause , Their
, Leeuwenhoek llie world of liJtlc' ajlnBaktJfS ' fli
diagrams, premonition smd treatment , It also deals he called Them , represented only a curiosity of
with the response of the human hosr ro microbial nature . ][ was only some [ wo centuries 1 a[er that
f
Copyrighted materi r_ l
2 4 Texlbcc * al Microbiology *
microbes. This was then the subject of much Contributions and simitar institutions were
controversy. Needham, an Irish priest , had m 1745 established soon in manv other countries for the
published experiments purporting the spontaneous preparation -of vaccines and lor tlue investigation of
generation (abiogeneri -s ) of microorganisms in infectious diseases.
putrescible fluids . This view was opposed by An immediate apphcacLon ofRasteur’s work was
Spallanzani, an Italian abbot (1769). In a series of the introduction of ant isepliC techniques in surgery
classic experiments, Pasteur proved conclusively that by I . if ter (1867) effecting a pronounced drop in
all forms of life , even microbes, arose only from mortality and morbidiry due tu surgical sepsis
their like and not tie novo. In the course of these Lister's antiseptic surgery involving the use of
studies, he introduced techniques of steriliRation carbolic add was cumbersome and hazardous but
and developed the steam steriliser, hot air oven and was a milestone in the evolution nfsurgicalpradke
autoclave. He also established the differ mg growth from the era of 'laudable pus’ to modern aseptic
needs of different bacteria. E lis work attracted such techniques-.
attention and he attained such eminence In the While Pasteur in France laid the foundations
world of science that not only France hut all Europe
looked to him to solve mo jot problems in various
-
of microbiology, Robert Koch (1843 1910 ) in
Germany perfected bacteriological techniques
fields. Thus starred Ins studies on pebrine, anthrax , during his studies on the culture and life cycle of
chicken cholera and hydrophobia. An accidental the anthrax bacillus (1876 ). He introduced staining
observation that chicken cholera bacillus culture* techniques .mel method * of obtaining bacteria ill
left on the bench for several weeks lost rheir pure culture u -lng solid media. He discovered the
pathogenic property but returned their ability to bacillus of tuberculosis (1882 ) and the cholera
protect the birds against subsequent infection by vibrio (18S3 ) r
them , led to the discovery of the process of Pasteur and Koch attracted many gi tted dm i pics
attenuation and the development of Live vaccines. who discovered rhe causative agents of several
He attenuated cultures of the anthrax bacillus bv r
bacterial infections and enlarged the scope and
incubation at high temperature ( 42-43 °C ) and content of microbiology by their labours- In 1974
proved that inoculation oJ such cultures in animals Hansen described the leprosy bacillus; in 1879
induced specific protection against anthrax .. [’ he Neisaer described the gonococcus; in 1881 Og ton
success of such immunisation was dramatically discovered the staphylococcus; in 1384 Loefflcr ^
demonstrated by a public experiment on a farm at isolated the diphtheria bacillus; in 1884 Nicolaicr
Pbuilty- le - Fort ( 18 B1 ) during which vaccinated observed the tetanus bacillus in soil ; in 1886
sheep, goats and cows were challenged wirh a Fracnkcl described the pneumococcus; in 1887
virulent anthrax bacillus culture. All the vaccinated 11 nice identified the causatu'e agent of Malta fever,
a nil u ills survived the challenge , while an L- qu.d in 1905 Schaudmn and Hoffmann discovered the
pyrignte<
—-
i. P ria
iLI 1
* HiglOlkfil IrttrotiUCtinn 3
The causative agents of various infectious fine filters . Beijerinck ( 1898 ) confirmed these
diseases were being reported by different findings and coined the term virus for such filterable
iiivc ^ ri tutors nsuch prolusion that i t was necessity infectious agents . Locfiler and Frosch ( 1898)
'
.
to introduce criteria for proving the claims that a observed rhat the foot and mouth disease of cattle
niLcifi ( iri ;,mh: ii iliolited from a disease was indeed wan caused by a similar filter - passing virus . The
causally related to if . These criteria, first militated first human disease proved to have a viral etiology
by Hctilc, were enunciated by Koch and ate known was yellow fever, T*hc US Army Commission under
V a
as Koch 's postulates . According to these , a Walter Reed , invesrigating yellow fever in Cuba
microorganism can be accepted as the causative i. 19 Q 2 ) established not only that it was caused by a
agenr of an infectious disease only if the follow ing filterable virus but also that ir was transmitted
conditions arc satisfied: through rhe bhe of i i lfocted mosquitoes. 1 -andstc 11 rtt
1 . The bacterium should be constantly associated and Popper (1909 ) showed that poliomyelitis was
with the lesions of the disease . caused by a filterable virus and transmitted the
1 . It should be possible tu isolate the battenLiin in disease experimentally to monkeys. Investigation of
pure culture from the lesions. viruses and the diseases caused by them wasr
3 . Inoculation of such poire culture into suitable rendered difficult as viruses could not be visualised
laboratory animals should reproduce the lesions under the light microscope or grown in culture
ot the disease . media . Though the larger viruses enutd be seen
4 . It should be possible to nsisolite the bacterium after appropriate staining under the light
in pure culture from the lesions produced in m icroscope, det .i i led study of their morphology had
the experimental animals. to wait till the introduction of the electron
An additional criterion introduced subsequtndy microscope by Ruska ( 1934) and subsequent
requires that specific; antibodies to the hacteri . jjn refinements in electron microscopic techniques.
should be demonstrable in the scrum of patients Cultivation of viruses was possible only in animals
suffering from the disease . Though it may not or in human volunteers [ ill the technique of
always he possible to satisfy all the postulates in growing them on chick embryos was developed by
every ease, they have proved extremely useful in Goodpasture in the 1930s. The application of tissue
sifting doubtful claims made regarding the causative culture in virology expanded the scope of virological
agents of infectious dj - eases. techniques considerably.
By the beginning of the twentieth century, many The possibility' that virus infection cnuld lead
infectious diseases had been proved to be caused to malignancy was first put forth by KHerman and
by bacteria . But there remained a large number of Bang ( 1908 ), Peyton Rous ( 1911 ) isolated a virus
diseases such as smallpox , chickenpOJt , measles, causing sarcoma in fowls. Several viruses have since
ridiirm and the common cold for which no been isolated which cause natural and experimental
ci . utcri . il cause could he established. During his tumours in animals and birds . Viruses also cause
l Oves Ligation of rabies in dog , Pasteur had suspected ma!ignant transformation uf infected cells in tissue
^
that the disease could be caused hv a miemhe too culture . The discovery of viral and c e l l u l a r
small to be seen even under the microscope . The oncogenes has shed light or the possible
existence of such ultramicroscopic microbes was mechanisms of viral oncogenesis . After many
proved when Ivanovslqr ( L 892 l TV produced mosaic decades of futile search , positive proof of a virus
disease in the tobacco plant, by applying to bealtby
leaves juice from the diseased plants from which
-^
i ; j'. j . i :i human malignancy wafi established when
the virus of human I - ceil leukemia was isolated in
all bacteria had been removed by passage through 1980.
Twt » rt ( ISIS ) and d ' Hercllc ( 1917 ) 'aicxitw*.A spe ific humoral (actor or 'antibody*
independently dermic red a lyric phenomenon in was described by voci Behring and Kitasato (1890 )
bacterial cultures. The , Lt;enL > responsible were in the serum of animals which had received
termed bacteriophages — viruses that attack bacleri . L. sublethal doses of tetanus torin. Pfeiffer ( 1893)
Early hopes that bacteriophages may have demonstrated bactericidal effect in vivo by injecting
therapeutic appl :icafions had to be abandoned but live cholera vibrios irtraperitoneally in guinea pigs
these viruses have paid unexpected scientific previously Injected with killed vibtios. The vibrios
dividends. The essentia ] part of viruses is rheir core were shown to undergo lysis. The humoral nature
of nucleic acid which acts as the Cartier of genetic of such lytic activity was proved by Bordet ( 1895 },
:. nformation in the same manner as in Higher who defined the two components participating in
organisms. The discipline of molecular biology owes the reaction, the first being heat stable and found
its origin largely rn studies or the genetics of in immune sera (antibody or substance
bacteriophage;* and bacteria . scnsrfijitca trice) and the second being heat labile
It had hern noticed from very early days [hat aod identical with Buchner 's aJudne , subsequently
persons surviving an attack of smallpox did not named 'cnmplancfi t \ Soon a number nf other ways
develop the disease when exposed to the infix non were demonstrated in which antibodies react with
subsequently. This observation had been applied an11 JI ILS , such as agglutination , precipitation ,
"
for the prevention of the disease by producing a complement fixation and neutralisation ,
mild form of smallpox intentionally (variolation ). MctchmkolT ( 1883) discovered the phenomenon
This practice , prevalent in India , China and other
, of phagocytosis and proposed the phagocytic
am lent civilisations from time immemorial , was response as the prime defence against the microbi . il
introduced in England by Lady Mary Worthy invasion of tissues. This led to the cellular concept
Montague ( 1718) who had observed the custom of immunity. Polemics regarding the significance
in Turkey. Variolation was effective but hazardous, of the cellular and humoral mechanisms of
lenner , observing the immunity to smallpox in i mmunity were largely put to rest with the discovery
introduced by him were successful , the mechanism and Richet ( 19U2) , studying the effect of the luric
of protection afforded by them remained obscure . extracts of sea anemones in dogs made the
The cxplanat I on of the underlying mechanism came paradoxical observation that dogs which had prior
from two sources. Nuttall ( 13SS ) observed that contact with the toxin were abnormally sensitive
defibrinated blood had a bactericidal effect , and to even minute quantities of it subsequently. This
Buchner ( 1889) noticed that this effect was phenomenon was termed anaphylaxi* . Later, many
abolished by beating the sera for one hour at 55 °C. similar reactions were observed, both
The heat labile bactericidal factor was termed experimentally and in nature, of injury, disease or
Copyrighted materi3
* historical Introduction * 5
even death resuming from repeated contacts wirh to include natural defence against cancer Another
antigens . 11 M. importance of this phenomenon, in aspect of tti i s role of i mnrauniTy is in the reiec c i nn of
the pathogenesis of many human diseases, led to homografts. Understanding of the immunological
the development of the discipline of allergy. basis of transplantation, largely due to the work of
The characteristic feature of immunity, whether Medawar and Burnet , made successful transplants
if is protective or destructive (as in allergy), is its possible by elective immunosuppression and proper
specificity. As the mediators of humoral immunity selection of donors based on hbtooomparibility. The
(an til Jodies) are globulins, the explanation for the history of transplantation thus runs parallel to the
exquisite spedGdty ol the immunological reaction history of blood transfusion , which was
had to await the advances iin protein chemistry, The unsuccessful and even fatal before the discovery of
pinneeri ng work of I ,andsteincr laid the foundations blood groups by Landsteiner ( 1900).
of immunochen ilstry Chen i ists dom 11 rated the study In the early twertliertth century, attempts were
of immunity for several decades , and theories of made to exploit the immunological information
anl. i body synthesis were postulated by them , will i ch available by the development of vaccines and sera
sometimes ran counter to biological laws. In 1955, for the prophylaxis and treatment of infectious
ILTUC proposed the natural selection theory of diseases. Till Domagk ( 1935 ) initiated scientific
antibody synthesis which attempted to explain the chemotherapy with the discovery of prontosil ,
chcmit al specificity and HologjraJ basis of antibody antisera were the only specific therapeutic agents
synthesis, signifying i return ro the original views available for the management of infectious diseases,
of antilxxdy formation proposed bv Ehrlich (1 S9E ). Fleming (1929) made the accidental discovery that
Burnct ( 1957 ) modified thi ? into the clonal the fungus Fcnicilliurn produces a substance wludi
selection 1 he ore, a concept which, with minor destroys Etaphylococvi. Work on this at Oxford bv
alterations, holds sway even now. The last few Florey, Chain and their team during the Second World
decades have witnessed an explosion of conceptual War led to the isolation of the active substance
and technical advances in immunology . penicillin and its subsequent mass production. This
Immunological processes; in health and disease are was the begi nni ng of the antihiotk: era - Other sii n ilar
now better understood follow!ng the identification antibiotics were discovered in rapid mcecssion. With
of the two components of irnmuni fy - the humoral the sudden availability of a wide range of antibiotics
or andbody mediated processes and the cellular or with potent antibacterial activity, it was hoped that
manilest in separate
—
cell medi . Lted processes which develop and are
pathways.
bactcn.i] iidecbonii wmild he controlled within . i ,- hort
per,ixl. But soon the development oJ drug resistance
Til ] recently, a teleological view of immunity in bacteria presented serkius difficulties.
prevailed. It was considered a protective mechanism With the development of a wide variety of
designed ro defend the body against invasion by antibiotics active against the whole spectrum of
microorganisms . Based on the original suggestion path* ’jgenic bacteria, and of effective vaccines against
f Thomas- ( 1959) , Burnet ( 1967 ) developed the most viral ibseases, expectations were rinsed about
conceptof immvnaingiczl survriifancc , according the eventual elimination of alt infectious, d Meases.
to which the primary function of the immune The global eradication of smallpox inspired visions
system is to preserve the integrity of the body, of similar campaigns against other major pestilences.
seeking and destroying all ‘foreign’ amigenS , However, when new infectious diseases began TO
whether autogenous or external in origin . appear , caused bv hitherto unknown micro'
Malignancy was visualised as a failure of this organisms, or bv known microbes producing novel
function, and the scope of immunity was enlarged manifestations, it was realised that controlling
opyrighted materia
i
6 r Te ^ lbcck o1 Micros
microbes was a far more difficult task than was 1901 Emil A Von Behnng
imagined . The dimax came in 1901 when AIDS 1902 Ronald Ross
was identified in the USA and began its pandemic 1905 Robert Koch
spread. Unceasing vi ii appear? essential to protect 1907 CIA Lavcran
^
humans from microbes .
1900 Paul Ehrlich and Elie Metchnikoff
1913 Charles Richet
Apart from the obvious benefits such as specific
methods of diagnosis, prevention and control of
infectious diseases, medical microbiology has
^
1919 ulcs Bordet
1926 Johannes Fibiger
1920 Charles Nicoflc
contributed to scientific knowledge and human 1930 KaH Landsteiner
1939 Gerhardt Domagk
welfare in many other ways . Microorganisms 1945 Alexander Fleming, Howard Finney and EB
constitute the smallest forms of lining beings and, Chain
therefore, have been employed as models of studies 1951 Max Theiler
dl gentries and biochemistry. As nature's laws are 1952 SeLman A Waksman
universal in application, information derived from 1954 JF Endens, FC Robbins and TH Weller
the investigation of m icrobcs holds true, in the main, 1958 GW Beadle,Joshua Lederbergand EL Tatum
1960 MadarJane Burnet and Peter Brian Medawar
for humans as well. 1965 Francois Jacob, Andre Lwoff and Jacques
Studies on microorganisms have contributed, Monod
more than anything else , to unravelling the genetic 1966 Peyton Rous
code and other mysteries of biology at the molecular 1969 Max Delbruck, AD Hershey and Salvador
level. .Bacteria and their plasmids , yeasts and viruses Luna
are mutinely employed M vectors in recombinant
1972 Gerald Edelman and Rodney Porter
1975 David Baltimore, Renato Dulbeoeo and
DNA technology. They have made available Howard M Tcmin
precious information and powerful techniques for 1976 S Baruch , Blumbcrg and Carleton
genetic manipulation and molecular engineering. Gajdusek
They need to be used wisely and well for the benefit 1978 W Arber, D Nathans and HO Smith
of all living beings. I 960 Banjj Benacmaf, Jean Dausset and George
The number of Nohel laureates, in Medicine
Snell
1984 Niels Jcrne, Cesar Milstein and Georges
and Physiology awarded the prize for their work Kohler
in microbiology, listed below, is evidence o f the 1987 Susurnu Toregawa
positive contribution made to human health by the 1989 J . Michael Bishop and E Vaimus
science of microbiology. 1996 Paul Doherty and Rolf Zinkemagel
1997 Stardev PrusiTier
Further Reading
Btuaotmf B et al . I 960. A History oFBactscr.- niogy and Jmmunotogy, London; William Heinemann .
Clark PF 1 %1 . PTuueer JVfjtnjfrwk r rs of Amelia. Madison: University of Wisconsin Pftti.
'
^ ^
Collard P 1976. The Development of Mictoi' iohgy, Cambridge University Press .
deKri - f P 1958 . Microbe !iuntets. London: Hutchison.
'
fo' ii ' T WI ) 1970. A History of Medial Bacteriology and Immunology, London; Cox and Wyman .
Lethfivaller HA unci M Solotorovsky 1974 , Three Centuries of Microbiology'. frovtr Publications.
Marquardt M 1951. Aiuf Ehrlich N*w York; bchuman ,
[hripb HJ i 960. Victory with Van vies - The Story of Immunisation , Loudon: Livingstone .
Vail cry Radot R 1948. The I .: fe ofPaattut . i r u = : . by ftL Devonshire. London: Constahbe.
1
Wiicrsnn AP . mil L Wilkinson 1978. An Introducoon to the History of Virology. London: Cambridge University Preas.
Williams G i 960. FJrus Hcijinfrtf . London: Hutchison .
opyrigniec maiterial
Morphology and Physiology
of Bacteria
Copyrighted material
S * lEfK:bM (t (tf M < r.' Qb \Q ogy
The following types of microscopes ire employed a size of approximately half the wavelength of the
now, light used. With edible light, using the best optical
Optic III or 11. ;Kt ihiontUDpC: Bacteria may systems, the limit of resolution is about J500 nm . Tf
he examined under the compound microscope, light of shorter wavelength is employed, as in the
either in the Living state or after fixation and staining. ultraviolet microscope, the resolving power can be
Examination of wet films or ' hanging drops 1
proportionately extended,
indicates the shape, arrangement, motility and r\vO specialised types of microscopes arc 1) the
approximate -iic of the cells. But due to lack of interference microscope winch not only reveals cell
contrast , dcra: H cannot be appreciated. organelles bur also enables quantitative
Phase contrast microscopy : improves the measurements of the chemical consritucnts of cells
contrast and makes evident the structures within such as lipids, proteins and nucleic acids, and 2 }
cells that differ in thLckness or refracri vv i ndex. Also, the polarisation microscope which enables the Study
the differences in refractive index between bacterial of intracellular structures using differences in
cells and the surrounding medium make them birefringence .
clearly visible . Retardation, hy a fraction of a Electron mioroaoope: Ln the electron
wavclengrb, of rhe rays of light tint pass through mcroscope, a beam of electrons is employed instead
the obiecr, compared to the ravs pacing through of the beam oflight used in the opritaJ microscope.
the Surrounding medium, produces 'phase '
producing Light and dark contrast in the image. produces an image which is focused on a fluorescent
Dark field t Dark ground microscope: viewing screen. As the wavelength of electrons used
Another method of improving the contrast is the is approximately 0,005 nm, as compared to 500 nm
dirk field ( dark ground ) microscope in which with visible light, rhe resolving power of the
reflected light is used instead of the transm itted electron microscope should be theoretically
*
light used in the ordinary microscope. The essential times that oflight microscopes but in
parr of the dark field microscope is the dark held practice the resolving power is about 0.1 nm.
condenser with a central circular stop , which I he technique of shadow - casting with vapori ^cd
illuminates the object with a cone oflight, without heavy metals has made possible pictures with good
letung my ray of Light to fall directly on the contrast and three - dimensional effect. Another
objective lens. .1 .l.nht rays falling on the object are valuable technique in studying fine structure is
reflected or scattered on to the objective lens, with negative staining with phosphotuilgstic acid.
the result that the object appears self- Tumi -lOUS Gas molecules scatter electrons , and it LS
against a dark background, The contrast gives an therefore necessary to examine the object in a
illusion o: increased resold ion, so- that verv slender vacuum. Hence, only dead and dried objects can be
organisms, such as spirochetes, nor visible under examined in the electron microscope. This may lead
Copyrighted materia
« Morphology and Physiology of Bacteria * 9
to considerable distortion in cell morphology, A too rhin to he seen under the ordinary microscope
method introduced to overcome this disadvantage may he rendered visible if they are thickened by
.
is freeze- etching involving the deep- freezing of
specimens in a liquid gas and the subsequent
.
iinprcgtiatieinofsilvcron the surface Such methods
are used for the demonstration of spirochetes and
formation of carbon-platinum replicas of the bacterial flagella.
material. Since such fnJ7ei«l!s may remain viable , Differential atainst These stains impart
it is claimed that (reeze-erehing enables the study different colours to different bacteria or bacterial
of cellular ultrahtructure as it appears in the living structure'-. The two most widely used differential
state. The recent development of very high voltage stairs arc the Gram stain and the acid fast stain.
electron microscopes may render possible the The Gram slain was originally devised by the
eventual examination of live objects. The scanning histologist Christian Gram (1&84) as a method of
electron microscope is a useful in novation which staining bacteria in tissues. The staining technique
permits the study of cell surfaces with greater consists of JIJUT steps:
contrast and higher resolution linn with the 1 . primary staining with a pararosanilinC dye such
shadow -easting technique . as crystal violet, methyl violet or gentian violet;
2 . application of a dilute solution of iodine ;
S I AIMKII PHKFAK .v decolouriisariuii with an organic solvent such as
1 AVC bactc ri a do not show much strue tu ral dctiiI ethanol , acetone or aniline;
under the light microscope due to lack ol contract. J. counters!aining with a dvr * ft contrasting Colour,
Hence ir is customary to use staining Techniques to such as carbo ] tuebsin , sutranine or neutral red.
.
produce colour contrast Bacteria may be stained The Gram stain differentiates bacteria into two
in the living state, hut this type of staining is broad groups. Gram positive bacteria arc those that
employed only for special purposes Routine . resist decolonisation and retain the primary ttain,
methods fer tfainiDg of bacteria involve drying and appearing violet . Gram negative bacteria are
fixing smears, procedures that kill them. Bacteria decolourised by Organic sohcntl and, therefore, take
have an affinity for basic dyes due to the acidic the counremain, appearing ted, The exact
nature of [ heir protoplasm. The following are mechanism ofthe Gram reaction is not understood.
staining techniques commonly used in The G rjrn positive cells have J more acidic
bacteriology. protoplasm, which may account for their retaining
Niimph; ‘t uns: Dyes such as methylene blue or the basic primary dye more strongly than the Grain
basic TLICLLSIRI are used for simple staining. '1 hey negative bacteria. DeeolourisaiLon is not an all-or -
provide colour contrast , but impart the same colour ioftC phenomenon. Even Gram fjositive cells may
*
tn bacteria.
all he decolourised bv prolonged treatment with the
Negative aldirling: Here, bacteria are mixed organic solvent . Conversely, inadequate
wich dyes such as Indian ink or nigrosin that provide dccolouiisation may cause all «11 H to appear Gram
a uniformly coloured background against which the positive,I’hc Gram reaction may be related to the
unstained bacteria stand out in contrast. This is permeability of the bacterial ceil wall and
particularly useful in the demonstration of bacterial cytoplasmic membrane to the dye - iodine complex,
capsules which do not take simple stairs . Very the Gram negative, but not the Gram positive cells,
slender bacteria such as spirochetes that arc nor permitting the outflow of the complex during
demonstrable by simple staining methods can be dccolorisstion. The Gram positive bacteria become
viewed by negative staining. Gram negative when the cell wall is damaged.
impTugimlinn methods; Cells and structure ? Gram staining is an essential procedure used
Copyrighted material
10 * Textbook of Microbiology *
in the identification nf bacteria and frequently is resemblance to the radiating rays of the sun when
the only method required for studying cheit seen Jo tissue lesions ( from acras meaning ray
morphology. Gram reactivity is of considerable and myites meaning fungus).
importance as the Gram positive and negative 7 . Mycoplasma* are bacteria that are cell wall
bacteria differ rn > t mertlv in staining thanuteristhin deficient and hence do not possess a Stable
and in structure but also in several other properties morphology;They occur as round or oval bodies
HILL IL as growth requirement ;; , susceptibility to
'
and as interlacing Filaments . When cell wall
antibiotics and pathogenicily synthesis becomes defective , cither
The iCtd fast stain was discovered bv EhrticlL, spontaneously or as a result nf drugs like
whra found that after staining with aniline dyieSs penicillin, bacteria lose their distinctive shape.
tubercle bacilli resist decolouri -sarion with acids. Such cells are called protoplasts, sphenopla &ts
The method, a & modified by Ziehl and Neelsen , is or L forms.
in common use now. The smear is grained bv a
,
j
Bacteria sometimes show characteristic cellular
strong solution of tarhol fuchsin with the arrangement or grouping ( Fig - 2.2 ', . Thus, cocci
application of heat . It is then decolourised with may be arranged in pairs (diplococd ) , chains
20 per cent sulphuric arid and enunterstained with (streptococci ) , groups ol four ( tetrads ) or eight
a contrasting dye such as methylene blue. " Hie acid ( sarcina) , ot a grape - like dusters (sraphylococd) .
fast bacteria retain the fiachsin ( red ) colour , while Some bacilli too may be arranged in chains
the others take the countenstain . Acid fastness has ( streplohacilli ). Others are arranged at angles to
heen ascribed to the high content and variety of each other, presenting a cuneiform 01 ChiriCK letter
lipids , fatty acids and higher alcohols found in pattern ( curyoebacteria ) . The type of cellular
tubercle bacilli. A lipid peculiar to acid fast bacilli, arrangement is determined by Lhc plane through
i high molecular weight hydroxy acid wan which binary fission takes place and bv the tendency
containing carboxyl groups ( mycolic acid ) is acid of the daughter cells to remain attached even after
fast in the free state . Acid fastness is not a property division .
•
of lipids alone but depends also on the integritv of
the Cell wall. >
SHAPE OF BACTERU
Depending on their shape, bacteria are classified
into several varieties ( Fig . 2.1 ): 1
1. Cocci {from kvkkot meaning berry ) are spherical
or oval cells.
2, Bacilli ( from bacuhi ? meaning rod ) arc rod 3
shaped cells.
2. Vibrios are comma shaped , curved rods and
derive the name from their characteristic 4
vibratory motility .
4. Spirilla are rigid spiral forms. 5
5 . Spirochetes {from speira meaning coil and chaffie
meaning hair) are tlexunus spiral forms ,
Copyrighted material
* Morphology ano Physiology -
oF BacCe i LI n
T
i
O to
"•
'£V i
<#% $
3
4
/\
2
m
5 7
n
«- am W
5 < k
/ >
* A
*a
^ 3
w~?
c 1C
1
H
f r
11
.
Fig 2.5 Diagram ol an ideatlsed bacterial ceil :
. .
I Capsule 2 . PIN 3 Outer membrane 4 Division .
seplum & . Ribosome 6 ONA 7 Granular inclu-
Fig. 2.4 Arrangements a I curved bacteria : l vibrio sion; 3 . Flaoel - a 9. Fat globules 10 Mesoaome
2. spirilla 3. spirochetes II. Cytoplasmic membrane 12. Paptldoglycan
Copyrighted material
12 * Textbook of Microbiology s
imav iwssess additional structures. The cell may be their cndotoxic activity and O antigen specificity.
enclosed in a viscid layer, which may he a loose They were formerly known as the Soivin anitgen.
slime layer, or organised is a capsule. Some bacteria The LPS consists oi three regions. Region T is the
CJirTy filamentous appendages protruding from tlir polysKcharide porrion determining the O antigen
cell surface - rhe flagella which are organs nt specificity. Region II is tire core polysaccharide .
locomotion and the ilmbriae which appear io be Region III is the glycolipid portion (lipid A ) and
organs for adhesion. is responsible for the cndotnxk activities, that is ,
The cull wall: The cell wall accounts for the pyrogenicitv, Lethal effect , tissue necrosis ,
shape of the bacterial cell and confers OIL it rigidity .
anticamplcmcntaiy activity', & cell mitogenicily,
and ductili ty. T he cel] wall cannot he seen by d ireel ioiioiiiLoadjuvant property and antitumour activity.
light microscopy and docs not stain with simple The outermost layer of CJram negative bacterial
stain* - lr mjy he demonstrated by plasmolvsis. cell wj.ll j* called rhe outer m«nbriiwT which
When placed in a hypertonic Solution , the contains various proteins known as outer membrane
cytoplasm loses water by osmosis and shrinks, while proteins (OMP). Among these are porins which
the cell wall retains its original shape and sfv.c tor in trans- membrane pores [ bat serve as ditfusior
( bacterial glidEi ) . The cell wall may also he channels tor small molecules- They also serve as
demonstrated hv mierodissectiou, reaction with specific receptors for some bacteriophages.
specific antibody, mechanical rupture of the cell , Cell wall synthesis may be inhibited by many
difiiientiil sraining procedures or by electron fimttfa. Lysozyme, an en/yrtlC MtnuJjy present in
tniavnopfr Bacterial cell walls are about lCb-25 many tissue fluids, lyses susceptible bacteria by
nm thick and account for about 20-50 per cent of split Ling the cell wall inucopeptide linkages. When
the dry weight of the cells. Chemically the eel! lysoTvrne acts on a t iram positive bacterium in a
wall is composed of mucopeptidr (pcplidnglycan hypertonic solution, a protoplast is formed ,
oi mu rein ) scaffolding formed hy N icetyl consisting of [ be cytoplasmic membrane and its
glucosamine and N acetyl mumnic acid mnleailcH With Gram negative bacteria, the result
contents.
alter na ring in chains, which arc cross!inked hy is a spbemplust which differs from the protoplast
Table 2 ,2 Comparison of cell walls ol Gram positive and Gram negalivs bacteria
tX 'C'jT
cytoplasm . They arc more prominent in Gram
positive bacteria. They are the principal sites of
respiratory enzymes in bacteria and are analogous
.
Fig 2.6 Chemical situelure nl bacterial cell Mall to the mitochondria ot eukaryotes. JVfBOSCVTJCJ ate
in that some cell wall material n retained. often seen in relation to the nuclear body and the
ProtopLuts and spheroplasts art spherical , site of syn thesis of cross wall septa , Suggesting that
regardless of the original shape of the bacterium. they coordinate nuclear und cytoplasmic division
Celt Will deficient forms of bactEria may probably during binary fission .
hive a role in the persistence of certain chronic Inlnioytoplasnitu inclusions mav be of
infections such as pyelonephritis, various types, the chief of which arc volutin ,
f Jytopl:] mmc mtilihrjini." The cytoplasmic polysaccharide, lipid and crystals. They are
( plasma ) membrane is a thin (5-10 nm ) layer lining characteristic for different species and depend on
the inner surface of the cell wall and separating it the age and condition of the culture. Volutin
from the cytoplasm. JT aers as a semipermeablc granules (iTHtlchlHnatlC Of flilbes- Efflir gTUIllfar)
membrane controlling the inflow and outflow ot are highly refractive, strongly basophilic bodies
metabolites to and from the protoplasm - Passage consisting of poly me raphosp hate . They appear
through the membrane is nut suldv a function of reddish when stained with polychrome methylene
the molecular siise of the particles but depends, in blue or tohudine blue (ilietHchmmaFia ). Special
many eases, on the presence in the membrane of seaming techniques Such as Albert’s or Neisser’s
specific enzymes ( pomatset ). Electron microscopy demonstrate rhe granules more dearly . Volutin
shows the presence of three layers constituting a granules any charactvnsricallv present ill diphtheria
' unit membrane ' structure. Chemically, the bacilli , Their function is Uncertain. They have been
membrane consists of lipoprotein with small considered in represent a reserve ot energy and
amounts of carbohydrate. Sterols are absent, except phosphate for cell metabolism but they arc most
in mycoplasma. frequent in cells grown under conditions of
Cytoplasm The bacterial cytoplasm is a colloidal nutritional deficiency and tend to disappear when
system ut a Variety of organic and inorganic solutes the deficient nutrients are supplied. Polysaccharide
in a viscous waters solution . It differs from granules may be demonstrated by staining with
Copyrighted material
14 « Textbook ol Microti . olQQy *
iodine, and lipid inclusions wkh fat soluble dyes layer- Capsules too thin to be seen under the light
siu h as Sudan bl . uk . They Appear to be s l u r . i e microscopes are called m rmcajuules. The slime
'
organisms form the main basis for classifying them in its reftactivity. This: capsule swelling or Qiidlung
as prokaryotes and eukaryotes . reaction, described by Ncufcld (1902) was widely
Bacteria may possess extranuclear genetic employed for the typing of pneumococci in the pre-
elements consisting of DNA. These cytoplasmic SLilphonamide days when lobar pneumonia used to
carriers of genetic i nformati^ n are termed plasmids be treated with specific anticapsular sera. Capsules
or epfsomes [see Chapter 6). Besides being protect bacteria bum deleterious agents such as lytic
transm i tted to daughter cells during binary fissh >n, enzymes found in nature. They also contribute to
they may be transferred from one bacterium to the virulence of pathogenic bacteria by inhibiting
another either through conjugation or the agency phagocyrosH. Loss of the capsule by mutation may
of bacteriophages, They are not esserri ;:] for the render the bacterium avirulent. Repeated
life of the cell they inhabit but may confer on it subcultures in vitro lead to the Joss of capsule and
certain properties like tux igeni city and drui; aisu of virulence.
resistance which may constitute a survival Mage ) In: Motile bacteria, except spirochetes ,
advantage. possess "tie or more unbranched, long, sinuous
Siimu layer and capsule: Mary bacteria filaments called flagella, which arc the organs of
secrete a viscid material around the cell surface. locomotion. Each flagellum consists of threedistinct
When this is organised into a sharplv defined parts, the filament , the hook and the basal body.
structure , as in the pneumococcus, it is known as The filament is external to the cell and connected
the capsule . When it is a loose undemarcared to the hook at the cell surface.
scenerion, as in leuconostoc, it is called the slime -
The hook basal body portion is embedded in
opyrighted n iteria
* Morphology and Physiology ol Bacteria * 15
the cell envelope . The huok and basal body Are (lophotxichous) AS in spirilla or wirh flagella at
antigeoicaily different. Mechanical detachment of both poles (amphiitrichcnis).
the fJimtnt does not impair the viability of the Flagella arc less than 0-02 pm in thickness and
cell. The flagella are 3-20 pm long and arc of hence beyond the limit of resolution of the light
-
uniform diameter (0.01 0.013 pm ) and terminate
in a square dp. The wavelength and thickness of
microscope . They' may, in some instances, be seen
under dark ground illumination. They can be
the filament are characteristic of each species but visualised by special staining techniques in which
some bacteria exhibit hipliclty, that is, they have their triiclcntsh is increased by mordanting , or by
flagella of two different wavelengths ( Fig . 2 , 8) , electron microscopy ( Fig. 2.10 ) . Due to the
Flagella are made up of a protein (flagellin ) similar difficulty of demonstrating flagella directly, their
to keratin or myosin - Though flagella of different presence is usually inferred from the motility of
genera of bacteria have the same chemical bacteria. Motility can be observed by noting the
composition ,, they are antigenic ally different . spreading type of growth on a semi -solid Agar
Flagellar antigens induce specific antibodies in high
titres. Flagellar antibodies are not protective hut
.
medium. Under the microscope Active motility has
to be differentiated from the passive movements of
are useful in serndiagnosis. the cells , either due to air currents or due to
The presence or absence of flagella and thei r Brownian movement. Bacterial motility may range
number and arrangement are characteristic of from the slow 'stately ' motion ofpcriirichatc bacteria
different genera of bacteria ( Fig. 2.9 ), Flagella may [ for example, Bacillus ) to the darting movement of
he arranged all round the cell ( peritridirius} as in polar flagellated vibrios . The cholera vibrio may
typhoid bacilli, or situated at one or both ends of move as fast as 200 pm per second .
the cell ( pulaf ) . Polar flagella irtuy be single Fimhnw Some Gram negative bacilli carry very
( monorrlcltous) as in cholera vibrios, in tufts fine , hair -like surface appendages called fimbriae
or pili. They arc shorter and thinner than flagella
( about 0.5 pm long and less than 10 nm thick ) and
project from the cell surface as straight filaments.
%i
JESjjp * T- -
‘
W
, A* * "• r^ mjm ^
'
i" m the cell membrane. Fimbriae can be seen only under
the electron microscope. They are unrelated to
motility and are found qn motile as well as
nanmotilc cells. 'Hiey are best developed io freshly
isolated smirii and in liquid Cultures. They tend to
disappear following subcultures on solid media -
Fimbriae function as organs of adhesion, helping
'
%: '
the cells to adhere firmly to particles of various
kinds . This property may serve to anchor the
bacteria in nutritionally favourable m i c r o
environments. Fimbriated bacteria form surface
.
Fig 2.7 Pnfcumocacd negatively stained with India pellicles in liquid media. Many fimbriated cells (for
ink to show capsule example Fscherichia , Klebsiella ) agglutinate rod
Copyrighted material
16 + Textbook of Microbiology
z
l
J
the pentn 11 aciHus and C1«rrldium have rhe ubili ty
I 5 to form highly rc ^isLuni refit rig Stages called spores
] ,
Copyrighted materia
i Marpnatagy and P^iysioiocjy of Idadu^. f 17
at its core rhe nuclear body, su mounded by the spore should ensure that spores also are destroyed.
wall, a delicate membrane from which the cell wall Spoliation helps haetcria survive for long periods
of the future vegetative bacterium will develop, Outside under unfavourable environments .
this LS the thick spore COXtCX, which in turn i.s enclosed When transterred to conditions conducive for
by 2 multiJayered tough spore coat. Some spores have growth* spores germinate. The spore loses its
an additional outer covering called exDSfJorium, which refractiliiy and sw ells. The spore wall is shed and
may have distinctive ridges and grooves (Fig- 2, 11) . the germ cell appears by rupturing the spore coat
New amij trts appear on sporulatum. and elongates to form the vegetative bacterium.
^
Young s[K>res we seen attached to the parent Spores maybe seen in unstained preparation as
cell. The shape and position of the spore and its refract!le bodies. The fcrespore stains intensely,but
si ye relative to the parent cell ;Lre species once the spore envelope is laid down , the spore
characteristics . Spores may be central .
d<ies not stiiin readily. Spores appear as unstained
(equatorial), terminsil or suhtrrminnl . They may be areas in Gram stained preparations, hut being more
aval [ ]r spherical. Ifocy may or may not distend the acid fast than the vegetative cell-s, they can be stained
bacillary body (Fig. 2.12 ). by A modification of rhe Ziehl- Neclsen technique,
Bacterial spares constitute some of the most Pluouflorphiim and involution forms;
resistant forms of life. They may remain viable for Some species of bacteria exhibit great variation In
centuries,I ' ’hey are cxtrenvcly resistant to desiccation the shape and size of individual cells 1 his is known
and relatively so to chemicals and heat . Though AS pltomorph ism. Certain species ( for example,
some spores may resist boding for prolonged plague bacillus, gonococcus ) show -swollen and
periods , spores of all medically important species Aberrant forms in ageing cultures, especially in the
are destroyed by autoclaving at L 20 for 15 presence of high salt concentration. These arc
minutes- Methods of sterilisation and disinfection known as involution forms . Many -of the ceils may
H
..
:
J 3
1 1 12
3 E*
51 6
.
Fig. 2.12 Types of bacterial spores. 1. central, bulging , 2 central , not bulging. 2. suSterminaE. bulging,
4. subliminal . rwl bulging, 5. terminal, spherical 6. terminal, oval.
Copyrighted material
H Morphology and Physiology ol Bacteria » 19
media form colonic . Each colony represents a done In the plating method, appropriate dilutions are
of cells der. ved from a single - urcuC cell. In liqLud inoculated on solid media, either on the surface of
media , growth is diffuse . places or as pour plates. The number of colon ics that
Bactef d growth may be con > idered at two lcvds, develop after incubation give? an estimate ofthc viable
increase in the size of the individual odl and increase count . The method commonly employed is that
,:i the number of cells. The former is ordinarily limited described by Miles and Misra. [1938 ) in which serial
and when the critical si * is reached , the cetl divides, dilutions are dropped on the surface of dried plates
Except when cetl division is inhibited bv substances and colony counts obtained.
like pci lictQin or ac riflaii nc or bygrowth in magnesi um
deficient media . Growth in numbers can be studied ftxct HRIAL GROWTH CURV E
hv bacterial counts. Twii types ofhactcri.il counts can When a bacterium is seeded into a s - iirablc liquid
'
be Tuade— total counl and viable count . medi um and i ncuhated , its growth follows a defini:c
'Ihe total count gives the tota ] number of cells course, If bacterial counts are made at intervals after
in the sample , irrespective of whether they are living inoculation and plotted in relation to rime, a growth
or not. It can he obtained by curve is obt . Lined [Fig. 2.13). The curve shows the
1 . direct counting under the microscope u - ing following phases:
counting chamber:, Lug phase: Immediately following the seeding
2. counting in an electronic device as in the Coulter of a culture medium , there is no appreciable
counter, increase in numherH , though there mav be an
3 - direct counting u^ing stained smears prepared increase in the size of the cells . This initial period
by spreading a known volume of the culture is the time required for adaptation to the new
over a measured area of a slide , environment , during which the necessary en-zymcR
4. comparing relati ye numbers i 1 i smears ofthc culture and metabolic intermediates are built up in adequate
mixed with known numbers of other cells, quantities for mult ip lie at ion to proceed . The
5 . by opacity measurements using an absnrptfo dura non of the lag phase varies with the species,
meter or nephalometer, size of inoculum , nature of culture medium and
6 . by separating the cells by centrifugation or environmental factors such as temperature .
filrration and measuring their wet or dry weight , I ( ) J£ ( LOGFLRILHMIC ) OT exponential! plnisel
H
Copyrighted material
20 ^ Te * tbooK of Microbiology *
TWE
reactions and also forms an integral port of the
pUltOplilHlTL.
.
Fig 2.13 Bacterial growth curve. The viable count Racteriit L-an he classified nutritionally, based on
.
shows tag. log slationary and decline phases In . rheir energy requirements and on their ability to
the total count, ihe phase ol decline Ls not evidenl- synthesise essential metabolites . Bacteria which
nulritionfll exhaustion and toxic accumulation , LL !1 derive their energy from sunlight are called
death may also he caused :iuit ( i]ytir enzymes .
by piotofrophs and those that obtain energy from
When the total ex Hint is plotted , it parallels the chemical reactions arc called chcmorrophs. Bacteria
viable count up to the stationary phase hut it that can Synthesise all their organic compounds are
continue* steadily without any phane of decline. called autotrophs - Those that are unable to
With autolytic bacteria, even the total count shows synthesise their own metabolites and depend on
a phase of decline. preformed organic compounds are called
The various stages of the growth curve are , hercrofropfrs. Autotrophs arc able to utilise
associated with morphological and physiological atmospheric carbon dioxide and nitrogen. They are
alterations of the cells. Bacteria have the maximum capable of independent existence in water and soil
cell size towards the end of the lag phase. In the and are of no medical importance, though they are
log phase, cells are smaller and stain uniformly. In of vital concern in agriculture and the maintenance
the stationary' phase , cells frequently are Gram of soil fertility- Meterotrophic bacteria arc unable
variable and show irregular staining due ro the to grow with carbon dioxide ax the sole source of
presence of intracellular storage granules. carbon . The nutritional requirements of
Sporulation occurs at this stage. Also, many bacteria heterotrophs vary widely. Some may require only a
produce Secondary metabolic products such as single organic substance such as glucose , while
exofnjtins and antihintics. Involution forms are others may need a large number of different
common in the phase of decline . compounds such as ami noacids , nucleotides, lipids,
carbohydrates and coenzymes.
HACTRRIM Ml TRTTION Bacteria require a supply of inorganic salts,
I'hc bacterial cell has the same general the mi cal particularly the anions phosphate and sulphate* and
pattern as the cells of higher organisms. The the cations sodium , potassium , magnesium, iron,
principal const ] ruent of bacterial cells is water, manganese and calcium . These are normally present
pyrighted materia
k. l* l El
H Morphology and Fhy oiogy ol Bacteria > 2\
in the natural environments where bacteria live but The reason for the apparent toxicity of oxygen
will have to be supplied in culture media. Some
ions such as cobalt maybe needed in nacr amounts-
-
lor anaerobic bacteria i i not well understood. If
has been suggested that in the presence of oxygen,
Sume bacteria require certain organic hydrogen peroxide and other toxic peroxides
compounds in minute quantities . These are known accumulate . The enzyme catalase which splits
as growth factors or bacterial vitamins. Growth hydrogen peroxide is present in most aerobic
factors are called «»ntuf when growth docs not bacteria but is absent in the anaerobes . Another
occur in their absence, or accessory when they reason might be that obligate anaerobes possess
enhance growth, without being absolutely necessary essential enzymes that are active only in the reduced
factor is inoculated into a medium containing an hydrogen acceptors other than oxygen. Facultative
excess of all Other nutrients, it ? growth will be anaerobes may act in both ways. In the case of
proportional to the amount of the limiting substance aerobes , where the ultimate electron acceptor is
added. Within a certain range, the concentration atmospheric oxygen (aerobic respiration ), the
of the growth factor will bear a linear relationship carbon and energy source may be completely
to the amount of growth of the organism. This is oxidised to carbon dioxide and water. Energy is
the principle id microhiuLogical assays, which
provide a very sensitive and specific method for
provided by the production of energy rich
phosphate bonds and the conversion of adenosine
-
estimation of many aminoacids and vitamins, as in diphosphate ( ADP) to adenotine triphosphate
the determination of vitamin Bl 2 using ( Ad P ). This process is known as nxidptive
LactofwcitfufT fckhnwnnil .
phosphorylation Anaerobic bacteria use as electron
acceptors compounds such as nitrates or sulphates
D\
^3 EN REOUJKEJUENT AINU instead of oxygen (anaerobic respiration ), A more
M i -. r A i K i i . i s M common process in anaerobic metabnli ^ m may be
Depending on the influence of oxygen on growth a series of axidoreduccions in which the carbon
and viability bacteria arc divided into aerobes and and energy source acts as both the electron donor
anaerobes. Aerobic bacteria require oxygen for and electron acceptor. This process is known as
growth. They may be obligate aerobes like the fermentation and leads to the formation of several
CII^ -JCNL vihrii :., which will jp-fra- i >:ilv in ihc ptencriCr ^ 11 1:11:11 end prutiucl- K such as .11 i 4 - :LHIL .11 . phitif , .i -,
^ as of gas (carbon dioxide and hydrogen ).
' '
L
anaerobic bacien .i, what Is more important L 11-.11: growing at temperatures as low as -7 °C. They arc
the presence or absence of oxygen is the state of soil and water saprophytes and though not of direct
oxidation of the environment , 'fhc oxidising or medical i mpoitance, may cause spoilage of refrigerated
reducing condition of a system is indicated by the food. Another group of nonpathogenic bacteria, the
net readiness of all the components in that system
GO take up, oe parr with electrons. This is known as
thcimophrfcs, grow best at high temperatures, 55
90 UC, They may cause spoilage of under processed
-
— -
the oxidxtinn reduction \ redox ' potential of the
system. The redox potenti .d of a medium is best
canned food - Some thcrmophilcR [ like Baiv / Zus
STCAROTHERMOPHJ J U F ) form spores that are
'
'
estimated try measuring the elccliical putenti J exceptionally thermores istant. Extremely
difference set up between the medium and an thermophilic bacteria have been identified which
unattackable electrode immersed in it . This can grow at temperatures as high as 250 flC -
electrode potential ( Eh ) car be measured in Bacteria also differ in the effect of temperature on
millivolts.The more oxidised the system, the higher viability. Heat is an important method for the
the potential. A simpler , though less accurate, destmcri'- in of microorganisms (sterilisation), moist
method of measurmg the redox potential is the use heat causing coagulation and denafluratfon of proteins
of oxidation-reduction indicators such as methylene and dry heat causing oxidation and charting. Moist
blue, and noting the change in colour. heat is more lethal than drv heat . The lowest
temperature that kills a bacterium under standard
CARBON DIOXIDE conditions in a gircn lime is known as the themed
All bacteria require smalt amounts of carbon deathporrtf. Under moist conditions most vegetative,
dioxide for growth.Tics requirement is usually met mesophikc bacteru have a thermal death point
,
by the carbon dioxide present in the atmosphere, between 50 and 65 JC and most spores between
or produced endogenously by cellular metabolism. 100 and 120 *C .
Some bacteria like BtuCella abottui require much Ai low temperatures some species die rapidly
higher levels of carbon dioxide (5-10 per cent) for but most survive well. Storage m the refrigerator
growth, especially on fresh isolation (capnophilic). - -
(3-5 "C) or the deep freeze cabinet ( 30 to 70 “ C)
is used for preservation of cultures. Rapid freezing
TBMhfiftATt itH as wi tii solid carbon J iodide or the use of a stabil i her
Bacteria vary in their requirements of temperature such as glycerol, minimises the death of cells on
for growth. For each species, there is a 'temperature freezing,
'
range and growth does not occur above the
,
ma’i i 1 num or below the mini mum of the range. The Moira Dm INC
UK AND
temperature at which growth occurs best is known Water is an essential ingredient of bacrenal
,
as the "optimum temperarurr winch in the case of
F protoplasm and hence drying i * lethal to cells.
most pathogen- -.. bacteria is 37 VC , Batten :* which
,
'
However, the effect of drying varies in different
grow best at temperatures of 25^40 “ C are called species. Some delicate bacteria like Treponema
mesophihe. All parodies of warm blooded animals pallidum arc highly sensitux, while others like
are mesophilic Within the group of mesophuc staphylococci withstand diving for months. Spores
bactcri i, some like Pseudomonas aeruginosa have are particularly resistant to desiccation and may survive
1 wider -
range (5 43 *C ) t while others like the
-
: gonococcus have a restricted range (30 39 *C ).
ill the dry state for several dccadiB. Drying in vacuum
111 thecold ( &W 3rdiyitigar haphils$aQnn) is a method
Paychruphilic hacteria are those that grow best for the preservation ofbscteria , viruses and many labile
at temperatures below 20 aC , some of them even biological materials.
Copyrighted material
* Mofphafogy and Pfiy$i$logy o( Bacteria * 23
Further heading
.
Kl iir DF 1995. How bacteria
tease and swim. Anmrad flrv Mirrnbioi tf lN.
^
Dawes |W -snd W SudKrlupd LVNI . Mfcmbiat Phyjkrftjfi ) „ 2^ Ed. UnlunL: EJIKILWCLL
CiuuLd. GW and A Him£ |WJB Thf Rorlrrial Spprf . Um m: Acadflnic PtCH.
Mmat AG and JW Fcwter 19-95. jWirnpJwcj/ Phyiinlofry ,
'
* -
edn. NEW Vcirfc ; Wikv Liss,
Sanief RY e ( * \ IM Thr Microbial World* 5* edn. Ne Jersey: PrcrUke-HaJI.
*
Copyrighted material
Sterilisation and Disinfection
Microorgamitny are ubiquitous. Since they cause before sterilisation or disinfection, by removing soil
COntaiflination , infection arid decay, i [ becomes and ocher dirt and reducing rhe microbial burden ,
nccessuy to remove or Jestrov them from iQdfleflAh nukiiig seeriEisadon more efTaiiw- Deconianiinarian
cir from ureas. This is the obicct of sterilisation. refers to the process of rendering an article or area
The process of sterilisation is used in microbiology free of danger from contaminants, including
for preventing cun laminar ion h y ext raucous microbial , chemical, radioactive and other hazards.
organisms , in surgery for maintaining asepsis, in The various agents used in sterilisation cun be
food and drug manufacture for ensuringsafely from classified as follows :
contaminating organisms, and in many other
A . Physical a fit ms 1
Sierilhaiiwi is defined as the process by which normal pressure, steam under pressure.
an article, surface or medium is freed of all living 5. Filtration: candles , asbestos pads, membranes .
6. Radiation.
microorganisms cither in rhe vegetative or spore
state. Djarlfcftl'o/) rnflttU the destruction or rcntdval 7 . Ultrasonic and sonic vibrations -
of all pathogenic organisms , or organisms capable B Chemiunls
,
of giving rise to infection. The term dfltrfqxu is 1 . Alcohols: ethyl , isopropyl , irichlorobutand .
used to indicate the prevention of infection, usually 2. Aldehydes: formaldehyde, glutaraldchydc,
by inhibiting the growth of bacteria in wounds or 3. Dyes.
tissues. Chemical diisintectants which can lie safeiv *1
4. Halogens.
applied to shin or mucous membrane and are used 5. Phenols,
to prevent infection by inhibiting the growth of 6. Surface-active agents.
bacteria are called anfjstynfjcs. agents 7 . Metallic salts .
or art; those which are able to kill H . Gases: ethylene oxide , formaldehyde, beta
{
bacteria . facte rjfwtxnr agents only prevent the propinlaclone.
multiplication ot bacteria which may, however,
remain alive, A chemical which is bactericidal at a Si NLIQHT
particular concentration , may only be bacteriolttatic Sunlight possesses appreciable bactericidal activity
at ahigher dilution. and pi ays an important role in the spontaneous
L leaning, though a routine nontechnical sterilisation that occurs under natural conditions,
procedure , plays an important preparatory role fht action is primarily due to its content of
Copyrighted materia
* Steri Ionian and DiF. miRr.rian 25
ultraviolet rays , most of which , however, are surface. In the case of the spore, steam condenses
screened out by glass and the presence of ozone in on it, increasing : ts water content with ultimate
the outer regions of rhe atmosphere. Under natural hydrolysis and breakdown of the bacterial protein.
circumstances.
-
conditions, its sterili. ing power varies according to
Direct sunlight , ns in tropical
-
I n a completely moi ture-ffee atmosphere, bacteria ,
like many proteins, sir more resistant to heat. They
countryside where it is not filtered off by impurities are killed when ax ids Lion of the cell constituents
i:i the atmosphere, has an active germicidal effect due occurs and this requires much higher temperatures
In the comhined effect of ultraviolet and heat rays. than that reeded for coagulation of proteins .
Semple and Grieg showed LII . U in Lidia, typhoid bacilli The time required for sterilisation is inversely
exposed to the sun on pieces of white drill doth were proportional to the temperature of exposure and
killed in two hours, whereas controls kepr in, the dark can be expressed as 'thermal death time', which is
were still alive after six days- Bacteria suspended in the minimum time required ro kill a suspension of
water are readily destroyed by exposure to sunlight . organisms at a predetermined temperature in a
specified environment. The stei disarm time is
I ) \< 'i i :s t i related to the number of organisms in the
Moisture is esSenlial for the growth of haute tin. suspension, presence or absence of Spores, and the
Four-fifths of the weight of the bacterial cell is strain and characteristics of organism . The
due ro water . Drying in air has therefore , a recommended minimum sterilising times do not
deleterious effect on many bacteria . However, this include the lime taken tu reach the specified
method is unreliable and is only of theoretical temperature. 'I'he nature of the material in which
interest. Spores are unaffected by drying. the organisms are heated affects the rate of killing.
The presence of organic substances, proteins, nucleic
I l l- .iciils , starch, gelatin, sugar, fats and oils, increase
Heat i -; the most reliable method of iterilisarion the thermal death time. The presence of
and should be the method of choice unless disinfectants and high acid or alkaline pH hasten
contraindicated. Materials char may be damaged bacterial killing.
by heat can be sterilised at lower temperatures, lot
longer periods or by repeated cycles. The factors DRY HEAT
influencing sterilisation by heat are: Flaming: Inoculating loop or wire , the tip of
—
1 - nature of beat CITY heat or moist heat,
2. temperature and time,
forceps and searing spatulas are held in a bunsen
flame till they become red hat. Inoculalion loops
2. number of microorganisms present * canrying infective material may be dipped in a
4. character I its of the organisms* such as, species* disinfectant before flaming to prevent spattering.
^
strain , sparing capacity, and Incineration: This is an excellent method for
5. type of material from which the organisms have safely destroying materials such as contaminated
to be eradicated . clorh, animal carcasses, and pathological material*.
Tbe killing effect of dry heat is due Bo prate in Plasties such as PVC and polythene can be dealt
dcnatur .ition, oxidative damage and LIIC toxic effeci with similarly bur polystyrene materials emit clouds
of elevated levels of electrolytes. The lethal effect of dense black smoke and hence should be
of moist heat is due to the denaluration and autoclaved in appropriate containers.
coagulation of protein. The advantage of steam lies Elm tiir oven: This is the most widely used
in the latent heat liberated when it condenses on a
cooler surface , raising the temperature of that
method of sterilisation by dry heat, A holding pen uni
of 160 °c far are hour is used to sterilise glassware .
Copyrighted material
2 f> * Textbook of Microbiology
r - j
ct L
j
i .' I
j
A Brownes tube (green spat ) is available For
dry heat and is- eemvenient for routine use* After
proper sterilisation a green colour is produced (after
60 minutes at 160 UC or 115 minutes at 150 °C).
Thermocouples may also be used periodically.
MOIST HEAT
Temperatures below LOO *C
For pasteurisation of milk : Jht milk is ,
Ftg . 3.1 Ho! air awn heated at either 63 °C for 30 minutes ( the holder
forceps, , udpdj, all-gloss syringes , svrabi,
JcnoR method ) or 72 JC for 15-20 seconds ( the flash
some pharmaceutical products such as liquid process ) followed by cooling quickly to 13 °C or
paraffin , dusting powder. fats and grease. Hot air LS lower. By these processes all iionsporing pathogens
a had conductor at heat and Ltn penetrating power such as mycobacteria bruceliac and salmonellac
15 |tr/v- The OKU is usually li elite L! hy dectxidty arc destroyed- CojfjeWa humeri/ is relatively heat
with hearing elements in the wall of the chamber. resistant and may survive the holder method.
It must be fitted with a Fid to ensure even Vaccines at non sparing bacteria ate heat
distribution a I air Sind elimination of air pockets -inactivated jjp special vaccine baths at 60 :IC tar
( Fig. 3.1), It should not be overloaded . The [ lucerial one hour- Set lien or body fluids containing
should he arranged so as to allow free circulation cnagulable proteins can he sterilized by hearing for
of air in between the objects. Glassware should be one hour at 56 *C in a water bath on several
perfectly dry before being placed in the oven. Test successive dlavs
Jr
.
tubes and flasks should he wrapped in paper . Media auch as Lowcnstcin — Jensen and
Kubbcr materials, except silicon rubber, will not LocftlcTs scrum are rendered sterile by hearing at
withstand the temperature. At 18041 C cotton plugs DC lor half m hour on three successive davs
may get chaired . Cutting instruments such as those in an inspissator.
u&ed in ophthalmic imgrry, should Ideally he Though practically all mesophilic nonsporing
sterilised for two hours at t SO ' C . The British
"
bacteria are lolled by exposure to moist beat at (iQ “ C
PfadftKHtopotH recommends a holding time ot one for 3 U minutes, Staphylococcus aureus and
hour at 150 “ C for oils , glycerol and dusting Strcjiwocccuf facetlit require f > 0 m inn ten . A
powder. The oven must be allowed to coo! lowly
for about two hours before the door is opened, sin re
- temperature of SO °C for 5-10 minutes destroys
the vegetative forma of all bacteria , yeasts and
the glassware may crack due to sudden or JiiL- vcn monMs. Among the most heat resistant cells are
cooling the spores of CfoytriJiurn botulinum which require
Copyrighted materia
* Sterilisation and Disirof ration » 27
'
recommended I D: Sterilising of instruments lined
for su rgicftl procedures and should be regarded only
as a means of disinfection. Nothing short of
autoclaving it high pressure Cad destroy spores and
ensure sterilisation. Hard water should not be used.
Sterilisation may be promoted b) the addition of
29f» sodium bicarbonate to the water.
In rases where boiling is considered adequate,
the material should be immersed in the water and
boiled for 10-30 manures. The lid of the steriliser Fig.. 3.2 Steame#
should not be opened during the peri -. hi. in a favourable medium, will germinate and be killed
Steam til slmosphuric pressure on the subsequent occasions. Though generally
tlGO k An atmosphere of free steam is used to adequate * this method may tail with spores oi
^
' l
sterilise culture media which may decompose if certain anaerobes and thermophiles..
subjected to higher temperatures . A Koch or Arnold
,
Steam under pressures I he principle ot the
steamer is usually used . ( Laboratory autoclaves cart autoclave car steam steriliser is that water boils when
also be used for this purpose.) This is an itsvapour pressure equals that of the surrounding
inexpensive method . I’hc container and medium are atmosphere. Hence when pressure inside a closed
simultaneously sterilised , evaporation from the vessel increases* the temperature at which water
medium Is prevented and the apparatus requires boils also increases Saturated steam has penetrative
little or mi attention. power. When steam comes into contact with a
The steamer consists of a tinned copper cabinet cooler surface it condenses to water and gives up
with the walls duirahlv lagged . " I "he lid is conical , its latent heat to that surface ( 1600 ml steam at
enabling drainage of condensed steam , and a 100 “ C and at atmospheric pressure condenses into
perforated trav fitted above the water level ensures one ml of water at 100 *G and releases 518 calorics
that the material placed on it is surrounded by steam of heat). The large reduction in volume sucks in
( Fig , 3.2 ), A tingle exposure of ninety minutes more steam to the area and the process continues
usually ensures sterilisation hut for media containing till the temperature of that surface is raised to that
sugars or gelatin an exposure of 100 ,:C for 20 of rhe steam . The condensed water enwnes moist
minutes on three successive days is used . This is conditions tor killing the microbes present.
known as tvjuJai riarj'oji oi infemjTtrejj; sreriisaffon.
T
^
The principle is chit the first exposure kills all
Sterilisation by steam under pressure is carried
out at temperatures between 10B UC and 147 ^C.
vegetative bacteria , and the spores, rincc they are By using the appropriate temperature and time, a
Copyrighted material
28 < Textbook oi Microbiology *
out in the pail the discharge tap is closed. The mininrure autocUre and maybu used for srerilnirLg
steam pressure rises inside and when it reaches the small articles in climes and similar csublishmcNh.
desired set level, the salety valve opens and the A ivude variety of JLIRNCFIVES have been
excess steam escapes. From tliis point, the holding manufactured incorporating carious devices fur
period is calculated. When the holding period is overcoming these defects and other difficulties in
over, the heater is turned off and the aui^lave working.
allowed to cool till the pressure gauge indicates Sterilisation cofttrab Far determining the
that the pressure inside is equal to the atmospheric efficacy af moist heat sterilisation,spores offiflerffus
pressure . The discharge tap is opened slowly and stcurothamephilur are used at the ctsi arganisio,
air is let into the autoclave. If rhe cap is opened I his LS a thermophilic otpuniui wirh nn optimum
when the pressure inside is high, Liquid media will growth temperature of 55-60 C and irn spores
Copyrighted material
* Sterilisation and Disinfection » 29
testing water aainpLes for vhulera vibrinfR or typhoid says ate of die rtonionisiuig low energy type* while
bacilli. Bacteria ] toxins can be obrai]ied by passing gamma rays and high energy electrons are the high
cultures through filters. energy" ionising type.
The following types of filters have been used. Monionising radiation: Here electromagnetic
( Randle filters ; These arc manufactured in rays with wavelengths longer than those of visible
different grades of pnrositv anti have been used light are used. These are to a large extent absorbed
widely for purification of water for industrial and as heat. Hence infrared radiation can be considered
drinking purposes. They are of two types: (a) Un- as a form of hot air sterilisation. Infrared radiation
glued ceramic filters (for example, Charnberbm! is used for rapid mass srerilisation of prepacked
and Dimlton lliter ; 0> ) Dialnmaceoti* earth filters items: such as syringe* and catheters , Ultraviolet
( for example, Berlucfcld and Mandler filters). radiation is used for disinfecting enclosed areas such
,
Copyrighted material
- 30 * Textbook or M crobiotogy
vital constituents . They have very high penetrative concentration of the substance;
power. Since there is no appreciable increase in * time of action;
temperature in this method, it is referred to as cold pH of the medium;
sterilisation , Commercial plants use gamma * temperature;
'
radiation far sterilising irons like plastics, syringes, * nature Ot rhc organisms; and
swabs* catheters , animal feeds ^ cardboard , oils, presence of extraneous material;
greases , fabrics anti metal toils. Chemical agencs act in various ways. The main
1 ligh energy electron radiation ns a method of modes of action arc :
sterilisation in not widely used in medicine . 1. protein coagulation;
2, disruption of cell membrane resulting in
ULTRASONIC AND SONIC VIBRATION exposure, damage or Id&J of the Contents;
Ultrasonic and sonic waves arc credited, with 3, removal of free sulphydryt groups essential for
bactericidal powers but the rcs- ults have been Line functioning of the enzymes; and
variable. Mirftnrg niuiu vary in their sensitivity 4 . substrate competition : A compound resembling
^
tn them and survivors have been round after such the essential substrate of the amine diverts or
r
.
treatment Hence this method is of no practical misleads rhe tnymes necessary for the
value in sterilisation and disinfection. metabolism of the cell and causes cell death.
AtJEXTtt
( llTKM U L A l . Ai . ciiuni s
Several chemical agents are wed as antiseptics and Ethyl alcohol (ethanol) and isopropyl ulcoltol are
disinfectants. However little is known about the the most frequently used . They sire used mainly as
mechanism of action of many of these agents. An skin antiseptics and act by denaturing bacterial
ideal antiseptic or disinfectant should : proteins. They have no action on spares, To be
have i wide spectrum of activity and must be effective, they must be used at a concentration of
effective against all microorganisms , that is, 6( b-9fl per cent in water. Protein slows its action
bacteria including spores, viruses, protozoa and whereas 1% mineral acid or alkali enhances the
fungi; action. Isopropyl alcohol is preferred as it is a better
* be active in tile presence of organic matter; fit solvent, more bactericidal and less volatile . It is
"
be effective in acid -as well , LH alkaline media; used far the disinfection of clinic , LI thermometers .
* have speedy action; Methyl alcohol is effective against fungal spores
* have high penetrating jHivver: and is used for treating cabinets and incubators
* be stable; affected by them. The insides of the chambers arc
* be compatible with other antiseptics and wiped with liberal amounts of methanol A pad
disinfectants; moistened with rue than > 4 and a dish of water ( to
* not corrode metals; ensure hi h humidity ) are kept insidiCj and rhe
* not cause local irritation or sensitisation;
^
incubator is left at working temperature for several
* not interfere with healing; hours. Methyl alcohol vapour is roxic and
* not be toxic if absorbed into circulation; inflammable.
* be cheap and easily avail able; ard be safe and
easv to use. AUD - ll V lilts
Such an ideal chemical is yet to he found . Fbimidchj/de is active against the amino group in
The factors that determine the potency of the protein molecule . In aqueous solutions, it is
disinfectants arc : markedly bactericidal and sponodol and also has a
Copyrighted material
* Stanlisancm ana Disinfection > 31
lethal effect on viruses . It is used to preserve cell . I he sc dyes arc used in the microbiology
:i n ; Lh > iiiLL - J sped mens. and for destroying anthiU* htontny as selective agents in culture media.
sj>ores in hair and wook 10 formalin containing The acridine dyes ate mote active against Gram
0.5% sodium tetraborate is used to sterilise dean positive organisms than against Gram negative but
metal iiLHTnjmenrh. tire not av selective as the aniline dies . They are
Formaldehyde gas is used for sterilising affected very Little by the presence of organic matter.
instruments and beat sensitive catheters and for The more important dyes arc proflavine, acriflavine,
fumigating wards., sick rooms and laboratories. cuflavint and aminicrinc. They show no significant
Uradi i proper!v controlled Conditions, clothing,
'
di fferences in potency. J f impregnated in gauze, they
bedding, forniture and books can he satts.factcariLi' are slowly released in a moist environment , and
disinfected. hence their advantage and use in clinical medicine.
Tic gas is irritant ami toxic when inhaled. They ini pair the DNA complexes of the organisms
Surfaces which haw been disinfected by [ his agent and thus kill or destroy the reproductive capacity
may give off an irritant vapour for some time after of the cell.
.
disinfection Ill in can be nullified by exposure tea
ammonia vapour after disinfection has been HALOOFXS
completed , Iodine in aqiKOUl and alcoholic solution has been
fillutanaldchyde: This has an action similar to used widely as a skin disinfectant . It is actively
formaldehyde . It is specially effective against bjlCttfitidal, with modflltt action against spores.
tubercle bacilli , fungi and viruses. It is less toxic Ir is active against the tubercle bacteria and viruses.
and irritant to the eves and skin tk.ni formaldehyde. Compound* of iodine with nonitmic welting or
It has no deleterious effect on the cement or lenses surface active agents known HR iodophores arc
of instruments such as cyito &copes and claimed to be more active than the aqueous or
bronchoscopes, li can be safely used to treat alcoholic solutions of iodine.
corrugated rubber anesthetic tubes and lace mask -- , Chlorine and its compounds have been used as
pbstk endotracheal lubes, metal instruments and disinfectants for many years- Water supplies,
pulythene tubing. swimming pools , food and dairy industries use
chlorine for disinfection . Chlorine is used
I1 v \: s commonly as hypochlorites . Chlorine and
Two groups of dyes , aniline dyes and acridine dyes hypochlorites are markedly bactericidal. They have
are used extensively as skin and wound antiseptics. a wide spectrum of action against viruses . The
Both are bictfirioftalic in high dilution but are of organic chloramine* are u*ed an antiseptics for
low bactericidal activity. The aniline dyes in use dressing wounds,
are brilliant tfreen, malachire green and crystal violet.
They are more active agains- l Gram positive PH K .M t l L S
organisms than against (dram negative organisms. These are obtained by distillation of coal tar
They have no activity against tubercle bacilli , and between temperatures of 170 "C and 270 G , 1 ,ister,
fie nee i fie use of malachite green in rite the father of antiseptic surgery, first introduced their
LoWCt Lite in-Jen sen medium. Though they are usv in surgery [1365). Since then A wide range of
nonirritant to the tissues and nuntoxic, lliev are phenolic compounds hare been developed as
considerably inhibited hv organic material such at;
'
disinfectants [ fie Lethal effect of phenols is due to
,
pus. Their lethal effectR on bacteria are believed to their capacity to cause cell membrane damage,
be due to their reaction with the acid groups in the releasing cel] contents and causing lysis. Low
Copyrighted material
n 4 Texlhook of Microbiology »
chlorojtyphenois, though less toxic and irritant, are formaldehyde gas is generated by adding 150 g of
less active and more readily inactivated by organic KMnO, to 2E0 ml formalin for every 1000 LLL. ft
matter. fforh these gn >ups of substances arc relatively .
[ 2 S. 3- cu . ]Ti ) of ruom volume The reaction produces
inactive Against Pseudomonas . Various considerable heat , and so heat resistant vessels
combinations of these are used in the control of should be used. Alter starling generation of
pyogenic cocci in mrgicfll and neonatal units in tormaldrhyde vapour, the doors should be scaled
hospitals. Hcxachtorophene Is potentially toxic and ,rnd left
unowned fur 4S lumrs.
should be used with care . Chlorhexidine ( Hibitane) BetapropiuEnctone ( BPL ): This is a
is a relatively non toxic skin antiseptic most active condensation product of kerane and formaldehyde
against Gram positive organism* and fairly effective with a boiling point of 163 *C, Though as a gas it
against Gram negative ones. A Ueuus solutions are- lias low penetrating power, it is said to be more
used in the treatment of wounds. ^ efficient tor fumigating purposes than formaldehyde.
It has a rapid biocidal action but unfortunately has
G \ s i -. s Carcinogenic activity. Tor sterilisation of biological
Ethylene nxidc: Th]^ is a colourless liquid with products 0, 2% BPL is used , It is capable of killing
a boiling point of l 0.7 *C+ and ar normal temperature all microorganisms and is very active against viruses.
.and pressure is a highly penetrating gas with a sweet
ethereal smell. It in highly In flammable and in -
Si HL ACB ACTIVE AGE: \ TH
concentrations in a :ir greater than d % highly h Substances which alter energy relationship at
explosive. Ely mixing it with inert gases such is INTERFACEH , producing a reduction of surface or
carbon dioxide or nitrogen, to J Concentration ot intei-facial tension nrc referred to as surface active
10%, its explosive tendency is eliminated. agents - 'I'hey are widely used as wetting agents,
It* action is due to its alkylating tint amino, detergents and emulsifiers . Thcv are classified into
carboxyl, hydroxyl and stilphydryl groups in protein four mid ii groups, anionic, cationic, nonionic and
molecules . In addition , ir reacts with [ ) N A and amphoteric. The most important antibacterial
RN A, Its use as a disinfectant presents a potential agents are the cationic surface active agents. These
1
toxicity rn human beings, including mutagenicity act on the phosphate groups of the cell membrane
and carcinogen i dry, Tr is effective against all tvpcs and ako enter the cell . The membrane loses its
of microorganisms including viruses and spores. sc mi permeability and the cell protein ? are
Copyrighted material
SierilisaliQP and D\ s > 33
denatured. The cationic compounds in the form of Mercuric chloride , once used as a disinfectant is
quaternary ammonium compounds are markedly highly toxic. The <*rg& nic compounds, tfliinmeisal,
bactericidal , bung active against Gram positive phenyl mercury nitrate and mercurochrome, are less
.
-
organ i ms and ro a lesser extent on Gram negative
ones They haw no action on spores, tubercle bacilli
toxic and are used as mild antiseptics and have a
marked bacteriostatic, hut a weak bactericidal and
and most viruses. The common compounds arc limited fungicidal action , blivet salts in aqueous
acetyl tr: methyl ammonium bromide (Cetavion or solution have a limited use. Copper salts arc used
cetrimide) and benaalfcoftiuin chloride . These me as. fungicides.
most active at alkaline pH. Acid inactivates them .
Organic matter reduces their action and anionic TESTING OP DISINFECTANTS
surface active agents, like ordinary soaps , render There is no single reliable test available to
them inactive . The anionic compounds, c g, , . determine efficiency of a disinfectant. This is due
common soap , have moderate action - Soaps to the number of parameters which influence
prepared from saturated fatty ac kin (such as coconut disinfectant activity. Traditionally In such tests
oil ) arc more effective against Gram negative bacilli phenol is taken as the standard - In the Rideal
while those prepared from unsaturated fatty acids Walker ttM , suspensions containing equal number^
(oleic acid ) have greater action against Gram nf typhoid bacilli are submitted to the action of
positive and Nci -;5cri :i group of organisms . The varying concentrations of phenol and of the
amphoteric or ampholytlc compounds, known *4 disinfectant be tested. The dilution of the rest
'
JL ' UO ' compounds, are active agsains-t a wide range disinfectant which sterilises the suspension in. a
of Gram positive and Gram negative organisms and gLven nine, divided by the corresponding dilution
some v: Tuses. These , however, are no! in general of phenol is stated as the phenol coefficient ( Phenol
use . .
= 1 ) of the disinfectant This test does not reflect
natural conditions as the bacteria and the
METALLIC SALTS disinfectant react directly without any organic
Though all salts have a certain amount of germicidal matter being present . MudificiTumK have therefore
action depending on their concentration, salts of been suggested - In the Chick Martin test, the
heavy metals have a greater action - The salts of disinfectant acts in the presence of organic matter
i I wr, copper and mercury are used as disinfectants. (dried yeast Or feces). Even this modification falls
They are protein Coagulants and have the capacity short of dinul ? ring naturalconditions . Various other
to combine with free sulphydryl groups of cell modifications have been introduced, but no test is
enzymes, when used at appropriate concentrations. entirely satisfactory.
Further Reading
Block 55 1 :. ! 1991 . Chemical Disinfection in Hospitals, 2 cdn . London, E 'u'. - lu Health Labcuutiiry Service .
'
Gardner JK. and MM - Pcd 1991 . Irrmxknr .' tJil JU i> (trM .irjtjon End Disinfection* !?'1 cdn . Edinburgh ; Churchill Livingstone .
Russell AD er a]. 1992 . I '.- iiiLijikx aid Ptuciiix nl ' Di.^ i '. .- itL-titm , SteriH -ntiitn und PJT*TTVJI 1. . n . 2^ edn . Oxford: Blackwell
r
Si i -c nr fi < .
Copyrighted material
Culture Media
Bacteria haw KJ l >e j invn (cuJnireJj for Them iu bacteria . The use of agar to solidify culture media
^
be identified , a ? imlv rarely can they he recognised was suggested by Frau Hesse , the wile ul one of
hy rheir morphology atone. 'Hit study of bacteria tbe investigators in Kocb’s laboratory, wko had seen
involves the ;tudy of bacterial popukioiu rather her mother using agar for making jellies!
than of single bacterial cells. In tlLt. IUIIILATL and
1
Agar (or agar-agar ) is now universally used
animal bodies, as well as in dthcr natural sounxa , for preparing solid media. Agar is obtained from
bacteria occur as mixed populations. By appropriate some types uf seaweeds , its chief constituent is a
procedures they have to he grown separately long chain pripardiuidt- It also contains varying
(isofoted } on cufrufif media and obtained as pure amounts of inorganic Milts and small quantities of a
cuftirre? lor study.
Numerous culture media have been devised. The
-
protein like substance. lt has virtually no nutritive
value and is rot affix ted by the growth of bacteria .
originLil media used by Luuii PaBttUJ weft liquids Ag ; u is hydrolysed ac high temperatures, at high
such as urine or meat broth. Liquid media have acid i > r alkaline ; ill . Irs unique property is that it
many disadvantages. Bacteria grow mg in liquid melts LIL 98 -'C and usually sets Lit 42 "iS depending
media may not exhibit specific characteristics for on agar concentration Approximately 2% agar is
their identification. Jt is also difficult to isolate employed for solid media. The irl lining property
different types of bacteria drum mined populations, varies in different brands < if agart for example, New
using liquid media . However, ] ic|UIL ) mediia have Zealand agar has more jellifying capacity than
their uses, for example, tor obtaining bacterial Japanese agar. Agar is manufactured either in long
growth from blood water when large ^ illumes shreds nr as powder . There may be variations
have to be tested , and for preparing hulk cultures berween different hatches nf the same brand, apart
of antigens or vaccines. from rhi. differences due to a different source of
While bacteria grow diffusely in liquids , lliev agar.
produce discrete visible growth on solid media . 11 Another almost universal ingredient > > I common
inoculated in suitable dilutions, bacteria form media is jjeptune. It is a complex mixture of partially
monies, which are clones of cell * originating from digested proteins . Its constituents arc proteoses ,
a single bacterial cell . Oil solid media , bacteria have pnlyi >epiiJcs and aminoacids, ; L variety of inorganic
distinct colony morphology mul exhibit many other salr * including phosphates , potassium and
characteristic features -mil as pi grit en tar ion ur magnesium acid certain accessory growdi factors
hemolysis, making identification easy 'flic earliest
, such as riboflavin . Diiterenl brands of peptone show
solid medi nm was c < mktd ,mt pntuti > \ i -^d I'' k nbtrt
'
appreciable differences in romposirion and growth
Kocb Later ho introduced gelatin to solidify liquid
. promoting properties. There may be variations
media but it was pot- satisfactory as gelatin is between different batches of the same brand.
liquefied at 24 "C and also by mult proteolytic ''necial brand? of peptone such as ueopeplone
Copyrighted materia
•r Cullure Media * 35
In
%
¥ y
Ui
. -
E'ood agnr ; STfiep pyagorr es
showing beti hemolysis
w
Blood agar . Strap pyogenes magnified lo snow
small colonics surrounded try zones of dear hemolysis
m. 'VI
T
&V f
'W f - T_
r
* >
i
i * - _
r *
* *
* -#-
. S *
r w .
r:
Copyrighted material
36 4 TexflDflofe aJ Microbiology
_•
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Copyrighted material
* Culture Media * 37
and proteose peptone- arc available tar special UKS- Enrichment media: In mixed cultures or in
be utdL
- Meat
CoTHitK rfially available or digest broth can
jieptunes
extract Ls also available OOmnierdillr
materials containing more than one bacterium, die
bacterium to be isolated is often overgrown hv the
and is known as I J^ Lcmco. Blood, scrum and yeast unwanted bacteria . Usually the ntmpathogenic or
extract arc other common ingredients, commensal bacteria tend fo overgrow the
pathogenic ones , for example S . typhi being
Ti V K S overgrown by E . coll in cultures from teccs. In such
Media have bee it classified in many ways : situations, substances which have a stimulating
1 . Solid media, liquid media, scmisolid media . effect on the bacteria to be grown or an inhibitory
2 . Simple media , complex media * synthetic or effect on those to he suppressed are incorporated
defined media, semidefined media, special in the medium. If such substances art added to a
media. Special media arc further divisible into: liquid medium, the result is an absolute increase in
enriched media , enrichment media, selective the numbers of the wanted bacterium relative to
media , indicator or differential media , sugar the other bacteria . Such media are called
media, and transport media. enrichment media, for example tetrathinnate broth
3. Aerobic media, anaerobic media. where rhe tetrathlonirc inhibits colilbrm < while
Simple media ( basal mudiab An example allowing tvphnLd - paratyphoid bacilli to grow freelv.
is nutrient broth . It Lunisistsof pep; ' me, meat extract , and Selenite Fbroth tar dysentery bacilli.
SOdiutn chloride and water. Nutrient agar, made by Selective media : As in the above case , it rhe
adding 2% ugar to nutrient broth is the simplest inhibiting substance is added to a solid medium, it
and most common medium in routine diagnostic enables a greater number of the required bacterium
laboratories . If the concentration of ipr is reduced to form colonies then the other bacteria, tar example,
to 0.2-0.5%, semisolid nr sloppy agar is obtained desoxychofatc citrate medium for dysentery bacilli.
which enables motile organisms to spread. Increasing Such solid media are known as selective media.
the uonue otration of agar to 6% firevents spreading nr Indicator media: These media contain an
swanning by organisms such as Ptotcus. indicator which changes colour when a bacterium
Complex rued in: Ine have added ingredients grows in them , for example incorporation of sulphite
for special purposes or for bringing our certain in Wilson and Blair medium. S, typhi reduces
characteristic nr providing special nutrients required sulphite to sulphide in the presence of glucose and
for the growth of the bacterium under study. the colonies of £. typbi have a black metallic sheen,
Synthetic Or defined (tied in: These media Rttassimn tellurite in MtUod 'n medium is reduced
are prepared from pure chemical tuibfttanoes and to metallic tellurium by the diphtheria bacillus to
the exact composition of the medium is known. produce black colonies.
These are used for various special studies such as Differential media: A medium which has
metabolic requirements. Simple peptone water substances incorporated in it, enabling it to bring
medium , 1% peptone with 0.5% N »< ’ l in water, out differing characteristics of bacteria and thus
may be considered a scnndcfincd medium since its helping to distinguish between rhem . is called a
composition is approximately known.. differential medium. For example, MacConkey’s
Enriched media: In these media, substances such medium which consists oi peptone, lactose, agar,
as hl ( K >d, scrum , W Cgg are added to A basal medium . neutral red and taurochoiate shows up lactose
They are used to grow bacteria which are more fermenters as pink colonics, while nonlactosc
exacting in their nutritional needs, Examples arc blood fermenters arc colourless or pale . This may also be
agar, chocolate agar and egg media. termed indicator medium.
Copyrighted material
IS i Textbook of Microbiology *
Some of These terms for medi .L arc ( such as dysentery or cholera organisms in feces),
interchangeable. For example , the blood agar special media are devised for transporting the
medium i& 10 enriched medium but bacterii ly- mg specimens. These are termed transport media, for
nod cells show a clearing around their colonies. example, Stuart '& medium— a non-nutri ent soft agar
Thus, d is an indicator medium .LS well. There are gel cont.1ini:ig1reducing agent to prevent oxidation,
many special inedi .L for demonstrating particular and charcoal to neutral ir- e certain bacterial
characten -tLCH, like Nagler 's medium which enables inhibitors— for gonococci , and buffered glycerol
view letirhimase activity.
us to saline for enteric bacilli .
Sugar media: ' fhc term 'sugar In microbiology Anaerobic media: These media arc used to
denotes any fermentable substance. They may be: grow anaerobic organisms, for example, Robertson's
1. Monosaccharides — a) pentoses, e.g. , araKinose, cooked meat medium.
xylose, b) hexoscs, c .g,, dextrose, mannose Various media to test special properties like
2. Disaccharide& t e.g., saccharose, lactose urease production, and composite media for
3 - FblyMCL - luridcs, e .g., starch , innIin simultaneous demonstration of different features
4. Trisaccharides, e.g., raffmose have been devised . They are dealt with in the
5 . Alcohols, crg„ glycerol, sorbitol appropriate chapters.
b . Glucostdes, e.g., salicin, aesculln For identifying prepared media, a colour code
7 . Noncarbohydrare substances, e.g., inositol. i s usually adopted. This depends on the laboratory
The usual sugar media consist of 1% nfthe sugar or group of laboratories, One colour or a mixture
iii peptone water along with an appropriate of colours is used on the cotton stopper, or colour
indicator. A small tube (Durham's tube) is kept prints are used on the caps.
inverted in the sugar tube to detect gas production. Culture media used TO be prepared in
For organisms which arc exacting in their growth laboratories themselves , starting w i t h b a s i c
requirements ( for example pneumococcil , Hiss
'
ingredients. Not only was this laborious but it also
serum sugars arc used . They contain 3% serum. led to considerable batch variation in the quality of
Transport media: In the case of delicate media, With the ready availability of commcrci d .
organisms (like gonococt D which may not survive dehydrated culture media, the process of media
the time taken for transporting the specimen to the making has become Simpler and its quality more
laboratory or may be overgrown by nonpathogens uniform.
Further K end in a.
Atlaa PM . I 1.C Plaits 1999 . iVfnTofiwfcypni? Media, Maryland: CRC Pews,
Crci .:Wiaflt ti * 1 aL. 1975. Malicil MiiTvbihhjry Vol. II, 77« Practice afMedicai MiirobiologyA 12"' edn. London:
t hun- hill I .iv: MptHinj;.
AfjflUjt? tti f>*frnMiwd CWturc Media and ffej n-Ty, 1977, 9* h edn, Michigan: Difco Laboratories.
^
Oxoid Manual 1976.3rd edn. Basingstoke: Ojtoid [ h i .
Murray PR et a] 1999. AfanuaJ of Opnical Aftcmbtcdcg}-: edn. Washington I>C: ASM Press.
Copyrighted materia
to Culture Methods
Culture methods employed depend on the purpose The hwn or carpet cu turr provides a uniform
^
fix which they are intended. In ihn clinical laboratory, surface growth of the bacterium and is useful tor
the indications -for culture are mainly to: bacteriophage typing and antibiotic sensitivity
1. isolate bacteria in pure culture; testing (disc method ). It may also be employed
2. demonstrate their properties; when a large amount [if growth is required on Solid
3. oh ruin sufficient growth fur preparation oi media as, for instance, in the preparation of bacterial
antigens and tor other tests; antigens and VUdlKL Lawn cultures are prepared
4 . type isolate* hy methods HiK h as bacteriophage
'
by flooding the surface of the plate with a liquid
and haercriocin susceptibility; culture or suspension of tbe bacterium, pipetting
5. determine sensitivity to antibiotics; off the excess inoculum and incubating [ be plate.
6 . estimate viable counts; and Alternatively, the surface of the plate may be
7 . maintain stock Cultures. inoculated by upplyi ng a swab staked i n the bacterial
The methods of culture used ordinarily in the culture or suspension,
laboratory are the streak lawn , stroke, stab, pour Tbe stroke ciJnire is made in tubes containing
plate and liquid cultures . Special methods are agar atnpe (slant) and is employed for providing A
employed for culturing anaerobic bacteria pure growth of the bacterium tor slide agglutination
The jTreaJ; cuff tin? ( surface plating} method is and other di agnostic tests.
routinely employed for the isolation of bactena I:I htifh cultures arc prepared hy puncturing a
pure culture tnorn climca] Specunens. A platinum suitable medium such -,is nutrient gelatin or glucose
loop is charged wirh the specimen to be cultured. agar with a long, straight , charged wire , The
Owing to the high cost ot platinum* loops for medium is allowed to set, with the rube in the
routine work are made of Nichrome resistance wire upright position, providing a flat Surface at the top
{ 24 SWG size ) . One loopful of the specimen is of the medium. Stab cultures are employed mainly
transferred onto the surface of a well dned plate , tor demonstration of gclati n 1 iquefactinn and oxygen
on which it is spread ovci a small area at the requirement of the bacterium under study. They
periphery . The inoculum is rhen distributed thinly
'
ate also used in rhe maintenance of stock culturcs.
IWIT the plate hy -streaking it with tile loop in 1 For preparing pour plate culture , tube*
series of parallel lines-, in different segments ot the containing 15 ml of the agar medium are melted
plate . The loop should be flamed and cooled and left to cop] in a water bath at 45- 50 :: C,
between the different w:ts of streaks . On i neubitton, Appropriate dilutions of the inoculum (of I ml )
'
growth may be confluent at the site of original are added to the molten agar, mixed well and the
inoculation , but becomes progressively thinner, and concent* of the rubes poured into sterile Pfctri dishes
well separated colonics are obtained over the find and allowed to set. Alter incubatioin, colonies will
scries of streaks. be seen well distributed throughout the depth of
Copyrighted materia
40 i Textbook d Microbiology »
i In-medium and can be enumerated U - . MIL' colony incubating cultures in a vacuum desiccator, but
counters. The pour plate method gives an estimate the method is unsari -. factory as some oxygen is
of the viable bacterial con nt in a suspension and is always left behind. Fluid cultures may hoi I over
the recoin mended method for quantitative urine and the media mav get detached from the plates
cultures. in the vacuum produced- This method is not in
In the sweep plate method, the edges of the use now.
Petri dishes containing the culture medium arc .
2 Displacement of oxygen with gases such as
robbed over the fabric, with the medium ft- ing it. hydrogen, ni ' togcn, helium or carbon dioxide
'HlC dust
particles shired up Irom the cloth settle is somerimcH employed, hilt this method rarely
on the culture medium , and colonics develop on produces complete anaeiobiosis. A popular, but
incubation. They can he counted and estimates ineffective method is the candle jar . Here
made . inoculated plates are placed inside largea
Liquid cu/rtm*s in tubes, bottles ot flasks may airtight container and A lighted an idle kept in
he inoculated bv touching with a charged loop or it betore the lid is sealed. The bum : ng candle is
by adding the inoculum with pipettes or syringes. expected touse up all the oxygen inside before
Large lima:l.i can be employed in /.quid Cultures :l is extinguished, hut some oxygen is always
and hence this method is adopted lor blood culture left behind . The candle iar provides a
and for sterility tests, where the conccntrcuion of concentration of carbon dioxide wh : ch
bactcria i n the i -locul i is expected to be sm all. Liquid stimulates the growth of most bacteria.
cultures are preferable for inocula containing 3 . In the chemical or biologic J method, alkaline
antibiotics and other inhibitory' substances, as these pyrogallol absorbs oxygen . Thi - method, first
arc rendered ineffective by dilution in the medium . introduced by Buchner ( ISflE ) has been .
Liquid cultures are also preferred when large v n I . employed vnh different modifies irons for
are desired, the yield being enhanced by agitation, providing anaerobinsis . Pymgallic acid added
acral inn, midi Mon of nutrients and removal of toxic to a solution of sodium hydroxide in a large teat
metabolites (contnuous culture methods) . 'lTic tube placed inside an airtight jar provides
maim disadvantage of liquid culture i. s that :l does anaernbiosis hut a small amount of carbon
not provide a pure- culture from mixed inocula. mnnnridc, which L formed do ring the reatriun,
mav be inhibitory to some bacteria . The method
m a!
hole in the centre, The metal disc in attached to chopped vegetables. Aimerobiosis produced by
tbe bottom lialfof the Pfeiri dish with plubdne. such biological methods is slow and ineffective.
Through the -central hole , i few j'cllets ol sodium The most reliable and widely used anaerobic
hydroxide and 10 nil of . 1 10% solution of
H
-
method is tlte Mclntosh Filded MiiMemhie jar
pyrogallic acid -are added. The inoculated half ( Fig. !i. l ) . It consists of a stout glass Dr metal
of the Petri dish ! H then inverted < m the metal jar with a metal lid which can be damped air
disc and sealed tightly. tight with a screw. The lid has two tubes with
The method in common use employs A disc taps, one acting as the ga* inlet and the other «
of filter paper having the same diameter as a tlie outlet. The lid also has two terminals
lVtrii dish. If is placed on top of one half ot the which can be connected to an electrical supply.
dish and a mixture of pyrogallol and sodium Leading from the terminals and suspended by
carbonate, in dry powder form, is spread on it. stout wires on the underside of the lid is a small
The inoculated plate is inverted over the filter GROOVEL! porcelain spool around which is
paper and scaled tight with molten wax . The wrapped a layer of palladiniscd asbestos.
dry pyrogallol mixture in activated by the Inoculated culture plates are placed inside the
moisture within the closed syFCfim, and complete jur, with the medium in the bottOfll half of the
macrobiosis develops within about two hour^ ,
[ plates, and the lid damped tight. The outlet tube
Instead of alkaline pyrogallol, anaerobioau is connected to a vacuum pump and the air
has been produced within jars with ,1 mis hire inside is evacuated. The nutlet tap is then closed
ot chromium and Sulphuric acid ; Rosenth .il and the inlet tube connected to a hydrogen
method ) or with vtdlow phosphorous. supply After the j .u i -s filled with hydrogen, the
Absorption of oxygen from small closed electric terminals arc connected to a current
systems has been attempted by incubation along supply so that the ptlhdmised asbestos is heated.
with aerobic bacteria , germmating >eeds or This acts as a catalyst for the combination of
hydrogen with the residual oxygen present in
the jar. This method ensures complete
anacrobiosis but carries the risk of explosion,
•t A l Y f which may rarclv occur. This risk can he
eliminated by modification of the catalyst .
Alumina pellets coated with palladium Ju a
in . FT
gauze sachet suspended tnom the lid of the jar
act as a catalyst at room temperature, at long as
woer
the sachet Ls kept dry.
FL FllTF cj Tbe Gisynk. is now the method of choice
ltHMlUftLS CXIAiVST for preparing anaerobic jars . The tiaspik. is
commercially available as a disposable envelope,
Containing chemicals which generate hydrogen
and carbon dioxide on the addition of water.
After the inoculated plates are kepi in tlte jtt,
rhe Oaspak envelope , with water added , is placed
inside and the lid screwed right . Hydrogen and
carbon dioxide are liberated and the presence
.
Fig 5.1 Mclntesh-FUdes jar of 1 cold catalyst in the envelope permits the
Copyrighted material
42 i Textbook of Microbiology *
by the use of various reducing agents, including 2 Enrichment, selective and indicator medin are
1% glucose, 0.1% thinglyoolate , 0.1% ascorbic widely used for the isolation of pathogens from
a < id and 0,05% cysteine , Broth is an easily specimens such as feces, with varied flora.
prepared anaerobic medium imo which pieces d. Pure cultures may be obtained by pre-treatment
of . red hot mcCallii: iron are introduced , It is
of specimens with appropriate bactericidal
then fctvertd over with sterile vaseline - Rrnth suh stances which destrw the unwanted bactcria.
containing fresh animal tissue, such as rabbit This method is the standard practice for the
medium ) r supports the growth of many
-
kidney, spleen, testes or heart (Smirh Noguchi isolation of tubercle bacilli from sputum and
other clinical specimens, by treatment with
anaerobes. alkali, acid or orher Substance* to which must
Thiogiycolatc broth with hemm and vitamin K commensals- ate suucepLible but tubercle bacilli
is an enriched liquid medium for culturing
anaerobic and microaemphi I ic bacteria .
-
are rc istant.
4 Obligate aerobes and anaerobes may be
.
Addition of a small quantity of agar enhances separated by cultivation under aerobic or
the anaerobic capacity of the medium byslowing anaerobic conditions. Shake cultures in Veillon
the diffusion of oxygen in it. tubes were in use formerly but are now obsolete.
This consists of a glass tube open at both ends.
Robertsons cooked meat medium is probably
the most widely used fluid medium for the culture
One end is closed with a rubber stopper and
molten glucose agar in which the inoculum is
of anaerobes. It consists of fat - free minced cooked
evenly dispersed is poured into the tube and
meat in broth, with a layer of sterile vaseline over
it . lr permits the growth of even strict anaerobes
allowed to set in a vertical position. The top of
the tube is closed with a cotton plug . On
and indicates rheir saccharolytic or proteolytic
incubation , the bacteria in the inoculum
activities, b ^ the meat being turned fed or blade,
differemiace depending on their oxygen
respectively.
requirement - The obligate aerobes grow at the
For fastidious anaerohes , particularly for top and the anaerobes at the but tom . while the
quantitative cultures, pre- reduced med: . L and an facultative hactefia grow throughout the
anaertibic chamber ('glove box’ ) may be used . The column . The entire medium can be extruded on
anaerobic chamber is an airtight, glass - fronted to a plate and the different colonics fished out.
cabinet filled w:lh inert gas, with an entry luck fur 5. Separation of bacteria with different
the introduction and removal of materials, and temperature optima can be effected by
gloves for die bands. incubation at different temperatures. Only
Copyrighted materia
* Culluro M -alhods * 43
thermophilic bacteria grow at 60 “ 'CL A mixture bacteria alone traverse the agar and appear at the
containing N* meningitidis and iV. top of the medium outside the central tube.
can be purified by incubation at 22 *C vdhen A IJ - tuhe also serves the same purpose, inoculation
only the latter gram. being perfumed in one limb and the subculture
A. Ry hearing a mixture containing vegetative and taken from the other. This method can also be
spore forming bacteria, at 80 *G the former can used to obtain phase variants in Satmundla species.
be eliminated . This method is useful for the 8. Pathogenic bacteria may be isolated from
isolation of tetanus bacilli from dust and similar mixtures by inocidatian into appropriate animals -
sources. Anthrax bacilli can be distinguished from other
7 Separation of motile from nonmotile hacteria
r , aerobic speculating bacilli by inoculation into
can be effected using Cnigie s tube. Tlxis conriits mice or guinea pigs. Anthrax bacilli produce a
of a tube of scrnisolid agar,. with a narrow tube fatal septicemia and may be cultured pure from
open at both ends placed in the centre of tile the bean blood.
medium in such a way that it projects above the 9 Bacteria of differing sizes may be separated by
?
Level of die medium. The mixture is incwrulated the use of selective filters. Fdters are widely used
into the central tube. On incubation, the motile for separating viruses from bacteria .
Further llvudmji
CoILceJG *
ei il . 19%. PlracticjJ Medical Microbiology. 4 edit. , Edinburgh: Churchill Livingstone .
Murray PR ct d . 1999. Manual of Clinical Microbiology. 7" cdn. Washington DC: American 5-ociety of Microbiology
*
Copyrighted material
Identification of Bacteria
Once a bacterium has l >ren obtained in pun.' culture , or may rot he present . Hanging drop preparations ,
it has- to be identified.The following characteristics , lark gnou nd i I Lum i na tinn, phase eon trust or electron
have to he studied in the proccss.
, microscopy all help in these studies,
MLIRPHOI nf . l S H I M M . Kb U T I O I M S
The morphology of the bacterium depends on a The age of the culture is important . In older
number of factors auch at- the st ^iin studied , nature aiImres , staining characteristics eichei vary or ate
of the uilmre medium, temperature and time of nol hroughr out well . Simple stains bring out the
incubation, age ot the culture and the number of morphology best - Differential and special stains ait
subcultures it htis undergone , The characteristics necessary to bring out characteristics like flagella ,
noted arc shipe, size, arrangeme nt, mot i I i ry, fl agdin , capsules, spores and metachromatic granules. The
sports and capsules. All these cannot he made our ( i ram stain divides bacteria Into linam positive and
in a single medium. The shape may he spherical , ( Jnnn negative; the Zichl-Neclscn stain into acid
filamentous, rod shaped, comma shaped or spiral. fast and non-add fast . The fluorescent antibody P
The axis of the organism may be straight or curved . technique enables one to identify them according
The length and breadth may vary. The sides of the to their surface antigens.
organism may be parallel , eonvex, concave or 1 'ic study of morphology and staining
irregular. The end* may be cut straight rounded or
, characteristics helps in preliminary idcJltiticiLtion
tapering. Considerable variations in shape iind size of the isolate .
leading to club, navicular and swollen < > r shadow
or giant forms may be seen. They may be arranged Clit .i l : itAL C n AH v c r i '. K i s I i c s
singly. ; u pairs, in tetrads or in packets of eight , or These provide additional information for the
in chains, shaft or lung , in the ease uf cocci; bacilli identification of the bacterium. The characteristics
may he arranged at random, in short long chains , re veiled i n dlffene nt type > of media are noted. Wh i le
in Chinese letter patterns, .is palisades or in bundles ; studying colonies on solid media, the following
vibrios may he single or in or spiral fbrrn ^ They
,
features nrc noted ;
may be uonmotile, sluggishly motile, actively motile
O r may exhibit darting motility. They may be
without flagella, that is atfichitc, or raonotrichiite,
—
1 . shape circular, irregular, or rhtzoid ;
2 . size itt millimetres;
lo]ihotrichutcr littphi trie hate or peritrichane. The
spores, when present may be oval or spherical or
—
3- elevation ettosc , elevated , convex, concave,
umhonate or nmbilieate;
ellipsoidal and may be of the same width or wider —
4 . margins bevelled or otherwiset
th n that of the badllary hrxly. The spores may he
^
equatorial , suhtcrminal or terminal t \ipsulcs may
, papillate
—
i . surface smooth , wavy, rough , granular ,
or glistening;
Copyrighted material
H Identification d Bacteria 45
6. —
edges cut Ire, undulate, erenatedf fimbriate or
curled ;
media . Acid production is shown by change in the
colour of the medium to pmk or ted , and the gas
7 . colour; produced collects in Durham’s tube.
—
S . structure opaque, translucent or transparent
9 . consistency— membra nous , triable , butyfOUJ or
2 . Litmus milk: There may he no change in
the medium, or acid oi alkali maybe produced;
viscid; Hotting of milk, peptortisabort or s .Lpmnficul n ML may
LII.emubiliability; and occur . The clot may be disrupted by the gas
1 Lwhether rhev are differentiated into a central produced {stormy fermentation ).
and a peripheral portion. 3. Indole production: This is tested in a
In a stroke culture, note peprone water culture after 48 or 96 hours
—
1. the degree of growth scanty, moderate, or incubation at .17 IC, This rest demonstrates the
|
-
Ii 11 L : 111. MJCAI . PmiritHTlhH solution of < r - miphthol in ethanol and 0.2 ml of
The more important and widely used lests are 40% K.OH to one ml of a glucose phosphate
described below: medium culture of the organism incubated at 30 't.’ '
I . Sugar fermentation: This is rested in sugar fur five days or 57 H C I nr 48 bouts. In a positive
|
Copyrighted material
46 H Taktbo& k o\ Microbiology
reaction, .i pirtk cal Du r appears in 2 -5 RliAUieE, feme ammonium citrate or ferrous acetate
jeetite ,
(tccpcniriff fo magenta or crimson in half an hnur. can be used. The organisms can he grown in
J ]i a negative reaction, it remainscolourless for half culture tubes. Between the cotton plug and the
in hour. Traces of pink colon ration should he tube insert a filter paper strip soaked in 1 Q% lead
ignored, acetate Solution and dried. Browning of the paper
fr. Citrate utilisation: Koser 's citrate medium indicates H;S production.
has citrate as the sole source of carbon . Abilin to 31 . Methylene blue reduction: One drop
use this substance is indicated hv the production nt of 1% aqueous methylene blue in added to the
turbidity in the medium. broth admix, and incubated at 37 “ C . Complete
J
Copyrighted material
* idem INCH lion oJ Bacteria > 47
composite medium is the Triple bugar Iron (TSl ) Bacterinphage and bacterincin typing:
medium which indicates whether a bacterium These enable intnuspedes typing of some bacteria ,
ferments glucose onlvi or lutOK and sucHrae also, Pathogen icily : Ptthogentcity teats by
with or without gas formation , besides indicating inoculation of the test organism into laboratory
HjS production as well . The medium is animals like the guinea pig, rabbit, rat ud mouse
distributed in Cubes, with a hurt and slant. After by intraderina ], subcutaneous , intramuscular,
inoculative]., it the slant remains red and the butt intraperituneul. intracerebral ur intravenous, Or by
becomes yellow, all die sugars - glucose, lactose oral or nasal spray were common procedures for
and sucrose - arc fermented , Bubbles in the butt identification nf isolates in the past , They arc rarely
indicate gas production und blackening of the used now because simpler in virro tests arc available.
medium shows formation of 11 5 , The TSI
^
medium facilitates prelim in an identification
' qf RAPII> IDENTIFICATION METHODS
L Irani negative bacilli . While classical phenotypic characterisation of isolates
Other tests such as fermentation of organic acids, takes days , auromared merhods are now available
oxidation of gluconate, ammoacid decaHtoaylation, which only take hours. liLenrificariun iv simplified
and hydrolysis of sodium hippumre arc sometimes by the detection of specific enzymes, H>x»isf antigens
employed . With increasing knowledge of the or metabolic end products of the isolates. For
metabolic processes in the growth of various example , many obiigate anaerobes can be identified
bacteria , the number of rests too is on the increase . rapidly by jps liquid chrumaloi raphy of tlie short
Special manuals have to be consulted for the derails ^
chain fatty acids pr-twluced bv ibem during glucose
and utility of these tests. fermentation. Molecular methods such as polymerase
Antigenic Structure; FSy using specific sera chain reaction and other amplification procedures
we can identify organisms by agglutination or other coupled with nucleic acid probes carrying specific
suitable serological reactions. Immunofluorescence DNA or RNA base sequences arc now widely tised
test is useful ill SOfOt CUtJ
- for identifying microbes.
Further heading
Cowtn ST ed, 1978. f VtnMfcy fifMcdictl 'ft aotnji London Oxford l Inivttiiiy Ptuxx ,
^
Cowan ST and KJ Srcd 19fi 5. AJanuaJ /ur the Identification of MotfkxJ Bacteria . London . Cambridge UnntrsiTV Press .
Flnegold SM and EJ baron 19 B 6-. BJUJCJ and Sojff 's Zftj nosrjL’ Bacterioiogi . 7* edn Si . Louis: C Vr Mosby.
^
'
Copyrighted material
Bacterial Taxonomy
-
Bacterial taxonomy or t vstematic - comprises three
1
kingdom?, plant and animal . Bacteria bad been
components : placet! in the plant kingdom and designated as
1. Classification , or The orderly arrangement ol' Schizomycctcs ( fission fungi ). But as bacteria
units. AgroupofunifsiscaHei J t^.vriiT (pl r. .vaj,
j
present feahI res common to btJfb pin nts 5 m! a rumal S,
irrespective < >r its hie muchic level . it has been proposed that a new kingdom, \ loners,
'
-
projxjsed by Mignis ( L 89I) . As more information is vagu L- and i11 dei i ned. 1 hit to the Lihsence of fnssiI
became available on the pbvsiologLc .il and remains in bacteria , the evolutionary status ot
biochemical properties of bas teria , those were species cannot be established . Morphological
employed in proposing new systems of bacfrrial differences are insufficient fui the definition of
classideation by Knight ( 1936 ) , KJuyver and van bacterial species. The general absence of sexual
Niel (1936 ) and others. reproduction in bacteria prevent ? the use ot
Bacterial classification, presents special pnohlcms . inbreeding as a test tot differentiation between
Linnaeus (173 ? ) divided ail living beings inLo two species .
Copyrighted materia
1 Bacter'ial ’Trixpr'
ipmy » 49
In spite of these difficulties, the concept of system iscalled phylogenetic because it implies an
specIcs provides a convenient unit in bacterial evolutionary arrangement of species. Here some
taxonomy. Betide* morphological features, criteria characteristics are arbitrarily giver special
useful for the definition of bacterial species arc weightage . Depending on the characteristic so
physiological , biochemical , antigenic and chosen, the classification would give different
pathogenic properties . As Species' is a genetic patterns. Far example, the intestinal Gram negative
concept, definitive information can be obtained by bacilli have been traditionally classified depending
comparison of the nucleotide base ratios, which on whether they ferment lactose or not. While this
are constant far ary OAC species but may he difiercnt provides a useful distinction between the pathogenic
in different species. Generic homology can be and non pathogenic groups of these bacilli, a
demonstrated by 1> NA hybridization between different hut usdiil classification could be obtained
different individuals of the same species . ufing fermentation of sucrose as the criterion .
Comparison of rRNA sequences helps ro arrange While classification based on weighted
bacterial species into a phylogene tic tree. 16SrRNA characteristic is a convenient method, it has the
sequencing ha* emerged as useful tool for serious drawback that the characters used may not
identifying many new unculturahle pathogens (e g . he valid . Fermentation of lactose, in the example
BartnneHa henselae ) . cited , is Hot an essential and permanent
An important difference between tbe characteristic. It nuv be acquired nr lost, upsetting
classification of bacteria and that of higher the system of arrange me nr.
organisms is that in the former, the properties of a Adontonian classification : Fhf Adansortian
population am studied , and not of an individual. A rlanifiration , so called after Michael AdflUOIl who
population derived by binary fission from a single introduced it in the eightenth century, avoids the
cell is called a clone . A single bacterial colony use of weighted characteristics . It makes no
represents a done . Though all the cells in ; L clone phylogenetic assumption but merely takes into
arc expected to be identical in all respects, a few of account all the characteristic* expressed at the time
them mav show differences due to mutation. A
Jr
of study. Hence it is called a phciietic system - It
population of bacteria derived from a particular gives equal weight to all measurable features, and
source, such as a patient, is called a strain. groups organ is ms on the ba -si ^ of similarities of
The general absence of sexual reproduction in several characteristics. Thc availability of computers
bacteria serves to maintain their character constant - has extended [ lie scope of phenetfc classification
But bacteria possess several features that contribute by permitting comparbons of very large number*
to some degree of heterogeneity in then populations. of properties of several organisms at the same rime.
Their short generation rime and high rate of mutation This is known a*. numericri tnccf >OWy
lead to tile presence, in any j'opulatLun, of cells with M o L t c u E t i r n r gefleLic uliissifitillictn : T h i s
altered character^. Methods ofgenetic exchange such is baaed on the degree of genetic nelatedtlCSS of
is transit in i tar mi i , transduction and conjugation cause different organisms Since all properties arc
,
diflerertces; in cliarucDer. Propliage and plasmid DNA u l t i m a t e l y hased on the genes present, this
can induce new properties. classification is said to be the most natural or
Phylogenetic classification: There are two tundamental method . ON A nelatedness can he
approaches to bacterial classification . The tested by studying the nucleotide sequences of ] 3 NA
iLKTarLhical i - iaasification represents a branching andby DN A hybridisation or recombination methods.
tree like arrangement, one characteristic being The nucleotide base composition and base ratio
employed for division at each branch ui level. Tliis —
( Adenirie-ThymlniciGuanLne CvtuiLne ratio.) varies
Copyrighted material
so * Textbook of Microbiology *
Further Reading
Cowan ST ed . 1978. DidJOtfLuro/Medical Tamoinr. London: Oxford Uditttttar Press.
r p r
Cowan ST and KJ Strel 196:5 .. AJkouaJf for the Identification of Medical Bacteria London; Cambridge University Press.
Finegold SM and EJ Baiun Bailey and Scott s Dhtgoostk Bacteriology, 7* edn, St. Louis: CV. Mosby
Goodiellmv M jnd RG Board ed. 1980 Microbiological Classification and Identification. London: Academic Press.
.
Murray P ea: ilF ds. 1999. Manual of Clink dl Microbiology' . 7' edn. Washington: American Sociery for Microbiology.
1
Copyrighted material
Bacterial Genetics
Genetics, i name coined by the llnrish biologist Even the existence of a bacterial nucleus was a
William Bateson in 1906, is the study of heredity subject of controversy. The differences in
utd variation , seeking to understand the causes of morphology and other properties were attributed
the resemblances and differences between parents by Nagcli ( 1 £77 ) to bacterial pltotaorphitm,which
and their progeny. Like other ogmnnu, Wteria postulated the existence of a single , or perhaps a
^ IR £ 1 breed true and maintain their characteristics tew species, of baCieria , whieh possessed a protean
from generation to generation , yet at the same time, capacity for variation , With the development and
exhibit variations in particular properties ir> a smaEI application of precise methods df pure culture , it
proportion of their progeny. Though heritabiliry became apparent that different type* of bacteria
and variation in bacteria had been noticed from retained constant form and function through
the early days of bacteriology, it was not realised successive gene rations. This led to rhe concept of
then that bacteria too obey the Laws of gtiwtics. monomorphim, proposed by Cohn and Koch,
which admitted of tittle potential for variation and
separated bacteria into species based upon single
character differences.
It was ( mlr - 11 icc the 1940s that principles of
genetics were applied to bacteria and their viruses.
ITihas led not merely to a better understanding
of the generic processes but also to fundamental
advances in biology ; ul biochemist tv and to the
hjrrh of a new h ranch of science , molecular biology.
G c
T
BASIC rhismrLES OF MOLECULAR
# lilOl . OCY
The 'central dwgma * f molecular hiolngy is that
* c deoxyribonucleic acid ( DNA ) carries genetic
T ri information, winch i - [inscribed onto ribonucleic
acid i RN -\ ) and then translated as the particular
polypeptide ( D\ A -> RNA — > polypeptide). As
the nature and timer ions of a cell .ire basically
determined by the pecific polypeptides that
constitute it - proteins and enzymes , it is evident
that the essential m .itcrial of heredity is I 3 N A
Fig . 9.1 DNA double helix which is the storehouse of all information for
Copyrighted material
52 < Te *tbook of M cfobiology *
protein synthesis. ( An exocpticm exists in rhc case unwinding at one end to form a fork, each strand
of some viruses in which the genetic material is of the fork acting as a template for the svm thesis of
RNA instead of DNA ) a complementary strand, with which it then forms
The DNA molecule is composed of tvifl chains a double helix.
of nucleotides wound together in the form of a Basically, RNA is structurally similar in DNA,
'double helix’ ( Pig . 8.1). lunch chair has a backbone except for two major differences. It contains the
of deoxynbose and phosphate residues arranged sugar I ibose ( instead of deoxyribose which is
'
alternately. Attached to each deuxjfibose is one of present in DNA) and ( he base uracil ( instead of
four nitrogenous bases , the purines, adenine ( A) thymine which is present in IJNA). Three distinct
and guanine ( G ), arid the pyrimidines, thymine (T) types of RNA can be distinguished on the basis of
and cytosine {G ) , The cWbic‘stranded nature of structure and (unction: messenger RNA ( mRNA ),
the molecule is stabilised by hydrogen bonding ribosomal RNA ( rRNA ) and transfer RNA
between the bases on the opposite strands in such (iKNA ). DNA acts as the template far the synthesis
a manner that adenine is always linked to thymine, of ni RNA and, therefore , the bases in the two will
and guanine to cytosine ( Fig. S.2 ) . .
be Complementsiy to ouch Other Adenine, guanine,
Adenine and thymine thus form a cytosine and uracil in mRNA will be
complementary base pair , and guanine and cyltisiiLL' complementary to thymine, cvlosine, guanine and
form another. A molecule of DNA will, therefore, adenine, respectively, in the DNA -
contain as many units of adenine as thymine, and Geneiie information is stored in the DNA as a
of guanine as cytosine but the ratio of each pair of code, the unit of the code consisting of a sequence
bases [A + T) /(G + O , though constant for each of three bases, that is, the code is triplet. Each triplet
species, varies widely from one bacterial species TO ( codon ) transcribed un mRNA sjtecifies for a single
another. The DNA molecule replicates by first aminoacid hut the code is ^ degenerate so that more
DEOXYR1BOSE ADENINE * - a
THYMINE DEOXYRIROSEi
/ \
PHOSPHATE PHOSPHATE
\ /
DEOXYRIBOSE
/
GUANINE E
CYTOSINE — DEOX YRIBOSE
\
PHOSPHATE PHOSPHATE
\ /
DBOXYRIBOSE CYTOSINE m GUANINE [ JEOXYRIliOSE
/ \
etc .
ONE STRAND
DOUBLE-STRANDED DNA
opyrighted material
* Bacterial Geneves 53
Than cme codon may exist for the uirc immoadd. their ability to transfer genes from one cell to
Thus, the triplet AG A codes for arginine but the another, plasmids have become important vectors
triplets AGO, CGU, CGC, CGA and CGG also in genetic engineering. Plasmids may also be seen
code for the same a mi noacid . The code is non - in yeasts.* which are eukaryotes. Plasmid DNA
overlapping, each triplet being a distinct euiiiy, and same times may be integrated with chromosomal
no base in one endun is employed as part nf the DNA* The name epifinme employed for such
message of an adjacent codon.Three codons (UAA, integrated dorms * though this distinction is not
UAG and UGA) do not code for any iminoadd usually made now.
and arc called ' nonsense codons\ They act as Plasmids have been classified .in many ways ,
punctuation marks (stop codons) termi ranting the depending on whether they arc self ^ tmnsmissiblc
message for the synthesis ot ' a polypeptide . or non transmissible ( nonccmjugating ) , ora the
A segment of DNA carrying cottons specifying property encoded (sex , drug resistance, etc.) * by
for a particular polypeptide is called a A UNA restriction endonuclease fingerprinting or other
molecule consists of a large number of genes* each criteria . An important method of plasmid
ol which contains hundreds of thousands of classification in by imornp:itihilinr typing,- Clofely
nucleotides. The bacterial chromosome consists of related plasmids do nor coexist stably in the same
-
a double stranded molecule of DNA arranged in a
circular form. When straightened * ir is about 1,000
bactcri al cell, wli 11c unrelated plasmids can . On this
basb, plasmids have been classified into different
jim in length . The length of DNA is usually incoapttibiHty groups. They have alio been
expressed is kilubases ( 1 kb 1 ,00i ) base pairs). clashi bed based on the types nt conjugation tuhe
linctcrini DNA Is about 4*000 kb and the human induced, which determine the susceptibility of the
genome about 3 million kb long. host bacterium to lysis by some virulent
In higher forms of life, several stretches of DNA bacteriophages.
that do not appear to function as codons occur
between the coding sequences of genes . These
GENlVTiPHJ AMI PHKNtlTVPIC
V M t i Ml I O N S
Apparently useless noncoding intrusions are caller ]
mfrcuis, while the stretches of coded genes are The sum Loial of the genes that make up the genetic
called exons. During transcription * the genome is apparatus of a cell (genome ) establishes its genotype,
copied in its entirety, both introns and crons. The which is rhe hereditary constitution of the cell that
irtrons arc then excised from the RNA copy before is transmitted TO its progeny. The genory ]>c includes
being translated by the ribosome* Into proteins. the Complete generic potential Ol the cell, all of
which may or may not be expressed in a given
E \|l t \ t:H l t O.M ( > K ( > M V I G l i l V l i T t C
J
environmental situation .
E I F M l? N T S
Die phenotype [ piiaenv meaning display ) is the
In addition to chromosomal DNA .. most bacteria physical expression of tbe genotype in a given
possess extrachromosomal genetic elements . These environment . It billows, therefore , that cell mav
are not essential for the normal life and functioning exhibit different phenotypic appearances in different
of the host bacterium but may confer on it si tuitions; fur exam pic , the typhoid bacillus is
properties such as drug resistance and trudgen icity normal ly flagellated fejt when grown in phenol agar,
leading to survival advantage under appropriate the flagella ate not synthesised. This is only i
conditions. PlAtmidt are circular DNA molecule* phenotypic variation determined by the environ men!
present in the cytoplasm of bacteria , capable of and is reversed when cubailtured from phenol agar
autonomous replication ( iraJepemJenf nepheorw). By into broth. Another example ol environmental
Copyrighted material
54 i Tyxlt?OQk Ol MiCrOOiUlogy
influence .* the synthesis by E, coil of the enzyme not* all mutations are - - Some
TL > CL!. mutat iiurts
-
beta galactosid ase , necessary for lactose
fermentation. JlTic bacillus possesses rhe genetic
involve vital functions , and such mutants arc
notifiable { lethal mu ran on ) , A type of lethal
information for the svnthcsls
.11
of the enzvme Hut
Dill
mutation which is of great interest is 'conditional
the actual nyntlutit takes place only when it is mutatiim’- A conditional lethal mutant may be able
grown in a medium containing lactose . When to live under certain contritions ( permissive
grown in a medium containing glucose only, the conditions). The commonest type of conditional
enxyme is not synthesised. Such enzymes which mutant is the temperature sensitive ( nt) mutant,
are synthesized only when induced by the suhstrate which is able to live at the permissive temperature
are called induced enzymes * as opposed to ( say, .35 JC), but not at the restrictive temperature
,
cspjfjfijtjvf enzymes , which are synthe -used ( say, 39 C ).
L
irrespective of the presence or absence of the Each gene undergoes mutation with a fixed
substrate. frequency. Mutation rates ol individual genes m
Phenotypic variations are influenced bv the bacteria range from 10^ to 10 ]U per bacterium per
"
environment, Limited in range by the genotype, division. The molecular mechanism of mutation is
temporary and not heritable. Variations are s::. id to rhat during DNA replication, some 'errori creeps
be genotypic when they arc due to alterations in in while the progeny strands are copied. For
the genome . Genotypic variations arc stable,
heritable and not influenced by the environment.
-
i n lance, i i istcad of thym i ne bond i ng wii h adenine,
it may, due to tautomerism, sometimes bond with
They may occur by mutation or by one of the guanine. Though mutation occurs Spontaneously,
mechanisms of genetic transfer or exchange, such : rs frequency can be . nercased by Ftcveral agents
'
cause cjitcnrvc chromosomal rearrangements . A For example, an E. coli mutant that loses its ability
misienre mutation is one in which rhe rripler code to ferment Lactose can be readily detected oil
is altered so as to specify an .linin ' u , id different MacConkey agar but is unrecognisable on nutrient
from that normally located at a PARLI. LII .LT position agar. Mutation is of vital importance when it
in the protein. l>cletion of a nucleotide within a confers a survival advantage. If a streptomycin
gene may cause premature polypeptide chain resistant mutant of the tubercle bacillus develops
term mar ion by generating a nonsense cudon , i.e . r in a patient under treatment with the drug, it
nonsense umtitiosi. Irailsvcrsiun is substitution of multiplies selectively and ultimately replaces the
a purine for a pvrami . hne and vice veiss. i. IL base original drug sensitive population of bacteria. But
pairing - ifcippmsormutiarion is reversal of a mutant in a patient who is not on treatment, the mutation
Copyrighted materia
1 Bacterial Genetics * ss
populating, observed in the presence of a selective practical importance of bacterial mu radon lies
environment , were formerly rnriRidercd to be mainly in the field of drug resistance and the
'adaptations'. By a post hoc, ergo propter hoc development of live vaccines.
reasmiing, the environment and the variation were
believed to have a ciuse-ind-eflfeet relationship, TRANSMISSION OF CFNRTIC
bin ll induced variations were considered heritable
' MATERIAL ( GENF. TRANSFER )
in the lamarckian sense, k was the demolition of Traasfumialmn: Transformation is the transfer
this concept of adaptation in the 1940s that of genetic information through the agency of free
established bacteria] genetics oil , L firm scientific UNA. 1 r was the first example of genetic exchange
basis. in bacteria nt have been discovered, Griffith in 1928
The proof that bacteria undergo spontaneous found that mice died when injected with i mixmre
imitation in Jejscndcnt of the environment was- tirsl of live noncapsulated ( R ) pneumococci and beat
provided he l . uria and De lb ruck (1943) bv the killed capsulatcd ( 5} pneumococci * neither of
' fluctuation trsr ' , They found that very wide wllicb separately proved fatal. If in the experiment,
fluctuations occurred in the numbers of the live ( R ) pneumococci weft derived from
bacteriophage resistant E . coli colonics when capsul .tr type II and the killed ( S ) strain from type
samples were plated from several separate small II 1, from blood cultures of the mice that had died,
volumecultures* as compared tri samples tested from live type III capsulatcd pneumococcus could be
a single large volume culture . Statistically, this isolated, showing that some factor in the heat killed
indicated that mutations occurred randomlv in the type 11 ] pneumococcus hud transferred the
separate small volume cultures, some early and some information tor capsule synthesis to the live rough
late, resulting in the Wide fluctuation. In the targe
volume cultures, fluctuations were within limits of
-.train. Such transformation was subsequently
demonstrated in vitro also . The nature of the
sampling errorr However, the logic of [ Iris transforming principle was identified as DNA by
experiment was not widely appreciated by Avery, Mavleod and McCarty ill 1944.
iHObiologitfl) probably due to the complicated IVUIIIKDLICLEOII: The transfer of a portion of the
statistical interpretation . Ir was the simple but DNA from one bacterium to another by. a
elegant 'rcplica-platlng’ technique ofLcderbcrg and bacteriophage is known as transduction *
1-edeiberg (1952) that proved the point beyond doubt. Bacteriophages are viruses that parasitise bacteria
Using i velvet template, they were able ro transfer and consist of a nucleic acid core and a protein
inocuta from colonies on a master plate, onro a number coat. During the assembly of bacteriophage progeny
of other plates, retaining die relative positions of [ he inside infected bacteria * ' packaging errors' may occur
colonies in all the plates. By such replica^pbling on _ occasiu nally. A phage particle may have at: its core,
vulture plates with and wbrltnut baercrinphuges, [1 LL V betide* its (I'VTI nucleic idd, a segment of the h*»l
were able to show that bacteriophage resistant mutants DNA. When this particle infects another bacterium,
appeared without the bacteria ever having had contact DNA transfer is effected and the recipient cell
with a selective agent. acquires new characteristics coded by the donor
Mutation may affect any gene and hetiee may DNA. Transduction may be ‘generalised ", when it
modify BnydiuBCteriEtk of the bacterium . Mutants involves xnv segment tfthfi ilonor UNA it random,
may varv in properties such as nutritional or it mav be restricted ' , when a specific
'
Copyrighted material
56 * TexlDook of Microbiology »
prophage Lambda is inserted in iHt bacteria!! the same or related phages ) and antigenic
chmrnoso-mq only between the genes d-Ct crrnin. iirig
'
character] sties. Of great mcd: cal importance is the
galactose utilisation (jja!) and. biotin synthesis { bio) lysogenic conversion in diphtheria bacilli , which
and. therefore it transduces unlv either of these. acquire roxiger. i . ity {and therefore virulence ) by
TnnsdLKriQin is not confined to transfer of lysogcnisation wi. rh the phage beta . Elimination of
chromosomal DNA , Epi - omes and plasmids may the phage from a toxigenic strain renders it
a I - be transduced . The plasmids delermi ni n r rontuxigenic.
pcnicillin resistance nt staphylococci. arc transferred^ f .onj u g a t i n n : Conjugation is the process
from cell to cell hy transduction. wherein a ‘mule’ or 'donor ' bacterium 'mates Of
'
I’ransduction appears to be the most widespread makes physical contact with a ‘female or 'recipient'
mechanism of gene transfer among prokaryotes and bacterium and transfers genetic elements into if .
provides an excellent fool tor the genetic mapping This has been considered to be rhe bacterial
of bacteria . Any group of bacteria for which equivalent of sexual mating in higher organisms
bacteriophages exist can be subject to transduction. bur rhe analogy is irrelevant as , following
It has been reported that transducrion may
conjugation , the female bacterium is in turn
occasionally be effected in eukaryotic cells also. converted into a male cell ! Bacterial conjugation
Transduction has been proposed as a method of was first described hy Lederherg and 1:itum (194b )
gene t ic engi need rj: i n the treatment of some i nborn in a stni ! n of £ , coYs called K12 and has been most
errors of metabolism .
extensively studied in this strain.
Lysogenic conversion: Ratter in phages Conjugation takes place between a male cell and
exhibit two types of lifecycle. In the virulent or lytic
cycle, large numbers of progeny phages arc built
- -
a female cell. The malcncss or donor C .L I U of a
cell is determined by the presence in it of a plasmid
up inside the host bacterium , which ruptures to which codes for specialised fimbria { sex pihi* }
release them. In the remans re or nuriiWe cycle h which proiects from the surface of the cell. The
the host bacterium is unharmed. The phage DNA plasmid DNA replicates and a copy of ir passes
becomes integrated with the bacterial chromosome from the donor to the recipient cell, probably along
as the yrrvjphagie, which multiplies F Tchtonouslv the sex pilus (cwy rogation tube). As a result, the
^
'
with rhe host DNA and is transferred to the recipient attains donor status and can in turn
daughter oelll. This process is called Ivsngcny and conjugate with other female cells. The maleness in
bacteria harbouiing prophages are called iysogenic bacteria is thus a transmissible or ‘infcctioni’
bactena Lysogeny IS extremely frequent in nature.
In lysogenic hacteris , the prophage behaves as
characteristic , Along with the plasmid DNA .
an additional scgmcjtt of the bacterial chromosome
-
Coding for new characteristics . Thi . process by
. portions of the host DNA also are sometimes
transferred to the recipient. The donor DNA then
combines with the DNA of the recipient, effecting
which the prophage DNA confers genetic genetic recombination. It was in E. coli K 1 2 that
infotmari "n to a bacterium is called Jvsc emc or the role of plasmids in conjugation was first
phage eanversfon. In transduction , the phage acts^ necogn i sed. The plasm id responsible was termed
the 'stx factor or 'fort liry ( F) factor '. When other
1
only as a vehicle carryirig baCtenal genes Ircim one
cell tO another but in lysogenic conversion the similar plasmids were also discovered, the term
phage DNA itself is the new genetic element . ' transfer factor came to he used lor .ill such plasmids
Lysogenic conversion influences susceptibility to which conferred on their host cells the ability to
bacteriophages (immunity to supcrinfcction with act as donors in conjugation .
CoDvriahted materia
* Bacterial Genetics » 57
Thu l: factor: The F factor is i transfer factor Resistance transfer factor ( RTF It This
cLi .Lt contains the genetic information necessary for plasmid is of great medical importance as it leads
the synthesis of the sex pilus and for seif- transfer to the spread of multiple drug resistance among
hut is devoid of other identifiable genetic markets bacteria .
such as drug resistance. Cells canying the F factor This extrachromosomal mechanism of drug
( F* cells ) hove DO distinguishing fcarutes other than resistance was first reported by Japanese workers
~
their ability to mate with F cells and render them ( 19? ) investigating the sudden increase in
F. The F factor is actually an eplsome and has the ^
infections caused hy the Shigella strains* resistant
jhility to exist in some cells in the ' integrated state’ simultaneously to SulphonaiiudeS, streptomycin ,
or inserted into the host chromosome . Such cells chloramphenicol and tetracycline . They observed
arc able to transfer chromosomal gen to recipient that patients excreting such Shigella strains also
Ceils with high frequency and are known as Hrr shed in fheir fcecs £, coli strains resistant to the
cells. Following conjugation with an Hfr «11, an same drugs. Transfer of multiple drug resistance
F only rarely becomes F*, though it receives was demonstrated between E. eoti and Shigella
chromosomal geues from the donor. strains both in vitro and in vivo. The resistance is
This conversion of an FH cell into the Hfr state plasmid mediated and is transferred hy conjugation .
[ JILS mechanism of drug resistance is known as
is reversible. When the F fector reverts from the
transferable, epi tnal or infectious drug resistance.
integrated state to the free stare* it may sometimes ^
carry with ] L some chromosomal genes from near This plasmid consists of two components the-
transfer factor called the RWfiHXV transier factor
its site of attachment . Such an F factor
( RTF ) which is responsible for conjugation ]
'
Copyrighted material
58 * Textbook ol Microbiology *
IwCTEItlAL GENETICS
bxciiiun
F
+
MATING
F F
o# ^ o
Fig. 8.3 The integrated F liclur ot n Hfr cell may revert to ihe cytoplasmic slate. During excision
Sejtduclion .
some host genes may be incorporated in the F factor F \ Whan an F cell males wilh an F cell the host gene-
'
,
Transferable drug resistance in now universal several, including decreased permeability to the drug,
in distribution and involves all antibiotics in development of altenative mctabolic pathways, altd
common use. IK incidence is directly proportional production of enitymes inactivating the drugs .
to the frcqueocy of use of antibiotics in the area ,
bacteria carrying H factors can He transmitted from Mutational resistance is mainly of two types:
animals to mao . Hence indiscriminate use of (1) the stepwise mutation , as seen with penicillin,
antibiotics in veterinary practice or m animal feeds where high levels of resistance arc achieved only
can also Lead to an increase of multiple drug hy a series of small- step mutations; and (21 the
resistance in the community- The addition of
m LL > ne-slep' mutation, as seen with streptomycin,
antibiotics in animal feeds has for this reason been where the mutants differ widely in the degree of
prohibited by legislation in some countries . resistance , some exhibit lot; low resistance , while
Widespread resistance has considerably diminished others may be highly resistant, and some even
the clinical efficacy of most antibiotics. streptomycin dependent.
Copyrighted material
* Bacterial GenelioS S9
treated with streptomycin alone, initially the bacilli resistance to mercuric chloride and erythromycin.
die in large numbers but soon resistant mutants Transferable drug resistance mediated by the R
appear and multiply unchecked, IF two or more factor is the most important method of drug
antituh ^ rculnus dnigb arc used for combined resistance, Acquisition of an R factor simultaneously
treatment , rcpopulafion by resistant mutants docs confers resistance to several drugs and therefore
not occur, as a mutant resistant to one drug will be treatment with a combination of drugs is not usefu l .
destroyed by Tin other drug. The pKissibilitv of a
1
The resistance is due to die production of degrading
mutant exhibiring resistance TO multiple drills enzymes, and the level of resistance is usually high.
simultaneously is so remote as to be virtually
J V
Resistance mav be transferred between bacteria of
lumoasteoL This Is the talkMile behind combined different taxonomic groups, While resistant mutants
fre tment in tuhcrculnsifi . However,, inspire of this usually hive a lower growth rate and reduced
^
knowledge, inadequate or inappropriate treatment
"
3 (
c
T
Su
s +
MATING
1
(3)
HTF RTF
T c T C
<u S
Su S
: Fig . B .4 Transferable drug resistance * The R cell carries the R lactor , consisting of RTF and r determinants
1
Its transfer to a sensitive Ft bacterium converts the recipients Into a resistant Ft * cell.
60 < TeiftbooK Of WnCr &biaJ& Sy *
humans; mid animals, making antibiotic therapy ot Transposons attach at certain regions of
infections ineffective. chromosomal , plasmid or phage DNA . Insertion
In the laboratory, R factors may sometimes be of a transposon lead ?; to the acquisition of new
eliminated by treating bacteria with acridine dyes characteristics by the recipient DNA molecule.
or etliidium bromide. Bur m rhe communin ', tltc Unlike plasmids, transposons are not self replicating
Dnjy wav to prevent widespread dissemination ul .
and depend or chromosomal or plasmid DNA for
-
multiple resistance in to resti-ict the use of ultibiotuis. replication
to the essential minimum. bv transposition, a segment of the DNA can be
transferred from a molecule to another molecule
THARUSPUS ANT . K GHNP.TH : KI . KMENTS that has no genetic homology wit ft either the
Certain structurally and genetically discrete tnmpoubk clement or with the donor DNA. Jn
segments (>f DKA have been identified that have this it differs from recombination . As. sizeable
,
Copyrighted m ria
* Bacterial Genetics
* 61
is cocicetTrtd with the maiyju and ourripuJatitm CJI desired protein in putt form, in large quantities
UNA u^ing biochemical and microbialogiaJ and at J reasonable coat.
technique!*. 1r lias been staled that these techniques Different strategics have been employed for
have revolutionised the study of bid logy rtn d - obtain ing the dosi red genes , For very small protci ns,
medicine f probably more than any technique since such as the pituitary hormone somatostatin whose
the development of the light mi otoscopes. Some complete aminoarld sequences are known, [be genes
techniques and applications of molecular genetics can be synthesised in the laboratory. With larger
are discussed beluw. proteins ^ this is not possible . The DNA can be
Genetic engineering: The most important cleaved by specific enzymes called res friction
application of molecular genetics in biotechnology'
, endonucleases and the fragments containing the
is genetic engineerlug or recombinant DNA desired genes isolated. This does not work with
( rDNA ) technology. This consists of imitation of DNA of higher OTganisms as they contain i citrons.
the genes ending for any desired profcin from In such eases, rln - messenger RNA concerned can
microorganisms or tinm cells "t higher forms of
'
be isi < h trd fit ' ni eel I s pr < iducing the dedred protein.
-
Life including human hi;in:: and their Introduction
, A DNA copy :s made from the mRNA using the
in Co suitable mlcrcjtn anismSj in which [he genes enzyme ttverse transcriptase. The double-stranded
^
would be functional, directing the production of DNA gene is then prepared using DNA polymerase.
the specific protein, buch cloning of genes in This it incorporated into suitable vectors or currier
microorganisms enables the preparation of the such as plasmids or temperate bacteriophages, for
^
o
OTSOTET * O T C C A r
©
I C_5 M
V
T A C C 7 G A
~ V V, Ji * '
§ 6 ©
A
CL
—-
—
——
A
T
C
o— Ol
T A
C =G
— c
-
m
~
-©
®
5
T“ A
IL
C
T st
A
—— r
A ^ -
Fig.i.S Diagrammatic representation. The figun above shows a iransposon along 1he course of a DNA
molecule. consisting of a gene in the middle and inverted repeal sequences of rtud&olidtai at either end, The
1
figures below represent me stem and loop structure (armed by each strand of the iransposon, The loop
consists of the gene and the steinm 1$ formed by hydrogen bonding between the terminal repeal sequences.
The slem and loop form can attach to Insertion silos on recipient DNA. 1 Transposon 2. Inverted repeat . .
-
.
of gene. 7. Double stranded stem formed by bonding of terminal Inverted tequmeft
- .
sequences . 3. Gene. 4. Inverted repeat sequences. 5 Double stranded DNA R fHngto ilrandtd loop consisting -.
62 * Textbook od Microbiology »
insertion Into microorganisms. The microorganism are their high degree of specificity, ability to derect
commonly employed is E. coli Kl2h though many minute quantities of complementary DNA even in
other bacteria and yeasts have also been used. the presence of other microbes, and the capacity to
Genetic engineering has become an established recognise microbes that arc either difficult or
branch of biotechnology with great scope for impossible to culture- DNA probes for the detection
commercial exploitation. Cloned human insulin , of many pathogens ate now commere ially available.
interferons, somatostatin, growth hormones and Blottii 4 techniques DNA fragments obtained
many other biological!have already been marketed. by restriction enzyme digestion and separation on
Safer vaccines can be produced by cloning the gel can be transferred from the gel by blotting to
protective antigens of pathogens, as has already been nitrocellulose or nvlon membranes that bind the
done, as in the case of foot and mouth disease, and DNA . The DNA bound to the membrane is
hepatitis B and rabies viruses . This versatile denatured {convened to the single-stranded form )
technique ins many extramedical application* also.
Restriction endonucleases: [restriction
-
and treated with radioactive single stranded DNA
probes - These will hybridise with homologous
enzymes ) are microbial enzymes which cleave -
DNA To form radioactive double stranded segments,
-
double stranded DNA at specific ohjrpmicleotide -
which can be detected on X ray film. This highly
sequences. Many such enzymes which act at sensitive technique for identifying DNA fragments
different nucleotide sequences (for example, Ecu by DNA : DNA hybri Libation is called 5ourficro
RI , Hind III, Taq I ) have been recognised . The blotting, after EM Southern who devised it. This
natural function of restriction enzyme* in bacteria technique has very wide applNation in DNA
may be the destruction of foreign DNA that may analysis.
enter the bacterial cell An analogous procedure for the analysis of
Restriction enzymes split DNA strands into RNA has been called northern blottiOg ( a opposed
fragment ^ of varying lengths . These can be to southern blotting!). Here the RNA mixture is
separated by gel electrophoresis and stained with separated by gel electrophoresis , blotted and
ethydium bromide and photographed. identified using labelled DNA of RNA probe;.
I ) N . \ probes The specificity of the Interaction A similar technique for the identification of
in base pairing during DNA or RNA synthesis proteins (antigens) is called immuitoblorfriig [or,
enables the production of specific DNA probes. in conformity with other blotting techniques,
These art radioactive, biotinylated or otherwise .
western blotting ) Here the protein antigen mixture
labelled copies of cloned single-stranded DNA
-
-
is separated by SDS PAGE (sodium dodecyl
fragments, usually 20 25 nucleotides long and
containing unique nucleotide sequences which can
—
sulfate polyacrylamide gel clectrophotesi s), blotted
on to nitrocellulose strips and identified by
be used (hr the detection of homologous DNA by radiolabelled or enzyme- labelled antibodies as
hybridisation . DNA probes are being used probes. The western blot test has received wide
increasingly in the diagnose of infectious diseases. publicity as the confirmatory test for the diagnosis
Probes cont & i n ing sequences un i que to the microbc of HIV antibody in sera. The specificity of the test
( 5tm i n, species or group ) to be detected can be added depends on its ability to separately i d e n t i t y
to microbial cultures, body fluids, tissues at other antibodies d irocted agai nst different antigens of the
materials suspected to cont .tin the microbe nr its pathogen ( for example, against the surface, core
DNA, The DNA probe hybridises with the and reverse transcriptase antigen* of HIV ).
complementary specific sequences on the microbes Polymerase chain reaction ( PCR ): This
DNA.The advantages of DNA probes for diagnosis is a rapid automated method for the amplification
Copyrighted materia
< Bacterid Genetics 63
of specific DNA sequences {or genes), invented by diseases , in forensic in vesti gallons , L:I
Kary B Mullis in 19B3, for which he wnn the Nnhd arch cubic I logical studies of ancient specimens
Pri. iL in Chemistry in 1993 . L(_ R consists of several
1
B
and i n the exam i nation ui phylugeiietic relatioi:sllips
cycles nf sequenrial DNA replication where the in evolution.
products of the first cycle become the template for Based on the principle of PCR, other target
the next cycle . IT makes available Abundant quantities amplification systems have been developed . One
of speed ic DNA sequences starting from sources
containing minimal quantities of the same.
-ucli, Transcj i pi ion mediated j my I i i i cal ion i Tb1A )
which amplifies ribosomal RNA instead of DNA
The technique LS as follows TWO oligonucleotide has been applied as a rapid diagnostic technique
primers complementary to the flanking region of the for infections such as tuberculosis where cultures
DNA sequence to be amplified are incubated with are difficult or delayed .
the target DNA, nucleotides and DNA polymerase. MbletiuLer epidemiology: One offshoot of
The reaction consists of three essential steps: molecular genetics is molecular epidemiology Here
1. heat demtuiat i Dn of the sample DNA to si n L I L
^ - molecular methods such as plasmid profile analysis,
strand; genomic fingerprinting and PCR are used for the
2. annealing of sequence-specific oligonucleotide idcnriiiLdrinn and matching of microbial isolates
primers Co the boundaries nfthe DNA segment; for epidemiological purposes.
and Genetic mappinj: As a result of the
3. extern- ion of the primers by DNA polymerase remarkable advances in molecular genetics it has ,
to form new double-stranded DNA across the been possible to delineate the complete genomic
segment by sequential addition of sequences of bacteriophages and other viruses,
deoxvnuclcutides.
"
bacteria and their plasmids, and even of some
These three steps constitute one cycle of the eukaryotes including mammals. Quite- apart from
,
merlon. These cycles arc repeated several times, the useful information it has provided in
usually for 20-50 cycles in the thermocytler, at the microbiology ^ its success emboldened the
end of which hundreds of thousands of copies of international scientific community to venture on the
the original target sequences are available. AH the " human genome project', the most expensive and
reaction steps take place at high temperature [50- ambitious scientific project so far undertaken in
heat -stable polymerase, such as Taq 1 has biology. The results of this mammoth study became
to be employed. available by the dawn of the rwenty -first centuiy
With its enormous capacity to . Lmplitv DNA, and have opened vistas in human biology and
PCR is a versatile tool useful m diverse areas such medianeh as well as controversies and dilemmas
as diagnosis, of infectious, genelic or neoplastic that transcend medicine.
Further Reading
Hardy K . 19fJ 6 . KacIVraiJ Plasmids- 2nd cdn . W* : n-bi^ld. .
Innis MA it 1 1990. PC’R IVotorak, San Disf i Academic £*TVH -
MulLin KH . 1 99 ) The pilymrme chain n- . u lion ^. Nitl'i- l Lecture , SiuckhciLm.
Sambroak J er aL 19119. Mofcaifar Cloning. Cold Spring Harbour Laboratory
Tompki HE LS. 1992 . The use of molecular methods in infocticniE diBcaaet , .Vav EnglJ Med. 127:1290.
Towner KJ and A Cockayne 1993. MftfecLilir AfedioiJs for Microbe ] JtfejiriificaTran and Typing. London: Chapman - Hall .
V/arson JD et a]. 19H3. Rtcomh i lanr DJVA. New York: Scientific American BookE.
Copyrighted material
05 Infection
living microbes that subsist on dead or decaying When a new parasite sets up an infection in a
organic matter They are found in soil and water host whose resistance is lowered by a preexisting
and play an important role in the degradaTLon of infectious disease, this is termed secondary
organic materials in nature. They arc generally ijiftct/ on. The term focal infection ( more
incapable of multiplying on living tissues and appropriately focal sepsis) indicates a condition
therefore are of little relevance in infectious where, due to infection or sepsis ar localised sites
disease. Exceptionally, hewerer, some saprophytes such as appendix or tonsils , generalised effects
like B. iubalin may infect devitalised hosts whose are produced. When in a patient already suffering
natural re id stance i s greatly reduced ( npportvin i she from a disease a new infection is act up from
infection ) . Parasites are microbes that can establish another host or another external source, it is
themselves and multiply in hosts . Parasitic termed cross-infection. Cross-infections occurring
microbes may be either pathogens ( horn Greek in hospitals are called nosncutnial infections [from
pathos suffering, and gen produce, that is, disease- Greek nosocomjon hospital ). The term iatrogenic
producing) or oormnemais (from Latin com with; wiicctiii.' j refers co physician - induced infections
and men&t, table, i.e. , living together ). Pathogens resulting from investigative, therapeutic or other
are microorganisms that arc capable of producing procedures. Depending on whether rhe source of
disease in the host. Commensal microbes live in infection is from the host 's own bodv or from
jt
Complete harmony with the host without causing external sources, infect ion s arc classified as
any damage to it. The normal bacterial flora of cndt eiious or otogenous, respectively. Based on
the body consist largely of commensals. Many ^
the clinical effects of infections, they may be
commensals behave as facultative pathogens in classified into different varieties Inapparent
,
that they can produce disease when the host infection is one where clinical effects arc not
-
rcRii lance is Inwered -
It is necessary to di s r i ngu i sh between the term
apparent. The term subclinical infection is often
used as a synonym . Atypical infection is one in
‘infection ' and infectious disease '. The w h i c h the typical or characteristic clinical
"
lodgement
and multiplication of a parasite in or on the tissues manifestations of the particular infect! i ms disease
of a host constitute infection. It docs not invariably are not present. Some parasites , following
result LH disease . In fact, disease is but a rare infection, may remain in the tissues in a latent or
hidden form proliferauiig .inJ producing- dmLeal caused am called arthropod - home diseases. 1 nsects
di- >icuHe when the host resistance is lowered. Thi5 such as mosquitoes, ticks, mite?, flics, flea? and
is termed Intent infection. lice that transmit infectiiins are called vectors.
Transmission may be mechanical (for example,
S * * i RCBS UJP INFECTION transnlira. ion of dysentery or typhoid h& L ills bv
'
I Inmans: The commonest source of i nicer ion the domestic fly ) . Such vectors arc called
for huituiti at* hmmni themalvts Tht partite mechanical vecrors. In other instances , the
may originate from a patient or a carrier. A earner pathogen multiplies in the body of the vector, often
in a person who harbours the pathogenic undergoing part oi its developmental cycle in if .
microorganism without suffering from any |
effect because of it. Several types of carriers have
i. l - Such vector? are termed biological vectors (for
example, Aedes aegypri mosquito in yellow fever .
been identified. A healthy carrier is one who Anopheles mosquito m mataiLa ). Biological
liarboui $ the pathogen bait lias never > uffered from vectors transmit infection only after the pathogen
the disease caused by the pathogen , while a has mulLiphed in them sufficiently or has
CUJTVXJESCERF tamer is one who has recovered undergone a developmental cycle. I'hc interval
from the disease and continue? to hwbour the between the time of entry of the pathogen into
pathogen in hi? body. Depending on the duration the vector and the vector becoming infective is
of carriage , carriers are classified as temporary called the exmraic incubation period ,
and chronic, J’he temporary' carrier state last ? less Besides acting as vectors, some insects may
than six months , while chrome carriage may last also act os reservoir hosts (for mm pic , ticks in
L
-
for several V:; LT and sometimes even for the rest
of one’s life , llic term contact carrier is applied
relapsing fever and spatted fever) . Infection is
maintained in such insects by transovarial or
to a person who acquires the pathogen from i uansstadial passage.
patient, while the term paradoxi.- al carrier refer? Soil and water: Some pathogens can survive
to a earlier who acquire? the pathogen from in the soil for very long periods. Spores of tetanus
another carrier. bacilli may remain viable in the KCMJ for several
Animals: Many pathogen ? arc able to infect decades and serve a ? the source of infection. Fungi
both human beings and animal?. Animal? may, ( Histopiasma capsuhtuio , Nocardia astcroides )
therefore, act as sources of human infection. In and also parasites such as roundworm and
some instances, the infection in animals may be hookworm survive m the soil and cause human
asymptomatic. Such animals serve to maintain the infection.
parasite in nature and act as the reserve nr of human \ V. Lcer may set is the source ot infect ion ei ther
infections. Thcv .ire, therefore, called reservoir due to contaminaiion with pathogenic
hosfr . Infecti ' vs diseases transin itted I n in i an i mals microorganisms ( cholera vibrio , infective heparin ®
to human beings are called zoono e*. Zoonotic virus ) or due to the presence of aquatic vectors
^
diseases may be bacterial ( plague from mrs ) , viral (cyclop^ m guineiworm infection ).
( rabies from dogs), protozoal ( toxoplasmosis (tom Ftuni: Cun turn mated food may act as a source
cats), helminthic ( hydatid disease from dogs ) or of infection. The presence of pathogen? in food
fungal (aoophilic dermatophytes from cats and may be due to external contamination ( food
dogs). porioiuiLg by staphylococcus ) or due to pre -
In sects; Blood sucking insects may transmit existent i nfoctii > n i n meat or other am mol product
pathogens to human beings . The diseases so (salmonellosis ).
trantmiiried diseases such as syphilis and arboviruses injected by ] nsectvectors are examples.
gonorrhea illustrate spread by direct contact . The Infection by inoculation may be iatrogenic when
term ccrt £i£ii < HJSdisease ILJI.J been used KJT diseases unstetile syringes and surgical equipment are
transmitted hy direct contact, distinct from employed . Hepatitis 13 and the Human
infection disease signifying ail other modes of
*
Immunodeficiency Virus ( HIV ) may be
transmission.This distincrior is now not gcncraUv transmitted through transfusion of infix-red blood,
employed. Indirect contact may be through the or the use of contaminated syringes and needles,
agency of tomitez, which are inanimate objects particularly among addicts of injectable drugs.
such as clothing* pencils or toys which mav be Insects; Insects may act as mechanical or
r
contaminated by a pathogen from one person and biological vectors of infectious diseases.
act is a vehicle for its transmission to another . Congenital: Some pathogens are able to cross
Pencils shared by school children may act as the placental barrier and infect the fetus in utcro.
fomites m the transmission of diphtheria, and face This is known as vcrT?W transmission This may
,
.
towels in trachoma . result in abortion , miscarriage or stillbirth. Live
infants may be born with manifestations of a
Inhalation: Respiratory infections such as disease , as in congenital syphilis . Intrauterine
influenza and tuberculosis are transmitted by P
infection with the rubella virus, especially in the
inhalation of the pathogen. Such microbes are first trimester of pregnancy, may interfere with
shed by the patients into the environ men t, in organogenesis and lead to congenital
secretions from the m >se or throat during SflC«ingh malformation. Such intections are known as
speaking or coughing. Large drops of such tmtqgenic infections,
secretions fall to the ground and dry there . Ifltrogeriic amt laboratory infection ;
*
Pathogens resistant ro drying may remain viable Infection may sometimes be transmitted during
in the dust and act at sources of infection. Small administration of injections, lumbar puncture and
droplets* under 0.1 mm in diameter, evaporate CltheterigltUHl , il meticulous care in asepsis is
immediately to become minute particles or dropJc T lacking. Modern methods of treatment such as
-
nuclei ( usually 1-1 Q pm in diameter ) which exchange transfusion, dialysis, and organ transplant
remain suspended in rhe air for long periods, surety have i ncreased the possibilities for iatrogenic
acting as Sources of infection . infections. Laboratory peraannet bundling infectious
Ingestion: Intestinal inlecriors arc generally material are At risk and special care should be taken
acquired hy the ingestion of food Or drink to prevent laboratory infection.
contaminated by pathogens. Infection transmitted The outcome of an infect ion will depend on
by ingestion nuv be waterborne { cholera ) , die interaction between microbial fector? which
foodborne ( food pOLSonLng} or hartJborne predispose to pathogenicity and host fact ora which
(dysentery ). The importance of fingerborne contribute to resistance .
transmission is being increasingly recognised , not
only in the case of pathogens entering through FACTORS PREDISPOSING TO
the mouth, but also those that enter through the MlcHUBIAL PATH o u t f i t:t T v
nose and eves. The terms 'pathogenicity * and Virulence* refer to
Copyrighted materia
* Infection 67
the ability of a microbe to produce disease or tissue of many infections is the attachment of the bittern
injury bur it is c&nvenient to make a fine to body surfaces. This attachment is not a dunce
distinction between them . " Pathogenicity ' is
, event but a specific reaction between surface
generally employed to refer to the ability o\ a receptors on host cells and adhesive structures
nicrobinl species rn produce disease, while the f /rgandsjon the surface of bacteria. These adhesive
lent ] Viralem* is applied to tile same property structures are called .idhesuis. Adhesins may occur
in a attain of nnicrchorganisiia . Thus the species as organised structures, such as fimbriae or
\i. tuberculosis or the polio virus is referred to fibrillar and pill, or is colonisation factors. This
as being pathogenic. The pathogenic species specific adhesin may account for rhe tissue
AJ . rubcrLt/ loirs and the polio viruscontain strains tropisms and host specificity exhibited by many
of varviog degrees of virulence including those pathogen 5- Adhtsins serve as virulence factors,
which arc avirulent, such as the vaccine strains . and loss of adhesins often renders the strain
The virulence of a strain is not constant and may jviruleni. Adhesins are usually made of protein
undergo spontaneous or induced variation . and ate antigenic in nature. Specific immunisation
Enhancement of virulence is known as exaltation with adhesins has been attempted as a method of
and can be demonstrated experimentally by serial prophylaxis in some infections, as for instance
passage in susceptible hosts . Reduction ot against E. ctyli diarrhea in calves and piglets, and
virulence is known as jttemudofl and can be gonorrhea in human beings.
achieved hv passage through unfavourable hosts, Lnvasiveiiess: This refers fo the Ability of a r
repeated cultures in artificial media, growth under pathogen to spread in the host tissues after
high temperature or in the presence of weak establishing uifetbon, Highly invasive pathogens
utiseptie, desiccation, or prolonged storage in characteristically produce spreadi ng or generalised
culture.
Virulence is the sum total of several
-
lesions (fcg p streptococcal septicemia following
wound infection ) , while less invasive pathogens
determinants, as detailed below. cause more localised lesions ( erg p staphylococcal
«
Adhesion; The initial event in the pathogenesis abscess ). Some pathogens* though capable of
Copyrighted material
hi * Te*ttwok a1 Micnotnology *
causing serious or even fatal diseases , lack luting tor several hour*. The pyrogenic effect of
invusiveflW allugetlijer (e.g, , [he LeLunUS buillut fluids used for intravenous administration is
which remains confined r< > the site of entry and usually due to the presence of endotoxins from
produces the disease by elaborating a potent contaminant bacteria . Intravenous injections of
tutiji) - large doses ot endotoxin uni massive Gram
Toxijerucity: Huc- retix produce two types of negative septicemias cause cnUntmcic shock
—
toxins exotmt i ns and endotoxi ns,
Ejtotoxins ATC heat labile prutrirts which are
marked by fever, leucnpenia, thrombocytopenia,
significant tall in blood pressure, circuiaCcry
secreted by certain species of bacteria and diffuse collapse and bloody diarrhea leading to death
readily into die surrounding medium. They are ( Tables 9.1, 9.2 ) .
.
highly potent in minute imounts and constitute PtasniiJic Genes coding for some virulence
some of the most poisonous substances known. characteristics may be plasmid borne. Lixamplcs
One mgrst tetanus or botulinum toxin is Sufficient of pilimid-barnc virulence factors are surface
to kill more than one million guinea pigs and it antigens responsible for the colonisatiem of
has been estimated rhit 3 kg of botulinum toxin intestinal mucosa by F . coli and enteroioxin
cad kill .LL ] the inhahi runts of the world - Treatment production by E, aoSand .Staph. aureus. Multiple
of cjiotoxins with formaldehyde yields toxoids dmg resistance (R) plasmids increase the severity
which are notonc hut retain the ability to induce
.a
of clinical disease by their resistance to antibiotic
Antibodies ( antitoxins}. They exhibit Specific tissue therapy.
Affinities and pharmacological Activities, each Llacteriophagei: The classical example of
toxin producing a typical effect which can he phage directed virulence is seen in diphtheria. In
made out by characteristic clinical manifestations diphtheria bacilli, the gene for toxin production
or autopsy appearances . EJKUKKUU aie generally LV present in beta or richer tux ' ooiyncphages.
formed hv Gram positive bacteria hut may also Communicability: The ability' of A parasite
be produced by some Gram negative organisms to spread from one host ro another is known as
such as Shiga’s dysentery bacillus, vibrio cholera communicability. This property does not influence
and enterotoxigenic E. coti . the production of disease in an individual host
Endotoxins arc heat stable lipopoEysaccharidcs but determines the survival and distribution of a
{ EPS ) which torm jn integral pjrt of the cell parasite in a commumcy. A CWIcUtlOXl need not
wall of Gram negative bacteria. Their toxicity exist between virulence and communicability. In
depends on the lipid component (lipid A ) . They t .LCt. a highly virulent parasite may not exhibit a
are not secreted outside the bacterial cell and are high degree of communicability due to its rapidly
released only by the disintegration of the cell wall . lethal effect on the host . In general , infections m
They cannot he coxoided . They an? poor antigens which the pathogen is shed in secretions, as in
and their toxicity is not completely neutralised by respiratory or in test in si diseases , are highly
[ lie homolijguui Antibodies. They are active oulr communicable . In sortie instances, as in
in relatively targe doses. They do not exhibit hydrophobia, human infection represents a dead
specific pharmacological activities. All endotoxins, end, there being an interruption in the spread of
whether isolated from pathogenic or the pathogen to other hosts.
nonpathogen ic bacteria , produce similar effects. Development oi epidemic and pandemic
Administration of email quantities of endotoxin diseases requires the strain of pathogen to possess
in susceptible animals causes an elevation ofbody high degrees of virulence and communicability.
temperature manifested within \ 5 minutes and .
Other bacterial prciducttt Sr: me bacterial
Copyrighted materia
* Inlection » 69
products other than toxins, though devoid of or death , rcapectively, in a sustcprihlc animal
under
intrinsic tonicity, nwy contribute to virulence by standard conditions . As animals exhibit
inhibiting the mechanisms of host resistance. considerable individual variation in susceptibility,
Pathogenic staphylococci produce a ihrombin - these doses are more correctly estimated as
like enzyme conge!use which prevents statistical expressions, ID SO and I ,D 50, as the
phagocytosis by forming a fibrin barrier around dose required to infect or kill 50 per cent of the
the bacteria and willing off the lesion , animals tested under standard conditions .
lnhrinnlyritls promote the spread nf infect inos hv Route of infection: Some bacteria, j^ h ns
breaking down the fibrin barrier in Einhues. EtTCptOcocci , Can initiate infection whatever he the
HvaJuronidascs split hyaluronic acid which is a mode of entry. Others can survive and multiply
component of intercellular connective iissue and only when introduced by the optimal routes .
thus facilitate the spread ot injection along tissue CbdLen vibrio* arc infective orallvhut arc unable
r
leucocytes. Many pathogens produee hemolysins This difference is probably related to mode? by
capable of destroying erythrocytes but their which different hacterij are able to initiate tissue
significance in pathogenicity is not clearly damage and establish themselves. Bacteria also
understood , differ in their rites of election in the host body
Bacterial appentiniest Capsulitcd bacteria after introduction into tissues. They also differ in
such pneumococci , /C, ponuDcunt and / / '
the ability' to produce damage of different organs
influenzae in nor readily phagocyiosed. Some in different species of luiimaJs. Tubercle bacilli
bacterial Hiirtace antigens such as the Vi antigen injected into rabbits cause lesions mainly in the
of 5, tvphi , K antigen* of Er anti also help rhe kidney and infrequently in the liver and spleen,
bacteria to wirhstind phagocytosis and the lytic but in guinea pigs the lesions are mainly in ibe
activity of complement liver mil spleen , the kidnew being spared. The
Infecting dose; Successful infections require reasons for such selective multiplication in tissues
that an adequate number of bacteria should gain are largelv obscure, though they may he related
entry into the host. The dosage may be estimated to the presence in rissucs of substances that may
as the minimum infecting dose ( MID ) nr selectively hinder or favour their multiplication,
minimum lethal dose ( MLD ) which are ,
respectively, the minimum number of bacteria -
TVIM I M OP INPKCTIOU DISKASKS
*
required ro produce clinical evidence of infection Infections diseases maybe localised or generalised.
Copyrighted material
70 * Texibook of Microbiology *
Localised infections may hr: superficial deep or depending or their spread in the community,
seated Generalised infccrinn involves the spread
. infectious diseases may be classified into different
of the infecting agent from the sice of entry by
contiguity, through tissue spaces or channels,
-
types . Enticmi : diseases arc those which are
consrandy present in a particular area. Typhoid
along the lymphatics or through the bloodstream. fever is endemic in most parts of India.An tpicfemte
Circulation of bacteria in the blood is known as disease is one that spreads rapidly, involving many
bacteremia . Transient bacteremia is a frequent persons in an area at the same time . Influenza
event even in healthy iruiividud; and may occur causes annual winter epidemics in the cold
during chewing, brushing of teeth or straining at countries. A pandemic Ls an epidemic that spreads
Stools, The bacteria are immediately mapped up thmugh many areas of the world involving very
by phagocytic cells and arc unable to initiate large numbers of people within a short period.
infection, JJactemrus ofgreaterseverity and longer Influenza , cholera, plague and cntcroviral
duration in seen during generalised infections as conjunctivitis are pandemic diseases. Epidemics
in typhoid fever, Sepricerma is the condition vary in the rapidity of spread. Waterborne diseases
where baden .1 circulate and mill [ i ply i n the blood, such us cholera and hepatite may cans? explosive
form toot ic products and cause n igh, swi nging type
of fever. Pyemia is a condition where pvogenic
outbreaks, while diseases which spread by person
to- person contact evolve more slowly. Such
-
bactena produce septicemia with multiple creeping or smouldering epidemics, as that of
abscesses 1 n the 1 niernal organs such as the; spinn, cerebrospinal fever, are termed pnosudemiL-
liver and kidney. diseases*
Further HonJinjl
Mint* CA- 1?S7. The /1ithqprne.sjs of Infectious Disease. 3*1 edn. London: Academic PTTJ*.
Rati ick S and MJ Larkr: 1995 . /FflJiWFKiAqpraJ and Molecular aspects of Bacterial K'imSence. Chichester: Wiley;
]
Copyrighted material
72 4
-
Te Jtibaok of MjcrobioJogiy
than the Caucasians- to tuberculosis. But such Some infections l i k e poliOntyel 11 i s and
comparisons are vitiated by menial influences such chickenpox tend to be more severe in adults than
as differences in socioeconomic levels . An in young children, due to hypersensitivity that
interesting instance ot genetic resistance to causes greater tissue damage. Conversely hepatitis
Plasmodium fakipanjm malaria is seen in some B virus infcctionl in the newborn are usually
parts of Africa and die Mediterranean coast. A asymptomatic because clinical disease requites
hereditary abnormality of ted cells ( sickling ), adequate immune response wliuch. is lacking at that
prevalent in the area , confer immunity to infection age. Hovrever, the virus multiplies unrestrained and
by the maJari .d parasite and may have evolved from such neonates end up as chronic viral carriers, often
the survival advantage conferred by it in a malari . ii developing late hepatic complications. Old persons
environment . arc highly susceptible to infections due to the
The differences in innate immunity exhibited waning of their immune responses and other
bv. different individuals in a race is known as infirmities like enlarged prostate leading to urinary
indjvidudimfn iiyThc genetic basis of individual
'
stasis.
^
immunity is evident ffnm studies on the incidence Hormonal Influence );: Endocrine disorders
of infectious diseases in twins. It is well documented such as diabetes mellirus , hypothyroidism and
that homozygous twins exhibit similar degrees of adrenal dysfunction are associated with an enhanced
resistance or susceptibility to lepromatous leprosy susceptibility to infections. The high incidence of
and tuberculosis. Such correlation is rtOT seen in staphylococcal sepsis in. diabetes maybe related to
heterozygous twins . the increased level of carbohydrates in tissues.
Several factors influence the level of innate Cort icostcroi Js exert an i mportant i influence on the
immunity in an inLlividuaJ: response to infection. Thev depress the host’s
A.fce: The two extremes of life carry higher resistance by their anr I inflammatory and
susceptibility to infectious diseases as compared antiphagocytic effects and by the suppression of
with adults. The fetus in uttro is normally protected antibody formation and hypersensitivity. They also
from maternal infection by the placental barrier. haw a beneficial effect m that they neutralise the
But some pathogens cross tins harrier causing harmful effect of bacterial products such as
overwhelming infections resulting in fetal death - endotoxins The elevated steroid level during
Smnc , such as rubella , herpes cytomegaloviruses pregnancy may be related to the heightened
and ,r . : injJ .^ .' ii . i IFONTJJJ , le .Lcl to congenital susceptibility ol pregnant women to many
malformations . 1"he hv ightencd susceptibility of the i nfections. The reported effect of stress in iitcreasing
fetus to infection -s related to the immaturity of its susceptibility to infections, may in some measure
immune apparatus . Newborn animals are more be due to the release of steroids.
susceptible to experimental infections than older Nutrit «m: The infraction between malnutrition
ones . Coxsackie viruses cause fatal infection in and immunity is complex but 1 in general , both
melding mice but not in adults. humoral and cell mediated immune processes are
Increased susceptibility in the young nuty, in some reduced when there is malnutrition. Cell mediated
instances , be due to hormonal influence . Tinea capitis immune responses such as the Mantoux test become
caused by Microsportim audouiitii frequently negative in severe protein deficiency, as in
undergoes spontaneous cure with the onset of puberty kwashiorkor. Because of its wide prevalence,
The susceptibility of the vaginal epithelium in malnutrition may well be the commonest cause of
prepubertal girts to gonococcal infection is another i n mu ii i identic in:v.
iff
instance of the effect of sex hormones on resistance . Paradoxically [here is some evidence that certain
Copyrighted materia
* Immunity » 1$
infections may nor became clinically apparent in Pianides. that manage to reach the pulmonary alveoli
the severely malnourished . Malarial infection in are iogcsrcd bv the phapjotytic cells present there.
the ranine stricken may nut induce fever but octet Tire mouth is constantlv bathed in saliva which
chcir nutrition is improved , clinical malaria hn.s an inhibitory effect cm many miLTacsTgam ^ iTis.
develop, fr has also been reported that some viruses Particles deposited in the mouth arc swallowed and
may not multiply in the tissues of severely subjected to the action of the digestive juices. The
malnourished individuals. high acidity of the stomach destroys most
microorganisms. The pH becomes progressively
MECHANISMS O F INNATE IMMUNITY alkaline from the duodenum to the ileum. The ifcum
ftpithelin! surfaces; The intact skin and contains a rich and varied flora and in. the large
mucous membrane covering the body protect if intestine,. the bulk of the contents is composed of
considerably against invasion by microorganisms. bacteria .
They provide much marc rhan u mechanical The intestinal mucosa is covered bv a bcelike
barrier. Healthy skin possesses bactericidal activity network of mucus. Particles get enmeshed in the
In which the presence of high concentration of sail mucus and form small masses which ire propelled
in the drying sweat, the sebaceous secretions and by peristalsis.
the long chain fatty acids and soaps contribute. The conjunctiva is freed of foreign particle* hy
When cultures of typhoid bacilli placed on healthy the flushing action of lachrymal secretion*. 'Hie
skin and on a glass surface are sampled at intervals, eyes become susceptible 10 infection when
the bacteria on the skin are seen to be killed within Lachrymal secretions are absent. Tears contain the
minutes, while those on glass survive for several antibacterial substance lysozyme, first described hy
hours. The bactericidal activity of skin secretions
JF Fleming (1922), This is a thermolabllc low
is illustrated by the frequent mycotic and pyogenic molecular weight basic protein which acts as a
infections seen in persons who immerse their hands inuramiiudasc - Lysozyme is present in tissue fluids
ip soapy water for l -: mg pnods OCCUpatLLipallv. and in nearly all secretions eveepe cerebrospinal
Though the skin frees itself readily of bacteria tluid , sweat and LLriue. It acts by splitting certain
deposited or it ( transients ), its reactions are different polysaccharide components r ? t the cell walls of
to the bacterial flora normally resident on it . susceptible bacteria. In the concentrations seen in
Resident flora arc not easily removed evert hv tears and other secretions, lysozyme is active onlv
washing and application of disinfcctant*- against & nmc nnnpsthngenic fltani positive
'
I hc mucosa of the respiratory tract has several
'
bacteria. However, it occurs in phagocytic ceils in
innate mechanisms of defence. The very architecture concentrations high enough to be lethal to mint
of the nose prevents entry of microorganisms to a pathogens .
Urge extent, the inhaled particles being arrested at The flushing action of urine eliminates bacteria
or near the nasal orifices. Those that pass beyond from the urethra. Spermine and zinc present in tbc
flic held by the mi LOLLS lining the epithelium, and nemen carry out antibacterial activity. The acidity
arc swept hack to the pharvmt where they tend to of the adult vagina, due to the fermenration ul
be swallowed or coughed out. The cough reflex LS glycogen in the epithelial cell* by the resident
an important defence mechanism of the respiratory' aciduric bacilli , makes it inhospitable io many
tract. The cilia on the respiratory epithelial cells pathogens .
propel particles upwards. Nasal and respiratory Antibacterial substances in hi nod and
secretions contain mucopolysaccharides capable of tisSUCSl The complement system possesses
combining with influenza ailJ certain other viruses. bactericidal activity and plays ,in important role in
Copyrighted material
74 * Textbook ai Microcnology >
the destruction of pathogen / bacteria that ; nvade and the monocytes in the blood. A major fund ion
the blood and Tissues (see chapter 14) , of the reticuloendothelial system is the removal of
Several substances possessing antibacterial foreign particles that enter the body. Phagocytic
properties have been described in blood and tissues. cells reach the -. ites of inflammation in large
These include ( 1 } beta lysi nh a relatively numbers, attracted by chemocactic substances, and
thermostable suhstance active against anrhricf and mgest particulate materials . Capsulated bacteria ,
related bacilli ; ( 2 ) basic polypeptides such as such AS pneumococci are not readily phagocytoscd
,
Lukins extracted from leucocytes and pkkins from except in the presence of opsonins. They are more
platelets; i -i I acidic substances, such as lactic acid readily phagocytosed when trapped against a firm
found in muscle tissue and in the inflammatory. surface such as the alveolar wall than when they
Tores; And ( 4) lactnperoxldase in milk. While these are free in [ issue fluids. Bacteria are phagocytosed
substances possess antibacterial properties into a vacuole (phagosome ), which fuses wirh the
demonstrable experimentally, their relevance in the lysosomes round in the cell to form the
natural context is not clearly understood . phagolysosome . The bacteria are subjected to the
A method of defence ag . iinst viral infections is action of the Ly 1 :c enzymes in the phagolysosome
the production or : utcHeror by cells sri - uulated by and ate destroyed. Some bacteria, such as brucella
live or killed viruses and certain other inducers. and lepra bacilli resist intracellular digestion and
,
Interferon has been shown ro be more important may actively multiply inside the phagocytic cells.
than specific antibodies in procecdon against and Phagocytosis in such instances may actually help
recovery from certain acute \ iral infections/! :" sues to disseminate infection to different parts of the
'
free animals to all types of infections . Jnllammntinn: Tissue ir- iury or irritation ,
Cellular factor* in innate immunity; initiated by the entry of pathogens or other irritants ,
Natural defence against the invasion oiblouJ and leads to iuflanimation, which is an important ,
Tissues by microorganisms and other foreign nonspecific defence mechanism. The arterioles at
particles Is mediated to a large extent by phagocytic the site constrict initi . dly and then dilate leading to
cells which ingest and destroy them. Phagocytic ai L i ncieased h ]ood flow. There i s a slowing of blood
cells, originally d / eovered by Mctchnikoff ( 1883) , flow and margination of the leucocytes , which
were classified by him into microphages and escape into the [ i ssuc > by diaped*:sis and accumulate
macrophages. Micro phage a arc polymorpho - in large numbers , attracted by the chrmntACfii;
nuclear leucocytes . Macrophages consist of substances released at the site of injury.
hi * riocytcs which ate the wandering ameboid cells Microotgamsms arc phagocytosed and destroyed .
seen in issues, the fixed ft- iu- ubenduthelLaJ cells [ here is an outpouring of plasma, which helps to
opyrighted materia
i Immunity 75
.
dilute die roxic produces present A fibrin barrier is cite antigen combines with any pie outing antibody
bid, serving ro wall off the site of infection. and lowers im level in circulation. Once developed,
I ' ever A rise of temperature following infection the active immunity is long- lasting. If in individual
L:- .1 natural defence mechanism. it not merelv helps who hi? been actively immunised against an antigen
ro iccekntt the physiological pcDcene but m^v, experience? the same antigen subsequently, the
in somf eases , actually destroy the infecting
* immune response occurs more quickly and
pathogens . Therapeutic induction of fever was abundantly than during the first encounter. This is
employed for the destruction of Treponema known ;LH .tfeondarv respond. Besides the
pallidum in the tissues of syphilitic patients before development of humoral and cellular immunitv,
penicillin became available . Fever stimulates the active immunity is associated with Immunological
pnxductiun ofinterferon and aids recovery trum viral memory. I '’
his implies that the immune system is
infcctions- able to retain tor long periods the memory of a
Acule phase proteins: Infection or Injury prior antigenic eKposure and ro produce a secondary
lead? to a sudden increase in plasma concentrations type of response when it encounters the same
of ccTfflin proteins, collectively called acute phase antigen again , Active immunisation is more
proteins. These include C reactive pmtcil ( CRP), effective and confers better protection than passive
mannose binding protein, alpha -1'me id immunisation .
glycoprotein, strain amyloid P component rod many The resistance that is transmitted passively to a
otheir. CRP and some other acute phase proteins recipient in a ’readymade' form is known as pltnfe
activate the alternative pathway of complement. murium'ty. Here the recipient 's immune system plays
rrhcy arc believed to enhance host resistance, prevent no active role. There is no antigenic stimulus;
tissue injury and promote repair of infiam maton 1
Copyrighted material
76 * Textbook ol Microbiology *
.
Ti 1 - -
i : immunisation i that H . kL immediately
and , therefore, can be employed w'hen ' instant '
Examples of vaccines are
1 . Haute:ial vaccines
as follows:
-
immunity is de ired I. I .ible 10.1).
Active immunity may be natural or arrifieiaL
a. Live ( BCG vaccine for tuberculosis)
b. Killed {Cholera vaccine )
Natural active immunity results ITUKI either a c. Subunit i ryphuid Vt antigen I
clinical or Jin inapparent infection by a microbe. A d. Bacterial products (Tetanus taw ; id )
--
pn r T I who lias recovered from an. attack of measles 2. Viral vaccines
develops natural active immunity. The huge majority a. Live ( Oral polio vaocinc^Sabin )
of adults in the developing countries possess natural b Killed {Injectable polio vaccinc-Salk)
active immunity to poliomyelitis due to repeated
, c. Subunit { Hepatitis 13 vaccine)
inappsrent infections wi. rh polioviruses during r ive vaccines initiate an infection without
childhood . Such immunity is usually long-lasting causing any injury or disease. The immunity
but the duration varies with the type of pathogen. Following live vaccine administration therefore
The immunity is lifelong following many ' iral parallels that following natural infection though it
diseases such as chicken pox or measles. In some may be of a lower order. The immunity lasts for
viral diseases* such as influenza or common cold, several years but booster [loses may be necessary'.
the immunity appears to be shortlived. Influenza Lice vaccines may be admi -listened orally (as with
can recur in an individual after a few months or a the Sabin vaccine for poliomyelitis) or paicntcrally
year hul this in not SD much due to lack of the (as with the measles vaccine ). Kilted I' sccines are
i menu n :ising capact ty of the virus as to : ts abil 1 ry to generally less immunogenic than live vaccines* and
undergo lAti mr variation so that immuniiy prnteclion lasts only 1 or a short period. They have,
^
following the first infection is not effective against therefore * to be administered repeatedly' generally
the second infection caused by an antigcmcally novel at least two doses being required for the production
virus . In common cold , the apparent lack of of immunity'. The first is known as the primary
immunity is because the same clinical picture can dose and the subsequent doses as booster doses.
be caused by infection with a large Humber of Killed vaccines may' be given orally but this route
diflfcrcn t viruses.The i mniulity follow 11 ig bactci i al is generally not effective. Parenteral administration
infection is generally less permanent than that provides humoral antibody response, which may
following viral infections. Some, such as typhoid be improved by the addinon of ‘adjuvants’ (for
fever , induce durable protecti ^ n. In syphilis , a speci .d example* aluminium phosphate ).
type of immunity known ss ' premunition’ i seen .
I lere, the immunity to reinfection lasts only as long
- Natural passive immunity is the resistance
passively transferred from mother to baby. In human
JV the original infcc’ Jon ncmnins active. Once the infants , maternal antibodies arc transmitted
disease is cured, the patient becomes susceptible to predominantly through the placenta, while in
the spirochete again. In chancroid, another venereal animals such as pigs* transfer of antibodies occurs
disease, caused by Haemophilus ducreyi, there does mainly orally through the colostrum - The human
not appear to be any effective i mrnuii Lev as the pat i mi colostrum, which i? also rich in IgA antibodies
may develop lesions following reinfection even resistant to intestinal digestion , gives protection to
while the original infection in active. the neonate.The human fetus acquires some ability
Artificial active immunity is rhe resistance to synthesise anybodies ( IgM ) from about the
induced by vno lines . Vaccines arc preparations of twentieth Week of life but LCE- immunological
I ivc or k i I led n i icrootgan i sms or thei r products used capacity is still inadequate at birth. It is only by
for immunisation . about the age nl three months that the Infant
Copyrighted materia
» Immunity
* 77
acquires SUIUE measure qf immnnnhig Lea I origin arc now recommended only where human
independence. Until then, maternal antibodies giivt ] ! reparations are not available ( and gas gangrene
passive prelection against iriJbctknis dlKUc4 to the and anti botulimim scrat antivenoms ) .
infant - Transport of antibodies across the placenta CoautlciCClK sera (sera of patients recovering
is an active process and therefore the concentration from infectious diseases) contain high levels of
of antibody in the fetal blood may sometimes be Specific aittihudy. Pturletl human gmutiggiobuUa
hiirhui tllin that seen in the mother Profcr rion so (gammaglobulin from pooled sera of healthy adults)
afforded Will ordinarily lie adequate against all the contains antibodies against Jill common pathogens
.
common infectious diseases in the locality It is for prevalent in the region, Convalescent sera and
this reason that most pediatric infections arc DKHE pooled human gammaglobulin were used tor
common after the age of three months than in passive immunisation against some vims infections
younger infants. By active immunisation of mothers (like viral Hepatitis A). Human gammaglobulin is
during pregnancy, it is possible to improve the also used in the treatment of patients with some
quality of passive immunity in the infants.
Immunisation oJ pregnant women with tetanus
-
immunodeficiendcS Gammaglobulin tends to
aggregate and when injected intravenously may
toxoid is recommended for this purpose in cause anaphylactic reaction due to complement
communities in which neonatal tetanus is common. activation. They are therefore to be given only
Artificial pawivt immunity is the resistance intramuscularly. It has to be ensured that all
passively transferred to a recipient by the preparations from human sera are free from the
administration of antibodies . The agents used for risk of infection with hepatitis Bn hepatitis C „111V
this purpose are hyperimmune sera of animal or nd other infective agents.
human origin, convalescent sera ami pooled human PSSHVC immunisation is indicated for Immediate
gamma globulin. T hese are used for prophylaxis and temporary protection in a nonimmune host
and therapy, Equine hyperimmune sen such as faced with the threat of an Infection, when there L 5
antitetanus scrum and ATS prepared from insufficient time for active 1minimisation to take
hyperiimmunised horses used to be extensively effect, It is also indicated for the treatment of Home
cm ployed. They gave Temporary protection but infections , Passive LTD mu nidation may also he
carried the disadvantages of hypersensitivity anti employed for rhe suppression of active immunity,
-
immune elimination Human hyperimmune when the latter may be injurious. An example is
globulin ( for example , tetanus immune globulin, the IISC of RJl immune globulin during delivery to
TIG) is free from those complications and also prevent Immune response to the khesus fectnr in
gives more lasting protection. Antisera of animal Rib negative women with Kh positive bablCS-
Sometimes a combination il active and passive protective annijert, serological attempts to measure
methods of immunisation is employed. This is i mmunity arc at best only approxi mati i ms. In some
known ns combined immunisation. Ideally , instances, as m diphtheria where pathogenc^ is is
whenever passive immunisation is employed for due to a well-defined antigen ( the toxin ) , the level
IITIMO- Liiatc protection, combined immunisation is of immunity can he assayed by in vitro or in vivo
- -
to IT- L p re hr rred , a.- i n the prafeefi i > n of a non ' mrnune
1 |Schick testi methods . Where protection is
individual with a tetanus - prone wound . The associated with cell-mediated immunity, skin tests
method is to inject TIG in one arm and the fust for delayed hypersensitivity and in vitro tests for
dose ill tetanus toxoid in the other. This is fallowed CM! ariord an indication ot i mmun i ty.
by the full course of phased tetanus toxoid Injections.
TIG provides the protection necessary till the active LOCAL I MUI NITlf
immunity is able to take effect. fhe concept of local immunity, proposed by
A special type oJ immunisation is the injection Besredka (1919-74), has gained importance in the
of immunoloj-ricaJlv cumpereiit lymphocytes. Tics .
treatment of infections wiii - h arc localised or where
is known as .u /oprnc immunity and does nut have il is operative combating infection at the site of
lit
general application. Instead of whole lymphocytes, primaiy entry of the pathogen . In poliomyelitis, for
'
MEASUREMENT OF IMMIMTI
-
Thi - is achieved by the local intestinal immuniry
acquired either as a result of natural infection or
'Hie tnily valid measurement of immunity is to test immunisation wirh the ILVC oral vaccine. In
-
the re .btance of an individual to a challenge by influenza, immunisation with the killed vaccine
-
the pathogen.Thi IH, however, nor applicable since
the challenge itself altera the state of imitiunily. it
elicits a humoral antibodv response. But the
antibody litre in respiratory secretion? is often not
is, therefore, rot possible to measure accurately the h igh enough to prevent infection, N atural infection
level of mmunity in an individual. Estimates of or the Lve virus vaccine administered intranasally
immunity are generally made by statistical methods provides local immunity. A special class of
using large numbers n ) individuals. immunoglobulin ? ( IgA ) forms the major
A simple method of testing immunity is to relate component of local immunity.
its level to some convenient indicator, such as One type nf IgA antibody called secretory IgA
demonstration of the specific antibody. This i > not is produced locally by plasma cells present on
always reliable as the immune response to a mucosal surfaces or in secretory glands. There
pathogen consists of the formation of antibodies to appears to be a selective transport of such antilxhlics
several antL ens present m it , as also to the between the various mucosal surfaces and secretory
^
production of cellular immunity. The antibodies glands . Thus, following intestinal exposure to an
maybe demonstrated by a variety of techniques such antigen, the Specific IgA anubody and the plasma
as agglutination, precipitation, complement fixation, cells (ormiogsuch an anribody can bedemonstrated
hemagglutiItalian inhibition, neutralisation , Kl . iS.A in breast milk - This indicates the existence of a
and others. In the absence of exact information as common mucosal or sccmroij immune system .
to which antigen of the pathogen constitutes die Besides providing local defence against
Copyrighted materia
n I nmunhty 79
-
l uirth gr itctfcliiiij
.
- 1
—
Copyrighted material
1
Antigens
An antigen has been defined as any substance or sensitised cells (immunological reactivity ). Based
which . when introduced parentcralU into the body, on the ability to carry out these two functions,
st i mutates the product in of an an t Lbody with w] i i eh antigens may be classified into different types. A
^
u reacts specifically and in an observable manner , complete antigen is able to induce anlibudy
-
Thi ; traditional description of an antigen is no
longer comprehensive enough in the light of current
formation and produce a specific and observable
reaction with the antibody so produced. Haptens
nancepts about the immune response. Some antigens are substances which are incapable of inducing
may rot induce ant i bodi LLS but may sens i r i sc spec ific antibody formation by themselves but can react
lymphocytes lead nig to «11 mediated immunity or specifically with ami bodies. (The term hapten is
may .. uisc i mmunologi cal tolerance. derived (TOITI the Greek h&ptcin which means ' to
The word ' parenteral' ( meaning, outside the fasten’. ) Haptens become immunogenic (capable of
intestinal tract ) is used in the definition because inducing antibodies ) on combining with a larger
orally administered antigens arc usually denatured molecule carrier. Haptens may be complex or
by digestive enzymes and their antigenicity simple; while compkis; haptens can precipitate with
destroyed, so that no sntiiwdv formation takes place. specific antibodies , sim /ife haptens are
When given parenwrally, antigens do nor undergo nonprecipitalirtg. Fhey can inhibit precipitation of
any such inactivation and can induce antibody specific antibodies by the corresponding antigen
production. However, there are exceptions and Home or complex hapten. Complex and simple haptens
antigens can be immunogenic when given orally, have been described as polyvalent and univalent,
such as oral vaccines . respectively, since it is assumed that precipitation
The word 'specifically’ in the definition is requires the antigen to have twn or more antiiiody
important as specificity is the hallmark of all combining sites,
immunological reactions. An antigen introduced [ iic smallest unit of antigenicity is known as
into the hndy reacts only with those particular the anf jgenic determinant or epitope . The epitope
iinmunocytcs ( B or T lymphocytes) which cany
the specific marker for that antigen and which
^
is that small area on the antigen, usually consisting
of tour or five aminoacid or monosaccharide
produce an antibody or Cells complementary to that residues, possessing a specilie chemical structure,
antigen only. The antibody so produced will react elect ] ical charge and stone [spatiid ) configuration,
cully with that particular antigen and with no other, capable of s e n s i t i s i n g a n immunocyte and of
though immunological cross reaction may occur reacting with its complementary site on the specific
between closely related antigens. antibody or T-ceU receptor. Epitopes may be present
The two attributes of inti genicity are (1) as a single linear segment of the primary sequence
induction of an immune response ( immuno
genicity) , and ( 2) specific reaction with antibodies
- [sequen t ial or linear epitope ) or formed by bringing
together on the surface residues from different sites
V- opyrighted materi3
M
* Anlfcg & ns SI
of RLIL:peptide :II:L n during :LH folding into [ Lie .Susceptibility to tissue enzymes: Only
L ]
tcrtiair ttniCtVR (conformational epitope ) . I cellf substances which arc metabolised and arc
recognise sequential epitope* while B cells identity susceptible to the action of tissue enzymes Liehave
*
the tertiary Configuration of the conformational as antigens . Antigens introduced into the body are
epitopes . The combining area on the antibody degraded by die host .it Lto fragments of appropriate
molecule, corresponding to the epitope , it; called size containing the antigenic determinants .
the pnnur>iK - llpitopcs and paratopes detemninc tire Phagocytosis and intracellular enzymes. appear to
,
specificity of immunologies! reactions . Antigens such play an essential role in breaking down antigens
at bacteria or viruses carry many different types of into immunogenic fragments , Substances
cpitofies,presenting an antigenK mosaic.!he presence unsusceptible to tissue enzymes such as polystyrene
nf the ^mc or siniilar epitopes on different antigens Latex are rot antigenic. Substances very rapidly
accounts for one ty|>e of antigenic cross- reaction. broken down hy tissue enzymes arc also not
antigenic. Synthetic polypeptides , composed ofD-
DETERMINANTS A NTIUENUJIT Y
OF aminom-icls which are not metabolised in the body,
A number of properties have been identified which are not antigenic , while polypeptides consisting of
make a substance antigenic but the exact basis of [ ,- ainmoacida are antigenic ,
1
antigenicity is; still not clear. hordfintiests: Only antigens \ v ]iich arc “ foreign
Siv.arr Antigenicity is related to molecular size. Very to the individual ( nonself ) induce an iulmuile
large molecules, such as hemocyaruns {MW h . 7 s response . The animal body contains numerous
millioo), are highly antigenic and particles \i i rh antigens which induce an immune response when
lots molecular weight ( less than SOUO) arc introduced into another individual or species. An
nonantigcnic or feebLv HO . LOW molecular weight individual tides not normally mount an immune
substances maybe rendered antigenic by adsorbing response against Liis (iwn normal constituent
them on Large inert particles such as bentonite or antigen'- "Hiis was first recognised hy Ehrlich who
kaolin . Some Low molecular weight substances proposed rite concept oThorror autotoxieus ' ( which
(such as picrvl chloride , formaldehyde and means fear of self 'poisoning) . ToLeraTLce of Self
penicillin) may he antigenic when applied on the antigens is auditioned by contact with them during
skin, prohablv hv combining with tissue proteins. the development of the immune apparatus .
'lTev are haptens of Low inimnnogenicitv, effective Ifeeakdown of this homeostatic mechanism result ^
in some persons only and related to hypersensitivity, in autoimmuniiaiion and autoimmune disease .
Chunriunl nature: Most naturally occurring Io general , the antigenicity of a substance is
antigens are proteins and polysaccharides, Lipidi related to the degree of its foreignness. Antigens
and nucleic acids are Less antigenic . Their from other individuals of the same species ;irc less
antigenicity is enhanced by combination with antigenic than chose front other species. Antigens
proteins. A certain degree of structural diversity is from related species are less antigenic than those
required for antigenicity. That probably explains from distant species.
why proreins which are composed if about 20 Antigenic specificity': The basis of antigenic
different jmiiiuAetds better antigens than
ire specificity is stereochemical , as was fim
polysaccharides which have Only four or five demonstrated by Obermajer and Pick and
monosaccharide units. However, not all proteins confirmed by Landsteitier. Using haptens such as
arc antigenic , A wed- known exception is gelatin, :Lt ( > xvi coupled with protein , it was shown that
Copyrighted material
82 * Textbook oF Microbiology
radical. The importance ot the position of the have been supplanted by the more discrimiuatoiy
antigenic determinant group in the antigen DNA fingerprinting tests . Blood groups find
molecule was evidenced by the differences in application in anthropology.
specificity in ootopfliods with the group attached Histocompatibility antigens arc those cellular
at the ortho, mefa fir /wa positions . The infinciue determinants specific to each individual of 4 species.
of spatial configuration of rhe determinant group Thcv arc recognised by genetically d Life rent
was shown by differences in antigenic specificity individuals of the same species when attempts arc
of the efejftro, leva and meso comers of substances made to transfer or transplant cellular material from
such as tartaric add. one individual to another [described in Chapter
Antigenic specificity is not absolute . Cross 15 ) .
reactions can occur between antigens which hear Autoapecifioity; Autologous or self antigens
stereochemical similarities - In some instances, are ordinarily nunantigenii: but there are exceptions.
apparent cross reactions nuy actually be due to the Sequestrated antigens that are not normally found
sharing of identical antigenic dctcrminants hy free in circulation or tissue fluids [ such as eye lens
different antigens . protein normally confined within its capsule ) arc
The specific] tv of natural tissue antigens of not recognised as self antigens. Similarly, antigens
animals may be considered under different categories that are absent during embryonic life and develop
as species, iso-, auto- and organ specificities. Later {such as sperm ) arc also not recognised as self
Specie *peuificit > i Tissues of all individuals antigens .
in a species contain iperia-specific antigens. There Organ specificity : Some organs, (inch as the
-
exists some degree of cross reaction between
antigens from related species. This immunological
brain, kidney and lens protein of different species-,
share the same antigen. Such antigens, characteristic
relationship parallels phylogenetic relationships. It of in organ or tissue and found in different species-,
has been used in tracing evolutionary relationships are called organ specific antigens. The
between species. If also has. forensic applications neuroparalytic complications following jLHtirabic
in the identification of the species of blood and of vaccination using sheep brain vaccines arc a
seminal stains. Phylogenetic relationships are cumequence of brain specific antigens shared by
reflected in the extent of cross reaction between sheep and human heiogs. The sheep brain antigens
antigens from different species that cause induct immunological response in the vaccinees,
hypersensitivity. An individual sensitised to horse damaging their oemus tissue.
scrum will react with serum from other equities 11tie rotfe no He ( heterophile) specificity:
bur may not do so with bovine serum.
Jr
'
Hie same or closely related antigens may sometimes
I &oapecificilv: lsoantigcm arc antigens found occur in different biological species , classes and
in some but not all members of a species. A species kingdoms. These are known as heterogenetic or
may be grouped depending on the presence of heterophile arttigenfiT best exemplified bv the
different isoantigens in its mem hern . The hest Forssman antigen which is a lipid carhuhydrate
examples oJ isumltigens are the human erythrocyte complex widely distributed in many animals , birds,
antigens based on which individuals can be plants and bacteria. It is ahsent in rabbits, so anti -
classified into different blood groups. 'These are Forssman antibody can be prepared in these
genetically determined . They are of clinical animals. Other heterophile antigens are responsible
importance in blood transfusion and in for some diagnostic serological reactions in which
isoimmunisation during pregnancy. They were of antigens unrelated to etiological agents are employed
help in determining disputed paternity cases, hut [ heterophile reaction ) . The Weil — Felix reaction in
Copyrighted materia
4 Anttgena * 83
typhus fever, the Pau ] Bunncl test in infectious pneumococcal capsular polysaccharide , bacterial
mononucleosis and the cold agglutinin test in lipopolya ace ha rides and the flagellar protein
primary atypical pneumonia are examples. flagd I in . Their immune response is critically dose
dependent . Too little is non iinmunogtnkp while too
liLOLOGicAL CLASHES OF ANTIGENS much results in immunological tolerance rather
Depending on their ability to induce antiihody than immunity. Their antibody tesponse is usually
formation, antigens are clarified asT cell dependent limited to IgM and IgG 3. They do not show
(TD) and T cell independent (TI ) antigens . immunological memory. I [ antigen:- dn not appear
Antibody production is the property of B to require preliminaiy processing by macrophages.
lymphocytes. For the full express ion of rh i --. function, They are metabolized very slowly and remain in
however, the coopcrarion of T lymphocytes is the body for long peril ids.
necessary. Some antigens can directly snmulate TD antigens, on the other hand, are structurally
antibody production by B cells, without the apparent more complex, such as erythrocytes, scrum proteins
participation of T cells. Such antigens are called TI and a variety' of protein-hapten complexes. They
antigens. Others that require T cell participation to are immunogenic over a wide dose range and do
generate an immune response are called TD antigens. not cause tolerance read illy. They induce the full
Several important differences exist between — IgM. IgG, IgA
gamut of immunoglobulin isotypes
TI and TD antigens. TT antigens arc structurally and IgE They show immunological memory,
, '
simple , being composed of a limited number of require preliminary processing, and are rapidly
repeating epi ' opcs , as- in the cane of the metabolised in the body.
I urthrT Rndiii
^
1
Copyrighted material
im Qe
not
n
A <
J
ble
* Antibodies — immunogioOins » S5
immunoglobulins, but :ill immunoglobulins may not called the Fab (ar? riitrcr? binding ) fragment. Each
be antibodies. molecule n1 immunoglobulin is split 1» papain into
'
Immunoglobulins constitute 30-2S pcx cent three partu, one Fc and two F.ih pieces, having a
the rord serum proteins . Based on phyticcchemid sedimentation coefficient of 3.5 S. When treated
and antigenic difKilcnc4s , five classes fit with pepsin , a 5 S fragment in obtained, which i*
bnmtmoftEobulj ns hive been recognised— IyG , I gA* composed essentially of two Fab fragments held
IgM, [ LfO and. IgE . ( Both I;LT and y are accepted together in position . It in bivalent and preripiliLteS
abbreviatione for immunoglobulins!. ^ vith the jiutgcn . This fragment is tailed Ffab72-
rrhe Fc portion it digested by |>cpsin into smaller
S rklJ ^ TtJKE fragments ( Fig. 12.2).
Studies involving rite cleavage of tluL Immunoglobulins arc glycoproteins, each
immunoglobulin molecule, pointered by ftirter, molecule consisting of two pairs of polypeptide
Edtdmim, NinttoS and their colleagues, have led chains ot Jiflereul sizet. The smaller chains art
to ,i detailed picture of its structure . Rabbit IgG called ' light' ( L ) chains and the larger ones 'heavy 1
antibody to egg iThumin, digested by papain in the ( H ) chains. The I. chain has a molecular weight
pRKQCe of cysteine , W; LH split into two frmtionji- -— «1 apprnximitdy 25/100 and the H chain ( lt 50,000.
an insoluble fraction which crystallised in the cold The L chain is attached to the H chain by a
(called JjC for cn-ntalhx.ihlr ] , .ind . I soluhle fragment disulphide bond. The two H Plains arc joined
which, while unable to precipitate w i t h egg
1
together by 1-5 b- S bonds, depending on the class
album in, could still bind w i t h it . This fragment in of immunoglobulins ( Fig. 123).
LCHAiN
3 H -CHAIlV
M
a * iK C
( Gairboxyte rnnmus )
2
2 ^-
a.
LL
U
I AmmolorrTwiLjs j
\ 1 ///
+1
!
fab
Copyrighted materia
86 * Tesihook of M b
the constant region '. Only two sequence patterns and CHI- Beiween CH' and CH: is the hinges region
'
.
3 re seen in the constant region - those determining I he LuiiinoiLfid sequences of die variable rejpons
the Js/ jpi and lambda specificities. On the other of the J.. and H chilin > arc not uniformly variable
hand, the aminoacid Sequence in lltC ammo!union at along their length , but consist of relatively invariable
half of the chair is highly variable , the variability and si mat highly variable zones. The highly \ ariable
determining the immunological specificity of the zones numbering three m the L and four in die H
antihody molecule. It is therefore called the variable chains .uv known .is hypervuriable regions {or hot
region * "Hue H chain also has 'constant ' and Variable' -.puts l and arc involved with the formation of the
ivgiuns. Wlule in the L chain the rwo regions are of antigen hinding sites.The sites on the hypervarinble
equal length, in the H chains the variable region regions that make actual contact with the epitope
constitutes, approximately onEy a fifth of the chain and arc called ‘complementarity determining regions'
is located at its aninotcrmunis. The infinite range of or CDKs .
the antibody sjjeeificUv of immunoglobulins dejiends rite Fc fragment Is composed of the
on the variability gf the aminoacitl sequences at the cathox terminal portion of the H chain .. If does
^
not possess antigen combining activity hui
-
Variable legions' of the H and L chains which form
the antigen combining sites. determines the biological properties of the
I
M
-
* Antibodies -immurmgleibifls 87
+
+
—
*++
-
— — ——
+ + " " "
Selective secretion by
seromucus glands
Heat stability ( 56 °C)
>
—
+ +
t-
— — ——
IRA may occur m 7S, 9S and. 11 S forms .
Copyrighted material
89 i T& itjGOOK n ( Microbiology »
is normally transported a-cioss- the placenta and glycopepfide termed the J chain ( J For joining ).
provide ^ natural passive immunity in the newborn „ This is colled the secretory IgA ( SJgA ). Dimeric
It is not synthesised by the ictus in any significant SIgA is svnthrsised by plasma cells situated near
amount. lgG binds to microorganisms and enhances the mucosal or glandular epithelium , llie j chain
their phagocytosis Eitmcdhdar killing of r .irgct is also produced in the same cells. J chains are
cells coated with IgC antibody is. mediated through present also III other polymeric miinunogLobulms
recognition of the surface Tc fragment by K cells such ns IgM ( Fig. 12.4 ) .
lieming the appropriate reCfptOfit Interaction oi lgG SlgA OtJUftains aiiothct give int rich polypeptide
ootripIcKes with platelet be receptors probably leads called the secretory component u secretory piece.
to aggregation and vasoactive amine release. IgG This LS not produced by lymphoid cells but bv
.1 le i -.e, .Li Lie 11 L” hu m*i L I I nmunoglohljl] i LK , can : i itse 11 mucosal or g .mdnlar epithelial cells Dimeric IgA
to guinea pig skin , but the significance ot this binds to a receptor on the surface of the epithelial
property Is not known . IgG participates in most
immunological reactions such as complement
-
Ctlls laid ii cndOLytostd and ran porlrvl across the
cells to rhe luomul surface . Durin this process, a
fixation , precipitation, am! neutralisation of toxins part of the receptor remains attached to the IgA
and viruses . It maybe considered a general purpose dimer . This part is kuem - n as the secretory
antibody, protective against tltnse infectious agents component. The secretory piece is believed to
which are active in the blood and tissues. Passively protect IgA from denaturatiem by bacterial
administered IjG suppresses rhe homologous proteases in sites such as the intestinal mucosa
antibody synthesis by a Feedback process. This which have a rich and varied bacteria flora , SlgA
property is utilised in the isoumminissiiun ofwnmen is a much larger molecule than scrum IgA ( 1 IS;
hy the administration of aatijKh ( D} IgG during MW about 400,000).
delivery. With most antigeot, IgG is a late antibody SlgA is selectively concert trilled in secretions
and makes its appearance after the initial immune
response which is IgM in nature.
Fatu subclasses of IgG have been recognised
( JgGU IgG 2 , lgG3, IgG 4) , each possessing ,i
distinct tvpc of gamma chain , identiliable with
specific antisera. The four IgG subclasses are 2
diitributcd in human scram in the approximate
proportions oft 5 per cent , 23 percent , 8 pci cent i
Copyrighted material
• Antibodies— Immunoglobins * 69
heavy molecule (19S' mol, m 900,000 to 1 ,000,000, are usually IgM, as also antibodies to typhoid 'O'
hence called ‘the millionaire molecule!, IgM antigen ( endotoxin) and re agin antibodies in
molecules are polymers of five tbur -pcplidc subunits, syphilis .
each bearing -an extra CH domain (Fig. 12.5). As The unique structural features of IgM appear
with IgA, polymerisation ot the subunits iiepends particularly suited to the biological role of
upon the presence of the J chain . Though the providing protection against microorganisms and
theoretical valency is ten , this is observed only with other large antigens that have repeating antigenic
small haptens. With larger antigens , the effective determinants on their surface, A single molecule
valence bills to five, pruhably due Do stone hindrance. of IgM can bring ahnut immune hemolysis ,
Most of IgM ( 80 per cmr ) is intravascular in whereas 1000 IgG molecules arc required for the
distribution. Phyloge n erica!IY, IgM is the oldest same effect. IgM is also 500- 1000 rimes more
immunoglobulin class . Ir is also the earliest effective diau IgG in opsonisation, 100 times more
immunoglobulin to be synthesized by [ he fetus, effective in bactericidal action and about 20 times
beginning by about 20 weeks of age , As it is not in bacterial agglutination. In the neutralisation of
transported across the placenta, the presence oflgM rmins and viruses, however it is less active than
,
in the fetus or newborn indicates intrauterine IgG . Being largely confined to the intravascular
infection and its detection is useful in the diagnosis space , IgM is believed to be responsible tor
of congenital infections such as syphilis , rubella, protection against blood invasion by-
HIV infection and tOXOpfrvMYlOJU IgM antibodies microorganisms. IgM deficiency is often associated
are relatively short lived, disappearing earlier than with septicemias .
IgG, Hence, tlitirdemonstration in serum indicates Monomeric IgM is the major antibody receptor
recent infection. Treatment of serum with 0.12M on the surface of H lymphocytes for antigen
2 - nt cre a p toet h a n t> ] sel c c t i vel y d c s r roy s IgM recognition .
Copyrighted material
W * Textbook of MicrotiiDlogy
Copyrighted materia
* Annbadios — ImmijnoglQtrin* * 91
Further Reading
(
JW . ] W?1 . I m muno globulin sirwiurc and 1 . n r: i . V" c d n. In fljjji and Clin ..* } Immunology.
1
Antigens u) jj antibodies, by definition, Combine neutralisation of toxins and other biologically active
with each other specifically and in ar observable antigens, fixation of complement, immobilisation
manner [ l i e nuiiuHi between antigens and
, of motile organisms ami enhancement ot
intibodici serve several purposes. In the body, they phagocytosis . When such reactions were discovered
fomi [ JLL basis of antibody mediated immunity in one by one, cl was believed that a different lyjie of
inlKtUUI diseases, or of tissue injury in wine type* anti hotly was responsible for each type of reaction
of hypersensitivity and autoimmune diseases. In and the antibodies came to be designated by tire
die laboratory, they help in the diagnosis of infections , reactions they were thought tu produce . Thus, the
in epidemiological surveys , in the identification of antibody causing agglutination wits called
infecrious agents and of ntminfectnuE anrigens such agglutinin, chat causing precipitation precipitin* and
as enzymes. ITL general tliese reactions c.m be used
, so on, and the corresponding antigen, agglutinngcn ,
lor the detection undtjuu nfitutjo n of either antigens precipitinogen, and so on . By the 1920s , this view
or antibodies. Antigen — antibody reactions in vitro was replaced by Zinsser's Unitarian hypothesis
are laHWD Ss scstiiogicd DCScdtUs. which held that an antigen gave rise to only one
"
J HLL reactions hetwrer antigens and antibodies antilWy, which wat- capable uf producing ill the
occur in rh ree The primary stage is rhe in i ri al different reactions depending on the nature of the
interaction between rhe two, without anv visiblt antigen and [ he conditions id the reaction . Both
effects. [’[ us reaction IS rapid , occurs ever at ttjw these extreme views are fallacious. While it is true
temperatures .uni obeys rhe general laws of physical rhdT a single antibody can cause precipitation,
chemistry and thermodynamics. The reaction is agglutination and must of the other serological
reversible , the combination between antigen and reaction *, it it also true that an antigen can stimulate
antibody molecules being effected by the weaker the production (it d i f f e r e n t closes id’
intermoJeculsr forces such as Van der Waal 's forces, immunoglobulins which differ in their reaction
ionic bonds and hydrogen bonding, ruber ih ; m by capacities as well as in o th L- r properties [Table 13.1 ) .
the firmer covakm bonding. The primary reaction
can bedet ecced tnv estm iati riLi; free and boimd antigen ^ -
Some anUj ei i .U Ltilxidy reactions occurring in vivo
initiate chain reactions that lead to neutralisation or
nr antibody separately in rhe reaction nurture by a DESMICIM > n of injur LOUS tu i ligens, i IT to tissue damage.
number o 1 physical and chemical methods , These an; tertiary' reactions and include bumonil
-
including die use oi markers inch as radioactive i mm 111 Li ry against infectiousdiseases* as well as. clinical
isotopes , fluorescent dyes ^ r ferritin. .dleigy id other immunological diseases.
In mutt instances, but not all , the primary stage GSNEHAI FflATI liKS OF
LS followed bv cite secondary stage , leading to ANTLGBM-ANTHBODY REACTIONS
demonstrable events s u c h a s precipitation , Antigen -antibody reactions have the following
agglutination, lysis tit cells, killing of live antigens, general characteristics:
Copyrighted material
* /Wigen Afilibody readions. 93
Table 13.1 Comparative efficiency ol the immunoglcbulin classes in dilterent serological reactions
Reaction IgG IgM IgA
Precipitation Strong Weak Variable
Agglutination Weak Strong Mode rate
Complement fixation Swing Weak Negative
1. The reaction is specific, an antigen combining though IgM molecules may have five or ten
only with its tromoiogous antibody and vice combining sites. Antigens may have valencies
.
versa The specificity, however, is not absolute UpTO hundreds.
and 'cross-reactions' may occur due TO antigenic
simiLiritv or rdatedness . MEASUREMENT nr AVTKIEX AND
r
Copyrighted material
94 * Texttwok 01 Microbiology
+ i
-a- ,-
*
--.
V
*r
- -- nraJii *
- II
B
b. j
-
r *
-
«i J*
* P * * *
- m m ,r
l
l
s r
* + +
* ::fef r
fN "«
.- -*- XV
^e
i
A
.
Fig. 13 1 A quantitative precipilalion teal snowing : A prozone ( zone of antibody excess] , B . zone o1
equivalence , C - zone of antigen excess
opyrighted material
* Arligen- -Antibody reactions * 95
antibodies in vnyuig proportions, depending on the The following types of precipitation and
-
antigen antibody ratio in the reacting mixture.
Precipitation result * when a large lattice is formed
flocculation tests arc in. common use:
"
Fig, 13.2 Mechanism 0i preclpitallon by lattice formation. In K (antigen excess) and C lantibody excess),
lattice formation does not occur. In B ( zone of aquivafonea), lattice formation and precipitation occur optimally.
The dark spheres indicate antigen and ihe spindles bivalent antibody molecules.
Copyrighted material
9
* * Tejdtjoo
* Q{ Wicrooiology
,’1
aggg
n% © o
i 2
4
©
o o o o o o
A B c
Fig. 13.3 Dillerent types ol imiminodltruslon ; t . Single drHusmn in one dimension t Double 11 uSian
In one dimension jQakley-Fulihorpti 3. Radial immunoctiHusion 4 . Double dirJu & mn In two dimensions
( Quchlffrior ) , ghpwing ruction p-f mill | ui lelflluLfm l M: :^, [ i r
ICf , The ^
wells marked $ contain n id n ? J conlaln ar > :
inirsa
Copyrighted material
* Antigen— Anribody reactions * 97
around the well. The diameter of the halo gives and diffusion allowed to proceed for 13— 2-4 hours .
(in estimate of the concentration of the antigen . The result! nu precipitin Lines can be photographed
Thr- method has been employed for the estimation and the slides dried, stained and preserved for
of the i : 111111. . i > glubuii. n classes in sera and fur record. Over 30 different proteins can be identified
screening sera lor antibodies to influenza viruses,
, by this method in human serum. This is useful
among others. for testing for normal and abnormal proteins in
4 . Double diffusion in t wo dimensions serum and urine ( Fig . 13.4).
{Oochterlonv procedure ): This is the Hkclroimmunodi ffus t > n: The development
. inmunodiffusion method most widely employed of precipitin lines can be speeded up by electrically
and helps To compare different antigens and driving the ,miLgen ami juilibody. Various methods
antisera directly. Agar gci. is poured on a slide and have been dewrihed combining electrophoresis
wells are cut using a template. The an i scrum is with diffusion . Of these, otic-dimensional double
placed in the central well and different antigens electroimmunodiffusion ( counterimmuno -
in the surrounding wells. If Two adiaccnt antigens clcctrnphnrc Ls ) and one - dimensional single
-
are identical , the lines of precipitate formed hy electroimmunodiffusion ( rocket electrophoresis )
them will fuse. If they are unrelated, the lines are used frequent!v lit the clinical laboratory.
will cross each other- Cross- reaci ion or partuE J. CoHnferimmunw/MtTopfroreNis (C/E. counter-
identity is indicated by spur formation ( Fig . 13.3 ) . cmreiif i mmu si oelec rropVi arts is h This involves
A special variety of double diffusion in two simultaneous electrophoresis of the antigen and
dimensions is the Elek test for toudgeniciTy in antibody in gel in appetite directions resulting in
diphtheria bacilli . When diphtheria bacilli arc predpitatiiin at a ptint between them ( Fig. 13.5).
streaked at ri . ht angles to a filter paper strip This method produces visible precipitation Writes
^
carrying the antitoxin implanted on a plate of within thirty minutes and is ten times more
suitable tncdijm, arrowhead shaped lines of sensitive than the standard double diffusion
precipitation appear on incubaiion, il the bacillus techniques . The clinical applications are for
'
Copyrighted materia
9S 4 Tsstiijagk gl Microbiology
*
t
> -x- irajs
AGGLUTINATION REACTION
m
; When a particulate antigen is mised with its antibody
in fhc presence ofelectrolytes ai a suitable temperature
and pi lh the panicles are dummied nr agglutinated.
- . .
"i? f
-
Agglutination is more sensitive than prpt ij iration for
' '
13
inhibiting agglutination by the complete antibody
added subsequently.
H .
'r j
m APPLICATIONS AGGLUTINATION
OF
. Y
* ’T REACTION
4 v
5 v
i*
is facilitated by mixing the antigen and the
- — antise rum wirh a loop or by gen tly HK hi ng t he slidtr
Depending cm the titrenfthr scrum, agglutination
. may oeeur instantly or wkbin seconds, CLuitiping
Lv-
: v- xi
x
V
*
to have on the gumc slide a control consisting of
0 i S . - . -.
in
"' ' 'ri'i' the antigen suspension in salineh without the
antiserum, to ensure that the antigen is not
Fig . 13.4 Immunoelectrophoresis 1 Samisolld agar autoagglutinable. AggluitiLatum is usujiElv visible
layered on 1+ie glass siide A well lor aniigen and to the naked eye but may sometimes require
a trough For anliserum cul out oF agar.I. Antigen confirmation under the microscope , Slide
well Filled wllh human swumj Serum separaled . agglutination is a routine procedure for the
Ay electrophoresis 4. Annse rmx trough filled wllh . - . idcntificatinn nf many bacterial isolates from
antiserum to whole human serum . 5, Serum and
antiserum allowed to diffuse into agar 6, Precipitin . .
clmictl specimens It is also the method used for
lines rcrm Tor individual serum proleing . blood grouping and cross- matching.
* Ap1 *gcn— Antibody raaclions » 99
©HI=©
tuhtt -
n Le tube agglutination COST tor brucellosis may
"
opyrigniea mstGf
100 H Tonttwok al Micros - ology *
Precipitin
arc
Antibody
in gel
t
r o
+
Staring waH
Fig .13.7 -
tockal daclraphotesls jtwo dlmenBlonal nimun&elcctrophoresl ? |. FIr run: antigens
Lagrpll' j variant ol
are separated on Ihe basis or el«ErophofeUc mobility. The second run ai dghi angles to " first drives the
antigens into the antiserum confining gel to form precipitation pej](s. The ares of me is proportional
to Ihe concentration 01 the antigen.
the antibody globulin arc washed tree of all to the surface of carrier panicles, it is possible to
unattached protein and treated wirh a rabbit convert precipitation rests into . luuiiaiiu ] LCSLS,
antiserum against human gammaglobulin which are more ronveiiient anti n sensitive lor
( antiglobulin or Coombs serum ) , the cells are the detection ot Antibodies. > LU . tents are known
agsdul iniled . 1 in > is the pfiiunplt: of the intiglobolm as Aauiir Ugfutuation re.srs.
test ( Fig. 13.8) .
The Cfjombs te:» t muv be direct nr indirect. In
-
The Lonmionly u ed carrier t > .i r t . . - are red
cells, lutes particles or bentonite . II . or sheep
the direct Coombs test , rhe sensitisation of the erythrocytes adsorb a variety of antigens
erythrocytes wid |mcuiTtplele antibodies fatuii place
in vivo, as in the hemolytic disease or [ be newborn
Polysaccharide antigens may be , . -
Ibv l . npi .
mixing with the cells, tor adsorption of protein
due to Rh incompnidbilitj, When the red cells af antigens, tanned red cells are
-
tty thru bias Lot ic EEiiuntH an washed free of
unattached protein and then mixed with :L drop of
A special type of passive hei lagghnJiiimori test
is tile Ruse Waaler test. In rheumati EI arthritic,
Coombs serum, agglutination results. The direct -
art auluanribcidy ( RA lacttsr ) ,ip . . in the SC rum,
Coombs [esl is alien negative in Siemulvtie disease
due to ABO incmnpiribilitv.
which aers as an antibody m cUnUm . . 11 > . TTle
RA factor LS able to m red Cetls Coated
-
In the indirect Onmibs test, sensitisation of ted with globulins. Th -e antigen ised for the test is a
cells with the anrihndy globulin is performed in suspension uf sheep erythrocytes s c n s i t j-s cd with a
vitro . Originally employed for detection of anti - subagglutimating dose ot rabbit antishccp
Rh Antibodies, the Coombs test ES useful tnr erythrocyte antibody (amboceptor) .
demonstrating any rype of incomplete or Rllyslyiene latex, which can be manufactured
nonagglutinating antibody, as, for ex Ample , ir
brucellosis.
.
as uniform spherical pArtictcs O -S-l m in diameter,
can adsorb several types of antigens . Latex
PA $S[V« :iggltiilin:iliim lest : I he only agglutination tent - ( latex fixation tests) are widely
difference between rhe requirements for the employed in the clinical laboratory tor the detection
precipitation and agglutination tests is rhe physical of ASO, CRP, RA factor, HCG and many other
nature ot die antigen. Bv ACL aching Soluble antigens antigens.
' iQhfl
* Antigen- Antibody tactions 101
J 3 HSH ivt ugglu t i nat ion tests are very sensitive and 'fix ' complement is made use of in the complement
yield iiigl ] tltres,. but mayjtb' C false positive results. fixation test (CFT") . This is ; L very versatile and
When instead of the antigen , the antibody is sensitive test , applicable with various types of
adsorbed to currier particles in tests for estimation antigens and antibodies and capable of detecting »
of antigens, the technique is known as reversed little as (1.04 mg erf antibody nitrogen and 0.1 mg
passive agglutination. of antigen - CFT is a complex procedure consisting
COMPLEMENT! FIXATION TEST (CFT]
—
of two steps and five reagents antigen, antibody',
complement , sheep erythrocytes and amboceptor
Complement takes part in many immunological { rabbit antibody to sheep red cells). Each of these
reactions and is absorbed during the combination of reagents has to be separately standardised.
antigens with ihrir :intihK >dieH. In the pfCKUOC of the The antigen may be soluble or particulate . The
appropriate antibodies, complement lyses eiythnxytes, antiserum should be healed at 5b "C ( inactivated )
kills and, in some oases, lyses hactcria, immobilises for half an hour before the test to destroy any
motile organisms, promotes phagocytosis and immune complement activity tile serum may have and also
adherence and contributes to tissue damage in certain to remove some nonspecific inhibitors of
types of hvpersensitivity. complement present in some sera
The ability' of antigen antibody [ ompieKes to (anticomplementary activity ). The source of
#
2
’ O. I I
+
4!i
1
4 <2
5
ojo
Fig . 13.0 Anilgtabulln ' Coombs ; teat . Rh positive erythrocytes ( 1 ) are mixed with Incomplete mlibody ( 2 ) The
antibody coats the cells ( 3) but , being incomplete , cannot produce acjgtuiination . On addition of
anllglobullri « rum ( 4 ) which Is complete antibody to immunoglobulin, agglutination takes place .
Copyrighted material
102 * Textbook of Microbiology
complement is guinea pip scrum. AH- complement used up in the first step and, therefore , the serum
activity is heat labile , rhe serum should be freshly contained the antibody ( positive CFT) ( Fig. 13-9).
drawn , nr preserved either in the b'ophDised or Appnopriate controls should be used, Including
frozen stare or with special preservatives , as in the following: antigen and serum controls to ensure
Richardson's method. The guinea pig serum should that they arc not anticomplemertary, complement
he titrated fur complement activity. One unit or control ro ensure that the desired amount of
minimum hemolytic dose ( MHD) of complement complement is added, ami cell control to see that
is defined as the highest dilution of the guinea pig sensitised ervthrocvtes do not undergo Ivsls in the
serum that lyses one unit volume of washed sheep absence of complement.
erythrocytes in the presence of excess hemolysin ln direct complement fixalinn lent:
(amboceptor) within a fixed time ( usually 30 or 60 Certain avian (for example, duck, turkey, parrot ) and
minutes ) at a fixed temperature ( 37 " C ) . The mammalian ( for example, horse, cat ) sera do not fix
amboceptor should be titrated for hemolytic activity. guinea pig complement. When such sera are to be
One MHD of amboceptor is defined as the least tested, the indirect complement fixation rest may he
amount (or highest dilution ) of the inactivated employed. Here the test is set up in duplicate and
amboceptor that lyses one unit volume of washed alter rhe first step, the standard antiserum known to
sheep erythrocytes in rhe presence of excess fix complement is added to one set. If the test serum
complement within a fixed time ( usually 30 or 60 contained antibody, rhe antigen would have been used
minutes) at a fixed temperature (3? ::C ).The diluent up in the first step and there lore the standard antiserum
used for the titrations and for CFT is physiological added subsequently would not he able to fix
saline with added calcium and magnesium ions . complement, Therefort, in the indirect test, hemolysis
The classical example of CFT is the indicates a positive result.
-
Wagscrmann reaction , formerly the rout i in method ( lon lutinatirtg complement nhsorption
^
luMt : For systems which do not fix guinea pig
for the senodiagnosis of syphilis , The test consists
of two steps. In the first , the inactivated scrum of complement , an alternative method is the
the patient is incubated at 37 fur one hour with corglulinating complement absorption test. This
the Wasscrmann antigen and a fixed amount ( two uses horse complement which is nonhemolytic. The
Units ) of guinea pig complement . If the Serum indicator system is sensitised sheep erythrocytes
contains syphilitic antibody the complement will mixed with bovine serum. Ravine serum contains a
be Utilised during the antigen antibody interaction . beta globulin component called conglutinin, which
If the serum docs not contain the antibody, no aers as antibody to complement . Therefore,
antigen - antibody reaction occurs and the conghitinm causes agglutination of sensitised sheep
complement will therefore be left intact. Testing erythrocytes ( conglutination ) if they have
for complement in the postincubation mixture will combined with complement . If the horse
thus indicate whether the scrum had antibodies or complement had been used up by the intigen-
,
not. This constitutes the second Step irt the test and antjjoody interaction in the fiisl step, agglutination
consists of adding sensitised cells ( sheep of sensitised cells will not occur.
erythrocytes coated with 4 MHD hemolysin ), and Other complement dependent
incubating at 37 °C for 30 minutes. Lysis ot rhe serological tests: When some bacteria ( for
erythrocytes indicates that complement WJS not example , Vibrio WioJerse, Treponema pallidum )
fixed in the first step and , therefore, the scrum did react with the specific antibody in the presence of
not have the antibody ( negative CFT). Absence of complement and particulate materials such as
erythrocyte lysis Indicates that the complement was erythrocytes or platelets , rhebacterii arc aggregated
Copyrighted material
1 Anligen — Arlib ay reactions
& 103
and adhere to the cells, This is known as humans Toxin neUtraliiiHLinn: Bacterial QQtO)d(U are
adherence. The 'rtirnobjJrj- ation tejfr is another
j good antigens and induce the formation oi
turn filcmc nt dependent inKtUOi In the Treponema neutralising antibodies ( antitoxins) which arc
pallidum inuoohiliatioa rest , A highly specific test important clinically, in protection against anal
formerly considered the 'gold standard' for the recovery from diseases such an diphtheria and
uerodiagnosis of syphilis * the tMt serum i > mixed tetanus. The toxicity of endotoxins is not neutralised
with a live motile suspension of T. palbdum in the by antisera.
presence of complement . On incubation, the specific Train neutralisation can be tested in vivo or ifl
antibody inhibits the imutility of treponemei. vitro. Neutralisation tests in animals consist of
Cytolytic Of cytotidoJ Jests are also complement -
injecting toxin antitoxin mixtures and estimating
dependent. When a suitable live bacterium, such as the least amount of antitoxin that prevents death or
the cholera vibrio, is mixed with its antibody in ihe disease in the animals. With the diphtheria toxin,
presence of complement , tlie bacterium Ls killed and which in small doses Causes a cutaneous react ion,
lysed. This forms the basis of thevjhriocidal antibody neutralisation Tests can he done on rabbit skin . Tlie
test lor the measurement of anticholera antibodies. Schick test is based on the ability of circulating
antitoxin to neutralise rhe diphtheria toxin gimi
NEUTRALISATION TESTS iniridem:ally and indicates immunity or
Virus neulralilstion tests: Neutralisation susceptibility to the disease. Toxin neutralisation
of viruses by their antibodies can be demonstrated in vitro depends on the inhibition of some
in various systems. Neutralisation of bacteriophages demonstrable toxic effect . An example is the
can he demonstrated by the plaque inhibition test . antistreptolysin O test, in which antitoxin present
When bacteriophages are seeded in appropriate in palienl sera neutralises the hemolytic activity of
dilution on tawn cultures of susceptible bacteria, the streptococcal O hemolysin,
plaques of lysis are produced . Specific anti phage
serum inhibits plaque formation. Neutralisation of OPSGNISATION
—
animal viruses can be demonstrated in three s ^ -stems
animals, eggs and tissue culture .
W
The name 'openin' was originally given by Wright
(1903 ) to a heat labile substance present in fresh
+ COMPLEMENT
RESULT-HEMOL Y SIS
+ HEMOLYTIC SYSTEM NEGATIVE CF TEST
.
Fig 119 Complement fi Kalian test
Copyrighted material
104 * Textbook flf Microbiology *
normal sera, which fat i Litated phagocytosis, This
'
antibody test for the diagnosis of syphilis, i [ere, a
factor was subsequently identified as complement. drop of the rest serum is placed on a smear of T
A heatstahle scrum factor with similar activity wa paJirddm on a slide and after incubation, the slide
harttnutripin'. TliLt appears to be a specific
called ‘ b washed well to remove all free serum, leaving
.
antibody The term opsonin is nqw generally used behind only amibody globulin, if present, coated
to refer both these factors. Wright used the
to on the surface of the rnrponemei ' The smear ii then
'opsonic index' to study the progress of resistance treated with a fluorescent labelled antiserum to
during the course of diseases. The opsonic index human gammaglobulin. The fluorescent conjugate
was defined as the ratio of the phagocytic activity 1
reacts with antibody globulin bound to the
of the patient s blood for a given bacterium, to the [reponemes. After washing away all the unbound
phagocytic activity of blood from a normal fluorescent conjugate, when the slide is examined
individual- It was measured by incubating fresh under ultraviolet illumination, if the tesr is positive
citrated blood wirh the bicteriiL] suspension at Mid trepnemp will be s«n ,LS bright objects .L .Linst
37 “C for 15 minutes and estimating the avenge a dark background. If the serum does not have
^
number of phagoLytosed bacteria per polymorpho ami treponemal antibody, there will be no globulin
nuckar leucocyte [phagocytic index) from stained coariiig (in the Ireponemes and therefore they will
blood films. not take on the fluorescent conjugates, A single
anti human globulin fluorescent conjugate can be
IMMUNOFLUORESCENCE employed for detecting human antibody to any
Fluorescence is the property of absorbing li ht rays antigen ! Rg_ 13.10).
^
of one particular wavelength and emitting rays with Fluorescent dyes may also be conjugated with
a different wavelength. Fluorescent dyes show up .
complement Labelled complement is a versatile
brightly under ultraviolet light as they convert tool and can be employed for the detection of
ultraviolet into visible light . Coons and his .
antigen or antibody Antigens also take fluorescent
colleagues (1943) showed that fluorescent dyes can labelling bur not as well as antibodies do. For
be conjugated to antibodies and that such labelled' detection of anti bodies by immunofluorescence, the
antibodies can be used to locate and identity sartdwfcA technique can be rfrtpli > ved. Thc autiboJv
antigens in tissues. This ‘
fluorescent antibody ' or IN first allowed to react with unlabelled antigen,
immunofluorescence technique has several which is then treated with fluorescent labelled
diagnostic and research applicates. antibody A sandwich is thus formed the antigen
n its simplest forms ( direct immuno - being In the middle and libelled and unlabeUed
fluorescence ,
it can be used for the
test) antibodies on either side .
idcntificarion of bacteria, viruses or other antigens, The fluorescent dyes commonly used are
using the specific antiserum Labelled with a fluorescein isochiocynate and lissamine rhodamine,
fluorescent dye . For example, direct immuno- -
exhibiting blue green and orange-red fluorescence,
fluorescence is routinely used as a sensitive method .
respectively By combining the specificity of
of diagnosing rabirs,by detection of the rabies virus serology with the localising capacity of histology,
antigens in brain smears . A disadvantage of this immunofluorescence helps in the visualisation of
method is that separate fluorescent conjugates have antigen-antiihody reactions :.n situ. It thus an
to be prepared against each antigen to be tested. mmunohistochemical technique The major .
The " indirect immunofluorescence wf overcomes disadvantage of the technique is the frequent
this difficulty by using an antiglobulin fluorescent occurrence of nonspecific fluorescence in tissues
conjugate. An example is the fluorescent treponemal and other materials.
Copyrighted materia
* Ariiigen- — Anlibody reactions * 105
RADIOIMMUNOASSAY (R|A ) [bt hinder. The first reaction rjI this; Ivjir wan
Besides tluorvscenl JvesH many i>t t ] t;r distinctive radioimmunoassay ; 1UA) described hi- Rerson and
YalloW ! [ ] 1 59. RIA jjennits thfi measureme nl of
'labels’ nlsn can he conjugated to antigens and ^
analytes upto piengmm (10 11 g] quantities . RIA
antibodies . 3 he most commonly used Libels arc
' '
radioisotope ami enzymes. A variirlv of tests have and its modifications have versatile applications in
*
been devised for the measurement of antigens and various areas of biology and medicine, including
.
antibodies using such labelled reactants The term the quantitation of hormone $ „ drugs, tumour
hindcr -ligtfld -assay lias been used for the HI: markers, IgE and viral antigens . The importance
reiactLoniH. The: substance ( antigen ) whose ol RIA was acknowledged when the Nohcl Rrizv
-
L- om-mt ration i-s ro be derL rmiued ]S termed the
awarded ro Yallnw for ITS discovery in 1977 .
ftFUt/vre Or ligdiul . d lie binding protein (ordinarily, RIAcompetitive binding assay in which
LS a
the antibody) which bunds to the ligand in called fixed amounts of antibody and radlolahelled antigen
Hfe& -r f f
A
1 + - *"
J
J. I
.
-
s«a»
2
A
+ is \ \} s
1
!
11
'x /
*i
Fig 1 J . 1 C ImmunoHuoneacMiee. 1. Direel lest: Am - cjtn ( A ] Is mixed with fluorescem- conjugaled anlibody (B),
The anHgen- antlfccdy complex is fluarvscenl. : Cj. 2 Indirect lesi : Antigen ( A ) IB mixed with antibody
iBi .The antigen - anti body complex (C ] is treated with fluorescent conjugated enllglobulin serum (D), The
final jjrodud is fluorescent (E|.
106 i Texlhaok of Microbiology Y
react in the presence of H labelled antigen - "fhc for assay of haptens such as drugs and not for
labelled .L' I -LI unlabelled antu ens compete fur the microbial antigens and antibodies. An example of
^
limited binding sires on the ant . body This '
. homogeneous El A is enzyme multiplied
competition it determined by the level of tlte '
i mmurtuaraay rec/imgue ( EMIT ), which is a simple
unlabelled ( test ) antigen present in the reacting assay method Tor small molecule drugs such as
system. After the reaction * the antiijen is separated opiates* cocaine, barbiturates or amphetamine in
i uto Tree* and ‘bound ' frac r i i > ns and their radioar rive serum.
counts measured. The concentration of the test Heitj eriet - i-r!- * El A requites the separation of
antigen can be calculated from the ralio nf the ^
the free and hound fraction* eithe r by centri fugation
bound and total antigen labels, using a standard or by absorption on solid surfaces. and washing. It
dose response curve. is therefore a muliisrep procedure * with reagents
For anv reacting system , the standard dose added sequentially. l*hc major type ofhcterogcncous
response or calibrating curve has to be prepared El A is Enzyme Linked Immunosorbent Assay
first - This is done by running the reaction with ( ELISA ) .
fixed amounts uf antilxody and labelled antigen * and Enzyme Linked lmmunosorbenf Assays
varying known amounts of unlabelled antigen. The ( ELISA ) .
fating [ > j bomul : total labels i B : 1' mfio ) plotted E1TSA is so named because the technique
agaiost the analyte concentrations give the standard
calibration curve. The concentration of antigen
involves the use of an immunosorbent an
absorbing mareiul specific lor one of the
-
in the test sample is computed from the R : T ratio .
components of the reaction the antigen or antibody.
of the test by interpolation from the calibration This may he particulate, for example cellulose or
curve. -
agarose Or i solid phase such as polystyrene,
ENZYME IMMUNOASSAYS (ELA )
polyvinyl or polycarbonate tubes or microwells
or membranes or discs of polyacrylamide , paper or
-
Enzyme labelled conjugates were first introduced plastic. ELISA is usually done using 96- well
in 19*6 for local iwr ion of antigens in tissues, assn TTiic- roritre plates miii table for automation . The
alternative to fluorescent conjugates . In 1971, principle of the test can be illustrated by outlining
enzyme labelled antigens and antibodies were i is applies do ci for the detection of rotavirus andgen
If the t« r
t« die antibody
^ mplc crsn ruins
H rniavinj ,
i r i s Fured
* -
contains anti HIV antibody it will form stable
,
complex with the HIV antigen the plate . A
a
coatinf; the weLls , When the enzynvc on goat
Labelled antibody is added subsequently, it is in turn antilmmafi immunoglobulin antibody conjugated
fixed. The presence of residual enzyme activity, with horse radish peroxidase enzyme is added and
indicated by the development of vellow eolour, Lncubated for 30 minutes. After thorough washing,
i here tore denotes i positive test ( Fig . 13.11), If the -
the substrate O phcnylene diamine dahydrochloride
is added and after 30 minutes , the Colour that
sample is negative , then: is no significant colour
change. An ELISA reader provides quantirativc develops is read using a m i L roansay plate reader,
colour recordings. Positive and negative controls should invariably be
'IllC detection of antibody by EJISA can be used with test sera ( Fag. 13.12),
iLLu & rrated bv the anti - 111 V antibody tesr . Purified
i
The examples described above are of simple
inactivated HIV antigen is adsorbed onto microassay noncompetitive sandwich ELISA. The test can be
plate wells. Test serum diluted in buffer is added to made more specific by making scram antibody and
the well and intubated aC: 37 JC for 10 minutes . enzyme labelled antibody compete for the binding
The well is then thoroughly washed. If the scrum sites on the antigen (compefidw ELISA ). Tests
U 1 2
*
..
©
Fig. 1111 ELISA far dalecticn of rotavirus in leces 1 MicrMssay plate coaled with goal antibody to rotavirus.
1When local suspension is added and incubated , rotavirus if present, will absorb to Ihe coaled anttbedy.
1 After thorough washings add guinea pig antirotavirus antibody conjugated wllh alkaline phosphatase
. .
enzyme If relaviruj is present the conjugate will compter with It 4 - Alter washing add Ihe substrate,
.
paranitrophenyl phosphate, S II Ihe enzyme conjugate is present, Itie SUbitrate Will be $p1h 10 term yellow
.
coloured product This indicates a positive test In a negative lesl liters will be no significant cotour formation .
s
w
JQ 'ft
..
Fig. 13.12 ELISA for HIV anlibody in serum. 1. Micruaasay plate coated with HIV antigen 2 Tesl serum 1$ added
. .
and incubaled Anli-HIV antibody 11 present In the serum will Mach to Hlv aniigen. 5. After washing, add goat
amihurnin immunoglobulin antibody conjugated with horse serum peroxidase enzyme. The conjugate will
- .
attach lo anti HIV antibody in a positive test. 4. Alter washing, add substrate OPD. 5 A colour develops in Ihe
.
positive tesl white teere will be no colour in A negative lesl.
Copyrighted material
108 * Textbritk oh MiCrcbioioqy f
tor specific immunoglobulin classes {for example, chcm idittincsccnt compounds (such as luminpl nr
'
visually. Inbuilt positive and negative controls are lb ) blotting nl the elcctrophorcscd Ligand fraction
usually provided foe validation of the test procedure. on nitrocellulose memliraric strips; and (c) enzyme
An example of cassette ELISA is the one used immunoassay (or radioimmunoassay) ro ( 1) detect
for the detection of HIV type 1 and 2 antibodics- antibody in [ r*.c seta against the various ligand
Speci fir type 1 and 2 antigens are immobilised at fraction bands; or ( 2) probe with known antisera
separate freed dtes on the nitrocellulose membrane against specific antigen bands . The Western Blot
ML the cassette . Test serum is added on the rest , considered die definitive test for the
membrane and allowed to tiller into absorbent sercwliaj nosis of HIV infection, is an example of
material placed befow it in the cassette basc-
^
the immuDQcIntroblot technique (see chapter 62
Antibody, if present in the sc runt will bind to the for details).
appropriate antigen . After washing to remove
unbound antibody, enzyme labelled antihuman IMMUNOCHHOMATOGRAPHIC TESTS
immunoglobulin antibody :is added . After additional A one- step qualitative iinmunochroiTurtographic
washing to remove unbound conjugate, u substrate technique h ^ s found wide application in
yielding a coloured product is added . A positive '.crodiagrosis due to its simplicity, economy and
result is indicated hy a coloured spot developing at reliability. A description of its use for HBsAg
the site of the antigen against winch antibody is detection illustrates the method. The test system is
present in the serum . Human immunoglobulin , a Km .ill cassette containing a membrane
immobilised at a spot on the membrane acts as a impregnated with ami - HbsAg umiljody-cuLLoidaJ
control for the test procedure , as shown by the gp]d dye conjugate. The membrane is exposed it
development of colour at the site. three windows on the cassette. The test serum is
dropped into the first window. As- the serum travels
CHEMILUMINESCENCE IMMUNOASSAY ( CLIAj upstream by capillary action , a coloured band
Chemiluminescence refers (o a chemical reaction appears ar the second window ( test site ) if the serum
emitting energy in the form ( if light just as
, contains HbsAg, due to the formation of a HbaAg-
radioactive conjugate $ are employed in RJA antibody conjugate complo/Thii is the positive
fluorescent cojugares inIFA and enzymes in ELISA. reaction . Absence of a coloured band at the test
Copyrighted materia
+ Amtig & r} — Anubody reactions » H)9
sire indicates A
-
negative reaction Simultaneously A
coloured band should appear tn every cue at rhe
Itnmunnferritin lesl: Ferritin (an electron -
dense substance from horse spleen ) can be
third window, which form* an inbuilt control , in conjugated, with antibody , and such labelled
the absence of which the te^C is invalid . The Belt LH antibody reacting with an antigen caul be visualised
claimed to be ready as sensitive and specific as under tire electron microscope.
EIA tests. Immunnenzyme lost: Some stable enzymes ,
SDCfa as pSOOCidasc, can La: coningateJ with re i Moodies.
IMMUNOELECTRONMlCflOSCOPlC TESTS
Tissue sections carrying the cunesfiording antigens
[ rnmur ] m:lectri}niiiicr[) \copy: When viral are treated with peroxidase labelled antisera - The
particles mixed with specific antisera arc observed perraddStsC bnifld to the arti Ti can he visualised under
under the electron migrowope, they are seen tn he ^
the election mkTOKOpC, by rnicrohistochemical
clumped . This finds application in the study of some mcthock Some o&cr enzymes, suchasghicose oxidase,
viruses Such as the hepatitis A virus and the viruses phosphatases and tyrosinase, may also he included in
causing diarrhea . imrmLnncnzyTTie tc^ts -
Furlher Heading
Bairns A ct al. 1991 . AJanmJ . / Opnjfff / . Witrn fcrn
' , gy-Wp.^>iLn tcni DC; Amakin Swiety of Mkrtbaologji
f
Copyrighted material
The Complement System
The term Lcumplement ' (C ) r L-: L C to a system of its rule LIS a mediator and an i pii lie r of many i mmunc
factors which occur in normal scrum and arc and inflammatory reactions recognised ,. The
-
activated characteristically by antigen antibody complement system belongs to the group of
interaction and subsequently mediate a number of biological effector mechanisms (called triggered
biologically significant consequences, enzyme cascades) which also includes coagulation,
Towards the end of the nineteenth century; it fibrinolytic and It in in systems. Such biological
was noticed that bactericidal , bacteriolytic and cascades havedistinct advantages. For example, each
hemolytic actions nf the appropriate anti bodies enxyrrie in the cascade i * able to activate many
requi ned the part ic i pari on of a heat labile component molecules of the succeeding component providing
present in the normal sera of human beings and for amplification of the response at each step. Every
animals. Buchner (1889) was the first to observe step has its own control mechanisms so that the
that the bactericidal effect of scrum was destroyed cascade can be regulated with prevision.
by heating at 55 aC ( br one liour . Pfielfer (1B94)
discovered that cholera vibrios were lysed when GEN Eft AI PhopHftTiEa
injected intraperi toneallv into speci Really Complement is present in the seta of all mammals
immunised guinea pigs ( bacteriolysis in vivo or and also in that of most lower animals, including
PFitfiet 's yihenonienort ). Bordet ( 1895) extended birds, amphibians and fishes . It is a nonspecific
these observations and established that immune
,
Copyrighted materia
* Ths Complement System * 111
Copyrighted material
112 * Toxlbook of Microbiology t
its l > i riding sires. One molecule of IgM or two be due to other stimuli, such as DNA, C-reactive
molecules of IgG can therefore initijirc rhe protein, trypsin - like enzymes or some letrovi ruses.
process. Clq binding In the presence of calcium
ions leads to sequential ICtiviuioilot Clf mid S. ALTERNATIVE C PATHWAY
2. Activated t ’ I s is an esterase (CI a esterase ) , one The Central process in the complement cascade is
molecule at which can cleave several molecules the activationofG , which is the major component
ofC4 - an instance ot amplification. C4 is split of Cr In the classical pathway, activation of C3 is
into C 4 an which is an anaphylatoKin , and C 4b achieved by {J 42 (classical C3 convertase). The
which binds to cell membranes along with Cl . activation ufC3 without prior participation ofCl 42
3. C 14 b in the prese ncc of magnevhun Lonl delves is known as the Haitemitive pathway'.
C2 into C3a , which remains linked to cell- The first example of the alternative pathway was
bound C 4b, and (.! 2 b which is rclcasd! intofluid the demonstration by EJilLemer (1954) of the
,
phase. C 4 bia has enzymatic acriv Ltry and is ‘properdin system ' aii a group of serum proteins
referred to as the classical pathway Cl Contributing to antimicrobial defence without
requiring specific antibodies. The activator in this
4 Cl convcrtase splits C3 into two fragments :
r system was zymosan , a polysaccharide from the
C3i which is an anaphylattmn, bud C3b which yeast cell wall, but many other substances can also
remain* celt - bound along with C4 b2i to fom acrivatc the pathway. These activators include
a trimokcular complex C *lb2a 3 b which has bacterid endotoxins, IgA and I ), the cobra venom
entymatic activity and is called C5 conwrtasc . factor and the nephritic factor ( a protein present in
5. The membaae arrack phase of complement the serum of glomerulonephritis patients) .
activity begins at this stage with C.5 ecatvotue The liftl step in llie alternative pathway is the
cleaving C 5 into C5 a, :in aruphytatovin which binding at C3b to an activator. C3b is continuously
is released into the medium, and CSb which generated in small quantities in the circulation but
continues with the cascade. C6 and C 7 then in the free state it LS rapidly inactivated by the serum
join together. A heat stable IT i molecular complex protein factors II and I. Bound C3b which is
C5b 7 3S formed, part at which binds to the cell protected from such inactivation interacts with a
membrane and prepares it for lysis by C 6 and srnmi protein called Fact nr R ( also known as ' C3
Copyrighted material
« The Complement System * 113
_ CI M. ri .
FA
' -
LA
EACH
/
S
^ '
*
CIi Di. ^
EAC w
,
CTTtl YSlS -
exhaustion of the
—
Fig. 14.1 Complement cascade the classical pathway
complement system but also activating factor or KAF), provides homeostatic
strioua damage to tissues. Several inbuilt control control of Co activation , particularlv bv the
mechanisms reflate the complement cascade ,n alternative pathway.
different steps. These are mainly of two kinds: 2. Another beta globulin Factor H acts in concert
inhibitors which bind to complement components and with Factor I modulating C3 activation ,
halt their further function,. and inactivators wluch are j. Anaphvlatoxin iomctivMtor is an alphaglobulim
emmtiEH that destroy complement proteinh. that en / vmari tally degrades Co .i . C4a and C5a
,
Copyrighted material
114 * ToutLnck cl Microbiology *
vhcmotactiv and also brings about reactive lysis. coagulation and t b mm btnylope ri In. Li ram negative
L participates in the cytotoxic (Type II) and
"
true E" 5h
by
/' TbN
" * FjICiDfN Jl jrid f
l in cirrulubmi I
- hcfn nifnhined
^“ HHUMHH
' nil ivuiiH t.fl.
i flC[
Wi
, cynuimn
CJhs B FatliW II *
ri C 3h
Fflfwr D
C3
Gusudc
/
Fig. 14,2 Complement cascade Ihr alternative pathway
Copyrighted material
* lira Complement System * 115
Table 14.1 Clinical syndromes associated with ganelic deliciencies ol complement components
*
hound Cl . Though conglutinin behaves an an ( Cl ) , macrophages (C 2, C4), spleen (C5» CS) and
antibody to C, ir is not an immunoglobulin and liver (C3, CO, C9 ). C is, to some extent , an ‘acute
requires CaH + for its activity. Antibodies with phase substance' and rise in C levels ( particularly
corgluririn -like activity' (immunoconghrinin, IK) C 4, C3, C5 and Cb ) is observed during the acute
car be produced by immunisation with complement phase of inflammation .
coated materials . They may also occur frequently
in human. beings and other rn .mmuh an DEFICIENCIES O F THE COMPLEMENT
autuanlilmdies to fixed C.. The times of tenim IK SYSTEM
rise in conditions such as infections and Complete or partial deficiencies of all the classical
autoimmune diseases associated with mere used complement components and several of the C
fixation of C in v[vo, High IK levels have been inhibitors have been described in humans or
noticed in the saliva and jejunal secretions. They animals. Some are associated with severe diseases,
are IgA antibodies whose significance is not while in others clinical manifestations arc sporadic .
krurwn . C deficiencies result in the host being unable to
efficiently eliminate the microbial antigens or
QUANTITATION OF C AND ITS circulating immune complexes. Recurrent bacterial
COMPONENTS and Jungal infections and collagen diseases also occur
Complement activity of serum is measured by ( Table 14.1) .
estimating the highest dilution of scram lysing Deficiency of the Cl inhibitor is associated with
Copyrighted material
116 H TcKlb& oii 01 Microtoology
Further Reading
l.tncw y CA - ind PTTPVFT* I* edn - London: CTirrmt Biikij y-
^
Trim-lin^on 5 199
- - J , CrHpphmtpt ( Ip/ pfl
'e i7rrohanJ -
1"
-
*m *, C NIT Op hnmvnnf , 5 , 33
^
DM and J Snewirr 19V 7. Immunoiapy . If* edn . Edinburgh : Churchill Lim ingstune.
Copyrighted material
Structure and Functions
of the Immune System
Hie lymphnrcHcular system is a complex ( primary) and the peripheral (secondary ) lymphoid
organisarion of cells of divert morphology organs . The central lymphoid organs arc
distributed widely ill different organs and tissues of lymphoepithelia ] structures in which the precursor
-
the body i es}Hi[isibk for immunity Lyrnpharediuilar
cells consist: of lymphoid and reticuloendothelial
lymphocytes proliferate , develop and acquire
immunological capability, The thymus and the bursa
components, with clearly dems reared functions. The of Kabricius itl birds ait primary lymphoi J organs,
lymphoid cells - lymphocytes and plasms cells
arc primarily concerned with the specific immune
- being responsible tor the cellular and humoral
immune responses, respectively. The equivalent of
response. The phagocytic cells, forming part of the the avian bursa in mammals is bone marrow. After
rcticukjCndothriiall system , arc primarily concerned acquiring imnumncompe fence , the lymphocytes
With the 'scavenger' functions of eliminating etfete migrate along blood and lymph streams, accumulate
cells and foreign particles. They contribute to ill ihc peripheral lymphoid organs and , following
noi ispcdtic immun Ley by removing microcupui i s i ns antigenic stimulus, effect the appropriate immune
frurrt blood and ti-smies. They ilsn * play a role in response. The spleen, lymph nodes and mucosa-
Specific immunity, both in The afferent arid efferent associatcd lymphoid tissue ( MALT ) ccmsiitute the
limliK of rhe immune response. mayor peripheral lymphoid organs. Lymphoid tissue
The functional anatomy ufthe lymphoid system in the gut , lungs , liver and bone marrow and
can be appreciated only against the background of lymphoid collections in the adventitious tissue of "
1
llic ' Lvro component concept of immunity. The all organs also form part of rhe peripheral lymphoid
inti [ tune response to art antigen, wli ateveT irs nature, system .
—
can he ot twn broad types the humoral or antibody
mediated immunity ( AMI ) and the cellular- or cell GB ^ TRAI . ( PRIMARY ) LYMPHOID
mediated immunity [CMI ) . Humoral immunity is Oftt:
mediated by antibodies produced by plasma cells Thymust rite thymus milage develops from the
and present in blood and other body fluids ( hence epithelium til the third and fourth pharyngeal
the nunc luimoal' from fun nor ' the old term for pouches at about rhe sixth week of gesrarion . By
body fluids). Cellular immunity is media ted directly the eighth week , mesenchymal stem cells
by sensitised lymphocytes. Cells for each of these [ precursors of lymphocytes ) from the yolk sac., fetal
enmponen ts develop th rough separate du nine Is and liver and born.- marrow reach the thymus and
remain independent , though they may also interact differentiate into the thymic lymphoid cells
in some instances ( Fig. 15.1 ). ( thymocytes ) . It is. thus the tirer < h|-gan in all animal
The lymphoid system consists cjf the lymphoid species ro become predominantly lymphoid. In
cells (lymphocytes and plasma celle) and lymphoid human bemgi, the Thymus reaches ire maximal
organs. Based on the different roles they perform, relative size just before birth . It continues to grow
lymphoid organs cut be classified into the cental till about [ lie twelfth year. After puberty, it undergoes
Copyrighted materia
118 * Textbook o1 Microbiology *
Ir
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Flgr 15.1 Development of T and E cell systems
spontaneous progressive involution, irudicJltiog that only about one per cent leave thq thymus . The rest
ii functions best in early life. are destroyed locally. T h e ' . t ^ - I f " 1 pparent!}
i
I he thymus in hunted behind the upper part of wasteful process is not known . In 1 Sit thvi - . I lie
the Ettraum. Aberrant thvmie tissues are often found lymphocytes acquire new Surface citigen: Thy'
in neighbouring sires , The thymus has. two loben antigens ) , Lymphocytes >ndidoited in the thy -
surrounded by a fibrous capsule. Septa arising from are called ^ thymus (T) dependent Ivniphocvio or
the eapsulc divide the gland it ] CO lubiiks which HT cells'. Unlike in rhe ; pheral . r. .. - . lymphiK-ytc .
are differentiated into an outer cortex and an inner proliferation in the thymus is - i- n dependent on
medulla . The cort -eit is crowded with actively antigenic stimulation . In tact, jteriphersd untigeclic
proliferating small Ivmphncvtes-. The medulla Stimuli do not lead to any in m m* response in the
consists mainly of epithelial cells and mature thymus. Antigen introduced (End tly intot n- : : .
lymphocytes in the middle of which arc the .LV lead
[ TI local Immune i r
to a
Hawaii ' s corpuscles , which .ire whorl - like The rbvmus confers MI ii '. . : i - u - . ter
aggregations of epithelial cells. or tbc lymphocytes during theii stay in the organ .
Till the l%Os, the thymus was an organ without Fret hymic lymphocytes are not mpermit
.LI LV (ecqgmsfid Iunction.The for tui coos obscr virions In the thymus they arc educated
' chaL th •
bv Good ( 1954) of thymoma and impaired become capable ot mounting cell media' d irm tuj c
:
immunity in a patient, and by Miller ( 1961) of response against appropriate antigens . The
mi memo della envy in neona tally rhymectomi&ed importance of thymus in km pi i< . vr . proliferation
mice, paved tlie wav for the understanding of the and development of CMI is evident from the
pivotal role of rhe organ in the development of cell lymphopenia, Jelk-lenr graft rejection ami [ he so
mediated immunity. TEie primary function <it the called ‘runt disease * seen in neonatally
thymus i -, the production of thymic Lymphocytes. thymccromised mice, Deficient t Mi is also seen
It is the major site tor lymphocyte proliferation in f
in congenital aplasia thymus ithumanbeings.
rhe body. Howc^r, nf rhe lymphocytes produced , (DiGeorge syndrome ), and in mice) (' nude mice') .
!opyrEoht&d material
* Structure and Functions ol the Immune System * 119
T lymphocyte jure selecris e!y seeded into certain In humans and other mammals , the bone
aite^ in the peripheral lymphatic tissues, being found marrow acts as the bursa equivalent. All
in the white pulp of the spleen, around the central lymphocytes originate in the hone marrow; While
arterioles, and in the pararorticnl areas of lymph T lymphocyte develop in the thymus , B
nodes. These regions have been termed 'thymus
*
lymphocytes develop in the bone marrow itself. In
dependent as they are found grossly depleted after rhe human fetus , Pejer's patches develop and
neonatal thymectomy. While thymectomy affects himphnid cells appear in the spleen and lymph nodes
CM!primarily, it also diminishes antibody response bv the 20th week of gestation. From then on the
to many types of antigens { thymus dependent fetus is able to produce IgM and IgJJ . It receives
antigens ) such as sheep eryrhrocyrcs and bovine maternal IgC , but IgA and Igt arc nor present. At
serum albti m in, FI uni oral response to or her antigens birth IgM production is enhanced , hut IgC level
is unaffected. tldis steadily, [o reach minimum levels by the 3 rd
LiurMJ of FabrirauK This is a Lvmphocpithdiil mo nth . IgCi production then picks up and becomes
organ arising as a pouch from the dorsal part of adequate by 2-5 years . Full immunocompctcnce is
lliu cloaca ill birds . It becomes J lymphoid organ attained only at ter tin first decade of life .
m
independent areas' , Following appropriate antigenic antigen . In the medulla , the lymphocytes, plasma
stimulation , B lymphocytes transform into plasma cells and macrophages arc arranged as elongated
cells and secrete antibodies . branching hands (medullary cords). The cortical
The vital role of the bursa in humoral immunity follicles and medullary cords contain B lymphocytes
was discovered accidentally by Click and Chang and constitute the bursa dependent ureas. Between
( 195b) who found that chickens bursectomised i[ the conical follicles and medullary cords, there is
batching failed to form antibodies wlien challenged a broad, ill- defined intermediate zone (paracortical
with a bacterial antigen . IrnnmmHrojnpetence is urea ) which contains T lymphocytes and
conferred on the lymphocytes by the bursa in inrcrdigiraririg cells, "Hiis constitutes the thymus
stages , Competence for IgM production is dependent area (Fig, 15 , 2),
acquired early ( about day 14 of embryonation ) Lymph nodes act as a Biter for lymph, each
and for IgG late ( about day 21 ) . Birds group of nodes draining a spec i lie part of the body,
burscctomised On IS to 2U days synthesise IgMh They phagocytose foreign materials including
but not fgG- microorganisms. They help in the proliferation and
Copyrighted material
120 « Textbook of Microbiology 1
»
circulation ofT and R cells. They enlarge following lymphoid tissues in the gut, from the adenoids and
local antigenic stimulation . tunsils to the follicles in the colon , are called the
Spleen; The spleen is ilie larges L oi the Lymphoid gut associated lymphoid tissue (GALT} smd those
organs tr has a capsule from which trabeculae
, in the respiratory tract, the bronchus associated
descend , dividing die organ into seven! lymphoid tissue ( HALT ).
in te rco mienl -ed compartments. The brandies ol the MALT contains lymphoid as well a± phagocytic
splenic juxery travel along the trabeculae, and on cells liorb and T cells arc present. While the
,
Leaving iheui branch again to form the central predominant immunoglobulin produced in rite
arterioles, which are surrounded by a sheath of mucosa is secretory IgA , other immunoglobulin
-
Lymphoid tissues. This part is known as LILL white
pttlp of the spleen and may uonstirure about bait to
.
classes , IgG IgM and lgE are also formed locally.
There appears In he a free traffic of antigen -
three quarters of the organ, hi [lowing antigenic specific effector lymphocytes between die various
stimulation.The central arterioles- proceed onto [1111 mucosal and secretory areas , so that an antigenic
red pulp , so called because at the abundance ot red exposure at one site may cause production of the
blmid cells in it . specific antibody ai the other nuicosal and secretory
Tbe periarterial Lymphoid collections in the sites also. I'bis indicates the existence ol a common
white pulp n ( the spleen are called the Malpighian mucoeal or weretory immune system anti explains
corpuscles or follicle*. Germ in til centres develop the superiority of oral or nasal immunisation over
following antigenic stimulation. Surrounding the the parenteral nuUCt for many enteric and respiratory
germinal centre is a ' mantle layer ot lymphocytes. tnfoctions-
Immediately outside the periarterial lymphatic
sheath and separating it from the red pulp lies the Cei - l -3 O F THIi t. V M F H O R H T I C i : I AH
marginal wnc . The lymphatic sheath immediately SYSTEM
surrounding the centra ) arteriole is the thymus Lymphocytes: Lymphocytes arc small, round
dependent area of tbe spleen. The perifollicular cells found in peripheral blood , hi up Is, Lvinphoid
region , germinal centre and munrte layer form the organs and in many other [issues . In peripheral
bursa dependent ( thymus independent} areas ( Fig.
15.3}.
-
blood , they constitute 20 45 pet cent of rhe
leucocyte population, while in lymph and Lymphoid
The spleen serves as the graveyard for effete organs they form the predominant cell type. The
blood cells, as a reserve tank and settling bed for human body contains about 10l! lymphocytes,
blood and as a systemic filter for trapping circulating
hlondluirne foreign particles . The immunological ^
approximately lC of them being renewed daily.Only
about one per cent of the total body lymphocytes
function of the spleen is primarily directed against
btoodbCHK antigens.
aie present in flic blood . Rliriieh ( 1J17 ) who
Copyrighted materia
4 Structure and Functions ol llie Immune System w 121
chromatin and a thin ricu of cytoplasm, containing in a process known as ‘lymphocyte recirculation' .
scattered ribosomes but virtually devoid of There is a constant traffic of lymphocyte through
endoplasmic reticulum or other organelles. They rbc blood, lymph , lymphatic organs and tissues .
*
arc capable of slow motility and during itHVHtlttlt This recirculation ensures that following
assume a 'hand minor1 form, with the nucleus in introduction of antigen into any pari of the body,
Front and the cytoplasm as a tail behind. lymphocytes of appropriate specificity would reach
Depending on their lifespan, they can he the site during their ceascles-s wandering and mount
classified as short-lived aid long-lived lymphocytes- an immune response . A Lymphocyte compiles one
ll human beings, the short lived. LymphOrytes Liave cycle of recirculation in about one or two days .
a lifespan of about two weeks , while the Long lived Recirculating lymphocytes can be recruited by ( Lie
cells miy last for three years or more, or even lor lymphoid tissues whenever necessary. Recirculating
life. Short lived lymphocytes are the cflTeuL-Oc cells lymphocytes are mainlyT cells , U cells tend to be
in immune response, while the long lived cells act more sessile. Chronic thoracic dnet drainage will
as the storehouse for immunological memory Long therefore result in selective T ceil depletion.
lived cells arc mainly thymus derived . A lymphocyte that has been ' educated' by the
Lymphopoiesis Hikes place mainly in die central central lymphoid organs becomes an
lvrnphold organs where they differentiate and immttnoJcjgically competent cell' (ICC), Mature
mature before entering the circulation and then the L and B celts , before they encounter antigens are
"
peripheral lymphoid organs Rnd tissues Like a called naive cells. Such cells, though not actually
polLcemait on bent patrol. These populations of engaged in an immupnLogical response , are
lympfuKTTes do not remum distincthue mis: together nevertEieless fully qualified to undertake such a
AFFERENT
LYMPHATIC
CAPSULE
TRABECULA
GERMINAL
- l CENTRE
CORTICAL
FOLLICLE
MEDULLARY
vv V SIN JS
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.
ms* v
r
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CORDS
- f EFFERENT
LYMPHATIC
.
Fig 15.2 Diagrammatic section of lymph node (arrows indicate the path ol lymph flow) .
opyrighted material
122 ^ Texttoo ol Microtvclogy
*
mpansibiliiy when appropriately stimulated bv an prepared the antibodies, the same marker came to
antigen . LTicy subserve the fcllining timedons—
1
hr known hy different names (T 4r Leu3 , and so
recognition of antigens , storage of immunological on ) , Order was introduced at the 'International
memory and immune response to specific antigen ^ Workshops for Leucocyte Differentiation
Lj!rnplKKytCS have antigen recognition mechanisms
"
Antigens' bycomparing the tpeofitities ofdifferent
on their surface, enabling each eel! to recognise antisera.. When a duster of monoclonal antibodies
only one antigen , I ’ hr reaction of an was found to react with a particular antigen, it was
immunocompetent cell ro its specific antigen may defined as a separate marker and given a CD (cluster
>
be induction of either ltoleniHt or the immune of differentiation) n umber. Over 15D CD markers
response. The nature of immune response depends have been identified so far. Tabic 15,1 lists a few
on whether the lymphocyte is a 11 or T cell . CD markers with their eel! association and
*
Stimulated T cell * produce certain activation previous designations fin spite of the CD
,
products (lymphoktnes ) and induce CM1, while nomenclature, some popular old names continue
Stimulated B cells divide and [rsQifbqn ill Co plasma to be iti use, tor example, T*l and TS are still in use
cells which synthesise immunoglobulins . for Cl >4 ( helpcr/ induccr) and CDS ( suppressor/
A number of surface antigen or markers have cytotoxic) cells) .
*
been identified on lymphocytes and other Leucocyte* The irust e!ea r\ut dilfercnti atinn between' \ and
by means of monoclonal antibodies These , B cells is by fheir surface markers, for example, by
marker * re fleet 1li c :--1JLTLU ot ditterenfiatiun ami demonstration of CD!) tin T cells and Ig on R OCILH,
,
functional properties of the cells. As they were given Many other tests help in their differentiation ( Tabic
different designations bv rhe investigators who 15.2). These include:
TRABECULAR-
VESSEL
-
£77*
TP 4BEOAA
RE & PULP
Q $IMJ$Q|D
WHITE PULP
MALPIGHIAN CORPUSCLE
GERMINAL CENTRE
MANTLE LAYER:
MARGINAL ZONE
Vi .
m.--
HFP 1*1LF
i
i+
%
CENTRAL ARTER JU
^
SWJE« ARTERV
riqhted
4 Structure and Functions of the Immune Syslem > 123
filamentous surface, with numerous microvilli . two major subsets CDS 4* or CD4 S' . Mature
COS 4 ' TCR ftp cells are hclper/inducer cells,
T CELL M VTURATION inducing R cell differentiation , stimulating
T cell precursors From the yolk sac, fetal liver and pro]] forarion of CDS' cytotoxic cells, producing
bone marrow migrate to the thymus during the lymphokines and regulating certain stages of
embryonic and postnatal stages. The earliest erythropoiesls . CD4 8 ' TCR Ctfi cells ane
"
identifiable cells of T lineage arc the CD7+ pro-T suppresHjr/cytoToxic cells, inhibiting B cell antibody
cells, which acquire CD2 on entering the thymus. synthesi s and acting as cytotoxic effector cells . Minor
They synthesise CD3 in the cytoplasm and become subsets ofCD4 + cells and CDS' cells also exist.
prc-T cells, T cell receptor (TCR ) synthesis also Small numbers of CW 8* and CD4 8 cells arc " “
Copyrighted material
124 * Te*lbook of MfcrobioKjgy
Thi“ func cir of TCR "/fi cell? in not well marker and MHC class I r*LNtrk- ti on They down
^
understood, but they art believed to be immune
.
Sequential antigenic changes characterising T cell marker and MHC class I restriction. The* can
inanar-jcnm enable their enr identification . Tins has
P
, kill and lyse target celk earthing new or foreign
application ir defining T cell malignancies. Acute imtigetis- r including tumour, allograft and virus
cell malignancies such as Lymphoblastic leukemia and infected cells .
KTnphrKmjs involve carle f cells, pro -T cells anil other 4 . Memory cells Tin 1mth CI 4 and CT)S tells
immature forms - Chronic T cell malignancies like provide memory and anamnestic immune
^
mycosis Eimguides, peripheral T cell lymphomas and responses.
HTIV - 1 associated . i . Lu. Lt 1 ted leukemias involve
4
CD liujfiibej
’
Cell typc iHocfiQoii Former designations
CD 1 Thymocytes, Lingertuuii alls T6, Leu 6
CD 2 T evil SRBC receptor Til , Uu 5
CD 3 T cell antigen receptor complex T3, Leu J
CD I
CD 8
- . Helper T tell ( receptor fbr HJV )
Supprotor/cytotmic T cells
T 4 Leu 3
f
Tfi. Leu 2
CD B cells fU , Leu 12
Copyrighted materia
* Slnuoture and Functions ot the immune Syelern * 125
development, self toiuancc is developed by clonal producing cell, lymphocytes , lymphoblasts and
deletion or anergy. transitional cells may also synthesise Ig to home
O n contact with its appropriate antigen , the extent,
mutunt B L L ' 1 I undergoes donal proliferation , Home
'
A separate lineage of B cells , which arc
activated B cells become long-lived memory cells predominant in fetal and early neonatal life, express
responsible for the recall phenomenon seen on tbe 1 cdl marker CDS on their surface and have
'
subsequent contact with the same antigen. The been named as 111 ndh . Their progenitor cells move
majority of activated R cells ire transformed into from the fcr.il liver to rhe peritoneal caviry where
they multiply. rl hcy secrete Low affinity palyrcaciivc
"
plasma cells,
Plasma cell is the antibody accreting cell. It is IgM antibodies many ot them autoanti bodies." ITiey
,
Copyrighted material
126 i T& xiOOQk of Microbiology *
several azuroph i I ic granules , Composed of which are cytotoxic to a wide range of tumour cells
milpfhoiuiii .L , ribosomes, endoplasmic reticulum without affecting normal cells . LAK cells have
and Golgi apparatus , LGL arc a heterogeneous shown promise in the treatment of some tumours
group of cells with differences in rheir functional such as renal cell carcinoma . 1 L 2 also acts as a
and surface marker features. TILC [] iosl important growth factor fur NK cells.
member of This group is the natural killer ( NK ) PtiRgemylic evils: Phagocytosis is pliylo -
cell , Others arc the antigen dependent eytatnwc gtinctically the oldest defence mechanism in
cells. (ADCC) and the tymphalanc activated killer Animals . Originating in protozoa as a combined
( LAK ) cells, '[‘he term NK cell is sometimes used mechanism tor nutrition and defence, along the
as a common name tor all uu.ll tells. course of evolution, the phagocyte lost its trophic
Natural killer cells possess spontaneous functions with the development of digestive
cytotoxicity towards various target cells mainly enzymes - In higher organisms it specialised in the
malignant acid vims infected cells. Their cytotoxicity removal of foreign and autochthonous particles.
is not antibody dependent or MHC restricted - NK Phagocytic cells are the mononuclear macrophages
ICtfritv is ‘natural or 'rtOfii runjune' as it does not (of blood ami tissues ) and the polvmurjihnnuclear
require sensitisation by prior antigenic contact. NK microphages.
cells therefore form part of the innate immune set - The blood macrophages ( monocytes ) are the
up. Tile v belong ro a different lineage from T and largest of the lymphoid cells (bund in peripheral
B cdls and are therefore normailv active in 'severe
1*
blood (12 - 15 pm ). The tissue macrophages
combined immunodeficiencr diseases', in which ( histkbytes ) arc larger (15-20 pm ) . Mononuclear
rnuture T and l? cells arc absent . They hive CD16 macrophage cells originate m the bone marrow from
and CD56 on rheir surface . They bind to fhe pVKtmor cells and become monocytes in about SI
glycoprotein receptors on the surface of autologous day ^ Monocytes in circulation haw an approximate
as well as allogeneic target cells and release several half- life of three days. They leave the circulation
Cytolytic factors. One of these, perform, which and teach various tissues to become transformed
resembles complement component CN, causes trans - into macrophages , with morphological and
membrane ports tlirough which cytotoxic 1 actors ,
such as the tumour necrosis (actor beta, enter the
-
functional feature - characteristic of the tissues, such
as alvtokr macrophages in the lungs and Kupffer
cell and destroy it by apoptosis ( programmed cell cells ill the liver. Tissue macrophages survive for
death ). NK cell activity is augmented by interferon . months . Multinuclcaied celts and epithelioid cells
They arc considered to be important in immune seen in granulomatous inflammatory lesions such
surveillance and natural defence against virus as tuberculosis originate from mononuclear
injected and malignant mutant cells. macrophage cells.
A subpopulation of LGI . H possWCS surface The primary function ot macrophages is
receptors for the Ft part ( if Ig. They are capable of phagocytosis. These cells move slowly in a
lysing or killing target cells sensitised with ) gt! ponderous and purposeful manner , their abundant
antibodies . This antibody dependent cellular cytoplasm thrusting our restless pseudopod i . L that
CytOTOXicity is distinct from the action of cytotoxic glide harmlessly past normal body ceils but engulf
T cells, which is independent of antibody. ADCC effete cells And foreign particles. They accumulate
Cells were formerly called killer ( K ) cells but are in areas ot inflammation or tissue damage by
row rlaMifiwI with N K cells. chemotajiis- Particles sensitised by antibodies arc
ki iler ( LAK ) cells a re N K
] ,ymphokine activated phagocytosed more readily. The phagocytosed
lymphocytes treated with interleukin - 2 ( IL2 ) , particle is held inside i vacuole ( phagosome ), the
Copyrighted material
I
membrane of which inses W ich :L lysosome, forming morphological and functional changes as compared
a ' phagolysosome*, Lysosomal enzymes digest the with un &ti undated quiescent macrophage5- They are
particle , the rtituuntibtm | extruded from the cells . larger adhere better, spread faster on glass and are
While phagocytosis is an effective defence against
most microorganisms, some {such as the bacilli of
.
more phagocytic They secrere a number of
biologically active substances, including hydrolytic
typh < i id , bracictlo}. i * and tubcrculo-.: s) res: st digesti on .
enzymes, b:. ndl ng protci n s (fibronect i n transferri n ),
and may multiply in the cells and he transported in tumour necrosis factor {cachectinl , colony
them to other locations. stimulating factor ( CSF) and interleukin 1
Macrophages express many surface receptors (formerly called the leukocyte activating factor ).
including la proteins, those for the Fc part of IgG , Interleukin 1 acts as an endogenous pyrogen and
activated complement components and various also induces svnthcMS of interleukin 2 bv T cells.
CB nB
lymphokines. Mac 1 is a protein antigen found on Interleukin 2 facilitates the activation of 1’ cells.
mouse macrophages. A similar protein on human When stimulated by cytoplnLic antibodies and
1
macrophages has been named Afl marker. This certain lymphokmes, macrophages become ‘aimed .
appears closely related to CR3, a cell receptor for
C3 components.
Such armed macrophages are capable of antigen -
specific cytotoxicity, which is important in
Macrophages may participate in several ways antitumour activity and graft rejection.
in the induction and execution of the specific Microphages are the polymorphonuclear
immune response.They trap the antigen and provide
it , in optimal concentration to the lymphocytes,
—
leucocytes of the blood neutrophils, eosinophils
and basophlk Neutrophils arc actively phagocytic
Too high a concentralion of antigen may he and form the predominant cell tvpe in acute
tolerogenic, and too low a concentration may not inflammation . The phagocytic property of
be immunogen . It has also been shown that with neutrophils is nonspecific , except for its
^
SOrtie ancigensj prior processing by macrophages is augr.imlaliim by npsonns. They do not appear Co
an essential prerequisite for indue - ion of antibodies. have any role in specific immune processes.
The processing and presentation of antigen by Eosinophilic leucocytes arc found in large numbers
the macrophage to T cells reeju. i re that both the in allergic inflammation , para- itic infections and
cells possess surface determinants coded for by the around z\.‘igen-antibody completes. They primar il y
same major histocompatibility complex ( MHC ) inhabit tissues rather than the bloodstream. 'I'heir
genes. The T cell can accept the processed antigen distinctive feature is the presence of two types of
only if IT i presented by a macrophage carrying on
^
its surface the self-MHC antigens. When the
—
granules the small, round , homogeneous ones and
the large ovvid one*. The granules contain a variety
macrophage bears a different MHC antigen, it of hydrolytic enzymes which bring about extracellular
cannot cooperate with the 1 jells. This is A/f /C killing af large parasites . Eosinophils possess
r&trictii m. phagocytic activity but only In a limited degree.
The functional efficiency of macrophages can Basophil leucocytes are found in the blond and
be increased in many ways. They may be 'activated’ Tissues ( mast cells ). Their cytoplasm has large
hy lymphokmes , complement components nr numbers of prominent basophilic granules
interferon. Activated macrophages are not antigen- containing heparin , histamine, serotonin and other
specific. For example, activated macrophages from hydrolytic enzymes. Degranulation of mast cells,
animals infected with one microorganism are with release of pharmacologically active agents,
cytotoxic to Tumour cells as well as to many other constitutes the effector mechaniim in anaphylactic
microorganisms. Activated macrophagL - show - and atnpic allergy.
uu py righted
/“
material
i
128 * Tanthe&k l Micr & tHalm y >
^
ffiwini ' Hi R vm iMMUfJi : itrsTHM
BONE | STEM 1
MARROW
v!v
SURFACE
CD MARKERS
7 PPG - T
THYMUS 7, - 2 fPLASMIC
OTO \ PRE T
kV'
7, 3, 3 IMMATURE - f
1.4. K
TCP -p
EX I Si A THYMIC -
MATURE 1
7.2. A 7.2. 3 . K 7. X ?
TCR « P
HELFER/INDUCER
TCR |i
CYTOTOXIC/
- TCRTS
SUFPRLSiiOR
Copyrighted material
4 Structure and Functors <A Ito Immune System 129
Dendritic ccl ] r< : While ma crophagcs are the {dr mice and to becoded for by a closely linked
mi: i ir
anti gtit present!ng cells another type of cell
, muliiallelic cluster of genes, which was called the
known a & rhe dendritic cell also performs this major histocompatibility complex.
fui]triu[ ]. Dendritic cells are bone marrow derived The development of congcnic and recombinant
cells of a lineage different from the macrophages strains of mice by Snell enabled the detailed analysis
and T or R lymphocytes. I hey possess MHC class of the various loci of this complex. {The term
II antigens bur not Fc or sheep RUC receptors or L ongenich means animals which differ only at a
:
surface immunoglobulins. They have little or no single genrlic ItHius) . Dausset pioneered Studies Oil
phagocytic activity. They are highly pleomorphic, human leucocyte antigens, which were later found
with a small central body and many long needte- ro be the ma : or histocompatibility antigens in
libc processes, and are present in the peripheral human beings.
blood and in the peripheral lymphoid organs, The genetic basis of immune response, which
particularly in the germinal areas of the spleen and had been suggested by many early observa:; o ns, was
lymph nodes. Dendritic cells are specially involved proved by Qenacerraf and colleagues, who
in the presentation of antigens tod' cells during established that rhe ability to respond
the primary immune response. immunologically to an antigen was conditioned by
The B cell is another antigen presenting cell, specific genes called the Immune response (Ir)
pan iculaHy du ri i ig the secondary i mmune response. genes . For their work un MHL and the genetic
Langcrhans cells in the skin possess features of control of immune response, Snell, Dau &set and
macrophages and dendritic cells. They process :md bcnaccrraf were awarded the Nobel Prize for
present arvigens that reach the dermis. Medicine in I 9fl0.
Earlv- studies on MHC were carried uut in mice.
M ^ ioa HISTOCOMPATIBILITY However, all species of animals ( including human
COMPLEX ( MHC ) brings) examined subsequently were found to
,
The primary function of the immune system is the possess a -.iiuilar complex of genes on a segment of
recognition and elimination of foreign cells and one eliromosome pair, coding for three different
antigens that enter the body. Tissues and organs classes of proteins:
grafted from one individual to another member of L Class I protL - ins that determine histo -
the mini; species {'allogral K .l are recognised as compatibility, and the acceptance or reiocrion
foreign and rejected. Jt was the early workofGorer of allografts { tissues or organs from different
in rhe 1930s on the antigens respond iblc for allograft individuals within the same species);
rejection in : n.bred mice that led to the discovery of 2 . Class II proteins that regulate the immune
the major histocompatibility complex ( MJ 1C). response; and,
GofCI i JtritiJied tWO blood group antigen 3, Cl ASS [ ][ proteins that include some components
systems in mice „ one of which (antigen 1) was of the complement system and a few others.
common ro all the strains. Antijjen 2 was found I’hc name 'histocompatibility complex' arose
only in some strains and appeared to he responsible because its discovery was based on transplantation
for allograft rtiection , This was called the H-2 experiments, and only Later were the Other two
anligen ( H for histocompatibility; Chapter 20}. components of the complex identified - The major
The hiitocompatibilitv antigens are cell surface antigens determining histocompatibility in human
antigens that induce an immune response leading beings are alloantigens, characteristically found on
to rejection of allografts. ( H -2 amigen system was rhe surface of leucocytes. Human MHC anugens
found to be the major histocompatibility antigen are therefore synonymous with human leucocyte
Copyrighted
^8 1
material
130 i T& xlhOok ol Microbiology *
antigens ( HLA ), and the MHC complex of genes HLA class I antigens ( A , B and C) are found
with rhe HLA complex . nn the surface of virtually all nucleated cells. They
HLA complex: The HLA complex of genes is are [he principal antigens involved in graft rejection
located On the short arm of chromosome 6 {Fig. and cell mediated evtotysis. Class I molecules may
15 ,5), Ti consists of three separate clusters of genes: function as components of hormone receptors.
1 ) HLA class I comprising A , E and C loci ; HLA class II antigens are more restricted in
2) Class II or rhe D region consisting of DR , DQ _ distribution, being found only on cells of the
and DP loci , and
'
immune system— macrophages, dendritic cells ,
3) Class [ II or the complement region containing activated 1’ cells, and particularly on B cells.
-
gene t for complement components C2 and C4
of the classical pathway, as well as properdin
Class II antigens are hecerodimers, consisting
of an alpha and a beta chsi i n ( Fig. 1 $.7] . Each chain
factor B of the alternative pathway heat shock has two domains, the proximal domain being the
proteins and tumour necrosis factors a and |1. constant region and the distal the variable. The two
HLA loci are multiallelic , chat ish the gene distal domains (alpha 1 , beta 1 ) constitute the
occupying the locus can be any one of several antigen- binding site, for recognition by CD4
alternative forms (alleles ). As each allele determines lymphocytes , in a fash ion si rm I nr to the recogn i fi on
a distinct product (antigen ), the HLA tyiitem is of the Class 1 antigen peptide complex by CDS T
very pleomorphic . For example , at least 24 distinct cells . HLA class 11 molecules are primarily
alleles have been identified at HLA locus A and responsible for the graft - versus -host response and
50 at S. the mixed leucocyte reaction ( MLR) .
HI , A molecules HLA antigens arc rwcnchain Both class 1 and II molecules ate members of
glycoprotein molecules anchored on the surface the immunogicihi] I i n gene supetfain iiy. The i mmune
membrane of cells {Fig . 15 -6 )- response ( Jr) genes which control immunological
Ckss I molecules consist of a heavy pep ride responses to specific antigens arc believed to be
chain (alpha chum ) noncovalendy linked to a much situated in the HLA Class II region , probably
smaller peptvlc called beta 2- muroglobulin ( beta associated viLth rhe DR locus. It genes have been
chain ) . The beta chain has a constant aminoacid studied cxten -uvelv. in mice and located in the I
sequence and is coded for by a gene on chromosome region of mouse MHC. They code far la ( 1 region
15 . The alpha chain consists of three globoid Mociated ) antigens consisting of IA and IE
domains (alpha ] , alpha 2 , alpha .? ! which protrude proteins . However, the relevance of Jr genes in
from the cell membrane and a small length of humans is not dear.
transmembrane C terminus reaching into the HIA class III molecules are heterogeneous.
cytoplasm . The distal domains {alpha 1 and alpha They include complement components linked lo
2) have highly van able aminoacid setjuences and the formation ofC3 convertases, heat shock proteins
are folded to form a cuvi tv or groove between the m. and tumour necrosis factors . They also display
Protein antigens. processed by macrophages or polymorphism.
dendritic cells to form small peptides ait bound to The MHC system was originally identified in
this groove for presentation to CDS T cells - The T the context of transplantation, which is an artificial
cell will recognise the and gen only when presented event. In the natural state* besides serving as cell
as a complex with the MHC das& 1 molecule and surface markers that help infected cells to signal
not otherwise ( MHC restriction ) . When so cytotoxic and helper T cells , the enormous
presented the CDS cytotoxic killer cell destroys
, polymorphism of the MHC helps maximise
the target cell [for example , a viios infected cell ). protection against microbial infections . Bv increasing
Copyrighted materia
* Structure and Functions ol ttie Immure System * 131
STRUCTURE *NlJf \ r
^Ofr-SOFTllL IMML NtSTiTtiM
O H-1W I
CantmrnwTi QBM il O
'll p
A
n
nr &
*p
S'
ft *-1
'
InVj :V2
:,
0 1 *
LI
DO DP CiB 0 F C? THF H ft
13
P-
at
HHi
m2
a Chain [i Chain
i
)
Pi
|ij Mtcroglobulin J
-
r.1?1!
-
U 0
GOQH COOH
cytopiasjri C iopi asm
^
Fig . 15.& HLA Class I mulacult Fig. 15 ,7 HLA Class II moleeuls
132 * Textbook ol Microbiology »
HLA-typing m advanced centres, 'I'hcsc employ exhibit a he reditu rv tendency. Kor example, strong
restriction fragment lctigrl’i polymorphism ( RFLP) association has been found between antqrlosing
and gene sequence specific oligonucleotide probe Spot idyl ids and .HLA- H 2 7, rheumatoid jrtli ri t is an LI
*YP“ g f 11 ^ A - DR 4, and m any autoi m mu nc cond i tions and
The HIA antigens coded for by the HLA- DR 1 .
combination of alleles at each locus on one stfud
of a chromosome pair represent the haplotype. I'hc MHC RESTRICTION
complete FI [ .A type ol ml individual comprises [ tie The importance of MHC antigens in immune
antigen* represented on both strand *, of the diploid reaction is indicated by the finding that '1' cells
chromosome and so will consist of TWO haplotvpvS respond to processed antigens on the macrophages
-
(for example, HLA Al , -A2; - R 7, - Bi2i -Cw3,
-Dwfit L>w 4; -Dw7t -DRl ; -DR7; DQyrl ; Qw3-
and other accessory cells only when they are
presented along with the self - MHC antigen , This
-DPw 4; -DPwti ) , is known as MHC restriction. Both class I and class
-
lihn. to the extreme pleomotphism ol the HLA
system, delineation of the I1LA type provides a
11 antigens o]K.Lrale in this phenomenon. CytOtOXic
T lymphocytes from immunised mice arc able to
method of typing of individuals, that is far more kill and Ivsc virus infected target cells only when
discri mi nating than blood grouping. 11LA typing the T cells and target cells ate of the tame MHC
i $, used primarily for testing computihilitv between lvpeNso that theT cells can recognise clans I MHC
recipients and potential donors before tissue antigens on the target cells. I JclperT cells can accept
tnuriHjilantation. Tt also has applications in disputed antigen presented by macrophages unlv when the
paternity. As the prevalence of HLA types varies macrophages bear the same class II Ml 1C
widely between different human races and ethnic molecules on the surface. For T cells participating
groups, HI . A typing i n used in anllirof J O logical in delayed type hypersensitivity the antigen has to ,
Further liunding
Ahhds AX cr (L iy 4. CeJJuIirand MflArcrjAtrliFhinLino/cgT. 2*: edn. Philadelphia : Saunders.
^
Chapel H and M Haeney 1993. Essentials ofOinic tirimtino/t y. fllLedn. [.(melon: Htnckwell Jkwirce.
^
/mrnujKiiNoJoijv, 2 lI cdn . London : Currant Biology.
' ^
JanewayGA and P Travers 19%,
. -
'
.
IVakrTun M and [) VerRani 1997 Basic and Clini al fn? mimcJc>£p Edinburgh, Churchill - Livingstone -
Iflfeir DM a n d j Stewart 1997 . laUBUJKrlvgf' S* ndrt . t Lli ntwicgl:: CtlUIXhall-I . Lvidj -tniu:.
"
^
Klein JK and A 2000. The HLA system - NewEngf Nfeti 34.L702
Copyrighted materia
Immune Response
The specific reactivity induced in a host by an 1 . The cntTy of the antigen, its dis-Triburiun and fete
antigenic stimulus is known as the immune in the tissues and its contact wirh appropriate
response . In infectious disease it is generally equated immunocompetent cells ( rhe afferenr /djiab).
with protection against invading microorganisms . 2. The processing of antigen by cells and the
I5 ut the immune response has a much wider scope control of the antibody forming process [cenfruf
and includes reactions against any antigen , living functions).
OT nonliving. It may lead to consequence ^ that are 3. The secretion of antibody, its dislributicm in
beneficial, indifferent or injurious to the host. It tissues and body fluids and the manifestations
also includes die state of specific oonreictivity .
of its effects { efferent limb )
( rule ran ue 1 induced by certaini types of antigenic Antibody production follows a characteristic pit tern
—
Stimuli , The immune response rim be of two types
the humoral ( antibody n led i ated ) and the cellular
(cell mediated ) types. The two ate usually
developed together, though at times one or the
consisting of
a. A lag phase, the immediate stage following
antigenic stimulus during which no and body is
detectable in circulation.
other may be predominant or exclusive. They b. A log phase in which there is steady rise in the
usually act in conjunction but may sometimes act titre of antibodies.
in opposition. c. A plateau or steady state when there is an
Antibody mediated immunity' ( AMI ) provides equilibrium between antibody synthesis and
primarv defence against most L'xiraciellliLLr bacterial catabolism.
pathogens, helps in defence against viruses rbiit infect d. The phase of decline during which the
through lilt respiratory or intestinal tracts , prevents catabolism exceeds the production and the titre
recurrence of virus intections and participates in the falls {Fig. 16.1).
pathogenesis of immediate ( types l , 2 and > )
"
hypersensitivity and certain autoimmune diseases. Cell PRIMARY AND SECONDARY RESPONSES
mediated Immunity ( CM I ) proteurs against fungi, The antibody response to an initial antigenic
viruses and facultative intracellular bacterial pathogens, stimulusdiffers qualitatively and quantitatively from
participates in the rejection of hotnograftn and graft-
, the response to subsequent stimuli with the same
versus - host reaction , provides immunological antigen.The former is called the primary response
surveillance and immti uity against cancer, and mediates and the latter the secondary response ( Tig. 16.2).
the }twtliogenesis of delayed ( type 4) hypersensitivity The primary response is slow', sluggish and short-
and certain autoimmune diseases. lived , with a long lag phase and low ricre of
antibodies that does not persist for long. In contrast,
HUMORAL IMMUNE RESPONSE the secondary response is prompt powerful and
The production nf antibodies consist; of three stops: prolonged , with a short or negligible lag phase and
Copyrighted materia
134 * TGnttOOk Of Microbiology
Copyrighted material
4 immune Response 135
noniinrrunc phase during which the antigen is molecules , to the 1 cell carrying the receptor
cn Eled by the phagocytic cells broken down and (TCR) for the epitope is the T cell able to recognise
^ ^
eliminated . With the appearance of the specific i :. In the case of CD4 ( helper T/TK ) cells, the
antibody, rhe phase of immune elimiLiar ion begins, antigen has tn he presented completed with MHC
during which antigen — antibody complexes are Class II and for CDS (cytotoxic T/T . ) cells with
formed and arc rapidly phagocytosed, resulting in ^ possess
MHC class I molecules . B cells, which
an accelerated disappearance of the antigen from surface Ig and MHC class II molecules, can also
circulation. With soluble antigens , three phases can present Antigens to '1 cells, particularly during the
be recognised — equilibration, metabolism and secondary' response .
immune elimination. The phase of equilibration The TH cell requires two signals for activation.
cnn5 i -.ts of diffusion of the antigen to the extruvascular The first signal is a combination of theT «11 receptor
spam. During the metabolic phase, the Level of the -
[TCR) with the MHC class II completed andgen .
antigen falls due to catabolic decay. Dun ng the phase Hu.- second signal is iuterleukin-1 (1L1 ) which is
"
of immune elimination, them is rapid elimination of produced by the APC . The activated TH cell forms
the antigen with the formation of antigen— antibody i ritcrlcukm-2 and other cytokines required for B cell
complexes. Snell complexes can cause i issue damage stimulation. These include 1L4, IL5 and JLfr which
and may be responsible for ' immune complex act as E cell growth (actor ( ROGF) and the B cell
diseases' such as scrum sickncss - differentiation factor ( BCDF) that activate B cells
Thc speed of elimination of an andgen is related which have combined with their respective antigens
to the speed . u which it i & metabolised. Protein to donaliy prohleratc and differentiate iinto antibody -
antigens arc generally eliminated within days or sccre ring plasma cdls- A small proportion of activated
weeks , whereas polysaccharides which arc H cells , instead of biLing transformed into plasma
metabolised slowly, persist for months or years. cells , become long lived memory cells producing a
Pneumococcal polysaccharide, for instance , may secondary type of response to subsequent contact with
persist upto 20 yean in human beings, following a the amigen.
- ingle injection . Cyrormtic T ( CDfl /TC) cdls are activated when
they contact amigens presented along with MHC
PRODUCTION OF ANTIBODIES class T molecules. They also need a second signal
Immune response to an antigen is brought about Th
IL2, which is secreted by activated 3 cells. On
hy three types nf cells : antigen processing cells contact with a target cell carrying the antigen or
( APC— pri ncipally maemphages and dendritic cells ), Its surface, the activated Tc cdls release cytotox ms
'
T cells and E cells. The first step is the capture and that destroy the target, which may be virus infected
processing of the antigens by APC and their or tumour cells. SomeTc cells also become memory
presentation, In association with the appropriate MHC cells [ Fig . 16.3).
molecule, to T cells. While this step is essential for
most antigens (T cell dependent antigens such as MONOI :I ONAI ANTIBODIES
proteins and erythrocytes ), in the case of T cell A single antibody forming cell or done produces
independent antigens, such as polysLreharidcs and antibodies specifically directed against a single
other structurally simple molecules with repeating antigen or antigenic determinant only. However,
epitopes, antibody production does not requmeT cell antibodies produced ordinarily by infection or
participation. anti gens have multiple epitopes or antigenic
Only when the processed antigen i - presented determinants, each of which generates separate
on the surface of APC, in association with Ml IC clones of lymphocytes. This results in antisera
Copyrighted material
136 + Textbook or Microbiology
rviML nr RESPOND
w
c
* / ®
§k TCH
cw
Wtwkmi
I •"!
B.- ZHI
0 Hi.ItiF
IL - ?
©
CytaloMia
MHC - I
tO
^
.
CTrt
_wafh
'r |
Tc
M
4
- ©r
/ \
HC
®
f ‘\
Fig.16 3 : Outline stbameaNmimjneresponse a ainstTceltcfep^nddnl antigen
^
1. Anl ige n praso ntl ng ee lls ( AFC) proees & phagccytosed antigen { A ] in thair cytoplasm I present oi thscefl
*
s u rf ace imm u nog eni c c p hope s (E f com piexcd wilti M HC Class II molecules .
.
2 Signals forlhe activation of CP 4 ( THJ cells are a combination of the T Cell antigen -
rht MH(
Classilantigen complexes APC . and stimulation by I L-l released by A PC .
1. The activated TH celt spteutslL- ZteCBptgrssndi secretes cytgfcines hich act on g cells i’ lgfor
IheepHope , causing cellgrowlh gnrl dilterenlielmn ) ,
*
^
5. Some of the activated 0 c Ns become memory cells (6U1 , wh He outers ate transformed c :: i i r: .
*
S 0C teti ng irttiba dy against ( he anti gen ( 6 f .
i
4. Cytotoxic T cells are stimulated by IL- 2 released by the activated Th cell. On pitepa
complexed with WHC Class t mdeCutee On tire Surface ol target Cells , (he Tc cell Ins which
destroy the target cells ( 9 }.
id . Some Fc cells become memory colls tT:u|.
Copyrighted material
* Immune Response * 137
containing immunoglobulins of different da--- with produced - Hybridomas may he frozen far prolonged
^
specialities against different epitopes of the antigen. Storage . The discovery of tlie hyhndoma technology
On the other hand, when a done of lymphocytes or for the production of unlimited quantities of identical
plasma cells undergoes. selective prul iteration, LLH in [noiuxJnnal antibodies of die same lg das*, possessing
multiple myeloma, sinribodieG with a single antigenic uniform spedlieky, affinity and other properties ,
.
specificity accumulate Such antibodies produced by created a revolution in immiLnology by opening up
a single done and directed ; LJ HJU >[ a single antigenic numerous diagnostic , therapeutic and research
^
determinant are Called monoclonal antibodies. application*. Monoclonal antibodies against scvcrel
Monoclonal antibodies ire very useful tools for anrigens are now available orwnmervLlly.
diagnostic and research techniques . Murine monoclonnl antibodies, however, proved
An ingenious method for rhe luge scale unsuitable for human therapeutic use hecause they
production of monoclonal antibodies against any induced strong anti mouse immune response .
des- i red antigen was developed by Kr>h I < -T and MUsed n Moreover* the Pc piece of mouse [g could not
in 1975. In recognition of the great importance of initiate effector defence mechanisms in human
this hybfidoma technology, the Nobel Prj c for
^ beings . Various modifications were introduced to
Medicine was awarded to them in 19fl 4.1 Iybridonuts improve efficiency. Cleaved Fab fragments could
arc somatic cell hybrids produced by tiising anlilwdy be coupled TO various active substances like toxins,
fanning spleen cells with myeloma cells.TIK resultant enzymes, radionuclides or cytotoxic drugs. Mouse
hybrid retains the antibody producing capacity of the monoclonal* have been humanised by genetic
spleen cell and the ability of the myeloma cells to manipulation ro inalte chimeric antibodies
multiply indefinitely ( fig. 16.4). consisting of murine variable region# and human
Lymphocytes from the spleen of mice constant region*. Grafting of murine monoclonal
immunised with the desired antigen are fused with Gl > R loops cm a human Ig framework provides a
mouse myeloma «Dl grown in culture which di > virtually human molecule . ( The antigen binding
not form immunoglobulins and are deficient m the surface of an antibody is composed of six
enzyme hypOKanthinc phosphorihnsyl transferase hypervuiible loops of ami noadds. These are called
( HPRT ). The fused cells are placed in basal oaaplementanty determining regions or CDRs).
culture medium ( HAT medium containing I Jumati monoclonal antibodies have subsequently
hypoonthinc, ammopterin and thymidine ) which been developed. Genes for particular antibody
does not permit the growth of the enEyme fragments have been fused to hacrcriophagC genes.
deficient myeloma cells. Av normal lymphocytes Whole libraries of such antibodies have been built
cannot replicate indefinitely, only hybrid cells using bacteriophages. I Jige quantises of the desired
possessing properties of both the splenic antibody can be prepared by infecting bacteria with
lymphooytei anti myeloma Cells can grow in the appropriate bacteriophage. Such antibody
culture . These hybrid cells, called liybridomas* are engineering holds great promise for immunotherapy
cloned and examined for the production of
antibodies. Clones producing antibodies jigainst the FACTORS INFLUENCING ANTIBODY
desired antigen arc selected for continuous PELODI CTION
cultivation. Such hyhridomas can be maint .lined
P
Genetic factors: The immune response is
indefinitely in culture and will continue to form under genetic control The differences in immune
monoclonal antibodies. They may be also injected response to ( lie same antigen shown by different
intrapcriioireally in. mice and monoclonal antibodies individuals in a species is determined by genetic
may be obtained bv harvesting the ascitic fluid differences . The t e r m s ' responder 1 and
Copyrighted material
m 4 Textbook n\ MiCf &biOtogy *
IMMUNE RESPONSE
MPBI »g
I^RT lg 1-« H«
i
^ Hn
,V> :
r © t fW 1
l
\
'
- '
r_ - .-
r i' C zdv
.
"
Jjn&dliE^g [rDi-!
Hp-binh
^
lurnwa liidurliuii
Mm -
Cw liir
*
. .. .
W.:rT r r: -mi;
fliiliEudy
y 4^
SMcmri.mnBi
niibodv
Freer
| .-
*
> 4i.ILHIH ilH
. .
Fig 1 & 4 : Monoclonal antibody flr &ductlon by bybridoma
Copyrighted marorial
lmmun & Response t 139
Hnnn responder arc used to describe the individual's folic acid) have beer shown to cause a decrease in
capacity to respond to a particular antigen. The Ir antibody synthesis.
( immune response ) genes control this property. Route of administration: The humoral
Aj ,“ The embryo is immunologlcally immature. immune response is better following parenteral
^
The opacity to produce antibodies start only with administration ot antigen than through oral or nasal
the development and JrfieienTiatLon. of lymphoid routes . Large particulate antigens, such as bacteria
organs. The age at which embryos acquire or erythrocytes, are more effective when injected
immunological competence varies with different into tissues. The route of administration may also
specie?. When the potential immunocompetent cell influence the type of antibody produced. For
comes into contact with its specific antigen during production of IgA antibodies, the oral or nasal route
-
embryonic hie, the response i elimination of the
cell or induction of tolerance, This is believed to
is must -suitable . Inhalation ot pollen antigens
Most IgG antibodies to polysaccharides arc of the Size find number of doses: Artihndy
lgC 2 type, and lgG 2 producing 11 cells art the last response is., roan extent , dose dependent. An antigen
to mahm: during infancy . is effective only above a minimum critical dote .
N LI Li ' iE irpilul S to Los; Malnutrition
affects the Further increase in dose enhances the intensity of
immune response adversely, though serum the anTih«ty response hut beyond a certain level,
,
components necessary for immunity are conserved increase in the dose df antigen docs not improve
selectively till the nutritional deficiency becomes rhe antibody response but may even inhibit it and
marked . Protein calorie malnutrition suppresses induce tolerance. Mice injected with 0.5 pg of
both humoral and cellular immune responses, the pneumococcal capsular polysaccharide produce
latter more severely, Deficiences of ami noacids specific antibodies but those injected with 5Q pg of
{ tryptophan, phenyl alaninet methionine, glycine, the antigen not only fall to form antibodies but may
i sole neine ) and vitamins [vitamin A, and B group not respond even to subsequent doses of the same
factors rilujfljvine, pyridoxine, pantothenic acid . antigen. The massive antigenic stimulus appears
Copyrighted material
140 * Tgxibook of Microbiology *
swamp [ III annlxhl/ producingsystem and paralyse competition is important, from a practical point of
"
1
it. This phenomenon wasdesigniLtecTirninLinolLiLdcai view, in immunisation wuh polyvalent antigens For
, .
paralysis' by Felton ( 1949 ) . optimal effect, the nature and relative proportions
The increased antibody response ro a secondary of the different antigens in a mixture should be
antigenic tdfflulur lias already been noticed. With carefully adjusted.
repeated antigen injtctioru, the .11 .i;':KK!V response
' Adjuvants: The term adjuvant refers to any
increases progressively hot after a certain stage, no substance that enhances the immunogcnicity of an
further increase occurs. antigc n. Adjuvants may confer 1m munogcn icily on
The term "anamnestic re actio n' wan originally [icmantigcnic substances, increase the concentration
applied to the production, in response to an and persistence of the circulating antibody, induce
antigenic stimulus, of a heterologous but related or enhance the degree of cellular immunity and
untiHody that the ho*r had earlier produced hbr , lead to the production of 'udjLtvant diseases" such as
instance, A person who had been immunised earlier .
allergic disseminated encephalomyelitis A variety
against typhoid bacilli may sornetinio: produce of substances exhibit adjuvant acLivhv.
ant Ltypho id antilhodic > in response to i1 Lfeetion wiih Repository adjuvants such HE aluminium
some other bacterium. This may cause L- onfusiort hydroxide or phosphate and incorporation of
in the serological diagnosis ot typhoid tcver but
urcu-m nestle reaction can he differentiated from a
protein antigens in the water phase of a water - in
oil emulsion (Freunds incomplete adjuvant ), delay
-
true secondary response as it is transient. The term the release of antigen from the sire of injection and
'anamnestic reaction’ has been employed by some prolong the antigenic stimulus . Others Euch as -silica
ro refer to the secondary response as well, so there particles , beryllium sulphate and endotoxins activate
is some confusion in the use of this term. macrophages, The most potent adjuvant is Freund’s
Multiple antigens: When two or more antigens complete adjuvant, which is the incomplete adjuvant
art administered simultaneously, [he effects may along with a suspension of killed tubercle hue ill'..
wary. Antibodies may he produced against the Besides increasing the humoral immune response,
different antigens just as though they hardbeen given it induces a high degree of cellular immuni tv
separately or antibody response to one or the other .
{ delayed hypeftcnwtivitj) as well As It produces a
'
of rhe an tigers may he enhanced* or the response local granuloma, it is unsuitable for human use .
to one or more of them may he diminished The adjuvant effect of tubercle bacilli is due to a water
(antigenic competition). When two bacterial soluble peptide MDP (muramyi dipepiide) which
vaccines ( for example, tvph- uU and cholera vaccines) induces good antihc -ilv response without causing
,
are given in a mixed form, the antibody response to granuloma . Given in mineral oil or as tposomes, it
.
each is nor influenced by the other When toxoids also stimulates cell mediated immunity. Derivatives
Art giycn ALONGWILIL h.1,rkr - iI ¥
| CQnes ( for esample, of MDP are hc-int; developed for human use. Gram
triple vaccine containing diphtheria and tetanus negative bacilli show an adjuvant effect due to their
toxoids along with Bord pertussis vaccine) the lipif Htlvvtfccharide fraction.Bordrldla pertussis, which
response to the toxoid is potentiated. When -
has, in atkiirion, a Eyinphocytosis pnomoting factor
diphtheria unci tetanus toxoids Aft given together, anting on both T and K cells, act? as a gtxxl adjuvant
with one in excess, the response to the other is for diphtlieri-a and tetanus tmuids in triple vaccine
.
inhibited When triple antigen is givtn to a person Ollier adjuvants otnumunlv uaed with human vaccines
who had earlier received a primary dose of are alnrYiinium hydroxide or phosphatC -
diphtheria teweid, the response to the tetanus and L mmunoguppro strive agents: These inhibit
pertussis antigens will be diminished.Such antigenic the immune response lltey are useful in certain
,
Copyrighted material
4 Immune- Response 141
«11 division and differentiation necessary for humoral passively administered antibody on the humoral
and cellular immune responses. They inchide folic i mmu n c response has been applied i n the prevention
acid antagonises { methotrexate), alkylating agents of Kh sensitisation in Rh negative women carrying
( cyclophosphamide ) and analogues of purme {6- Rb positive fetuses . This L S achieved by the
mcnzaptopurinc, oxathioprine), cytosine {cytosine administration of anti - Kh globulin immedinrclv
arabi noside ) and uracil ( 5-fluorouracil ) . Many following delivery ( within 72 hours).
am ! metabolites find clinical application in the This effect is also relevant in the practice of
preveniiun of graft rejection. combined immunisation as in diphtheria and
The drug most widely used now for tetanus. In such eu«, the toxoid and Antitoxin
Copyrighted material
142 * Texl & ook o Microbiology r
'
should he giveo at separate sites, Adsorbed toxoid involve antibodies - for long the only demonstrable
'. bould be used as the inhibitory effect is much Less
r
facet of CM I was the phenomenon of delayed
than with fluid toxoid - hypersensitivity ( DH ) which resulted in injury
Inmu - m / js adm nihtiatnm cC immune gkAulin I:. L -
been shown to haw: immtmninrsdulatmy effete It has
-
lather titan protection The first description of a
CM! response was the observation by Jennet
been used in the treatment of many diseases of presumed ( ] ?V8) that inoculation of vaccinia virus in an
immune individual led to a local erythematous
^
TiiriuiHjpadi£ilpg etioli /jji such as thiwnbocyKjpeni- is
i[
the MHL’ molecules through the V regions ol lesion 48-75 hours after administration of the
TCR a and [1 chains, ? uperantigens bind directly
antigen . The lesion is an indurated nodule with
to the lateral aspect of the TCR |1 chain. Updo 50 infiltration hy mononuclear cells. DH was found
per cent of the circulating T cells may be so to be immunologically specific but IT did not have
activated , compared to conventional antigenic any relation to antibodies and could not be
r
stimuli which involve only about 0 -001 per cent of Transferred passively by serum. The cellular basis
them. This exaggerated T cell activation leads to
ofDH was shown by Landsteiner and Chase (1942)
massive outpouring of T cell cytokines , causing bv its passive transfer in guinea pigs through the
multisystem dysfunctions, such as seen lit injection of leucocytes ftom sensitised donors, With
staphylococcal toxic shock syndrome. the recognition of the two component concept of
MITOGENS immunity, DH and other types ofCMI were found
Mitogens are certain > ubscance & chat induce division to be medi .ited by T lymphocytes, A variety of
of lymphocytes and other cells. Some of these, like techniques are now available for the detection of
the lectin glycoproteins hind to sugars DO the surface CMI, though they lack the sensitivity and precision
i h antibody assays fbr humoral inmmnil y.
of responsive cells and activate them, causing a
polyclonal reaction. At low concentrations , they SCOPE OF ( 1 M 1
stimulate B cells without polyclonal activation .
Lipopolysaccharide is such a B cell mitogen. CMT participates in the following immunological
Some Large molecules with repeating epitopes, functions:
such as pneumococcal polysaccha ride directly i nteract 1. Delayed hypersensitivity.
with B cell surface imrmmogjobLilins, leading to an 2. Immunity in infectious diseases caused by
TgM immune response. Such T- i ndependent immune obligate and facultative intracellular para -.ites -
response with IgTvI antibody; is not associated cither These include infections with bacteria ( for
with Ig class switching or memory. example , tuberculosis, leprosy, listeriosis,
brucellosis), fungi ( for example, histoplasmosis,
CELLULAR IMMUNE fl ESPONSE coccidioidomycosis, blastomycosis ) , protozoa
The term 'tell mediated immunity (CM1) refers
1
( for example , leishmaniasis, trypanosomiasis)
to the specific immure responses that do not and viruses (for example, measles, mumps).
xJ opyrighted materia
[
* hvnu'ie R HSPO - i HE? * 143
or allograft cells), in association with MCH class endocrine hormones in being produced not by
1 molecules, secrete lymphokines and destroy the specialised glands but by widely distributed cells (such
target cells. as lymphocytes, macrophages, platelets and fibrobtets),
and acting not systemically but locally near the producing
C VTOJilNKS cells (paracrine effect ) or directly on the produdi cells
Biologically active substances released by activated ^
themsdves (autocrine effect]. They arc, in general,
T lymphocytes were called lymphokines. Similar pleintmpic, hati ng multiple effects on the growth and
substances produced by monocytes or macrophages differentia* ion of various cell types . There is
were called monokines. Initially they were given considerable overlap in the effects produced by
names based on rhe i r demonstrated biologi cal effects different cytokines . Cloning of cytokines and the
(Table 16,1 ) . As most lyirphoki ncs exhibit multiple availability of monoclonal antibodies against them
biological effects and rhe same effect may be caused have helped to characterise them better {Tabic 16.2),
Copyrighted materia
144 * Textbook of Microtuologv
A. INTERLEUKINS:
Mufl WWro« Major function*
1L- I ( H and p.) Macrophages and other cell types f Liferation and differentiation of T, B and other
^
Ctl ]a; pyrwprnio; induce acute phase jwntdflp; bone
IL 2
- T *
cell
-
mArrow < cU prolifentiun ,
PmiAOU i ywdi ; iid tiiffHHitiirimi ofT acu. l B
. ^
cells Cytotodrity ot T and N 6L. cells, secrethm of
other lymphokines .
1L-3 T cells Multi CSF
IJ . -4 T„« U* FroLiter at ion of F and MUDDCT cells: increase
IgCI and igE production; enhance MHC class II
SiElrd IgE rcMptOtfS.
1 L- S TM celt* Proliferation of eosinophils, stimulate IgA and IgM
,
production.
11 . fn TH, macrophages, fibroblasts Promote B cell difftraitkboa; IgG pnxiuctiun ,
acute phase proteins.
1L-7 Spleen , bone marrow stroma] cells B andT cell growth factor.
iL - e
IL 9 -
.
IL - 10
Macrophages, others
Ted )
T. n odls, macrophages
Neutrophil chemoractic factor.
T cell growth and! proliferation
Isiliibrt IFN production and mononuclear cdl functions.
iL n
-
IL 12
- Bone marrow ttroinil cells
T cells
Induce acute phase proteins,
Activate NK cdk
IL 13 T cells Inhibit mononuclear cdl fiinctikH]*.
B. COLONY ST1MU LATIN Cl FACTORS:
--
GM C5F T cells, macrophages, fibroblasts T cell and macrophage growth stimulation
CSF Fibroblasts, endothelium Granulocyte growth stimulation .
M CSF- Fibroblasts, endothelium
C . TUMOUR NECROSIS FACTORS:
Macrophage growrh stimulation.
Copyrighted materia
< Immune Response > 145
-
E
stimulation of T cells for the production of 11.2 growth factor II , 11 0 causes proliferation of
and other lympliokj nes, U cell proliferation and activated B cells . Ir also induces maturation of
,Lilt ] body synthesis , neutrophil rhcijiotaxb and eosinophils.
phagocytosis. h mediates a wide range of metabolic,
physiological, inflammatory and hematological
-
Inlcrlcukin ti; 1L6 is produced by stimulated
T and H cells , macrophages and fibroblasts . It
effects. I : v nclirlg on bone marrow, epithelial and induces immunoglobulin synthesis by activated 15
synovial cells, fibroblasts, osteoclasts, hepatocyica, cells and formation of 1L 2 receptors on T cells. It
vascular trulothelium ami other targets li . l is an
, has a Stimulator,’ effect on lueputocytes, nerve cells
important endogenous pyrogen . Together with the and hematopoietic cells . It acts as an .inflammatory
tumour necrosis factor (TNF), it is responsible for response mediator in host det enre aguinst infections.
'
many of the liematologicul changes in septic shodk Colony stimulating factors (GSFk These
ami also enhances the initial meningeal cytokines stimulate the growth and differentiation
inflammation in bacterial meningitis. Cytokine f pluripotent item cells in the bone marrow. They
inhibitors such an Jesimcrbasone have been found have been named after tike types of ceil colonies
In protect against the sequelae of such CWUSMVL -
meningeal inflammation . On the Other hand, ILl
-
they induce in soft agar culture for example ,
granulocyte (G ) t nr mononuclear (M) C5F ] L3
Has a beneficial effect in severe infections in which induces growth of all types of hematfjpoictic
immunocompromised hosts. cells is known as multi -CSF. In the bntlv they cause
-
Interleukin 2: The discovery in 1976 of a T
cell growth factor (TCG F) produced by Activated
nther effects also, presum .ibly by inducing cascades
of other cytokines. They arc responsible for adjusting
T cells., which induced 1’ cell proliferation and the rate of production ot blood cells according to
enabled their maintenance in continuous culture, requirements, for example , the massive granulocyte
contributed greatly to the understanding t>fT Cell response seen in pyogenic infections . Colony
functions. This cytokine, renamed 1L2, is a powerful stimulating factors have clinical applications for
muduLlor of the immune response. It LF the major treating hematopoietic dysfunctions in infections
activator of T and R cdls und stimulates cytotnic and malignancies.
T cells and NK cells. It converts some null cells Tumnur necrosis factors (TNF ): The
(LGL ) intotympbakioe activated killer ( J .AK ) cells rumour necrosis factor occurs as two types, alpha and
which can destroy NK rcsLstanr tumour Octli- TIrfo beta . A scrum factor found to induce hemorrhagic
property has been used in the treatment of certain necrosis in certain rumours Wt& mured the tumour
types of cancer. neciOtiLii factor. Tlte tame substance was independently
lnlerkuikiti-1 1L3 is a growth factor for bone _
described as ( itchcctin , a serum factor causing the
marrow stem cells . It stimulates multilincagc wasting syndrome (cachexia ) during chronic
hematopoiesis and is therefore known also as the infections.This has been reranvedTNFtx . It is formed
multicolony stimulating factor (multi- C 5 F ). principally hy activated macrophages; and
Interleukin- 4: Formerly known as the 11 cell monocytes . It resemble ^ 11.1 in possessing a very
growth factor -1 (BCGF-1), IL4 activates resting wide spectrum of biological activities* such as
B cells and acts as a H cell differentiating factor. Ir
,
participation in the manifestations of endotoxic
Copyrighted material
146 ^ Textbook pf Microbiology
Other cytokines , TNFP+ formerly known JX and negative feedbacks. A number of cytokines (for
iymphotcndrL, is produced principally by T helper example, ILl , 2 , 3, colony stimulating factors*
cells . Its effects are similar to chose of TNFa. interferons) have already found therapeutic
Interferons UFN ); Originally identified JH application, With better understanding of their
antiviral agents (see chapter 49), interferons are now properties, it is possible that many cytokines, their
clarified as cytdkinCE. There are three etsisHCi. of igofiuits and antagonists could eventually be used
IFNs, alpha produced by leucocyte*, beta produced in the m an age men r of inflammatory, infectious ,
by fibroblasts and gamma by T tells activated by autoimmune and neoplastic condition.
antigens, mitogens or expusoire Co IL2. IFNy causes
many immunological effects, such as macrophage DETECTION OF CM I
activation , augmentation of neutmphi and The original method fur delecting CM1 Wfll the
monocyte functions, and antitumour activity. 5 kin rest delayed hypersensitivity ( for example,
for
Othflr cyttikintHT The transforming growth the tuberculin test ). A number of In vitro correlates
factor beta (TGFp) was RO named because of its ofCMI have now become available . These include
ability to transform flbroblasts. He sides acting as a tlie lymphocyte transformation test ( transformation
growth factor for fibroblasts and promoting wound ot cultured sensifiwd T lymphocytes on contact
lieal ing* it also acts as a down regulator of some with the antigen ), target cell destruction ( killing
immunological and hematological processes . trf cultured alls by T lymphocytes sensitised against
ITic leukemia inhibitors factor ( LI F), produced them ), and the migration inhibiting factor test
by T cells, helps stem cell proliferation and which is commonly cm ployed. As uriginally
eosinophil chemotnxis- d t ihed, r i . i . iiisi 'icki oJ incubating in .i culture
Cytokine production is regulated by exogenous chambrt, packed peritoneal macrophages in a
stimuli such us antigens and mitogens, as well as cap ! Uy tube. Hue maUuphageB migrate 1 form a
by endogenous factors such as neuroendocrine
hormonal peptides (corticosteroids, endorphins )
-
bey, fun like pattern. If the macrophages are from
iguinea pig en ti cd to rubcrouloprotrin, addition
ami piKjducts of lipoxygenase and cyclooxygenase [ | tubcr-culin in
j ;
culture handle: will inhibit
pathway! .They i&o regulate each other by positive the migration ( Fig 1 m5) , Hi is h . i - been adapted
Fig. 16.5 Inhibition sT migration of macrophage cells lift Exposure to antigen or cells Irom Rensilfc &ed guinea
,
pigs Migration inhibited Right Control Exposure tt mtlgs r of cells iron- normal guinea pigs Show i
. . .
foam — like migration of macrophage*.
? materia
4 Immune Response 147
lor clinical use by IIIV. JI v. l i:ig human peri ] rtic- ral deficiency ( Wiskott-Aldrich syndrome). It has also
leucocytes in cap:. Hjiry tune - in culture chambers. been used in the treatment of disseminated infections
When an antigen to which the individual has CMI associated with deficient CMI {lepromatous leprosy,
is introduced into the culture medium, the leucocytes tuberculosis, imicocutaiieoLis candidiasis). IT has been
am prevented from migrating , By comparison with employed 111 the treatment of malignant melanoma
the control , it is possible to make a semiijiianritHtiw? and may be be nefivial i n other types of cancer as well.
assessment of the migration inhibition. Its use has been suggested in some autoimmune
diseases ( systemic lupus erythematosus, rheumatoid
TEIANS^ BR FACTOR arthritis ) and diseases of unknown etiology ( sarcoidosis,
Passi ve transfer of CMI was first achieved by the multiple sderusis).
injection of viable leucocytes from sensitised donors.
T -awrcncc ( 1954) reported transferof CMJ in human IMMUM0 LOGICAL TOLER AM CE
beings by the injection of extracts from leucocytes. Immunological tolerance or immunological
Thn extract is known as the 'transfer facror' (TF). unresponsiveness is tlie condition in which contact
The transferred immunity is specific in that CMI with an antigen specifically abolishes the capacity to
can be transferred only to those antigens to which mount ar. immune response agionat that partkiilar
the donor is sensitive. artCij rt when it ns administered subsequently- Hns
TF is a dialysable , low molecular weight ^
nonreactiVLty is specific to the particular antigen,
substance ( MW 2000 to 4000), repi -itant to trypsin, immune reactivity to other an - igens being unaffected .
DNAase , RNAase and freeze thawing. Tt LH stable The first example of immunological tolerance
for several years at — 20 C and in the lyophilised was the nhservatinn by Owen ( 1945 ) of erythrocyte
form at 4 ^C . It is i nacf i rated at 56 5C in 30 mi mites. chimcrism in dizygotic cattle twins, each of the
It is not anCii cniL . Chemically* it appears to be a twins having erythrocytes of its own and the other’s
-^
polypeptide polynucleotide . blood groups. As dizygotic twins are genetically
TF is highly potent , an extract from 0.1 ml of dissimilar, they do not ordinarily accept transplants
packed leucocytes lx Ing sufficient for transfer. The from each other but such transplants survive in
transferred CMI is SYSTEMLL: and. not local at the cattle twins. The reason for this tolerance was shown
:
iu ecte (i site alone . FollowingTF injection , DH and to be the sharing of the same placental blood supply
various in vitro correlates of CMI can be by the twins during intrauterine life . Based on this
demonstrated in the recipient . Hu morn I immunity observation, Burnet and Fenner (1949) suggested
is not transmitted by rF; TF transfers CMI to all that the unresponsiveiLess of individuals to self
the antiL enn to whi .: b the donor is sensitive, en antigens was due to the contact of the i rpmature
^
htne . It is possible to transfer CMJ from the immunological system with self antigens during
recipient to another in a serial fashion. embryonic life. Any anriLcn that comes . nro contact
The mechan i sm of action of the TF i s not known. wi:h the immunological system during embryonic
TF could be an informational molecule or a specific life would be recognised as a self antigen and would
gene dcrepressor capable of inducing antigenically not induce any immune response. They postulated
uncommitted lymphocytes to produce artigen - that tolerance could he induced against foreign
specitic receptors . TF activity was till recently ant i .L ens i f they were adm ini *tcred during embtyon ic
demonstrable only I n human beings but it has now ^
life. This was proved experimentally by Medawar
been reported in monkeys , guinea pigs and mice. and his colleagues ( 1953) using two strains of
TF has several applications. It has been used to syngeneic mice . When a skin graft from one inbred
restore immune capacity in patients with T cell strain of mice ( CBA ) is applied on a mouse of
Copyrighted material
148 A Textbook of Microbiology
another strain (A ), it is rejected. If GHA cells arc known ns 'high none’ and low mne tolerance
1
injected into frtll nr newborn Strain A mite , lesfJCLtively. A special type of high lOtle tolerance
however, the lamer when they grow up will freely is Felton's immunological paralysis. The duration
accept skir- grafts from CRA mice. The content of of tolerance iti variable.Tolerance can be prolonged
the self antigen appears to have been enlarged hy k... J ..
1 L: TL _e
contact with ti foreign antigen during embryonic administration th .tr best induces tolerance is that
life. This phenomenon is called 'specific whereby rhe antigen equilibrate* throughout the
immunological tolerance'. extra - and intravascular compartments. With
Development of tolerance is not confined to rhe antigen* tha t do lint LCJU i I ihra I e readi ly or ire rapidly
embiyo or newborn but can occur in adults also. eliminated , the route of choice is intravenous.
Tolerance may he total or partial* short-lived of Certain haptens that are immunogenic in guinea
long -lasting. The induction, degree and duration pigs hy the intraderm& l route are tolerogenic orally "
Copyrighted materia
Irnmuns Response L 49
*
interfered with or an 'efferent block ' in which the physiological significance of such receptors was in
*
antibody Evnthesihcd LR Hdltn£jxd or destroyed. T anchoring nutrients to cells before assimilation.
and B lymphocytes appear to possess differing When foreign anrigens arc introduced info the body,
sensitivity to tolerance induction, the former being they combine with those cell receptors which have
more susceptible. In general, high doses of antigen a complementary fit. This inactivates the receptors
induce H cell tolerance and negated minute doses and interferes with the absorption of nutrients, As
of antigen induce T cell tolerance. a compensatory mecbariitm, there Is an
Tolerance to humoral and cellular types of overproduction of the same type of receptors, which
immunity LHusually induceif simultaneously. S-plit '
spill over into the blood and circulate as antibodies.
tolerance can also occur where unrespon&ivenesa This was the first of the selection theories , ii
is established for one paiumeter' of the immune
, explain'd .i JugfulLv the specificity of the antibody
response and not to the other. In guinea pigs, i)H response. However, when 3 ,anJ.slcincrdemonstrated
tu tuberculin can. be inhibited, without affecting that aitihiJies could be formed not only against
the production of a circulating antibody, by the natural antigens but also against various synthetic
injection oftuberculoprotcin prior to immunisation chemicals, this theory was abandoned, Ii was
with BCG. believed that an Impossibly large number of
receptors would be needed to account for the
THEORIES OF IMMUNE RESPONSE seemingly endless scope of antibody specificity. It
A succession of theories h vc been put forward is, however , remarkable bpw closely Ehrlich
*
from time to time in order to explain the versatility, anticipated modern views on the immune response .
specificity, memory and other features oi the Direct template ihenries: Instructive
immune response. Theories of immunity' fall into theories were proposed by BrcinJ and I laurowita
two categories : instructive and selective ['lie
,
'
(1930), Alexander (1931) and Mudd ( 1932),
instructive theories p o s t u l a t e that a n According td these, the antigen (or the antigenic
immunocompetent cell is capable of SytttilCtibng determinant! enters antibody to ruling cells and
antibodies of any peeifi city. Tlae antigen encounters serves as a 'template ' against which antibody
*
an immunocompetent cell and instruct? lr to produce molecules arc synthesised so that they have
the complementary antibody. Instructive theories combining sites complementary to the antigenic
were proposed by chemists who were more determinant. 1’hese are lliuretore known as 'direct
concerned with explaining the physicochemical template' th iriw, J’sud i ng ( 1940) presented a more
aspects of specificity than with the biological detailed model suggesting that specificity was
principles of immune processus . Stltttivt theories , determined by the folding of ihe antibody
on the contrary,shift the emphasis from the antigen jicrlypcptrilc chains to form a tertiary structure fitting
to the immune competent cell. 'Hicy postulate that the antigenic determinant.
immunocompetent cells have only a rust rifted Indirect template theory, Burnet and
immunological range. The antigen exerts only a Tenner ( 1949) proposed this instructive theory " to
selective influence by srtrrmUring the appropriate explain rhe synthesis of antibody as an adaptive
-
immuno competent «11 to synthesise JIL antibody, .
protein They postulated that the entry of the
Side chain theory: The first plausible theory antigenic determinant into the antibody producing
of immune response was the 'side chain' theory cell induced in it i heritable change . A 'genocopy7
proposed by Ehrlich (1900) , Cells were considered of the antigenic determinant was thus incorporated
to have surface 'receptors' capable of reacting with in Lrs genome and transmitted to the progeny cells
substances having complementary 'side chains’. The fiudinecr template) . This theory explained specifidry
Copyrighted material
150 i Texlbqgk ol MipmbioJQgy *
pustulated that about £ million globulin (antibodv) Were eliminated during : niu l Such clones
molecules were fonned in embryonic life , which are called forbidden dcutcj. persistence or
covered: the full range of antigenic specificities . development in later ifchi mutation could
These globulins were the 'ualura] antibodies". When lead to autoimmune processes Each
ar. antigen was introduced, il combined selectively iniTnuiuHiompEtenC cell was capable of reacting with
with the globulin that bud the nearest tme antigen (ora small numlier antigens} which
complementary ' fit ' . The globulin , with the could recognise and > with antigens
combined antigen, homed in on the antibody introduced into [he body. The : - H : of the contact
'
forming cells 3 rul Stimulated rhem to synthesise the with the specific antigen was ; i r proliferation
'
same kind of antibody. Here; selection was to form clones synthesising the tntibods
postulated at the level of the antibodv molecule, It Tbe clonal Selection li is more widely
did not explain the fact that immunological memory accepted than the other theories, t ! n i - unable
resides in the cells, and not in serum. to account for all the features of the immune i&pi nue.
CONSTANT
* VARIABLE HEOlOH REGION
T
Genmhrce
tjflriaa
VM 1 1 DO Ol - J J l -6 C it TN yj if , . . 7*
.
f a,, ns
+ TRANSLOCATION /
^
B cell genes V5 D3 Jt 1
Fig. 1 S.6 ' llustration of gone shuffling to form a 5 cell encoding for lyGM heavy chain with Dy , J variable
region sequence (lidlRtr shuffling also occurs for light chains — nol shown)
jl -
A vari ntv of modificationsand altcrmri v? theoti L S have of the aJidhtidv molecule. As the constant regions
been proposed in recent timesblit rvnnr has succeeded .Lre identical for immunoglobulins of anyone type,
M L explaining all that is known of immunity. there need be only one gene or a few genes for
As an explanation for the mechanism of each constant region, as ag .ii. nst a very large number
regulation of antibody response, Jeme has pi ^ tulatcd of genes for the variable regions. For example , the
the network hypothesis. The variable region of an kappa L chain genes are composed of three separate
immunoglobulin : molecule currying the .ouL'cn - segments; V, J and C . There are about a hundred
combining site Is tliflfrrenl in tliflfrrenl antibudicH. ditfefrnt types of V ( variable ) domain sequences
''
J hc distinct ami noacid sequences at the antigen - and only one C (constant) segment, with some five
combining site and the adjacent parts of the variable J ( joining) segments in-between . By combining
region are termed idiorypes. The idiotype can , in different V and J sequences with the C domain, it
turn , act as an antigenic determinant and induce is possible to provide for antibodies with at least
antiidiotypic antibodies, These in turn can induce 500 different specificities . By p a l i n d r o m i c
antibodies to them and so on, forming an idiotype arrangement (sequences that can be attached by
network which 1s postulated to regulate the amount cither end ), it is possible to generate many times
of am i bodies produced and the number oLuitih ' idv - more different specificities. The lambda chain has
forming cells in action . For hi * theoretical additional C sequences. The H chain gene has also
contribution to antibody formation and regulation a C (diversity ) segment. By the shuffling of these
of the immune system, Niels It Jeme was awarded different segments of the C and EE chains, it is
the Nobel Prize- for Medicine in 19S4. possible to have antibodies with far mure than 10 '
The genetic basis of antibody diversity has been types of specificity, a total repertoire that can react
clarified recently. An individual has the capacity to with any conceivable antigen. The split gene
produce an estimated 1(P different antibody shuffling takes place during cell development and
molecules. Io have each such antlhodv molecule a mature B cell DNA w i l l have Only one
coded for by a separate gene would requi re millh ms combination of the different segments of the
of genes to be set apart for antibody production unmunoglobLilin gene and cati therefore produce
alone- This would obviously be impossible. The only one type of antibody ( Fig. 16. &),
phenomenon of splir genes explains this . The The discovery of split genes for
genetic informal Lon for the synthesis of an immunoglobulins demolished the long standing
immunoglobulin molecule is not present in a dogma of one jztnc-onc protein and has impunant
continuous array of codons. Instead, this information implications in biology beyund immunology. For
occurs in several discontinuous stretches of DNA this discovery, Susumu Toncgawia was awarded the
('gene segments') , each coding for separate regions Nobel Pp7e for Medicine in 19fl 7.
V u r L h e r Remit inti
Enjjfclhjpd VH . 1994 , Hcwoclh proms antipenE. Scimt American 44,
Jancway CA 1993.1 low the immune system recognises invaders. Scieni American 41 .
.
Jeme NK. 1985. The generative grammar of the Immune aysrem- Sriena 29,1057.
^
Llewellyn MB et al. 1992. Monoclonal antibodies, BMJ 305: 1269, 1348, 1424.
Remkk DG arid JS Friodfcunri 1997 . OytAfnn JJI hcaith zred fibnur . 2nd cdn. Base ]; MttCdFDefcker
Tonegarwa S. 1983. Somatic generation of antibody diversity, Nnuir 302 = 575 .,
Weir PM find J S(cw?Lrt 1 W7 finUuMJiqgpr. 8 , b cdn . EdmbiUKh: Chnrrhill - Iiringihane.
S
syndrome described by iStutor ( 1957 ) is the first The incidence of this condition has been
Copyrighted materia
* immunodeficiency Diseases * 153
Table 17 1 Classification of primary Immunodeficiency syndromes
per kg of hody weight in three doses followed by employed, the donors being tested for hepatitis and
monthly injections of 100 mg per kg. The slow other transmissible infections.
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154 « Textbocm ot Microbiology *
TH r M \ I IH. .
AM.U AC I.OBI LINI : .MLA dysgammaglobulirimias’ are common and haw
‘
and respiratory infections arc the. common diseases in many of these patients .
found ill thin condition . Spontaneous recovery occurs Selective IgM deficit; my has been found ro be
between 18 und 30 months of age. It maybe found associated with septicemia . Deficiencies of IgG
in iofaurs of both sexes . Treatment with subclasses hive been observed in relation with
gammaglobulin mij be required in some case; but chronic progressive bronchiectasis ,
it is not recommended [ imphylactically, as it may IramunodtiliciencieB with hypwp IjM: In -
this group of immunodefidcTtcics, some of which
contribute to prolongation ul immunodeficienCY bv
i negative feedback inhibition of IgG synthesis..
arc X-linked and some inherited as autosomal
recessive, tow IgA and IgG levels are seen with
( lOIIHtiN V ART ADI V elevated. IgM . " Hu.- Ig.\ l molecules appear to have
IMMLNODGKICIENCY normal structure and possess antibody activity.
Patients show enhanced susceptibility to infections
This wmmvr form of inoutriodefidcnt}' is also and ILL toil untune processes such as
known as late onset hypogammaglobulinemia [ hrumbocytopeuix, neutropenia , hemolytic anemia
because it usually manifests only by 15-35 years of ' and renal lesions . Some patient ? develop malignant
age. It is characterised by recurrent pyogenic infiltration with igM-producing cells. Elevated
infections and an increased incidence of IgM level with immunodeficiency is sometimes seen
autoimmune disease . MflllbtOrpfiori and giardiasis LU congenital rubella.
arc common . The tots I immunoglobulin level is TrqnsaohnluniLn II deficiency : In this
usually Less than 300 mg j'er 100 mb with IgG less disorder, inherited as autosomal recessive, patients
than 250 mg per 100 ml, U cdb may be present in show metabolic effects of vitamin B12 deficiency
circulation in normal numbers , but they appear including megaloblastic anemia and intestinal
defective in their inahiliry to dittenentiate into plasma villous atrophy. The associated immunological
cells and secrete iinmunoglorutlinK . Increased defects arc depleted plasma cells , diminished
suppressor T cell ml diminished helper T cell activity Immunoglobulin levels and impaired phagocytosis.
hsvc been proposed as a cause cif tKi -s disorder. Treatment with vitamin R12 hm been reported lo
Treatment is by administration ot" gammaglobulin restore hematopoietic, gastrointestinal and B cell
preparations inlrajuLLScularlv or intravenously. functions but not phagocytic activity.
StiLliCTi v i; h i m JNot : i n u t it . tN CKJ I . L I . All iMMUNOUEPICIEiNCIES
DrarrciRVLii's
Thymic hypoplusia ( Digeorgc **
In these conditions, there is selective deficiency of syndrom? }. This is i develop mental defect
one or more immunoglobulin cl asset, while ihe involving the endtidermil derivatives of the third
others remain normal or elevated . These and fourth pharyngeal pouches, It leads ro aplasia
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- Immunodeficiency
It docs not appear to be hereditary and does not uric acid may point to the diagnosis .
show a TIMIII . LI jjyijflKt It in probably due to Home
intrauterine infection or other complications, Jr is <. ^< iM IUrS ED lM MUNODEPJCIE NCI FS
usually associated with Fallot's necrology and other i T- iJu hi r irtrniuriodefiuiency with
anomalies of the heart and the great vessels, and a abnormal immunoglobulin synthesis
characteristic facial a ] i] >earj] icc . Neunatal tetany is { Nezelof syndrome): The term Nezelof
present. ftitients who survive the neonatal period show syndrome has been rather loosely applied to a group
.
enhanced susceptibility to viral fungal and bacterial of disorders, probably of varied etiology, where
infections, which ultimately prove fatal. depressed cell mediated immunity' is associated with
The immunodeficiency primarily involves cell
mediated immunity. The thymus dependent areas
•immunoglobulin
datively elevated, decreased normal levels of
of
. The consistent features are a
of lymph nodes and spleen are deplored of marked deficiency of T cell immunity and varying
lymphocytes. Circulating T cells are reduced in degrees of deficiency of B cell immunitv. rodents
number delayed hypersensitivity :md graft rejection
, are susceptible to recurrent fungal, bacterial , viral
.
are depressed The humoral immune mechanism .
and protozoal diseases Abundant plasma cells are
is largely unaffected. Antibody response ts> primary seen in the spleen, lymph nodes, intestines and
antigenic stimuli is normal hut secondary response elsewhere in the body, Thymic dysplasia occurs
to many antigens is impaired. Transplantation of with lymphoid depletion. Autoimmune processes
fetal thymus tissue has been reported l-o restore the such is hemolytic anemia are common. In spite of
immunological function. normal levels of immunoglobulins, antigenic stimuli
Chronic mucocutaneous candidiasis: do not induce anlibudy formation.
This constitutes an ahnurmal Lmmunological Histocumparible home marrow transplantation,
response to Candida albicans. Patients develop severe transfer factor and thymus transplantation have been
chronic candidiasis of the mucosa, skin and nails. used for treatment, with success in some cases.
rfhcy do not show increased susceptibility' to other Adequate antimicrobial therapy is essential.
infections but often have endoCrinupatlries. Cell Ataxia 11Inngiectasia: This is a hereditary
mediated immunity to Candida is deficient . In some condition transmitted in the autosomal recessive
cases there is a total failure of T cell response to mnde , where combined immunodeficiency is
any test antigen. Delayed hypersensitivity to Candida associated with cerebellar ataxia, telangiectasia,
antigens is absent hut circulating imLibudics to them ovarian dysgenesis andchromosomal abnormalities.
arc found in high litres, Intracellular killing of The earliest signs are ataxia and chorioathctold
Candida is defective. Transfer factor tbcrapv, along movemean which are usually noticed in infancy.
with amphotericin B, has been reported effective. Telangiectasia involving the conjunctiva, face and
Purine nuuduoside phusphnrylase other puns of the body usually appears at Five or
(PNP ) deficiency; The enzyme purine . -
six years of age Death occurs due to i uupu.t ] i ion nr v
nucleoside pho-sphoryliiHe is involved in the infection uarlv in life, or malignancy in the second
sequential degradation of purines Co hypo.tonlbine o: third decade . The majority of patients lack serum
and finally to uric acid. Patients who have PNP and secretory IgA and. some possess antihody to
deficiency as on autosomal recessive inherited trait IgA . IgE deficiency is also frequent. Cell mediated
show decreased cel ] mediated imum II I [ y and immunity is also defective, resulting in an
recurrent or chronic infections. They usually impairment of delayed hypersensitivity and graft
present with hypoplastic anemia and recurrent rejection. The disease is progressive, with both
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156 i TgxtbQOk L'l M Lrobiu C- qy
Affected boys rarely survive the first decade of life , In 1 5 H , Swiss workers reported
death being due to infection , hemorrhage or
^
agammaglobulinemia with lymphocytopenia and
lympboreticufar malignancy. Cell mediated severe defect in cell mediated immunity This has
immunity undergoes- progressive deterioration b e e r referred to as Swiss type aga /nma -
associated with cellular depletion oi the thymus ghhulincmig . The basic defect is presumed to be
and the paracortkd areas of lymph nodes, berum at the level of the lymphoid stem cell.
IgM level is low tun IgG and IgA levels are normal The most serious form nf combined
-
or elevated Isuhcniagglutinins ate absent in the immunodeficiency is the reticular dysgenesis of de
serum. The humoral detect appears rn he a specific Vagi , Here the defect is at the level of the
inability tu respond to polysaccharide antigens. Bone mnltipotenr hemopoietic stem cell, as a result of
marrow transplantation and transfer factor therapy which there is a total failure of myelopoic&LS leading
have been found beneficial . tolymphopenia, neutropenia, thrombocytopenia,
Immunodeficiency w i t h thymoma: This anemia and bone marrow aplasia. The condition is
syndrome, occurring usually In adults, consists of a invariably fatal in the first week of Life.
benign thymic tumour, impaired cell mediated AcfencMEJte deaminase (ADA ) deficiency is the
immunity and agammaglobulinemia. It is frequently first immunodeficiency disease associated with an
accompanied by aplastic anemia.This is of historical enzyme deficiency. ADA catalyses the conversion
importance as one of the experiments ot nature of adenosine to irosine , an important step in the
which Suggested tlie immunological function of the purine metabolic pathway: How this deficiency
thymus . causes L:11 oui no logical impairment is not clear, I "he
Immunodeficiency with short ' limbed range <> t immunodeficiency' varies from complete
dwarfism: The features of this condition arc a absence to mild abnormalities of B and T cell
distinctive form of short - limbed dwarfism , functions . The condition is associated w i t h
ectodermal dvsplasta , thvmic defects and enhanced chondrocyte abnormalities which can be discerned
susceptibility to infection . These defects arc radio logically.
appatendy inherited as autosomal recessivcs.
Epiioditi l y m p h o p e n i a with l y m p h o - DISORDERS OF COMPLEMENT
oytptoxin: In this syndrome there occurs ait Complement component deficiencies:
episodic but profound depression ofT cell (unction Genetic deficiencies have been detected for almost
by the action of a circulating cample ment dependent all the complement components in human beings.
Ivmphocy lotos in . The toxin appears to he an The defeers ate transmitted as autosomal recessive
antilymphocyte antibody . The p a t i e n t s lack Traits. Hemolytic and other functional activities are
'
immunological memory so the secondary antibody
1
completely restored by supplying the deficient
response is abolished . The disease is familial. factor. Complement component deficiencies have
Severe c o m b i n e d immunodeficiencies: been frequently associated with systemic lupus
T h e s e include many syndromes w i t h severe erythematosus . Recurrent pyogcnk infections WCft
CoDvriahted
rJ * material
* immunodeficiency Diseases » 157
found associated with C3 deficiency and neisserial their intracellular position , sec up chronic
in it.LL fi )]i H with deficiency of Cb, C ? und CS.
"
suppurative infection - The: diminished bactericidal
Complement inhibitor deficiencies: capacity' of the phagocytic cells is associated with
I leredirary angioneurotic edema is due to a genetic a decrease of some metabolic processes like oxygen
deficient of Cl inhibitor. This relatively common consumption , hexose monophosphate pathway
^
defect is transmitted as an autosomal dominant. activity and production of hvdrogen peroxide . The
Androgen H , aminocuproie acid and its analogue diminished HjOa production appears to be the
IranejtamiL- acid haw been found useful in the major reason for the bactericidal defect . The
management of this condition. Plasma infusions, leucocytes do not undergo degnmulation following
once recommended for treatment , haw been given phagocytosis . The delayed granule rupture and
up as they were found to worsen the condition in defective release of myeloperoxidase also contribute
some eases. to inefficient bactericidal activity. TreuctMtytes from
Tiie rare deficienci' of Cdb in activator has been the patients fail to reduce nitnablue tetrazolium
associated with chronic recurrent pyogenic lesions. (NBT) during phagocytosis. Thia property has been
used as a screening method { NET test ) for the
DlSQRDEflS OF PHAGOCYTOSIS diagnosis of chronic granulomatous diseasC-
Phagocytosis may be impaired by either intrinsic or Thc disease shows two types of inheritance-^
extrinsic defects, Intrinsic disorders may be due to the more common X-linked type seen in boys and
defects within [he phagocytic Cell, such as enzyme the rare autosomal recessive lype seen in girls.
deficiencies. Extrinsic disorders may he due to a Myeloperoxidase deficiency : In this rare
deficiency' of opsonic antibody, complement or other disease, leucocytes have reduced myeloperoxidase .
factors promoting phagocytosis, or to the effects of Patients are particularly liable to Candida albicans
drujp or antineutrophil autoantibodies. Phagocytic infectinn -
dysfunction leads lo increased susceptibility to Chediak -Higaahi syndrome: This is a
infection, rjmgntg from ntild recurrent skin infections genetic disorder characterised by decreased
to owrwhdming systemic infection. pigmentation of the skin , eycn and hair,
Chronic granulomatous disease: 'Ibis photophobia, nystagmus and giant peroxidase
familial disease manifests itself as rccumcnt infection positive inclusions in the cytoplasm of leucocytes.
with low grade pthegens, starring early in life, The The inclusions may be the result of autophagocytic
progress is chronic and the outcome fatal . Chronic activity. The leucocytes possess diminished
suppurative granulomatous lesions develop in the shin phagocytic activity. Patients suffer from frequent
itod lymph noties, along with hepahKplennmegaly, and severe pyogenic infections,
progressive infiltration of Lungs and granulomatous I ciicocytu G6PO deficiency: Ju this rare
septic osteomyelitis. Humoral and cellular Immune disease , leucocytes arc deficient in glucose 6
responses arc normal phosphate dehydrogenase and show diminished
The bacteria involved in the recurrent infections bactericidal activity after phagocytosis . The
arc catalase positive pyogenic pathogens such as condition resembles chronic granulomatous disease
staphylococci and coliforms . Catalase negative in reduced myeloperoxidase activity and .
pathogens such as streptococci and pneumococci susceptibility to micnjbi J agents, but the NBT test
are handled normally. Leucocytes from the patients ii Lav be normal.
f
are unable to kill catalase positive bacteria following Job s syndrome: This is characterised by
,
phagocytosis.The bacteria multiply in the cells and, multiple large "cold' staphylococcal abscesses
being protected from antihcidies and antibiotics bv containing large quantities of pus , occurring
1
Copyrighted material
158 * loxltxia ^ 01 \tiurOh uloQy *
Further Rending
Rosen FS CT ai. 1984. The primary Lmmunodeflcreiicits. New Engl J Med 311L23SN 300.
-
Fischer A and A Amtix Villcna 1995. Immunodeficiencies of genetic origin , Today 16:510,
Sriita DP and AJ Ten 199 L BASIC and Clinical Immunology. 7* cdn. Connccricur: Appleton - Lange.
Wizgcl H . 1993. Immunodeficiency. CLI /T Qpin Tmimanoi 5 :567 .
Copyrighted materia
Hypersensitivity
Immunity was originally considered a protective should have had contact with the antigjcn (alktgen).
process, helping the body to overcame infectious The initial contact sensitises the immune system *
agents and their tmira. This however is only one Leading to the priming of the appropriate R or T
aspect of thebroad phenomenon of tmmu nity wh ieh .
lymphocytes This is known as the 'sensitising' or
includes all manner of specific responses TO antigens. 'priming dose. Subsequent contact with the allergen
1
Copyrighted material
160 * Texttiook ol Microbiology
Copyrighted material
1 Hyper s- en sin vi1\ t 161
macrophages ^
survived a sublcthfll injection of a toxic extract of the species. Tissues or organs predominantly
sea anemones wen; rendered highly susorptitile to involved in die anaphylactic reaction are known as
minute doses of the toxin given days or weeks laici, Target tissues.' or 'shockorgans’. Other changes seen
instead of becoming immune to it - Theobald Smith in anaphylaxis arc edema, decreased coagulability
(1905) had noticed H similar phenomenon in guinea of blood, lull in blood pressure and temperature,
pigs, following widely spaced injections of toxin
antitoxin mixtures . Ehrlich named this the
— Icucopcnia and thrombocytojvenia.
There is considerable species variation in
Theobald Smith phenomenon ' and showed that it susceptibil i ty to anaphylaxis. Go i nta pigs are h ighly
wan independent of tile toxin and. unliLoxm used, susceptible and rats very resistant. Rabbits, dogs
since the phenomenon canid he induced with and human beings are of intermediate susceptibility.
normal serum also. Anaphylaxis can he readily induced in guinea pigs.
Sensitisation is most effective when the antigen If a small dose of egg albumin is injected
is introduced parenteraliy but may occur by any intraperitoreally, fallowed 2 - 3 weeks later by a
route , including ingestion or inhalation. In slightly larger dose of the same antigen
susceptible s-pecies, very minute doses Can sensitise intravenously, the guinea pig will exhibit a dramatic
the host . Antigens as well as haptens can induce sequence of events. Wilkin minutes the an [ mil
anaphylaxis. There should be art Interval of at 1e?st becomes- irritable, sneezes , coughs, experiences
2- 3 weeks between the sensitising dose and the respiratory distress , develops convulsions and dies.
shocking dose . Once sensitised, the individual The heart continues to beat for some time after the
remains so for lung periods- TliC shocking d(3K is respiration has stopped. At autopsy, the lungs are
mast effective- when injected intravenously, less marked ly emphysematous and do not collapse when
effective intrupcritoneully or subcutaneously and the thorax is opened or ever when they arc cut
least effective mcradcrmaMy- 'I'hc shocking antigen into pieces. The shuck organ is tile lung. Death LS
must he identical or immunologic a Ely closely due to the constriction of the smooth muscles of
related to the sensitising antigen , The clinical the bronchioles causing respiratory standstill.
features of anaphylaxis are the same with aHV antigen ID rabbits, death in anaphylactic shock is due
but vary between species. The clinical effects are- to constriction of the pulmonary' artery and its
due to smooth muscle contraction and increased brunches, leading to extreme dilatation of the right
mta 11 AT permeability. The organs affected vary with side of the heart - Respiratory movements continue
Copyrighted material
162 H Textbook ad Microbiology »
after the cessation of the heartbeat. In dugs, the intravenously 4-24 hours afterwards, there will be
reaction is slower and takes 1 -2 hours. There is an immediate blueing at the site of iutradermal
constri cti jn of the hepa Me venous system with gross injection due to vasodilaia : iun and increased
engorgement oftlie liver and profound fall of blood
pressure. In human beings, fatal anaphylaxis is
- -
capillary permeability ( wheal and flare reaction ).
PCA can be used to detect human Ig£J antibody
fortunately Symptoms and si ^ ns of
rare. which is heterocytotropic (capable of fixing to cells
anaphylactic shock begin with itching of the scalp of other species ) but not IgE which is
and tongue, flushing of the skin over the whole homocytotropic (capable of Fixing to cells of
body and difficulty in breatlung due TO bronchial homologous species only) .
spawn. There may he nausea, vomiting, abdominal
pain and diarrhea , sometimes with blood in the
-
Anaphyta \ i : i in vitroi Isolated tissues, such
as intestinal or uterine muscle scrips from sensitised
Stool. Acute hypotension, Loss. dfnonScinusness and guinea pigs, held in a bath of Ringers solution will
death follow. Human anaphylaxis , once commonly contract vigorously on addition of the specific
associated with heterologous scrum therapy, is now
seen mostly following injections of antibiotics or
antigen to the bath. This is known as the Schultz
Dale phenomenon. The reaction is specific and will
—
other drugs. Insect stings can also cause anaphylaxis be elicited only by the antigen to which the aiiiimal
m human beings, Prompt treatment with adrenaline is sensitive. Tissues from normal animals can be
tan be lifr -savi itg. Adrenaline is to be ad mi n istered , passively sensitised by treatment with scrum from
0.5 ml of a 1 in 1000 solutvm, subcutaneously or sensitised animals.
intramuscularly, the dose bring repeated upto a total Mechanism of anaphylaxis: The
of 2 ml Liver 15 minutes, if necessary. i immunologic basis for hypersensitivity is cytotropic
Cutaneous anaphylaxis; When a small IgE antibody Free IgE antibody in circulation is
shocking dose of an antigen is administered not relevant in anaphylaxis Thus, an animal
intradermally to a scnsiti'ied host, there will be a with a high litre of Liiculating antibody may be
local whcal-and -fUie response (local anaphylaxis). refractory to shock, while anaphylaxis may be
The wheal! i s a pale, central area of puffi ness due caused by cell fixed antibody, even in the absence
to edcmah which is surrounded by a flare caused by of detectable circulating antibody. While in human
hyperenu .i and subsequent erythema. Cutaneous beings, IgE is the cytophilic antibody, in the guinea
anaphylaxis (skin test for Type I hypersensitivity') pig and mouse the analogous mop hi I ir antibody
is useful in testing fur hypersensitivity and in is IgGl .
identifying the allergen responsible in atopic IgE molecules arc bound to surface receptor*
diseases. In highly sensitised individuals, even on mast cells and basophils. These cells cany large
the skin test may lead to serious and even fatal number* of such receptors called Fc ER receptors,
reactions. Hence a syringe loaded with adrenaline analogous to TCR receptors on T cell surface. IgE
should always be kept ready whenever a skin molecules attach to these receptors by their Fc end.
test is performed to detecr anaphylactic Following exposure to the shocking dose, the
hypersensitivity. antigen molecules combine with the cell bound
Ebisaive cutaneous aimphylavis ( 13 ( ’ \ > e .
IgE bridging the gap between adjacent antibody
Th is test developed by Ovaiy (1952) is an extremely molecules. This truss - linking increases the
sensitive in vivo method for detection of antibodies. permeability of the cells to calcium ions and leads
A small volume of the antibody is injected to ckgranukrion , with release of biologically active
intradermaJly into a normal animal. If the antigen, substances contained in the granules. The
along with a dye such as Evans blue, is injected manifestations of anaphylaxis are due to
LJ opyrighted materia
* Hyper^fisilivity 163
smooth muscle contraction In diverse [issues and powerful, but transient , bronchoconstrictors .
organs, including vasculature, intestines, uterus and Prostaglandins also affect secretion by mucous
especially the bronchioles. It also stimulates glands, platelet adhesion , permeability and
secretions (secretogogue effect ). dilatation of capillaries and the pain tlireshold .
1 . Serotonin ( 5 - hydroxy try ptamine): 2 , Platelet activating factor ( PAF ) t PAF
This is a base derived by decarboxylation of is a low molecular weight lipid released from
tryptophan. It LS found in the intestinal mucosa, basophils which causes aggregation of platelets
brain tissue and platelets. It Causes smooth and release of their vasoactive amines.
muscle contraction , increased capillary Other mediators nf anaphylaxir Besides
permeability und vasoconstriction , It 1 s the products Of mast cells and Other Leucocytes ,
important in anaphylaxis in rats and mice but several other biologically active substances have
its mle in human beings is uncertain. been implicated in anaphylaxis. These include the
2. llhcmotndic factors: The eosinophil anaphylatoxins released by complement activation
cbemohictit factors of anaphylaxis f F.CF-A ) are and bradykmin and other kioins formed from plasma
acidic tctmpepridcs released from mast cell kininogens .
granules which arc strongly chemotactic for An n p h y l u u t n i d react!cm: Inifivcfloas
eosinophils . These probably contribute to the injection of peptone, typ'd U and Certain Other
eosinophilic accompanying many hyper - substances provokes a clinical reaction resembling
sensitivity states . A high molecular weight anaphvlactic shock - TTiLs is termed 'anaphylactoid
diemoiactic factor has been identified, which reaction’- The clinical resemblance is due to the
attracts [leutrophils ( NCI7) . same chemical mediators participating in both
Heparin is an acidic mucopolvsacchjride . It reactions. The only difference is that anaphylactoid
Copyrighted materia
164 ^ Ts < rt>c 0i< of MiCrtSfcioloqy
shock has no immunological basis and is a 3. IgE is lmmi wmimpiv, that is, species specific.
nonspecific mechanism involving the activation of Only human IgE can fix to the surface of human
complement and the release of anaphylaiuodiiis.
Atopy; The term 'atopy (literally meaning nut
1
UJK ; reaction, which was the original method
-
cells. This is the basis of the Prausnitz Kustner
of place or strangeness ) was introduced by Coca for detecting atopic antibody. Prausnitz and
! 1* 23 ) to refer to naturally occurring familial Kusiner ( 1921) reported that if serum collected
^
hypersensitivities of human brings* typified by hay from Kustner, who had atopic hypersenativity to
fever and asthma.The antigens commonly involved certain species of cooked fish, was injected
in atopy are characteristically inhalants (for iotracutaneously into Prausnitz, followed 24 hours
example , pollen, house dust ; or ingest ants (for later by an iniracutaneous injection of a small
example, eggs, milk ). Some of them are contact quantity of the cooked fish antigen into the same
allergens, to which the skin and conjunctiva may- - -
site, a wheal and flanp reaction occurred within a
be exposed. These atopens are generally not good
antigens when injected parcntcrally but induce IgE
-
few minutes. As rcagjnic IgE is homo- cytotropic>
the test has to be carried out on human skin. It
antibodies, formerly termed as ' reapin .mtihodies.
'
carries the risk of transmission o1 infection and so
Atopic sensitisation is developed spontaneously- is no longer used.
following natural contact with atopens. It is difficult A . Unlike other antibodies, IgE is heat sensitive
-
to induce atopy ariiiicially. and inacl ivated at 56 nC in 2-4 hours .
Predisposition to atopy is gencncallydetennLncd, Hearing appears to damage the Fc part of the
-i n drably I i oked Do MHC j. jcnOtypc - . A [ Opy 11 LL rttfut IgE molecule, which is necessary for fixation
runs in families. What is inherited is not sensitivity tocells.
to a particular antigen , or a particular atopic 5- Atopic antibody docs not pass through the
syndrome but the tendency to produce IgE placenta.
antibodies in unusually large quantifies. All Atopic sensitivity is due to an overproduction
individuals arc capable of farming IgE antihodics of IgE antibodies - Tbi -. i -. otter associated with a
in small amounts but in atopies IgE response is deficiency of IgA. This association has led to the
preponderant - About 10 per cert of persons have suggicsrinn that IgA deficiency may predispose to
this tendency to overproduce IgE. Jt has beer atopy. The distribuiinn of lymphocytes capable of
reported that borlJefed infants tend to develop atopy synthesising IgA and IgE is closely parallel,
in later life more often than breastfed babies. especially m the submucosa. In normal individuals,
IgE differs from other immunoglobulins in the the inhalant and ingestant antigens are dealt with
following respects:. by JgA lini ng the respiratory and i ntestinal mucosa
1. It cannot be demonstrated by the conventional and therefore they do nut come into contact with
serological rcact > > nfi such as precipitation or the pntenti d JgE producing cells. When IgA is
complement fixation. 'l'he first in vitro method deficient, the antigens cause massive stimulation of
for IgE detection was the mdioailergosorbent IgE forming cells, leading to overproduction oflgE.
test ( RAST ) . Simpler techniques such as The symptoms nt atopy are caused by the release
ELlbA and passive agglutination have since of pharmacologically active substances following
lieen introduced. the combination of the antigen and the cell fixed
2. While atopy occurs commonly in human beings, IgE. l’he clinical expression of atopic reactions is
it is not easy to induce it experimentally in usually determined by tile portal of entry uf the
animals. —
antigen conjunctivitis, rhinitis , gastrointestinal
.
J*“ 4
Lj opy righted materia
4 Hyp«f sensitivity 165
symptoms and dermatitis following exposure molecules. This leads to increased vascular
through the eyes, respiratory tract, intestine or skin, permeability and infiltration of the site with
respectively. Sometimes the effects m»V he at sites neutrophils. Leucocyte— platelet thrombi arc formed
remote from the portal of entry, for example, that reduce the blood supply and Lead to tissue
tirtitdtia full owing ingestion of the allergen. neefosis . The Arthus reaction can be passively
Specific deseftsirisatiuti (hyposensitization) is often transferred with sera containing precipitating
practised in the treatment of atopy. antibodies (IgG, IgM) in high litres.
Arthus reaction forms a pathogenic Component
TYPE II REACTION : CYTOLYTIC AND CYTOTOXIC .
of many clinical syndromes For example,
These reactions involve a combination of TgG (or intrapulmonary Arthus ‘like reaction to inhaled
rarely IgM) antibodies with tire antigenic antigen , such as thermophilic actinomyceces from
-
determinants on tin surface of cells lending to *
mouldy bay or grain causes Farmer;' ; Jung mid other
cytotoxic or cytolytic effects, Kxamples are lysis of types of hvpcrsensi tivitv pneumonitis.
'
red cells caused by antierythrocvie j-ruilHidies in Serum sickness: This is a systemic form of
autoimmune anemias and hemolytic disease of the Type 111 hypersensitivity. As originally described
-
newborn Alternative!!', a free antigen or hapten mar
lie absorbed on cell surfaces . Subsequent reaction
hy von Ehrquct and Schick ( 1905 ), this appeared
7-12 days following a single injection of a high
of the combined antigen or hapten with its concentration, of foreign scrum such as the
corresponding antibody leads to cell damage. Many diphtheria antitoxin. The clinical syndrome consists
-
drugs may act in I hi manner, leading to of fever, lyniphadenopachy, splenomegaly, arthritis,
complement mediated Lysis of red cells, leucocyte* glomerulonephritis, endocarditis, vasculitis,
and platelets, causing hemolytic anemia, urticant] rashes, abdominal pit in, nausea and
agranulocytosis and thrombocytopenk; purpura. vomiting, 'llie pathogenesis is the tbrmation of
In some Type II reactions, the antihodv immune complexes (consisting of the foreign serum
combines with cell surface receptors and disrupts and antihody to it that reaches high enough litres
normal time I ion, either by uncontrolled activation by 7-12 days), which get deposited on rhe
-
( agonist effect a caused by the antibody ‘
1
long- acting
thyroid stimulator in Grans' disease ) or hy
endothelial Lining of blood vessels in various parts
of the body, causing inflammatory infiltration .
blocking (antagonist cftcer as in myasthenia gravis) . I'hc plasma concentration of ’ complement falls
due to massive complement activation and fixation
TYPE III REACTIONS: IMMUNE COMPLEX
DISEASES
.
hy the antigen antibody complexes The disease is
self limited. With continued rise in antibody
Arthus reaction: Arthus {1903 } observed that production, rhe immune complexes become larger
when rabbin were repeatedly injected and mote susceptible to phagocytosis and immune
subcutaneously with normal horse scrum, the initial .
dimination When all foreign antigen is thus
inject ions had no Local effect but with Later eliminated, and free antibody appears, the symptoms
injections, there occurred intense local reaction clear without any sequelae, The latent period of 7~
consisting ot edema, induration ami hemorrhagic 12 days is required only for serum sickness following
necrosis, lliis is known as the Arthus reaction and a single injection. With subsequent injections , the
is a Local manifestation of generalised disease manifests earlier, her uni sickness differs
hypersensitivity, The tissue damage is due to from other types of hypersensitivity reaction in that
-
formation ofantigenr Mitibody pretip'rtHes causing a single injection can serve both as the sensitising
dtjse amt the shocking dose. As heterologous serum
complement activation and release of inflammatory
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166 ^ Textbcon oJ Microbiology *
injections ire not used often now, the syndrome is hypersensitivity not only in the longer interval for
currently more commonly seen following injections appearance hut alio in its morphology and
of penicillin or other antibiotics , histology.
Immune complexes occur in many diseasest Tuberculin type hypersensitivity develops in
including bacteii .iL , viral and parasitic infections (for many infections with bacteria, fungi , viruses and
example , poststreptococcal glomerulonephritis , paruiles, especially when the infection is subacute
hepatitis type li , malaria ) , disseminated or chronic and the pathogen intracellular. A similar
imEgnifieiH and autoimmune conditions . The hypersensitivity is developed in allograft reaction
nephritis and arthritis seen in these conditions may and in many autoimmune diseases.
be caused by deposition of immune complexes . Cutancouf bnsophil hypenenaitivity; A
local reaction resembling the tuberculin response
TYPE IV REACTIONS: DELAYED may he produced by intradcrmal injection of some
HYPEflSENSmvrTY protein antigens. This is not a delayed
Type !. V hypersensitivity reset ions ( delayed hypersensitivity reaction as it Can be passively
hypersensitivity) constitute one aspect ot cell mediated transferred by serum. Its histology is different from
immune response. These are typically provoked by the tuberculin response, being characterised by
intracellular niLLTK dual infections or haptens like simple prominent basophil infiltration. This was formerly
chemicals applied on the skin, evolve slowly and known as the Jones-Motc reaction but is now
consist of a mixed cellular reaction involving ter met ] cutaneous basophil hypersensitivity. Its
lymphocytes and macrophages in particular.. The signiticance is not known .
reaction is nut induced by circulating antibodies hut
Contact dermatitis typer Delayed
by sensitisedJ 1' cells [Tdtb, "!hi „"I'hS ,1” c ) which, or
contact with the specific antigen, release cytokines
t h a t cause biological effects on leucocytes ,
macrophages and tissue cells, Delayed hypersemitivity
—
hypersensitivity sometimes results fiom skin contact
with a variety of chemicals metals such as nickel
and chromium , simple chemicals like dves, picryl
,
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< HyptruntitMly 167
the antigen they realeasc vari ous lymphoki ncs. Thl leucocytes which condition the site by release of
cell!-! secrete II - Ny and It ,2 which activate [ ysosomat enzymes damaging capillary walls ,
macrophages and other lymphocytes Th 2 cells Following the provocative dose, there occurs
release IL 4 h ILS, GM-CSF and other factors that intravascular clotting, the thrombi leading to
lead to an influx of eosinophils and tissue damage. necOisK of vessel walls and hemorrhage .
Acrivatcd Te cells mediate killing of target cells . If both the injections are given intravenously,
Contact with the allergen in a sensitised
individual leads to 'contact dermatitis', the lesions
-
the animal dies 12 24 hours after the second dose.
Autopsy showy hiiateral corneal necrosis of the
varying from macules and papules to vesicles that kidneys and patchy hemorrhagic necrosis in the
break down , leaving behind raw weeping areas Liver, spleen and othe r o rg.m > . An essent i .dly s im i [ j r
typical of acute eczematous dermatitis , phenomenon was dcscriljcd by Sanarcl ! i (1924) in
Hyperser.sitis iry is- detected by the 'patch test’. The experimental cholera. The reaction is therefore
allergen is applied to the skin under an adherent
dressing . Sensitivity is indicated by itching
-
called the Sanarelli Shwartzmati reaction or the
generalised Shwartzman reaction.
appearing in 4-6 hour ?. , and local reaction which It has been suggested that mechani -- ms similar
may vary from erythema to vesicle or blister to the Shwartzman reaction may operate in some
-
B CP MR
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I JF W
m HI Texlbook of Microbiology *
massive release TKF cytokines, IcJ-d-ing to the toxic origin but their pathogenic mechanisms jnEsemtle
shock syndrome. These tioadhionfl arc not of immune
jf
those in mumme inflammation.
K u r t her Keidini
Chapel HL and M Haemy 1993. Es&mtizls of CUnicil Immunology. 3rd edn. Oxford: Blackwell.
Even PW, 199®. AiuphyLaxis, iUWJ 316- 1442.
Frank M et ?il (eds ). 1995- louniJHafcgicif JDIWMJW- Boston: Little Emm.
.
Ho t ST mA MK Church 1993- Atksgy* Londoo:Gnu
^ *
Howanh PH. 1998. AltVjy ; Parbngtruc mechanism. New EqgJMtd 343:712,
LKIUDHII P et J1. 1993. OrUiVjJ ofJirtm-urkafr -. Oxfinrd: Blackwell.
Rmn IM. 1994. EsscrHeaJ Imatturnifc y. S*41 -edn. Oxford ^: Blackwell.
^
Copyrighted material
Autoimmunity
Self -antigens arc not ordinarily immunogenic . injection of self -antigens along with the complete
Ehrlich (1901) observed that goats produced Kreund’s adjuvant . The use of sensitive serological
antibodies against erythrocytes from other goats but techniques led to the demonstration of
rot against their own , and postulated the concept autoantibodies in several diseases and even in. a
of 'horror autotoxieus’ . Uur he did not regard proportion of healthy individuals ,. Some
a utoLminumsat La n JH jn impossibility and even auroantihodies, such as the anti idiotypic antibody
envisaged its pathogenic possibility. may even be essential for the normal functioning
Autoimmuniiy is a condition in illicit of the immune system.
structural or functional damage is produced bv the When the concept of auloimmuritv came to he
action of immunologic ally competent ecELs nr accepted as a pathogenic mechanism, a Large number
antibodies against the normal components of the of diseases were suggested to hive an autoimmune
body. Autoimmunity literally mcniH protection etiology, based on the finding ot autoantlbodies in
against self but it actually implies 'injury to self rbc patients . Thill was soon recognised to be
a nd therefor. it h as been c ri rictscd as a con tndicbo 11
1
tin ten able as nutuartibodies could be often
in terms . Autoallergy ' has been suggested as an incidental or the result , and not the cause of disease.
acceptable alternative but the term autuunmunity Criteria were proposed For proving the
has the sanction of wide usage. authenticity ot putative autoimmune diseases ,
The earliest example of autoimmunity wa * the similar to Koch's postulates in infectious diseases .
duiervanon by MeTsilmkjofF ( l 90()l that guinea fujp. The« were found to lx- not applicable in the case
injected with their own spermatozoa produced of such spontaneous and multi factorial conditions
sperm Immobilising antibodies, Donat h and as autoimmune diseases . It may be proper to restrict
Lindstciner ( 1904 ) identified circulating the rerm 'autoimmune diseases' to those where
autoantibodies in paroxysmal cold hemoglobinuria autoimmune processes , humoral or cellular, are
— a hemolysin which binds with the patients shown to be responsible for the pathogenesis , rather
erythrocytes low temperatures and produces
at than merely HMrirjptrrl Tfrii is not strictly adhered
complement dependent hemolysis on warming. to. Mrnuiiu, the border between autoimmunity and
This was the first description ot an autoimmune hypersensitivity is Largely ill -defined or evert
disease in human beings, Dameshek aid Schwartz nonexistent .
{ 1938 ) established the autoimmune basis of acute Diseases of autoimmune origin usually exhibit
hemolytic anemia. With the discovery of Coombs the following tenures:
test for incomplete lantilwHiies it became possible 1 . An elevaCcd level ot immunoglobulins.
to demonstrate globulins bound to the surface ot 2 . Demonstrable autuantibodics.
erythrocytes in this condition, Antoimmunisation 3- Deposition of immunoglobulins or rheir
could be induced in experimental animals bv derivatives at sites of election , such as renal
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170 * Textbook of Microbiology
ant igens altered by the i r comb i nation wi r h chert ical mimicry' which is due to the presence in some
allergens. Drug induced anemias, leucopenias and infectuig microorganisms and self - antigenn , of
thrombocytopenias often have an autoimmune basis. fij ' iwpes with identical peptide sequences ( instead
Infectious microorganisms, particularly viruses and of Similarities in 'cross- reactions ]. Examples of
1
other intracellular pathogens, may induce alteration such homologous sequences arc seen in
of cell antigens. Viral infections, such as infectious
mononucleosis , arc known to often precede
arih Erogenic Shigella flerneri and HLA B27 ,
Afyvotactenirm fubemritMis and joint membranes,
-
autoimmune diseases. Bacterial enzymes also induce Uoxsackie B and myocardium .
alteration of cell antigens. Neuraminidases formed Another hypothesis is polyclonal B cell
by myxoviruses and many bacteria act on activat i on. While an antigen generally activates only
erythrocytes releasing the 1’ antigen . The almost i t s corresponding B cell, certain stimuli
universal occurence of T agglutinins in human nonspeafically turn on multiple B cell clones. Such
sera is believed to represent a harmless autoimmune stimuli include chemicals ( for example , 2-
response following infecrions. NcoanTigens may also mercaptoethanol ) , bacterial products (PPD,
arise by mutation. Such mutant cells may be lipopolyweeharide], enzymes (trypsin), antibiotics
immunogenic. ( nystatin ) and infections with some bacteria
Immunological damage may result from ( mycoplasma ) , viruses [ EM virus) and parasites
immune responses induced by cross- reacting ( malaria) . Multiple nonspecific antibodies form
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« Autoimmunity » 171
during some infectious diseases, such ns anti human Jl hum JO autoimmune diseases and in animal
erythrocyte cold antibodies in mycoplasma models genetic factors appear to influence the
pneumonia and anti sheep erythrocyte antibody in development and fate of autoimmune states . Inspire
infectious mononucleosis.These polyclonal of so many different possible mechanisms proposed ,
antibodies are IgM in nature, similar 10 the 'natural their actual role in autoimmunity, if any, has not
antibodies’ produced by CCS * B cells. been established ,
-
Breakdown ot immunological borne stasis may
lead to cessation of tolerance and the emergence of
Many animal models of spontaneous and induced
autoimmunity have contributed to an understanding
forbidden clones of immunocompetent cells capable of this condition. Examples of spontaneous
of mounting immune response against sel antigens.
^
Autoimmunisation may result when tolerance to a
autoimmune diseases in animals arc autoimmune
hemolytic anemia in the New Zealand Black
self-antigen is abrogated, as for instance bv the ( NZR ) mouse strain, systemic lupus erythematosus
injection of the self- antigen with Freund’s adjuvant , in NZB X NZW cross, insulin dependent diabetes
Enhanced helper T cell and decreased mellitus in the nonobese diabetic ( NOD) mouse,
suppressorT cell functions have been suggested as and thyroiditis in the obese strain ( OS) chicken .
causes of autoimmunity Defects in the thymus* in Experimentally, autoimmunity can he induced in
stem cell development and macrophage (unction
have also been postulated as causes.
Certain self - antigens :tre present in closed
systems and are not accessible to the immune
—
many animal species by injecting tissue extracts in
the complete Freund 's adjuvant for example ,
experimental allergic L' liL-ephaJomyeiitis with brain
or spina] cord extracts, and thyroiditis with thyroid
,
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172 r Textbook of Microbiology
nowadays. Cold agglutinins arc also seen in [ primary the thyroid gland and symptoms of hypothyroidism
atypical pneumonia trypanosomiasis and bkekwater nr frank mneedema. Histologically, the glandular
fever. structure is replaced by lymphoid tissue consisting
Warm smtoantibodies arc generally incomplete, of Lymphocytes , histiocytes and plasma cells.
Ticinflggliiriniting antibodies usually belonging to Antibodies with different specificirics have been
the IgG class. They can be shown coating the found in this condition. They include antibodies
erythrocytes in the direct Coombs test. Warm that react with thyroglobulin, a second acinar
jinticrythrocytc antibodies are frequently seen in Colloid, microsomal antigen arid a thyroid cell
patients taking certain drugs sueli as gulphonamides,
, , surface component .
antibiotics, and alpha methyl dopa. 2. ThyrotoxicotiB { Graves ' disease)! The
In aiifnimmnrwi anemias , the red cells coated majority of patients with thyrotoxicosis possess
with antibodies arc prematurely destroyed in the antibody to thyroglohulin . Lymphocytic
spleen and liver. Complement dependent infiltration is common in thyrotoxic glands. 'I’he
intravascular hemolysis appears to be a rare event. immunological basis ot thyrotoxicosis is
Auto immune thrombocytopenia; supported by the identification of the 'long
7
Au roan LI bodies directed against platelets occur in acting thyroid stimulator ( LATS) which is an
idiopathic thrombocytopenic purpura. Sedormid IgG antibody to the thyroid membrane antigen.
purpura is an instance of immune response against Combination of LATS with the surface
drug induced neoantigens on platelets. This membrane of thyroid cells seems to stimulate
condition is traditionally considered an antibody excessive hormone secretion.
mediated hypersensitivity. Addison’g disease:: The immunological basis
Autoimmune luucupcniat Nonagglutinating of Addison * disease is suggested by lymphocytic
antileucocyte antibodies can he demonstrated in the i nfiltration of the adrenal glands and the presence
serum of patients with systemic lupus of circulating antibodies directed against the cells
erythematosus and rheumatoid! arthritis. of the zona glomerulus .L. Similar lesions Can be
produced in experimental animals by immunisation
LOCALISBP lUK ( i , \.N SPEtJFIt :) '
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* AulOimmun -i ty 173
contraction . Tbti thymus shows lymphoid adjuvant . The cncephalogenic protein has been
hyperplasia and numerous germinal centres. Infants identified sis the myelin basic protein ( MR?) which
born 10 affected mothers show symptoms of the shows no species specificity.
disease but [Boner spontaneously by the age of two Idiopathic polyneuritis ( Guillfiin- Ilarre
months, coinciding with the disappearance of syndrome) is considered an autoimmune response
maternal antibodies. This suggests that the against rhe peripheral nervous tissue. It tan be
pathogenic factor in neonatal myasthenia may be reproduced in experimental .LiiimaU by
the autoant ibody passively acquired from the immunisation with peripheral nervous tissue in an
mothc r. adjuvant
Autoimmune diseases of thu eye: Two Autoimmune diseases nf ihe skin : Three
types of autoimmune diseases arc seen in the eye- serious diseases of the skin art considered to
Cataract surgery sometimes leads to intraocular have an autoimmune basis . Pemphigus vulgaris
inflammation caused by the autoimmune response may he caused by an antibody to the inlcrceitular
to the lens protein . This is known as cement substance. In bullous pemphigoid *
phico&n&phjinxit' antilmdies directed against the dermal cpilhcli.il
Perforating injuries of the eye, particularly tlutSC junction have been demonstrated - Specific
involving the iris orciliaiyrbodies arc often followed antibodies in dermatitis herpetiformis have not been
by i/iupadteftc op/iEhaiirfa in the opposite eye . The identified .
disease can. he produced in experimental animals
by immunisation with uveal or retinal tissue in SYSTEMIC ( NONOROAN SPECIFIC )
Freund's adjuvant and can he passively transferred AI miMMtJNE DIHEAHEK
with the spleen or lymph node cells but not wirh This group includes conditions characterised by
serum. immune response against a variety of self-antigens
Pemiciriu * an ami a: Two types ot and damage to several organs and tissue systems.
auloantibodtes arc present in this condition. The Klemperer ( 1942) classified a number of diseases
first is directed against the parietal cells of the of unknown origin wrirh the common feature of
gastric mucosa . This is believed to cause connective tissue lesions as 'collagen diseases' .
achlorhydria and atrophic gastritis . The second type Iiieluded in this category are systemic lupus
of antibody is directed against the intrinsic factor erythematosus ( SLR ) , rheumatoid arthritis ,
and prevents absorption of vitamin 11 , , either by polyarteritis nodosa * Sjogren 's syndrome,
blocking its attachment to the gastric intrinsic facto: denoato myositis and scleroderma . All these
«r
or by binding to the intrinsic: factor complex conditions arc associated with generalised
and interfering with its uptake hy the intestinal autoimmune processes.
dfluOOH . Systemic lupus cry them AtOAUK This is H
Autoimmune disease* of the nervous chronic, multisystem disease with remissions and
i ) stern: The ‘neuroparalytic accidents' following exacerbations, Terminating fatally. Patients have , L
rabies vaccination represent injury the nervous
to variety of iutOantibodiev directed against Cell nuclei ,
system hy the immure response against the sheep intracytoplasmic cell constituents , immune
nervous tissue in the vaccine, which cross reacts glohulins, thyroid and other organ specific antigen ^ ,
with human nerve tissue . An essentially similar biological false positive reaction is scon in standard
condition, experimental allergic encephalomyelitis tests for syphilis. The abundance and variety of
( F, AE ) . can be produced in animals by autoamibodies suggest a breakdown in tlie central
immunisation with nervous tissue in Freund s control of immunological homeostasis.
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174 * Textbook ot Microbiology
The fnsr immunological feature identified in Lymphocytes and plasma cells. A striking feature is
SLE was the LE ceil phenomenon described in the presence of a circulating autoantibody called
1948 . The LE cell is a neutrophil containing a the ' Rheumatoid factor' ( RE) . This is usually a 19s
large* pale, homogeneous body ( LE body) almost IgM , though IgG , and IgA RF have also been
ELI mg the cytoplasm - [ III LE body is the demonstrated - RF acts as an antibody agtirst the
immunologically damaged nucleus of a leucocyte. Fc fragment of immunoglobulins. They combine
Sometimes, instead of being intracellular * the LE usually with IgG though some types of RF are
body can be seen free, surrounded by a rosette of directed towards other immunoglobulin classes, FIE
neutrophils. The fact that LE cell formation in due reacts wirh autologous* isologous or heterologous
to an antibody ( LE factor) present in SLE can be immunoglobulins. RE is generally considered to
demonstrated by incubating normal blood with be an immunoglobulin behaving as anti body to
serum from an SLE patient. The nuclei of some determinants present in the patient's own I
leucuCytrs can be seen to swell and become pale molecules, though some configure - i "Hal alteration
and spherical. Neutrophils can be observed to of IgG may be required before its reactivity with
surround these damaged cells , strip away the RF becomes demonstrable.
cytoplasm and c; r:i;i:ll the free nucleus td form LE RF is detected by agglutination tests using, as
cells- Giemsa stained smears of blood or bone antigens , particles coated with globulins. In the
marrow can demonstrate LE cells, but its sensitivity Rose-Waaler test, the original technique for
is so low that thin test has been replaced by other detection of RE, sheep erythrocytes coated with a
antibody tests for diagnosis. subagglm inaiiiig dose of and erythrocyte antibody
Immunofluorcscent tests for anT : nuclear ( amboceptor ) are used M the antigen in an
antibody ( ANA ) show up different patterns of agglut i par ion test, J n modifications of the test, latex
staining, such as homogeneous (diffuse ), peripheral and bentonite are used as the carrier particles for
(outline ), speckled and nucleolar staining patterns. lgG. Antinuclear antibodies are frequently found
ANA tests are sensitive but not specific for SLE, 111 rheumatoid arthritis.
as they may be positive in many other autoimmune Polyarteritis nodata: T h i i s a nccrotising
conditions, viral infections, chronic inflammatory angiitis involving medium sized arteries , ending
processes, as well as in persons using certain fatal tv due to coronary thrombosis , cerebral
medicines and in the aged . hemorrhage or gastrointesrinal bleeding .
Anti - DNA antibodies are tested by R1A or Polyarteritis is seen as a component of serum
ELISA, Three major types of these antibodies are sickness and other toxic complex diseases. Immure
—
Seen - those reacting with single stranded fss ),
1
LJ opyrighted materia
< Autoimmunity 175
TRANSITORY AUTOIMMUNE PROCESSES large amount? of uutoanrihodics are regularly found ,
1 bese include Lund it lorni such as anemia , It has been said that but for the lepra bacillus,
thrombocytopenia or nephritis that follow certain lepmmatnus lepreuy may have been proposed as an
infections or drug therapy. The infecting agent oi autoimmune disease .
drug induces antigenic alteration in some self- The relative importance of humoral and cellular
antigens. Thc imimine response set up causes tissue immune processes in the etiology of autoimmune
damage. The disease is transient and undergoes diseases is not known. Antibodies may cause damage
spontaneous cure when the infection is controlled by the cytolytic or cytotoxic ( type 2 ) and toxic
or the drug withdrawn, complex ( type T ) reactions. They are obviously
important in hcmocytoljtic autoimmune diseases.
PATHOGENESIS OF AUTOIMMUNE DISEASE Another mechanism of autoimmune tissue damage
Manv diseases arc considered to be of autoimmune is by sensitised T lymphocytes ( type 4 reaction ). It
origin, based on their association with cellular or is likely that humoral and cellular i m m u n e
humoral immune responses against self-antigens. responses may act synerglsticady in die production
Autoanribodics are more easily detected than cellular 1' some a u t o i m m u n e disease;. For example ,
autosensitisation. However, the mere presence of experimental orchitis can he induced o n l y when
autoantibodtes during the course of a disease does both types of immune responses are operative,
not prove rheir etiological role. Autoantibody O ncc in i ri atedl, m ost autoimmu nc responses tend
formation may be a result of tissue injury Lind the to be self perpetuating . Their progress can be
antibody may help in promoting immune arrested by immunosuppressive therapy, though the
elimination of the damaged cell Of tissue elements. degree of response to such therapy varies in different
A typical example is lepromatous leprosy in which diseases.
I u rt h er It e ad i i\ £
'
^Etakman>MMandand] Stewart
\ fcirI
. 2
^
1997 hanwnohgy- 8 cdfL Edinburgh: Churchill Livingstone
.
:
.
U Vetgani 1947 Basic nd Clinical Immunology. Edinburgh Churchill Ljfingjtooe.
Rcitt J et al. 1999. Immunology. 5 " edrc London: Mosby.
Copyrighted material
Immunology of Transplantation and
S3 Malignancy
transplantation nr grafting becomes necessary tor Orthntopic grafts are applied in anatomically
the restoration of function. The tissue or organ ‘normal' sires, as in skin grafts . J letcrotopie grafts
transplanted is known as the transplant or graft. are placed in anatomically ‘abnormal' sites, as
The individual from whom the transplant is when thyroid tissue is transplanted in a
obtained is known as the donor and the individual subcutaneous pocket.
to whom it is applied , the reopjertr. 3. Transplants may he of fresh tissues and organs
Transplantation Is one of mankind s ancient or of stored ones.
dreams-. L’ himcras, fanciful creatures composed cit 4. Transplants may he of living or dead materials.
parts from different species, figure in the mythology Live grafts, such as kidney or heart, are expected
jmd pantheon of all ancient nations . Such to survive and function physiologically in the
transplantations across the species barrier, however, recipient and are called \dtaJ grafts'. Nonliving
do not succeed. It was recognised very early that transplants like bone or artery merely provide a
transplants survive onlv when the tissue or organ scaffolding on which new tissue is laid bv the
recipient. They arc called ‘static or ‘structural’
1
skin grafting for reconstruction of the severed nose, donor and the recipient ( Table 20, l ) An organ
r
using the patient's own skin - a technique or tissue taken from an individual and grafted
described in the SirshrufiF iiFm/irfa (circa 800 RC ) . on himself is an autograft. A gruff taken from
T1 lu reasons fur the rejection of exogenous grafts an individual and placed on another individual
were tor long suspected to he due to active immunity of the same generic constitution is called an
hut it was onlv in the 1940s that the work of
m isograft. Grafts made between identical twins
Mcdawar and bis colleagues conclusively proved or between ngeneic memhers of highly inbred
its immunological basis. ^
strains of animals are examples of isografts.
Grafts between rwo genetically non identical
Cr ASS I FIXATION OF TnANSPl AMTS members of the same species are called allografts
Transplants mav he classified in various wavs: (formerly called hoinografts). Grafts between
1. Hascd on the organ or tissue transplanted, they members of different species are called
arc classified as kidney, heart, skin transplant . xenografts ( turmerlv called heterografts).
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< -
l nmunology of Transplanlalion and Malignancy » 177
a skin graft from another donor will evoke only place. The graft tern airs pale and is rejected within
the first set responsc. hours without even an attempt ac vascukrisation ,
Table 20.1 Terminology o gratis
Donor Name Synonym*
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173 * Te? j< ihao * of Microbiology *
This is knwn as the ‘white grift response'. This I llSTi I C O M I ' VTIBII.I T Y A v j T I U E P J ^ i
type of hyperacute rejection is sometimes seen in Immune response against transplants depends on
human recipients of kidney transplants, who may the presence in the grafted tissue of antigens that
possess preexisting antibody as a resnJt of prior are absent in the recipient and hence recognised
transplantsiortr transfusion or pregnancy. The as foreign . It follows, therefore , that if the
glomeruli in such cases are choked by platelet and recipient possesses all LILC antigens present in the
leucocyte agglomerates . Donor specific blood graft , there will he no immune response, and
transfusions to recipients before kidney Transplants consequently no graft rejection, even when the
have been found to favour graft survival. This may donor and recipient art nut syngencif . The first
he due to the enhancing effect of antibodies to generation { Fl ) hybrids between two inbred
mismatched donor antigens , induced by the strains possess antigens representative of both the
transfusion. parent str .i i ns and will therefore accept grafts from
Humoral antibodies may someumes act in r iihtr of the parental strains. If the two parental
ppositK m to cell mediated immun i iy, by inhihit 11 Lg strains have genotypes AA and BB, respectively,
graft rejection . This phenomenon , called the Fl hybrid will be of genotype AB , It can
immunological enha /t cement was originally therefore accept tissues with genotvpc AA as well
described hy Kabss in tumour transplants. If the as BB, as it possesses both alleles.Transplantation
recipient is pnetreated with one or more injections in the reverse direct inn {from Fl to parent) will
of killed donor tissue and the transplant applied not succeed as strain AA will react against antigen
subsequently, it survives much longer than in B and strain BB against antigen A.
control animals. The enhancing effect can be While transplants between members of a
passively transferred to normal animals by an JILLOILV inbred strain of animals are successful, an
injection of serum from immunised animals , ,
except mr is seen when the donor is a male and
showing that the effect is due to humoral antibodies. the recipient a female . Such grafts are rejected as
The antibodies may bring about the enhancing effect the grafted male tissue (XY ) will have antigens
in various ways . They may combine with the determined by the Y chromosome which will be
antigens released from the graft so that they arc absent in the female {XX) recipient. Grafts from
unable to initiate an immune response {afferent the female to the male will succeed - This
nth 11 L' ion ). The antibodies may combine with the
'
unilateral sac linked li i sti > i na > m patibil ity is known
lymphoid cells of appropri . Lte specificity .md, by a as the EichwaldSilmseT effect
negative feedback influence, render them incapable Antigens that participate in graft rejection are
of responding to the antigens of the graft {central called transplantation or h istocompatihiI i, ty
inhibition ) . They may also cause 'efferent antigens. The blood group antigens are important
inhibition * hy coating the surface of cells in the in transplantation. The term 'major
graft so that sensitised lymphocytes are kept out of h ^TocoEnpatiliibcy system" refers to a system of
contact with them. cell antigens that exert a decisive influence on
-
Allograft immunity' 1 a generalised response
directed against all the antigens of the donor. A
the fate of allografts. Major histocompatibility
systems have been identified in different species
-
,
-
ieciu i L:I L sen ^ itised by a ho L graft wi II reicct by the
second set response not only another skin graft but
~ El 2 in mice, AgB in rats, B in chickens, HI in
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i immunology of Transplaniaiion and Malignancy 179
description of the 11LA system is presented in transformation, the intensity of the reaction being
Chapter 15 . a measure of the antigenic disparity between the
donor and recipient lymphocytes. The test, as
FACTORS FAVOURING ALLOGRAFT performed, is a one-way rest in which the donor
SURVIVAL lymphocytes arc killed and only the recipient
1- Next to AUO blood group compatibility;the most lymphocytes ire permitted to be transformed in
importMt factor in allograft iLLrvival it- HLA response to the incompatible antigens on the donor
ciinipatihiiity This is Gated bv HLA typing and cells,
tissue matching- III, A typing identifies the 2 . As allograft rejection is an immunological
ElLA antigens expressed on the surface of process, j /?imunosi! jppfcsL«jcinwL ]l inhibit ir . This
'
Copyrighted material
ISO * Textbook of Microbiology
C - F, hybrid receiving a transplant from any one rheir clinical incidence would suggest. This
parental strain. indicates that the immune system is able to deal
The major clinical features of the GVH reaction with malignant cells as they arise and that only
in animals arc retardation of growth , emaciation, some of them are able to overcome the defence
diarrhea, hepatosplenomcgaly, lymphoid atrophy mechanisms and develop into clinical cancer,
and anemia, terminating fatally. The syndrome has 4. Histological evidence of immune response
been called runt disease. against malignancy is provided by the presence
of lymphocytes, plasma cells and macrophages
IMMUNOLOGY OF MALIGNANCY infiltrating tumours . The cellular response
When a cell undergoes malignant transformation , it resembles that seen in the allograft reaction.
acquires new surface antigens. It may al«i kwe some Turnout? shewing such cellular infiltration have
normal antigens, This makes a tumour antigerucally a better prognosis than those that do not.
different from the normal tissues of the host. A tumour 5. If the immune system plays a natural role in
Copyrighted material
IP
immune response when the tumour is transplanted be demonstrated by the stimulation of DNA
in syngeneic animals. Such rumour specific antigens synthesis and lymphokiine production by the
which induce rejection of rumour transplants in patients leucocytes on exposure to the himour
immunised hosts are termed tumour specific antigens. The lymphocytes from the patients are
transplantation antigens ( TSTA ) or tumour cytotoxic to the Cultured tumour cells.
associated transplantation antigens (TATA ). Cell mediated immunity \$ believed to be the
In chemically induced tumours, the TSTA is mechanism of host defence against malignancy.The
tumour specific. Different tumours possess different humoral response may not be relevant, or miv even
TS FA, even though induced by the same be detrimental due to its facilitating tumour growth
carcinogen. In contrast, the TSTA of virus induced by the process of enhancement.
tumours is virus specific in thar all tumours
produced by one virus will possess the same antigen, IMMUNOLOGICAI SURVEILI ANCE
ever ifthe tumours occur in different animal strains The concept of immunological surveillance had its
or species. beginning in the observations of Ehrlich (1906) ,
A second type of antigen is found in some It was revived by Lewis Thomas in the 195th and
tumours . These are the fetal antigens which are developed by Burnet. It postulates that the primary
found :n embryonic and muiignant cells but not in function of cell mediated immunity is to "seek and
normal adult cells. The best known examples arc destroyf malignant cells that arise by somatic
alpha fetoprotein in hepatomas and the mutation. Such malignant mutations are believed
carcinoembtyonic antigen found in colonic cancers. to occur frequently and would develop into tumours
Their m i l [ lies is represents a dedifferenti .LtJon of but for the constant vigilance of the immune system.
malignant cells into more primitive forms. Inefficiency of the surveillance mechanism, either
The carcinoembryohK antigen is a glycoprotein as a result of arcing or in congenital or acquired
wh i ch can be detected i n the serum of many pat ic ots immunodeficiencies^ leads to an increased incidence
With carcinoma of the Colon, particularly in the of cancer.While this hypothesis is attract ive, it may
presence of rnetastaSes. However, it also appears in perhaps represent an ave r --- i.m p I i t’icaT it) n of a
some other condiLions such as alcoholic cirrhosis, complex ^ ituation -
and hcncc its diagnostic value i -. limited . If immunological surveillance iseffective, cancer
Copyrighted materia
p
should not occur. The development of tumours Specific acuvt immunotherapy by the in jeer ion
represents a lapse in surveillance. The mechanisms of tumour cell Vaccines' was tried carlv in last
a
of such lapses are not dear but several possibilities century but was given up as unprofitable. The
have been suggested. Due to the very fast rate of method has been modi hied recently hy using purified
proliferation of malignant cells, thev may be able tumour cell membrane antigens and tumour cells
to 'sneak through' before the development of an treated with neuraminidase to increase their
effective immune response and once they reach a immunogenic potential.
Certain mass may be beyond the power o f Nonspec ific active immunotherapy employs
immunological attack . Circulating tumour andgens BCG and non 1 iv mg Cutyfitbactefium parvu m.
may act as a 'smokescreen ', coaling the lymphoid Mat he , the leading proponent of cancer
cells and preventing them from acting on the immunotherapy has reported very good results in
tumour cells . The rumour antigens on malignant acute Irultem i a, fnliowi :ig combi ucd treatment with
cells may be inaccessible to sensitised cells, being BCG and allogeneic or autochthonous leukemia
covered by some antigeidually neutral substance . blast cells . 1 ntralesional BCG in malignant
Humoral antibodies may / ause immunological
i melanoma has been reported to induce complete
1
enhancement, ‘Blocking activity has been remission in a high percentage of pari curs [t has
,
demonstrated m humoral factors. This may be due also been used against mtradermaJ recurrence of
to the circulating anLigen1 an Li body or antigen- breast cancer following mastectomy.
art ilwdy completes. Some rumours maybe of low Dimtrochlorobenxene has been tried in the
immunogeniclry or may form cytokines like treatment of squamous and basal cell can-inomn of
transforming growth factor (3 (TGF^ PI which the skin. Clucanh a pyran copolymer derived from
suppresses cell mediated ! mmunity. microorganisms , and levamisule , originally
Introduced as an anthelmintic, have been tried for
lUMI NOTHERAPY O F CANCBA stimulating cell mediated immunity and
Different approaches have been attempted in the macrophage functions. Interferons have been
—
immunotherapy of -cancer passive, active and
adoptive immunotherapy, specific and nonspecific-
employed in the treatment of leukemias.
Specific adoptive immunotherapy has been
Passbe immunotherapy was the earliest method attempted with lymphocytes, transfer factor and
of cancer immunotherapy. Antisera prepared by 'immune RNA . The donors have been persons who
i m mu n is ing an i mils wi th tumour b i opsy specimens have been cured of their neoplasms or specifically
were used for the treatment of human cancer as immunised against the patient 's tumour.
carlv as in 1#95. This method was found to be of Lymphokine activated killer ( LAKJ cells obtained
no use and therefore abandoned. A special type of bv treatment of the natural killer cells with
serotherapy has recently been found beneficial in i nterkukin -2 have been found useful in the treatment
experimental tumours . Appropriate antisera that of certain malignandes1 such as renal carcinomas,
possess 'deblocking activity in V itro have been found
1
Immunotherapy is ineffective in the presence
IO Cause regress ion of tumours , apparently by of a large mass of tumour cells . Its role appears to
neutralising the circulating tumour antigens and be more in getting rid of the residual malignant
permitting the sensitised lymphocytes to act on cells after the gross tumour has been removed. The
tumour cells . Monoclonal antibodies to tumour best results in the treatment of cancer apparently
antigens may play a mle 35 carriers in transporting follow an integrated approach to therapy, employing
Cytotoxic or radioactive drugs specifically To the surgery, radiotherapy, chemotherapy and
tumour cells, immunotherapy.
Further Reading
-
Barrett AJL 1987. G ^ft - wm - hns-T reaction a nrvkm J Royal Sot Med 80:368,
lilihfip JML 1987. THe molecular generic! of canter. SbiflC B JDfi.
^ ^-
Lachmann PJ ec aj. 1*393, CGLEWA} AjpCCtl of JjttrhirjWt y. 5* ecln Oxford; Blackwell .
Roitt 1 rt aJr 1998, / mmuncA y, 51* cdn. London; Mosby,
^
^
RtwnbCTE SA et d . 19#B. I^ fw tpfhMdn to immunotherapy of cancer. Anmlr Jnt Med lft8: 853.
Armrtrorig A, 200 L CelluLar immunotherapy for cancer. BMJt 323’12S9.
Copyrighted material
Immunohematology
Blood has held a mysterious fascinat i on tor LLH from blood transfusion ? or mothers of infants with
the dawn of time. It was considered the essence of hemolytic disease. The main blood group system;
life and was believed to cure diverse diseases and with the dates of their discovery are shown below.
restore youth and vitality to the aged , Blood
transfusion has been attempted from very early antes ABO 1900 Duffy'
1950
bur such attempts were fruitless and often fraught MN 1926 Kidd 1951
with disastrous consequences. Blood transfusion P 1926 Dicco 1955
became sc ientitically feasible only after the discovery Rh 1940 Yt 1956
of blood groups by Landsteiner. Lutheran 1945 H 1962
In his original experiment, Landsteiner (1900) Lewis 1946 Do mbrock 1965
cross tested serum from himself and five of his Kell 1946 Colton 1967
colleagues against their red blood cells. Three
distinct patterns of agglutination were observed , Some antigens have been identified that occur
Cells which failed to agglutinate with any of the very rarely, being limited to certain individuals or
scrum sample? were des ignated group On while cells families.These have been termed 'private antigens .1
Copyrighted materia
Immunohematology * 185
powerfully hut A2 cells only weakly. About StO per commonly they result from the injection of
rent of group A blood is Al and 20 per cent A2, substances containing blood group- like antigens,
The subgroups of A antigen are represented ingroup such as horse serum or bacterial vaccin.es made
AR also. The recognition of group A subgroups from media containing horse or hog extracts.
increases the number of ABO phenotypes from four Immune isoantibodics are "albumin agglutinating'
to si*; Al , A2 t B , A2B, A1B and O, Other A IgG antibodies reacting optimally at 37 ‘t and
suhgroups ( A3, A 4 , A5 ) have also been described acting as hemolysins in the presence of complement.
but they arc not clinically rdevant- They are clinically more important than natural
Blood group antigens are inherited according IgM antibodies and may cause more severe
to simple Mendelian laws . Their synthesis is transfusion reactions.
determined by allelomorphic genes A, B and O. H uniigLti: Red cells of all ABO groups possess
Genes A and B give rise to the corresponding a common antigen , the H antigen or H substance
antigens, hut O is an JMORPH and dues nut produce which is a pieaireor for the formation of A and R
any antigen. The frequency of ABO distribution antigens, The amount of the 11 antigen is related
differs in different peoples. Group O is the most to the ABO group of the cell, group O cells having
cummon group and AB the rarest. The ABO the most and AR the least amount. Due to its
distribution in Britain Is approximately 0-47 per universal distribution , the H antigen is not
cent, A - 42 per cent , H - B per cent and AH - 3 ordinarily important in grouping or blood
per cent - In India, the distribution is approximately transfusion . Bhendc ct al {1952) from Bombay
- -
0 40 per cent, A 22 per cent, B - 33 per cent and reported the very rare instance in which A and B
—
AR 5 per cent,
Anti-A and anti-B isoantibodies appear in the
antigens as well as the H antigens are absent from
red cells. This is known as "Bombay’ or OH blood.
serum of infants by about the age of six months Such individuals have anti- A, anti-B and anti - H
and persist thereafter . These are called ‘natural " antibodies and their sen are i ncompatihle with all
antibodies because they seem to arise under genetic red cells except of those with the same rare blood
control without any apparent antigenic stimulation. group.
However, it is likely that they develop as a result of A, B and H antigens ate glycoproteins. They
unidentified environmental stimuli with the blood are not confined to erythrocytes but can be detected
group- like antigens present in bacteria or other in almost all tissues and Quids of the body While
sources. Natural anti - A and anti - B antibodies are these antigens are always present in tissues, they
IgAl saline agglutinating antibodies reacting are found in secretions (saliva, gastric juice, sweat )
optimally between 4 'HT and 1.B °C but less active at of only about 75 per cent of alt persons . Such
37 ' "C . Immune isoantibodics may develop following persons are called 'sccrctors' and those who lack
A BO incompatible pregnancy or transfusion . More blood group antigens in secretions arc called
Table 21.1 Dislribulion of ABO aniigens and antibodies In red cells and serum
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m * Textbook oJ Microbiology *
lnonsecretorsh , The secretion of ARH antigens in surface of erythrocytes:. Each agglutinogen is in turn
c&ncroUed by two allelic genes Se and ^c. Individuals .
m idc up of one nr morn antigens. Fisher, on the
who arc homozygous or heterozygous (or Sc arc other hand, postulated that Rh antigens are
secretory while rhose who are
iMQKCffetQCL
-
RC SC ;iie determined by three pairs. ol closely linked
allelomorphic genes , Cc , L> d and Ee . Every
A and El antigens are also found in certain individual possesses one member of each pair of
animals and pi Jilts. ITicy have Eieen extracted and ilieve j;eiiev derived from each parent. Each gene
purified commercially from the stomach of horses would be responsible for the production of a specific
and hogs. Blood group andbodio are al >o found in antigen* which could In: detected Eiy its Specific
some annuals. Substances spedfkiUy agglutinating antihody.
A or H antigens have lieen detected in HOfAt plants. The designations employed by the two systems
A potent anti A 1 agglutinin has been extracted tmm for the different Rh types arc as follows:
tWrvfnis bifltirux aru! ant ] Hfrom ( /fe.x eunj Kftus.
^
Blood group aggltitinins ol plant origin are known Fisher Wiener
Rh positive CDe Rhl
j -. icetins ' .
cDE Rh 2
cDe Rho
RH BLOOD GROUP SYSTEM CDE Rhz
Levine and Stetson { ] 939) demonstrated a new type Rh negative Cde rh/
of and body ni rhe serum of a WOtliatl who develo}>ed odE rh //
ede rh
severe re jet Lons- following trails! Uiion of her
CdE rhy
husband '1 ABO compatible bl 1 iod . Sh r had m-en tly
delivered a stillborn intacil with hemolytic disease. For routine purposes, the typing of pcfWOTS us
They suggested rh,u the woman may have been Rh positive or negative depends on the presence 01
sensitised by some antigen inherited by the fetus absence of antigen D ( Rim ) on red cells .and hence
from its father . [.arilsteiner aril Wiener ( 194!) )
identified iu the red cells ol the majority of persons
car lie accomplished hy testing with anti D {anti
RJL) serum. This is because D is the most powerful
-
tested, an arftigcn thatrac ted with rabbit antiserum RH antigen and acioun for die majority of
to Rhesus monkey erythrocytes. 'Ehis antigen was Rh incompatibility reactions. The distribution of
culled the ‘Rhesus' or Rh factor. The ‘new type o! 1
Rh positives difteni in different races. Among|jeople
antibody described by Levine and Stetson was of European descent , about 85 per Cent are Rh
identified as the anti-1th factor antibody. Wiener positive and 15 per cent negative. Among Indians*
and Rerers (1940) demonstrated anti-Kh antibody approximately 93 per cent arc Rh positive and 7
in some peisons who had received ABO compatible per cent negative.
transfusion. Levine and colleagues ( 1941) proved A variant of TJ is known as Du . fed cells of Du
that Rh sensitisation was the cause of hemolytic a
subtype react with some but not all tnti-D sera.
disease of the newborn. Though Du cells may nor be agglutinated by anti-
The Rh system is complex ami its study is 1 > scrj , they absorb the antibody on their surface.
complicated by the existence of two different The Du subtype can therefore be detected by
theories and nomenclatures for the genes und reacting reJ cell with anti - D Serum ami then doing
antigens . Wiener proposed that Rh antigens arc L direct Coombs test - For the purpose of blood
dete rm ined hy any one of several itllelk gene; which donation, Du cells are considered Rh positive. But
may appear at a single locus and govern the when it Du individual requires transfusion , it is
production of the appropriate agglutinogen on the advisable to use Rh negative blood hecause he is
Copyrighted materia
* Immunobematolcgy 187
capable of being immunised by standard Rh positive 2 . The donor plasma should not have any antibody
blond. that will damage the recipient 's red cells .
I’here arc antibodies in the
no natural unti ’ Rb !i. The donor tedcells should not have any antigen
scrum. They arise only as a result of Rh that is lacking in the recipient . 11 the transfused
incompatible pregnancy or transfusion . Cells posnevH a " foreign antigen', it will stimulate
an immune response in the recipient ,
OTHER BLOOD GROUP SYSTEMS ideally, the donor and recipient should belong
The Lewis blood group system consists of two to the name ABO group. It used to be belt ] that O
antigens LeJ and Le\ It differs from other blood group colls could be transfused to recipients of any
group systems lit that the antigens ate preient group as they possessed neither A nor li antigen.
primarily in the plasma and saliva . Red cells acquire Hence the O group wan- designated ;LH dlC 'universal
the antigen by adsorbing them front plasma- The domr ' . The inti - A and snti - B antibodies in the
Lewis phenotypes are doscly related ro the ABO transfused O blood group do not ordinarily cause
group and to the secretor status of an individual. any damage TO the ted cells of the A or U group
Narurally oreurring f ^wis antibodies arc frequently recipients because they will he rendered ineffective
found in the sera of persons lacking the bv dilution in the recipient ' plasma. But some O
*
corresponding antigen. group plasma may contain isoantibodics in high
In cbe MN system , using rabbit antisera, persons titres ( 1 :200 or above) so that damage to recipient
were originally classified into three groups M, — cells may result. This is known as the 'dangerous
N and MN . An antigen, S , was later added to this O group'. Tlie ami- A antibody in the C group blood
syitem. This system hte expanded to include at least is generally more potent than the anti - B antibody.
28 antigens. Hence the O blood group is more likely to cause
Blood grump systems other than ABO and Rh an adverse reaction when given to the A group
are of little clinical importance as they do not usually recipients than to those of tile .B group. While the
cause transfusion reactions or kcmolvtic disease. O group blood with low titre antibodies may he
They have applications in genetics, anthropology, transfused to a patient of any other group in dire
tissue typing and torertsi-c medicine. As blood group emergency, this practice should never be employed
antigens are inherited from the parents, they are as .a routine . Transfusion 0 I large quantities of the
often useful in settling oases of disputed paternity O group blood to persons of any other group may
cause adverse reaction s-
MEDICAL APPLICATIONS OF BLOOD GROUPS Due ro the absence of isoatitibodies In plasma,
BLOOD TRANSFUSION fhe AE group persons were designated 'universal
The existence of several different blood group recipients' . A IS group donors may not always be
.mrigeni: makes it almost impossible to obtain .
available -due to their rarity and ir may on occasion,
perfectly matched blood for transfusion. Bur in be necessary to use donurn of other groups. In Such
routine transfusion practice, only the AEG and Rh cases, group A blood is safer than group B , because
antigens are relevant . The other antigens ate H>0 the anti- A antibody is usually mare potent than
weak to he of importance. Safety ill blood the arti - B antibody.
transfusion requires that rbe following conditions Rh compatibility is important only when the
be satisfied LN choosing a donor recipient is Rh negative. An Rh positive person
1. The recipient 's plasma should not contain any may safely receive cither Rh positive or negative
anribody that will damage the dun.Or 's blood. But an Hh negative individual receiving Rh
erythrocytes. jjosiTive blood may form antibodies against the Rh
Copyrighted material
IBS * TewrtJCIJk d Mir.ro biology
-
antigen. A subsequent tTunsfu .iun wilh Rh p< reitiv(
blood mav then cause an advene reaction . An
excruciating headache, constricting precordial
discomfort and severe lumbar pain . Hypotension,
additional risk m women is Rh sensitisation le , LLLING cold clammy skin, cyanosis, feeble pulse and other
tci hemolytic disease of the newborn. Therefore it signs of collapse mav be seen. Jaundice, hematun.L,
is particularly Important that Rh negative women oliguria and anuria may follow.
who arc not past childbearing age receive only Rh Some transfusion reactions may be due to
negative bloi >d . immunological processes other than blood group
Besides ABO grouping and Rh typing of the incompatibility. Rigor, urticaria and other
donor and recipient, it k Lnvari.i.hiy necessary heftire manilestanons often occur due to the recipient bring
transfusion to perform a 'cross matching? to ensure hypersensitive to some allcigcn present in the donor
1 k . n the donor 's blood is compatible with the blood. Serious reactions follow when hemolysed
recipient' blood. The routine procedure used in or contaminated blood is transfused.
most blood b?nk^ LS a rapid cross match by the tile Whenever any react Lon OCCLITT, the transfusion
-
or slide method . Thi is done in two parts - the
major cross match where the dunor red cells are
should be stopped immediately. The remainder of
the demur blood should be sent to the blood bank
tested against the recipient's scrum , and the minor for investigation .
cross match where the recipient’s cells are rested The most common complications following
against the donor serum . One drop of a blood transfusion are of infectious origin .
suspension of donor red cells In saline is added to a Transfusion of blood contaminated by bacteria may
drop of the recipient's scrum on a porcelain tile or Lead to cndotnxiv shock or septicemia . Gross
a glass slide, mixed and observed for agglutination. contamination can be recognised in most cases by
Though in most cases agglutination occurs cariy, inspection of the blood before transfusion* as
it mav4 sometimes he delayed 4
. 'lire result is to be hemolysis is usually apparent. Such contamination
read , microscopically and under low power can be eliminated by proper Techniques of blood
micTuscope , after incubation in a moist chamber collection .MLJ storage .
—
for 10 15 nii notes at room temperature. In the The most important infections transmitted at
minor cross match , the same is repeated using present by blood transfusion are rhe HIV and
reel|'Lent cells and donor serum. Only the major bepatiri '- viruses- Several cases of transfusion*
cross match is done ordinarilv, !
J
induced AIDS have occurred before HIV screening
The saline slide test does not detect Rh and of donors became mandatory. However, screening
o t h e r m i n o r incompatibilities . The most may rot detect H ) V infected donors during the
discriminating method is the Coombs cross match window period when they are infectious. Hepatitis
where washed donor cells and recipient sen mi are B, C, D and possibly others can be transmitted by
incubatec in a water bath at 37 *C for two hours transfusion. Screening for the hepatitis B surface
and a direct Coombs test done. This detects all antigen can exclude most HBV carriers but the
incompatibilities, including incomplete annbotues. available serological tests against other hepatitis
Following an incompatible blood transfusion, viruses are not quite satisfactory.
the red cells may undergo dumping and Despite diligent Screening, there exists a small
intravascular hemolysin or they may be coated hy risk (about 1 in 300,000 ) risk of transfusion
antibodies, engulfed by phagocytes, removed from associated HIV, HBV and HCV infections. The
circulation and subjected to cxtravascular lysis.. vari . mt CJD fmon IS another risk in endemic arras
Incompatible transfusion may be accompanied by like the UK where it is mandatory to screen donors
symptoms Such as shLuring , tingling sensation , fbr the prior and to remove leucocytes from blood
Copyrighted materi3
* Immure hematology m
before nan* fusion, as a precautionary measure. may be started a month before the expected date of
Cytomegalovirus transmitted by transfusion may transfusion. Autologous transfusion eliminates not
cause in infectious mononucleosis-like svndromc. only infectious complications but also those due to
-I
S>phili s may be transmitted by transfusion of fresh minor blood group incompatibilities and
blood from an infectious donor but not if the blood hypersensitivity .
tins been stored fot three days or more before
transfus ion, Malaria is another disease transm issiblc HEMOLYTIC DISEASE OP THE
by transfusion. NBVBOftN
Whole blood transfusion is being replaced When an Rh negative woman carries an Rh positive
increasingly by blood component therapy which fetus, she may be sensitized against the Rh antigen
causes fewer com plications and results in more by the passage of fetal red crib into the maternal
npti m al uti I isa rion of hu man blood wh ich is a scarce circulation . Minor transplacental leaks may occur
commodiry. For example, in anemia , packed red cell any lime during pregnancy but it is during delivery
transfusion is more beneficial than whole blood LLLi that fetal cells enter the maternal circulation in Ljige
it provides greater oxygen carrying capacity' with numbers . The mother is usually sensitized only at
less circulatoiy overload and minimal clecrrolytc the first delivery and, consequently, the first child
disturbance . Frozen red Cel is are available for escapes damage ( except wllere the woman has been
transfusion in patients with rare hi nod groups. sensitised already by prior Rh incompatible
Similarly leucocyte and platelet concentrates are transfusion ) . During a subsequent pregnancy. Rh
available for specific needs. Plasma crynprecipi Cates antibodies of the IgG class pass from the mother
and Factor VII1 are routinely used in treating to the fetus and damage its erythrocytes. This is
hemophilia, Other coagulation factors are also the pathogenesis of the hemolvtic disease of the
available for different coagulation disorders. Such newborn , The cLin ical features may vary from a mere
plasma products are manufactured from pooled accentuation of the physiological jaundice in the
human blood and may transmit 11J V, hepatitis type newborn to erythroblastosis fetalis or intrauterine
11 or C virus and other infectious agents unless great death due ta hydrops fetalis.
care is taken in the selection of donors and in Hemulvtic disease docs not affect all the
manufacturing processes , An illustration of the offspring of Rh incompatible marriages, Its
enormity of the risk was the tragedy in France in incidence is much less than the expected figures.
the mid-1980s, when tainted blood caused HIV' The following fucrors influence the incidence of
infection in over 4000 persons., of whom hundreds hemolytic disease due ro Rh incompatibility:
died of AIDS in rhe next few years. Charges of rmmunuln ical unreispomiveneu to the
aHminai negligence were framed against the persons ^
Rh uiitigeiu Mot every Rh negative individual
considered responsible, From the Prime Minister forms Rh antibodies following antigenic
downwards. Though the Prime Minister was stimulation. Some Fail ro do so even after repeated
acquitted, the Health Minister, the Head of the injection qf Rh positive cells. Thev are called
National Blood Transfusion Service and many other noniesponders / J lie reason for this immunological
top officials wete cortvicted - unrcsponsivicnefts is not known.
Almost ail adverse reactions of transfusion can 1' etorunlcrnnl ABO incompntihility: Rh
be eliminated by autologous blood transfusion Immunisation is more likely to result when the
which Is rapidly becoming popular , Here, blood is mother and fetus possess the same ABO group.
collected from rhe individual himself and stored When Rh and ABO incompatibility coexist, Rh
for use during elective surgery. Blood collection sensitisation in the mother is rare , In this situation
Copyrighted material
190 ^ Texlbook ol Microbiology *
the fetal cells entering the maternal circulation are rbc fetus is Rh positive. In the case of hemolytic
believed to be destroyed rapidly by the ARO disease oi the newborn, the maternal serum will
antibodies be tore thev can induce Rb antibodies show Rh antibodies in the indirect Coombs test
Number of pregnancies: The first child and T1IL infantk red cells wQl give a positive m the
usually escapes disease because sensitisation occurs direct Conmhs test.
only during its delivery. The risk t o the icitaiU When hemolytic disease is diagnosed
increases with each successive pregnancy. antepartum , an intrauterine transfusion with Rh
Zygosity t> f the father: All individual may- negative blood may be indicated Red cells
be liomorygpus or heoeroiygoLis with respect introduced Into the fetal peritoneal cavity will find
to [} antigen. When the tathcr in hurt ' > £Yg( Jus their way into the circulation and will survive
all his children will be Rh positive . When he normally. Rremature delivery followed by
is heterozygous , half his children will he Kh transfusion may he necessary in some cases . When
positive (i baby i $ horn with hemolytic disease, exchange
Copyrighted material
* Imfnunoh&malolagy » 191
firstborn* without prior immunisation. AIJO to oume hom the infecting microorganism.
hemtdytic diseav? is much milder than Kh disease, Red cell suspensions contaminated with certain
probably because erythrocytes of the newborn ham: bacteria, such as Pseudomonas aeruginosa,
fewer A or ll antigenic sites; as tumpiLred to adult become agglurinablc by .ill blood group sera and
erythrocytes. The direct Coomhs Test is therefore even by normal human sera. This phenomenon,
often negative in this condition, while the indirec t IqlOWtl as the 77ionFsiejT— f ' renirirmchphcnamcium ,
Coombs test ( neonatal serum with type specific is due to the unmasking of a hidden antigen normally
adult erythrocytes ) Ls more commonly positive. present on ill human erytbnocjtCS , This is called
Pettphnral blood smear characteristically shows the T antigen. Anti -T agglutinins art normally
Spherocytosi H. present in human sera . Such panagglutiiLability cl
red cells has occasionally been observed in persons
BLOOD GROUP AND DISIMSI S - - suffering from systemic bacterial infections .
It has been shown that some diseases may influence Several investigators have attempted to correlate
blood group antigens. Blood group antigens have blood group and susceptibility to certain diseases.
been Imported CO become weal; in leukemia. The It has Keen shnwn that duodena) ulcer is more
reason for this is nor known- The acquisition of H frequent in persons of blood group O than in others.
antigens by Group A perrons his been observed An association has also been established between
following some infections. The antigen is believed group A and cancer of the stomach.
Further Heading.
Andtrsun K< nj PM Nesi eds. 1994. SdatiiiC Bitit uflrajw/usunn MttUcirte - Philadelphia; S^undeft -
*
Sdtfaher OB el aL. 1996. [he rixL tvf tr iLifiixiinn- tc? iL»iiiLt ( cd IITIL infocticmi. Afeir Pnfff / MpJ!
"
. S 4c1Ht5.
^
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CM Staphylococcus
CM
Staphylococci arc Gram positive cocci that occur was between virulence and production of the e nzvme
in grape-like clusters. hl hey are ubiquitous and form
"
.
coagulase and to a lesser extent fermentation of
the mo*t common cause of localised suppurative mannitol . Staphylococci were therefore classified
lesions in human beings. Their ability ro develop into two groups; Staph , aureus (formerly also called
resistance penicillin and other antibiotics Sraphpyogenes) containing scrams giving i positive
enhances their importance as a human pathogen, coagulate test , fermenting mannitol and usually
especially in the hospital environment. being pathogenic, and Staph - cpiiicrmkiis{formerly
Staphylococci writ first observed in human also called Staph . dhirr) containing coagulase
pyogenic lesions by von Recklinghausen in 1371. negative , mannitol non fermenting and usually
Pasteur ( 1880) obtained liquid cultures of the cocci nonputhogeuic strains. The genus Stapbpiocoecus
from pus and produced abscesses by inoculating is now classified into 32 species and 15 subspecies
them into rabbi ts. It was Sir Alexander Ogstun, a based on the chemical compoiition of their cell
.
Scottish surgeon who established conclusively the wall components and other properties . Besides
causative role of the coccus in abscesses and other Staph, jurrus, two cuagulaae negative species -
suppurative lesions ( 1880 ). He also gave it the name Staph epidermidis. Staph , baemohlraut and Stsph.
,
Staphylococcus ( Staphylc , in Greek , meaning saprophyticus - can also cause human disease. Some
‘bunch of grapes'; kokkos, meaning a berry ) due to other coagulase negative species such as Staph ,
the typical occurrence of the cocci in grape-like homink and SupA. capitus are part of the
dusters in pus and in cultures . Ogstort noticed [ hat LOrnoiensal flora of the human skin. Other species
norvirulcnt staphylococci were also often present arc parasitic on animals.
on skin surfaces.
Most staphylococcal strains from pyogenic &TAPHVL.OCQCCUS AUREUS
lesions were found to produce golden yellow Morphology: They are spherical cocci ,
colonies, anti the strains front normal skin , white approximately 1 Jim in diameter , arranged
colonics on solid media . Rosenbach (1884) named characteristically in grape-like clusters ( Fig. 22.1).
them Staph . aureus and Sraph . albus respectively. Cluster formation is due to cell division occurring
Passet ( lkS5 ) described a third variety, Staph. in three planes, with daughter cells tending to
c/tFRH, producing lemon yellow colonics. However, remain in close proximity. They may also be found
the association between virulence and pigment singly, in pains and in short chains of three or four
production was not found to be constant. cells* especially when examined from liquid culture .
Many other properties were proposed as Long chains never occur. They are nonmotile and
indicators of virulence. These included hemolysis, noftfiporiilg. A few strains possess microscopically
gelatin liquefaction , Lipolytic activity' produedon of visible capsules, particularly in young cultures .
urease, phosphatase and others, but none of these Many apparently noncapsulated strains have small
was found reliable. The most constant association amounts of capsular material on the surface. Thcv
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H StapMyloccccus
* 193
medium), and pcblvrnyxin . For primary isolation, Staph epidcrmkfa in mixed cultures, as the former
sheep blood agar is recommends! Human blood gives prompt phosphatase reaction , while the latter
should not be used ns it may cntitam antibodies or is usually negative Or only weakly positive .
other inhibitors. Staph- aureus strains usuallv exhibit the following
Biochemical react M i n s , They Ferment a characteristics: (1) cnagulase positive; ( 3) greater
number of sugars, producing acid bur no gas. Sugar biochemical activity, ferment mannitc ; (3 ) produce
ferine nratjon is > f no diagnostic value except for
[ clear hemolyris on blood agar; ( 4 ) produce a
mannitol , which is usually fermented bv Stitph. golden yellow pigment; ( ) liqucfr gelatin;
^
194 * Textbook ot Microbiology
even in 15% NaCI , These features are of also and cause outbreaks of hospital infection .
significance in food preservation. These strains have been called 'epidemic
They resist 1% phenol for 15 minutes . Mercury
perchloride 1% solution kills them in 10 minutes.
methicillin resistant Staphylococcus atireu ot
EMRSA (as metiiii.ilLin is ail unstable drug,
^
Many aniline dyes are strongly bactericidal crystal cloxacillin is used for sensiri vity testing I it -4ead ).
violet being lethal at a concentration of one in live 3. Development of tolerance to penicillin, by which
hundred thousand and brilliant green , one in ten the bacterium is only inhibited but not killed .
million. Staphylococci also exhibit plasmid-borne
Fatty acids inhihir the growth of staphylococci, resistance to erythromycins, tetracyclines.,
the highly unsaturated acids having a more aminoglycosides and almost all clinically useful
powerful action on cnigulasc positive than on antibiotics except vancomycin ,
Copyrighted materia
* Staphylococcus * 195
compkmentary effects. It also induces platelet usually form both coagulase and clumping
damage and hypersensitivity. Protein A binds factor. Coagulase test is the standard criterion
to the Fc terminal of IgG molecules (except for the identification of Sfcfph , aureus isolates,
IgGJ ), leaving the Fab region free to combine 2. Lipases. Staphylococci produce n. number of
-
with its specific antigen . Protein A bearing
staphylococci coated with any IgG antiserum
lipid hydrolases which help them in infecting
the skin and subcutaneous tissues ,
will be agglutinated if mixed with its d, Hyaluromidase breaks down the connective
corresponding antigen . This procedure known tissue. Staphylnkimse (fibrinolysm ), farcy acid
as Cnagglu h nation has many up I i cations, as for modifying enzymes and proteases help in
streptococcal grouping and gonococcal typmg - initiation and spread of infection.
Protciii A is a 11 cell mitogen. It has also been 4. Nuclease. A heat stable nuclease is a
used as a ligand for isolation of IgG. .
characteristic feature of Staph aureus.
2- Clumping factor, another surface protein is the 5. Protein receptors . Staphylococci possets
“ bound coagulase' which is responsible for the receptors lor many mammalian proteins such
‘slide onagulaae ' rest. When a saline suspension as fibronectinj fibrinogen, IgG and Clip These
of Staph , jureus is mixed on a slide with a drop facilitate staphylococcal adhesion to host cell
of human plasma the cocci arc clumped. The and tissues.
slide coagulase test is routinely used for the
TOXINS
identification of Srajih . isolates ,
unreins
Capsulatcd strains may sometimes show a Cytolytic toxins: Cytolytic toxins arc
negative test because the dumping factor may membrane - active substances, consisting of four
hemolysins and a leucocidin.
be enveloped by the capsutar polysaccharide
Alpha hemolysin ( Alpha toxin, lysin ) is rhe
EXTRACELLULAR ENZYMES most important among them - It is a protein
1 . Coagulase is an enzyme which hrings about inactivated at 70 DC, but rcactivarcd paradoxically
dotting of human or rabbi r plasma. It acts along at 100 °C. This is because at 60-70 °C, the toxin
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19 ft * extdc c k
"
-- or MicrotHO - cgy
Combines with 2. heat labile inhi bitor wlnth is Eight antigenic types of emerutoxin are currently
denatured at 100 °C, leaving the taxm free. known, named A , R , C _ , D, E and H . They
.. . aTe
Alpha tn^ in lyrics rabbit erythrocytes, bul is less formed by io:\ igenic strai 1 is, M 1 igjy or in combinatiim .
active against sheep and human red cells . It is also The toxin is believed to set directly on the
leucocldal , cytotoxic , dcrmonccrot it (on autonomic nervous system to cause the illness ,
i ntradermal inoculation in fu I hbili , neurotOMC and rather than on rhe gastrointestinal mucosa . The
lethal (on intravenous inoculation in rabbits 1. Il is
'
toxin is antigenic and neutralised by the specific
toxic to macrophages, lysusomes, muscle tissues, antimxin. Type A Euxin is responsible for must cases.
renal cortex and the circulatory system. Sensitive serological testa such as latex
£ t 'fa hemahxin is a sphingomyelinase, hemolytic agglutmai ion and ELISA are available for detection
for sheep cells, but not for human or rabbit cells . Lt of the toxin.
exhibits a ‘hot-cold phenomenon', the hemolysis The tewm i ^ potent, mkrogram amounts being
being initiated at 37 *C, but becoming evident only capable of causing the illness. Some cases of post
after chilling . antibiotic diarrhea are caused bv/ enterntoxin-
1
Gamma hemoh^.' Ji L > composed of two separate forming staphylococci . The toxin also exhibits
proteins, both of which arc necessary (br hemolytic pyrogenic, mitogenic , hypotensive, thrombo-
activity. cytopenic and cytotoxic effects .
Delta hemolysin has a dctergent -likc effect on Toxic shook syndrome toxin (TSST):
cell membranes of erythrocytes , leucocytes , Toxic shock syndrome (TSS ) is a potentially fatal
macrophages and platelets. multisystem disease presenting with fever ,
L^ucotrjd .- .T ( called the Panton-Valcnfine toxin hypotcnki m, myalgia, vomiting , diarrhea, mucosal
'
after its discoverer ) is also a two component toxin, hyperemia and an erythematous rash which
like the gamma lysin, hi' mg com poised of two desquamates Subsequently, Hi is is associated with
components ( S and P ) . Such bicomponent infection of mucosal or sequestered sites by TSST-
membranc -acfive toxins as the staphylococcal produving Staphr aureus strains usually belonging
leucocidin and gamma lysin have been grouped as to bacteriophage group 1. TSST type -1 ( formerly
svncfgnhymcnatmpk fnvins.
'
also known as enterotosin. type F or pyrogenic
I ' nicmluxin: This toviu is responsible for rhe exotoxin C) most often responsible , though
mamlestac 1 ons of staphylococcal food poisoning
nausea , vomiting and diarrhea 2 - h hours after
— enterotaxins B or C may also cause the syndrome.
TSS was first identified in 1978 in children and
consuming contaminated fond cunr . i i r. i ng adolescents, but became widely known only in 1980
preformed toxi :i . The toxin is relatively heat stable, following outbreaks in the USA in menstruating
resisting 100 for 10 to 40 minutes depending women using highly absorbent vaginal tampons.
on the concentration of the toxin and nature of the Thei r vag: nal swabs showed heavy growth of Staph ,
medium. About two - tlv. rds of Staph. aureus strains, aureus, though blood cultures were mvariahly
-
growing in carbohydrate and protein food secrete
the Mo; in. Meat and fish or milk and milk products
negative. TSST- 1 antihndy is seen in convalescents
This is protective and absence of TSST- 1 antibody
cooked and left at room temperature after is a factor in [ he pathogenesis of the condition .
concaminadon with staphylococci lor enough time
,
-
Though tampon related TSS is now rare, the
fbr the toiun to accumulate, arc the common items syndrome occurs in other infections of rhe skin,
responsible . The source of infection is usually a mucosa and other sites and also in some surgical
food handler who is a earner. I’hc illness is usually wounds.
self limited , with recovery in a day or so . Staphylococcal cntcrotoxins and TSST- 1 arc
opyrighted materia
< S:aohpiQcoccLS- » 197
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19G * Taxlbook ol Microbiology
Copyrighted material
< Staphylococcus t 199
;r
200 i Tentbook of Microbiology
Lesions, systemic antibiotics itiay Cot be fiflSSSary, but can cause disease when the host defences arc
Topics! applications of drugs not used systemic ally, breached. It is a common cause of stitch abscesses.
as bacitracin, chlotbaddiH or rnupirocir may he It ban a predilection for growth on implanted
suffioen[ . fore ign bodies such as artificial heart valves, shunts,
Some strains show the phenomenon of drug intravascular catheters and prosthetic appliances,
tolerance . These strains will be found to be leading to bacteremia . Hospital strains of Staph ,
susceptible in the disc sensitivity test but their cpidcftnkiis are usually multiple drug resistant . It
minim urn bactericidal concentration will be very J
can cause cystitis. Endocarditis may be caused ,
much higher than their minimum inhibitory particularly in drag addicts .
concentration . They are pot killed bv antibiotics in Staph bjprophyricus may be present on normal
,
the usual doses and persist, leading to failure in human skin and the periurethral area and r an cause
eradicating the infection. urinary tract infection, particularly in sexually active
The treatment of carriers is bv local Application young women. The infection is symptomatic and may
of suitable antibiotics such as bacitracin and involve the upper urinary tract also. Men are
antiseptics such as chlorhexidine. In resistant cases infected much ( css often, though it is sometimes wen
posing major problems, rifampicin along with in older persons. The infecting strains arc usually
another oral antibiotic may he effective in long term sensitive to most common antibiotics, except
suppression or elimination of die carrier state, nalidixic acid. Staph, saprophvticus is novobiocin
resistant.
OTHER COAGULASE POSITIVE STAPHYLOCOCCI Table 22.1 lists the features useful for
Hesides S/apll tuiHJS, a few other StAphyluccmcal
species are coagulue positive , c . g . Staph ,
-
distinguishing the major Species of - taphylooucci .
Staph ,
-
can produce human infections Staph epidettnidu,
,
Copyrighted materia
i Staphylococcus 201
HiaphylchicKci is the Hugh and [.ciffinn's oxidation— oxidative and staphylococci show fermentative
fermentation test in which micrococci show patterns.
Pufthdr rtetidimijl,
Cmnlcjy KB and GC Arvher. 1997 . Stapfiybaocal human disease. New Yurt. Chiirchill -Lmn bbuiie.
^
Easnion . CSF and C Adlsuvi 1983. Staphyimwii tuu! Staphylococcal Infeaitm : London: Academic Press.
. .
Kk*uti WE jndlTi. RaniKrEnin 1994. Cliulcsi! japiditajitE erf' nsaiguJiutE Motive sc hylnvwn. Ota MknsAiaf Res - 7 t 117
,
^
Mtnrvdc P inrl J Kippk 1990- StAphylococad enteruCmins and their irhtivw. Science 24B : 705.
—
Rupp ME and GL Archer 1994. Coagdase nptm scaphvUxued, ,4m / Mtd 94 ; 313,
Sheaprtn JN. 19S4. STapJsyloociCLLiK iurum EIW persisieni pdugen, iVew jEsgfjF Med, 310, 136R,. 1437-
Copyrighted material
CO Streptococcus
CM
| %
/Kw
categorised them into three varieties based un tlieir
growth in 5% horse blood agar pour plate eolturcs.
Alpha (ft ) hemolytic streptococci produce a
greenish discolouration with partial hemolysin
around the colonies. The zone pf tysis is small (1
or 2 mm wide ) with indefinite margins , and unlysed
<
\ erythrocytes can he made out microscopically
within tins zone. These are known as 'viridans
streptococci ' or .Streptococcus viridans (from
lviridisr meaning green ). The alpha streptococci arc
- normal commensals in the throat, but may
cause opportunist infections rarely. Pnemocoocus
fi fr. pneumoniae! is also an alpha hemolytic
'
streptococcus .
Bets ( [3 ) hemolytic streptococci produce a
sharply defined, dear , colourless zone of hemolysis,
Fig . ±3.1 Streptococci 2 ~ 4 mm wide , within which red cells are
loovricihted material
« Sneplacoceus » 203
msiBooK w MiCHuBioLOov
SlrppNH. ih cL
' '
ft I ^LjUETLTIICn!;
f
Ach ihf -i aiM.I r-K.ullalivc
'
Obligate uia^ mK.\ 1
Honulysih
I
—
11
j . AI phi
3. ( himmil
.
\ibt vintau freup.'} ( The cnDemonccyri grcHIp )
OiMififid unm fififctitt pliyiiB- Class-iFicd. into specie* by phy & iQ-
Inficnl And- tii'XtM:rnk:. al prvpcstic »- and Mncb lcil piuprrlics
2. Bela
cTlic hemolytic slrcpictLio i i
_
Smtajffcal GfCup tfKilk C Larhohvdraic aruigra
H r
-
Gnffuh types 41.2 , 3 etc , }
.
Fig. ZA Z Classification of streptococci
completely lysed.The term ' hemolytic streptococci ' infections belong to group A . Hemolytic
strictly applies only to beta lytic strains . Most streptococci of group A are known as iir./yogeMS,
pathogenic streptococci belong to this group . These may be further subdivided into types based
Gammfl (?) or nonhemolytic streptococci on the protein (M, T and R ) antigens present on
produce no change in the medium and so arc the cell surface (Griffith typi rig ). About eigh ty types
sometimes referred to is ‘indifferent StTCptOCOCd'- of Str. pyogenes hive been recognised so far {types
They include the fecal streptococci (en tenxacci, 1, 2 n 3 and so cm).
$tr faecalis ) and related species, They are called Table 23.1 shows the medically important
the 'enterococcus group'. streptococci and their characteristics.
Hemolytic streptococci were classified by
Lancefield ( 1933 ) serologically into groups based STREPTOCOCCUS PYOGENES
on the nature of a carbohydrate (C) antigen on the Mi ir p l u p h \ : The individual cocci are spherical
cell wall. These arc known as Lance field groups, or ova ] 0.5-1.0 pm in diameter Size variations
twenty of which have been identified &o far and result from cultural conditions, lor example, when
named A~V ( without 1 and J ). The great majority grown anaerobically they are somewhat smaller.
of hemolytic streptococci that produce human They arc arranged in chains, the length of which
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304 Textbook of Micmbiok y *
4
^
Table 23.1 Medically important streptococci and their characteristics
varies within wide limits and is influenced by the Streptococci arc nonmotilc and nonspoting.
nature of the culture medium , chains being longer Some strains; of Sir. pyogenes and some group C
in liquid tlum in aoltd media . Chain formation is strains hitve capsules composed of hyaluronic acid,
due to the cocci dividing in fine plane only & nd the while polysaccharide capsules are encountered in
daughter cells failing ro separate completely. 'ITierc members of group B arid D, These capsules are
is often an appearance nt pairing within the chains. best seen in very young cultures.
Sigtiificince was once attached to rhe length of Cultural characteristic*; It is an aerobe and
the chains , and streptococci had been classified a facultative anaerobe , growing best at a temperature
accordingly { SET. inruns and brews) but this has no of 37 ( range TL-X1 ^C)- It is exacting in nutritive
relevance to virulence or other properties ^ III fact, requirements , growth occurring only in media
some nonpathngenic streptococci term the longest containing fermentable carbohydrates or enriched
chains, for example, bnr saLViriuj. ( Fig . 23.1 ). w iiti blood or serum. On blood agar, after incubation
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* Streptococcus t 205
for 24 hours , the colonics are small ( 0.5-1.0 mm ) The cell will is composed of an outer layer of
circular scuu' ransparent, luw convex lines v. i 1 hi an
( protein and ( ipoteichoic .KHI , a middle layer of
area of dear hemolysis around them. Growth and group spec irk carbohydrate and an inner layer of
hemolysis are promoted by L 0 per cent CO?. pepddoglycan.
Virulent strains , on fresh isolation from lesions, The pepridoglycan ( mLiCoprotein ) is responsible
produce a ' matt ( finely granular ) colony, while for cell wall rigidity. It ban also some biological
a^ irulcut Rtr.iins form
'
colon ies. Strainswith properties such as pyrogenic and thrombolytic
well marked capsules produce 'mucoid' colon es, activity.
corresponding in virulence to the matt type. Very Serological grouping of streptococci depends on
rarely * nonhemolytic group A streptococci are the C carbohydrate. Sir. pyugeres belongs to group
encountered, which are typical of 5tr. pyrenes in A. As this antigen is an iutqgTal part of the cell
other respects. will , it has to be extracted for grouping by a
Tn liquid media, such as glucose « r semm broth, precipitation test with group antisera - For the test ,
growth occurs as a granular turbidity with a streptococii are grown in Todd-Hewiit broth and
powdery deposit. [Vo pellicle is formed. extracted with hydrochloric acid ( Lancefield's acid
Bioohemioal reactions: Streptococci ferment extraction method ) , or foritianiidc ( Fuller s
several sugars produt ing acid hut no gas. method ) or by an enTymc produced hv StnepfomyeeK
Streptococci .Ire catalase negative. They are not MISUS ( Maxted 's method ) or by autoclaving ( Rantx
soluhle in 10 per cent bile, unlike pneumococci. and Randall 's method ). The extract and the specific
,
Hydrolysis of pyrmlidoryl naphthylamule ( PYR antisera are allowed to react in capillary tubes.
test ) and failure to ferment ribose are useful in Precipitation occurs within five minutes at the
differentiating Su; pyogenes from other streptococci. interface between the extract and the homologous
Resistance: $fr- pyv encs is a delicate organism, nnti -icrum. Grouping may also be done by agar gel
^
easily destroyed by heat ( 54 for 30 minutes). It precipitation.
divs in a few davs .
Jr
in cultures- unless stored n a low
, r
, Several proton antigens have been identified in
temperature 4 C) , preferably in Robertson 's
( ?
the outer part of the cell wall, Stn pyogenes can be
cooked meat medium. It can, however , survive in typed , based on the surface proteins M, T and R .
dust for several weeks, it protected from sunlight The M protein : s the most important of these- Tt
It is rapidly inactivated by anfscptics. It is more acts as a viralcncc factor by inhibiting phagocytosis.
resistant to crystal violet than many bacteria, It is an i i.L- eiiic . The antibody to M PROIEIIL promotes
including Staph , aureus. Crystal violet (1 mg/ L ), phagocytosis of the coccus and is therefore
nalidixic avid ( 15 mg/ L ) and colisrin sulphate (1C protective. Tire M protein is hear and acid stable
mg/ T.) added tn blood agar provide a good selective but susceptible to tryptjc digestion . Tt car he
medium tor the ivolatii in of streptoeocs i . including extracted by the Lancefield acid extract!' in method
pnemnuLOccL. It ii susceptible to sulphonamides and typing is done with type specific sera. About
and many antibiotics but unlike Staph , aureus dues SO M protein types have been neoognised.
rot develop resistance TO < Irugs. Sens il ivi tv to bacitracin TheT prrnteir. is an fri id lahlie, ttyps i n n' -lstant
'
is employed as a convenient method fbrdiffcrenti .Lting antigen present in many serotypes of Str. pyqgenes-
Sir pyogenes from ocher hemolytic streptococci. IE may be specific but many different M types
Antigenic structure: Fig. 23, 3 illustrates the possess the same T antigen, lc is usually
disposition of the various anti gens in 5tr. pyogenes. demonstrated by the slide agglutinjtion test using
The capsule when present inhibits phagocytosis ,
trypsin.- treated whole streptococci . Some types of
k is not antigenic in human beings. i’fr pvogenej ( types 2 * 3 , 28 and 48 ) and some
Copyrighted material
I JF W
206 4 .
Tesctbr Dk Dt Mu;robiology
strains ofgroups B, C and G contain a third antigen, Toxins and other virulence factors; Srr .
the R protein.TheT and R proteins have no relation pyogenes forms several exotoxin ? and enzymes
to virulence . A nontype-spetific protein, associated which Contribute to its virulence. Besides these,
with the M protein, has been identified. This is the M protein also aoa as a virulence factor by
known is M associated protein ( MAP). inhibiting phagocytosis. hfhe G polysaccharide has
I lair - - ike pili (fimbria ) project through the
'
been shown to have a toxic effect on connective
capsule of group A streptococci. The pLLi consist tissue in experimental animals.
part ] / of M protein And are covered with Hemolysins Streptococci produce two
Lipoteictioic acid which is important in the hemolysins, streptolysin "O' and 'S'. Streptolysin
attachment of streptococci to epithelial cells. O is so called because it is oxygen labile. It is
Various structural components of $tr. pyogenes inactive in the oxidised form but may be
exhibit antigenic cross reaction with different reactivated by treatment with mild reducing agents.
tissues of the human body. Antigenic relations] lips On blood agar, Streptolysin O activity is seen only
have been demonstrated between capsular in pour plates and not in surface cultures. It may
hyaluronic acid and Imm mi synovial fluid, cell will be obtained in the active state hy growing
protein and myocardium, group A carbohydrate and streptococci in broth containing reducing agents
cardiac valves, cytoplasmic membrane antigens and such as sodium hydrosulphite . It is also heat Labile.
vascular intima , and peptidoglycans and skin It appears to be important in contributing to
antigens. It has bcc a postulated that these antigenic virulence. It is lethal on intravenous injection into
cross reactions may account for some of the animals and has a specific cardiotoxic activity. It
manifestations of rheumatic fever and other has leucotoxic activity also. In in biological action ,
streptococcal diseases, the tissue damage being of streptolysin O resembles the oxygen labile
mi immunological nature hemolysins of CL perfringens, Gf. reran! and the
pneu mococcus.
Streptolysin O is antigenic and antistreptolysin
V O appears in sera following streptococcal infection .
J
. Esti matitm of this antibody (ASO titre) is a standard
serological procedure for the retrospective diagnosis
of infection with Sir. pyogenes. The lytin is
inhibited by cholesterol but not by normal sera,
* Following certain chemical treatments or bacterial
contamination, sera may develop inhibitory activity
2m due to some changes in the lipoproteins. Such sera
2 p -2 arc unfit for the ASO test. Because of the
:
2c
complexity of the hemolysis inhibition test, ASO
1 rest is now done by the serological method of latex
agglutination. An ASO time in excess of 200 units
is considered significant and suggests either recent
.
Fig . 33.3 Antigenic structure of S t f pyogenes . or recurrent infeution with streptn-cocci -
.
1 Hyaluronic Kid capsule. 2. Cell wall comprising
£ A . peptldoglytan , SB. group Speelfic carbohydrErte Streptolysi n S and O are produced by groups A, C
and 2C . protein llpoteichcic acid fimbria 3 , Cyto- and G also.
.
:iasmic memtirane 4. Cytoplasm. 5 PHI cowed with Streptolysin S is an oxygen stable hemolysin and
Ipotdcholc acid. HO in responsible for the hemolysis seen around
Copyrighted materia
i Slreplococcus * 207
streptococcal .colon ics on the nufin of blood agar Deoxyribonucleases { Streptodurnu &e .
plates. Ic is called vtnptofywn S since it is soluble DNAase ); These cause dcpolymerisation of
in serum . It is a protein but la not antigenic . DNA . Pyogenic exudates contain large amounts of
Convalescent sera do not neutralise streptolysin S DNA, derived from rhe nuclei of necrotic cells.
activity. It is inhibited nonspccifically by scrum Strcptodornasc helps to liquefy rhe thick pus and
Lipoproteins. may be responsible fur rbe thin serous charterer ot
Pyrogenic exotoxin ( t> ytIm L nieh
Dick , tcnrlatinn.1 toxin ): This toxin was ^- streptococcal exudates . This proper tv has heen
applied therapeutics LI v in Liquelying localised
named 'erythrogeoie because its intradermil
1
collections of thick exudates* as in empyema. A
injection into susceptible individuals produced an preparation containing streptokinase and
erythematous reaction ( Dick test , 1924 ) . This test streprodornase is available for this purpose . Four
was used to identify children susceptible to scarlet antigcnirallydistinct I JNAases, A , R, C and I ), have
fever, ,t type of acute pharyngitis with extensive been recognised , of which type B is the most
erythematous rash , caused by the Sir. pyvfienes antigenic in human beings . Demonstration of anti -
strains producing this toxin . Blanching of the rash DNAase R antibody is useful in the retrospective
on lot a I injection of convalescent senim was used diagnosis of $tr. pyogenes infection , particularly in
as a diagnostic , test for scarlet fever ( Schultz skin infections, where ASO fitres may he low.
Charlton reaction, 191 H ). The Dick test and Schultz Strcptodornasc Ft and 1 ) also possess tihonudeasc
Charlton reaction are now only of historical value activity.
as scalier fever is no longer a common or serious Nicotinamide adenine tlinucleotidase
disease. ( NADaBet formerly drphosphopyridine
The primary effect of the toxin is induction of nudentidnse , UPNase ): This acta on the
fevtf and so it was ren amed Streptococcal pyrogen ic coenzyene NAD and Lihe rates nicotinamide tmm
r
exotoxin (SEE ). Three types of SPE have been the molecule. It is antigenic and is specifically
—
identified SPE ,ArR and C. Tvpes A and C are
coded for by bacteriophage genes , while type B
gene is chromosomal. SPE-s are ‘supefan tige ns’ ( like
neutralised by rbe aotibodv in convalescent sera.
The biological significance of NADasc is not
known * though it LH believed to be itUCOToxic.
staphylococcal entero toxins and TS-S toxin ), T cell Hynlumnidnn ^ This enzyme breaks down the
mitogens which induce massive release of hyaluronic acid of the tissues . This might favour
inflammatory cytokines closing fever, shock and the spread of inlection along the intercellular spaces.
tissue damage. Streptococci possess n hvalumnic acid capsule and
Stfcptokinafle (fibrinotysin ) ; This roxin
prom ores the Lvsis of human fibrin dots bv activating
—
also elaborate a hyaluronidasc a seem ingly self -
destructive process. IT is, however* found that strains
a plasma procursor (plasminogen ) . It is an antigenic ( hut form hyalutoiiidwsv ill targe qoutitHt (M types
prorein and neutralising antibodies appear in 4 and 22 ) ire noncapsulated . The enzyme is
convalescent sera. Anti-Streptokinase antibody antigenic and specific antibodies appear in
provides retrospective evidence of streptococcal convalescent sera.
infection . kihrinolysin appears to play a biological Serum npmtiiy fucior: Some M types
role in streptococcal infections by breaking down pyogenes produce a lipoproteinase which produces ^
the fibrin harrier around the lesions and fkicilitaring opacity when applied to aggr gel containing horse
the spread of infection - Streptokinase is given or swine scrum. This is known as scrum opacity
intravenously tor rhe treatment of carlv myocardial factor ( SOI 1).
infarction and other thrombocmboloic disorders . Many strains also produce proteinase ,
Copyrighted material
m -.
< TeKitwo of Microbiology *
Copyrighted materia
* SUepU) coccus 209
only in persons ntmirtlrtlune L :: Lht infecting M i ofect ion with Str. pyogenes and in mice by in; action
types. of sonic tysates of the COCL i .
Genital infections: Both aerobic and While rheumatic fever may follow Infection
anaerobic Rtreptococn are norma] inhab:i ; ants of with any serotvpe of Stt. pyogenes1 nephritis is
the female genitalia. Str. pyngcnesvms an important caused hy only a few 'ncphritogertic' types. In the
cause of puerperal Rep -. ii , with the infection usually tropics, skin infections are perhaps more important
being exogenous, The emphatic demonstration by in this respect than throat infect ions. The nephritis
Semmelweis in 1847 that hospital outbreaks of Is usually a self -limited episode that resolves
puerperal fever could be prevented by the simple without any permanent damage. The pathogenesis
measure of handwashing by those attending the may be due to antigenic cross-reactions between
labour wards remains a landmark in clinical the glomerular membrane antigen and cell
microbiology. Puerperal fever is now much more membranes of ncphiitogcnic streptococci, or more
commonly due to endogenous infection with often it may be an immune complex disease. This
anaerobic streptococci Streptococcal puerperal condition has been produced in monkeys and rabbits
sepsis used to take a tieaw tut! of lile before by repeated infection with type \2 Str. pyogenes or
antibiotic ? became available . injection of bacteri .d products, and i:i mije with
Other suppurative ! nfecth > n &: Str. soluble streptococcal products.
pyogenes may cause abscesses in internal organs Epidemiology: The major source of Str
such as the brain, lungs, liver and kidneys, and also
septicemia and pyemia.
pyogenes is the human upper respiratory tract
throat, nasopharynx or nose - of patients and
-
NonguppurfU i VC Cflinpliioatnjns; Sfr. earners . Carrier rates of up to 20 per cent have
pyogenes infections lead to two important been observed. Symptomless infection is common
—
nonsuppurative sequelae acute rheumatic fever and
acute glomerulonephritis. These complications
and helps
community.
to maintain the organism in the
Transmission of infection is cither by
ensue J -3 week? after the acute Infection so that d i rect contact or through contaminated finger*, dust
the organism may not be detectable when sequelae or fbmites. In the tropics, streptococcal infection
set in . They differ in their natural history in a of the skin is common and may be spread by
number of respects ('Table 23.2). nonbitliig insects, particularly the eye gnat
The pathogenesis of these complications is not Hippelares.
clearly understood. The C 5senti.il loio:i in rheumatic Streptococcal infections of the icsfur.Ltorv tract
fever is carditis , including connective tissue arc mote frequent i:i children 5-8 yeai* «f age than
degeneration of the heart valves and inflammatory in children below two years or in adults. They arc
myocardial lesions characterised by Aschoff more common in winter in the temperate countries.
nodules. Typically, rheumatic fever follows persi ' tent No seasonal distribution has been identified in the
or repeated streptococcal chroac infections with a tropics, Crowding is an important factor in the
strong antibody response . The lesions are believed tuns mission o I infection. Outbreaks ol infection
to be the result of hypersensitivity to some may occur in closed communities such a* boarding
streptococcal component. It has also been suggested schools or army camps.
that there may be an clement of autoimmunity Immunity is type specific and appears TO be
involved , and anrigcric cross* reach ' ms have been associated with antibody to the M protein .
demonstrated between streptococc i and heart Reinfections occur because of the multiplicity of
tissues. Lesions resembling rheumatic fever have serotypes.
been produced experimentally In rabbits by repeated Laboratory diagnosis; In acute infections,
Copyrighted material
210 4 Textbook of Microbiology *
specimen IS plated on blood agar and humbated at of antibody to strep EOLUCCAT toxins. The usual test
27 anaerobic ally or untier 5- L 0% CO , , as done is antistreptolysin (.1 titration. ASO titres
hemolysin develops better umbr these conditions, h ighc r thni n 200 arc in dicat i we of prior streptococcal
-Sheep blood agar is recommended b > r primary infection. High levels are usually found in acute
isolation because it is inhibitory for Haemophilus rheumatic fever but in glomerulonephritis, titles
hsemoJy/ii -EJff, colonies of which may be confused arc often low, Anti deoxyribonuclease B ( anti-
with those of hemolytic streptococci . I [cmolytic i > NAase b) estimation is also commonly employed.
streptococci are grouped by the LancefieJd Titres higher than 200 are taken as significant . Anti-
technique [’fie fluorescent antibody technique has
, i > NAasc B and iintihyaluronidase tests are very
been employed for the rapid identification of group Useful for the retrospective diagnosis of streptococcal
A streptococci A convcaienc method for the
* pyoderma, for winch ASO is of much less value.
identification of Str. pyogenes is baaed on M sorted 's The streptozyme test , ,i passive slide
ohRcrvation th ,i t they are more sens i five to baci tiiici n
P
hemagglutination test using erythrocyte* sensitised
than other streptococci . A filter paper disc dipped with a crude preparation of extracellular antigens
Copyrighted material
< Straplacaccus * 211
of streptococci , is a convenient sensitive and specific occurring vi thin a week of birth , and the late onset
screening tst. Il becomes positive after nearly all type developing between the second and twelfth
Iv yes of streptococcal infection!;, whether of the weeks of life. The more common early unset type
^
throat or the skin . presents as septicemia, meningitis or pneumonia ,
Evmphylavi& The indication for prophylaxis in and is often fatal- Infection is acquired from the
streptococcal infection is only in the prevention of maternal vagina during birth. In chc late onset type ,
rheumatic fever. This is achieved hy a long-term infection is more often obtained from the
administration of penicillin in children who have environment. Other Group B infections in neonates
developed early signs of rheumatic fever. This include arthritis , osteomyelitis , conjunctivitis,
prevents streptococcal reinfection and further respiratory infections, peritonitis, omphalitis and
damage to the heart. Antibiotic prophylaxis is not endocarditis. Group 1 ? streptococci may al -so cause
useful for glomerulonephritis as this complication adult infections , including puerperal sepsis and
follows a single streptococci! infection , and pneumonia.
reinfections do not occur. Their ability to hydrolyse hippurate acts as a
Treatment: All beta hemolytic group A presumptive identification method. They may be
streptococci arc sensitive to penicillin G , and most identified by the GAMP reaction (Christie , Atkins
are sensitive to erythromycin . In patients allergic and Munch- PcLCTjonh which can be deulon &CfLted
to jnrnicillin, erythromycin nr cephalexin may he as an accentuated / one of hemolysis when Str.
used . Strains resistant to erythromycin hive brer qgubctuc is inoculated perpendicular to a streak
reported. Tetracylines and snlphonarirides are not of Staph. a uncus' grown on blood agar. Occasional
recommended . Antimicrobial drugs have no effect strains are bacitracin sensitive. Human pathogenic
on established glomerulonephritis and rheumatic Group R strains possess a polysaccharide capsule
fever which appears ro confer virulence. Nine capsular
semtypes have been identified, antibodies to which
OTHER HEMOLYTIC STREPTOCOCCI confer type specific protection .
Besides Str, pyogenes, streptococci belonging to
groups B , C, D, FH C and rarely H , K . O' and R GROUP C
duty also t£ ii!ie human infections.
p
Streptococci of this group arc predominantly animal
Data from Streptococcal Reference LdbontOiits pathogens and may be divided into ftiur species
in India ( Lady Hardingc Medical College, New biochemically. Group G rtnins isolated from
l>lhi ; Christian Medical College, Vellore ) showed human sources usually belong to Strep, equisimiia
that while approximutely 45 per cent of hcinnllvt i c species . It can cause upper respiratory infections ,
streptococcal isolates tested belong to group A , ID- ,is well as deep infections such as endocarditis ,
15 per cent belong to groups K and C each , about osteomyelitis, brain abscess, pneumonia and
25 per cent to group G and 5 per cent to group K puerperal sepsis. Strain ^ arc often tolerant to
penicillin and serious infections may not respond
GROUP It to penicillin treatment. The addition of gentamicin
These are important pathogens of cattle , producing is recommended in serious cases. It resembles Stz
bovine mastitis ( 5rr. itgaLietnie}. From the lyfeOs, pyogenes in fermenting trehalose but differs in
Group B streptococcus lias assumed great clinical fermenting riboHe. It produce -? strcplulvsin OH
importance as rhe single most common cause of streptokinane ( antigcnicjilly distinct from that
neonatal meningitis in the West. Infection in the produced by Sir. pyogenes ) and other entraceltular
newborn is classified is the early onset type substances. .Str ctjaiumilis is the source of
Copyrighted materia
212 H Ifevttucifc of Microbiology »
streptokinase used for thrombolytic therapy in blue milk. On MacConkey s medium they produce
patients. tiny deep pink colonics, They arc relatively heat
resistant , surviving 60 C for .iO minutes .
GROUP F Enterococci typically appear as pair? of oval cocci,
These grow poorly on blood agar unless incubated the cells in a pair arranged at an angle to each
under CO .,. They have been called the 'minute other [Fig. 25.4). They are usually nonhemolytic,
K *
streptococci '. They are sometimes found in though some strains may show alpha or beta
suppurative lesions. One member of this group is hemolysis .
Sfrrpfoiucus MG which is an alpha lytic strain The identification of enterococcus species is
isolated from cases of primary atypical pneumom .L. made on biochemical grounds. E. Fsa^lii is the
Demonstration ofagglurinius to Streptococci MG enterococcus most often isolated from human
sources. E ikv. jj' s can be identified by its ability
'
in sera of patients had been used as a diagnostic j
test tor primary atypical pneumonia. to ferment mannitol, sucrose, sorbitol and aesculm ,
and to grow on tellurite blood agar producing black
GROUP G colonics.
These are commensals in the throats of human Enterococci are present in the intestine, genital
beings, monkeys nr dogs. 1 hey may occasiunally tract and saliva. They :uc frequently isolated from
cause tonsillidsn endocarditis and urinary Infections cases of urinary tract infection and wound infection.
in human brings. They may also cause endocardi us, infection of the
Groups H and K sometimes cause infective hi I iary tract, sept icen i ia, arid iiitraabdomiiial abscess
endocarditis. compEcating diverticulitis and peritonitis. Strains
Group O is isolated mainly from the healthy resistant to penicillin and other antibiotics occur
human throat and may cause acute tonsillitis and frequently, so it is essential to perform antibiotic
endocarditis. sensitivity for proper therapy.
Group R strains are natural pathogens of pigs. Nonenterococcal species of group D [ Str, bovk ,
They have beer reported from occasional cases of Str. njufnus) are generally susceptible to penicillin
meningitis, septicemia and respiratory infection in and are inhibited by 6.5 per cent sodium chloride
persons in contact with infected pigs or or bile . They may cause urinary infection or
contaminated meat. endocarditis rarely.
* 4.
J“
Ij opy righted materia
i Sirepwcoccus e 2U
I * y* ?
fift #
v
\ i*
/
tv 4 f V #-.is- c v 4
^
"
OO r < . .±
& <
*J ?oI P / r
Er
Gram stem of a pus smear . Plate stows Gram Gonococci In urethral discharge. Gram stain.
positive violet coloured cocci in groups.
{ Staphylococci). in chains ( streptococci) and
pink rods (Gram negative bacilli}. Pus cells
show up pinfc slaincd.
ft /
% 4
© 8? *•$i i*l y«J V
.
_
rjt ,
4
* 6
%?
•
4
, JT e
*
49 /
% $> * ® v,» > i*
4 »
Q
<*
*
Acid fast slain ( Ziehl-heelsen stain) cl sputum.
The red rods are M (crfiereulosis.
^ «8»
V leprae; ZwW -Nwisen slain of section of lepra netful? showing
liprn cells and characteristic arrangeoienl of lepra bacilli.
Copyrighted material
214 * Texiaoo
*. o < Microbiology *
%
t*
vo . Vu ••
h
9 •P a '* f
"(FT
&
^
<
* *r Jf i
- *
*^ '
0
' • f
I• !* •
‘
%
e
A AOA D 7 *
«
Vtersjnia pe$ft& Sm f frflrti enlarged lymph glartd Kegrr bodies in dog' s brain in rabies [hippocampus
from a cast of plague- Leishman slain of dog). Alcoholic eosin and methylene blue stain.
CJwactarislic bipolar &;a-nmg,
|
r\;*
f T
Allantoic memb W showing Variola packs. Ai arttoic mcmornna showing Vaccinia pocks. Note
^
Clear cut and white irregularity in shape and sire
Copyrighted material
' Streptococcus * 215
*" u J*’
w* *
can be employed for treatment .
Sir. mijriEm- (so called because It assumes a
bacillary form in ,tcid environments! is important
in the causation of dental caries. It break.; down
1
Fig. £ 3.4 £ nl«r«occiu. Ova ! calls arangcc in pairs al the dextrans bind together food debris , epithelial
an angle, or in short chains . cells, EiiLicus and bacteria to torm dental plaqt s,
^
They are ordinarily nonpathogenic bur can on which lead to caries, Experimental caries in
occasion cause disease. In persons with preexisting monkeys has been prevented by a £ fr. /nutans
cardiac lesions , they may cause bacterial vaccine, but its extenfion to human use is fraught
endocarditis., Srr. sanguis being most often with problems.
Further Eteuding
Brighton D et iL 1591, A scheme for identification ofviridaru streptococci , / Afn/ A-frcrutuoJ 35 ;367
L
BISRW AL - 1951. Gftttip A ntpptrNewe ) infections Hid Jcvtc Hwninnk; fever. Afcw t'-ngj Med 325;7fS3
* r
Bisno AL and DL Stevens 1996. Streptococcal infections of slcin and soft tissues. Ne\vEnglJ Med 334:240.
( .
Chaelet [ J and H Liratn 1936 . Suspiocrtecal puerperal atpsis ud JIIKICIILL InfecdtHi: A Instc rinL perspective. .-
L JJ ;
Copyrighted material
Pneumococcus
{ Dipiococcus pneumoniae: Sir:. pneumoniae)
<*% >n,
J
., i
anaerobes, the optimum temperature being 37 “ C
( range 25-42 °G ) and pi 1 7.3 ( range b. 5-8.3) .
#? —
Growth is improved by 5 10% CO .
On bhjod agar, after incubation ^ for 18 hours,
-
i L.'
*0 ' .« the colonies are small (0. -1 mm), dome shaped
^
and glistening, with an area of green discoloration
( alpha hemolysis ) around them , resembling
colonies of Stt, vitidsns. On further incubation the
Fig. 24.1 Pneumococci in pus
colonics become flat with raised edges and central
Copyrighted material
Pneumococcus 217
Copyrighted material
21 B * Taxlbook of Microbiology *
classified into types haned on the antigenic nature a nudeoprotein deep inside the cell and a somatic
of the capsular polysaccharide. Pneumococci ‘C’ carbohydrate antigen, both of which are species
isolated from Lobar pneumonia were originally specific.
classified info three types* I , II and Til , and a An abnormal protein ( beta globulin] that
heterogeneous group JV , Members of group ] V precipitates with the somatic ' C antigen of
were Later classified into types , and now more than pneumococci , appears in the acute phase sera of
% different serotypes are recognised, named 1, 2 , cases of pneumonia but disappears during
and so on. convalescence , li also occurs in some other
Typing may be carried out by (1) agglutination pathological conditions. This is known as the 'C-
of [ he cocci with the type specific antiserum ; rcactive protein' (CRP ). Its apparent antibody-like
( 2) prod pitation of the S5S with the specific Rcnim; relation to the f antigen of pneumococcus is only
or (3 ) by the capsule swelling reaction described fortuitous. It Is not an antibody produced as a result
hy Neufrld (1902). In the capsule swelling or of pneumococcal infection. It is an 'acute phase 1
of methylene blue solution . Ln the presence of the when the inflammatory reactions subside. CRP is
homologous antiserum , the Capsule becomes used as an index of response to treatment in
apparently swollen , sharply delineated and tefVactile. rheumatic fever and certain other conditions. CRP
The quellung test can be done directly with sputum testing, by passive Agglutination using Latex particles
from acute pneumonia cases. It used to be a routine coated with anri- CRF antibody is a routine
bedside procedure in the past when the specific diagnostic procedure.
antiserum was used tor the treatment ofpncumonia. \ iiH cititwr: On repeated subculture, pneumococci
The antigenic icy of the capsular polysaccharide
varies in different species. Ic is antigenic in human
--
undergo i smooth to rough (S-R) variation . In the
R form , the colonies are rough and the cocci are
beings and rabbits. But in mice, large doses noncapsulated, autoagglutinible and a virulent. R
( 500 fig ) induce no immunological response forms atix v spontaneous mutants and outgrow
(immunological paralysis), while small doses ibe parental 5 forms in artificial culture; in tissues,
(0.5 |uLg) are antigenic. such R mutants are eliminated by phagocytosis.
Pneumococci contain other antigens also — Rough pneumococci derived from cap&ukted
v* f. i
t
«
4
-
Fig . 24.3 Draughtsman appearance of pneumococcus colonies. Left: View Irom above to show concentric
rings . Right : side view In cross section .
Copyrighted materia
4 Pneumococcus f 219
cells of one serotype may be made to produce contiguity or through blood . Pneumococcal
capsule -5 of the same or different serotypes, on bacteremia may also lead to distant infections as in
treatment with DNA from the respective serotypes the heart, peritoneum or joints. Infection is
of pneumococci . Th i s transforms!ion, which may commonly endogenous, but exogenous injection may
be demonstrated in vivo or in vitro, was discovered also occur, especially wirh highly virulent strains.
by Griffith (1928) and is of consnlcrable historical The commonest pneumococcal infections are
interest as the first demonstration of generic oriris media and sinusitis. Prior respiratory infection
exchange of information in bacteria. or allergy causing congestion and blockage
Tooting and other virulence factors: predispose to these conditions. Serotypes 6, 14, J 9p
PneiiintMiDCL-: produce ait oxygen labile hemolysin and 1. ll ire eornTn -mi. lv/ encountered in these
'
i
and a Icucocidin bait these arc weak and make no conditions, :.n the West.
contribution to virulence . The virulence of Pneumococci are one -of the most common
pneumococci depends on irs capsule and the bacteria causing pneumonia, both lobar and
production of a toxin called pncumolysin . The broncbopncumnniiL. They also cause acute
capsular polysaccharide, because of its acidic and tracheobronchitis and empyema.
hydrophilic proper ics, protects the cocci from Aspiration of nasopharyngeal secretions
phagocytosis. Capsulated pneumococci are not containing pneumococci into the lower respiratory
phagocytosed efficiendy in fluid media or exudates. tract is a common event and may occur even in
1 :iey are however , Susceptible to 'surface sleep. Normal mucosal defence mechanisms such
phagocytosis', hung engulfed against a firm surface, as entrapment, expulsion and the cough reflex, aided
such as fibrin clot or epithelium . by the ciliary escalator effect prevent establishment
The enhanced VLTulencc of type 2 pneumococcus nf infection. When the normal defences are
is due to the abundance of its capsular maferial - compromised by viral infection, anesthesia, chilling
Noncapsuiatcd strains are av:.rulcnt. The antibody or other factors, pneumococci nultiply, penetrate
to che capsular polysaccharide affords protection the bronchi .il mucosa and spread through the lung
against infection. along peribronchial tissues and lymphatics.
Pncumolysin, a membrane damaging toxin Bacteremia is common during the early stage of
produced by pneumococci has cytotoxic and lobar pneumonia. Toxemia is due to the diffusion
complement ac - ivaiing properties and so may be a of the capsular polysaccharide into the btaod and
virulence factor. It is immunogenic. Pncumolysin tissues. The fall of temperature by crisis and relief
negative mutants show reduced virulence - o
'
Copyrighted materia
220 + Texibgak jiH. Microbiology
*
BioncKopneurfwnii is frequently a terminal cwnt may vary According to The virulence of the infecting
in aged ami debilitated patients. scrorypc. Type 3 is the most virulent,
MiiL'innoLocu are- cuautiuiilyuudued with the Lobar pneumonia is usually a sporadic disease
acute exacerbation? in. chronic bronchitis. The but c ] >i Jcinics i nav occur anKmg dosed communit ies
copious respiratory KcietkHU in chronic bronchitis a ? in army camp? - The incidence of
aid pneumococcal invasion. Another bacterium bronchopneumonia increases when an epidemic of
cnmmonlv associated with this condition is influenza or ocher viral infection of the respiratory
Haemophilus influenzae. tract occurs. Cases are more common in winter and
Meningitis in the most serious ot pneumococcal affect the two extreme age groups more often.
infections . ] r is usually secondary to other 1. Llhi >mttirjy di -. if{ iUiMi !lt The clinical diagnosis
pneumococcal infections such as pneumonia . otitis yt pneumonia is easy but as the disease may be
media, sinusitis or LOI ij Linctmtis but in a pn>portion caused by several different microorganisms ,
of ca?es , other foci of infection may not be etiological diagnosis should be marie hy laboratory
demonstrable . Pneumococcal meningitis occurs ir tests- d 'h is is of great importance in treatment.
11 ages. Untmltd case;; sue almost invariably fetal . In the acute phase of Lobar pneumonia, the
*Ever with antibiotic therapy, the case fatality nttt rusty sputum contains pneumococci in large
is about 2 ? per cent . numbers, with hardly any other kind of bacterium.
Pneumococci may also produce suppurative E’hev mav hr demonstrated bv Oram stain. In the
1 > ions in other parts ot the body — empvema , preanti biotic eta, direct sc retyping ol pneumococci
pericarditis , cniris media, sinusitis, conjunctivitis in wet films of sputum by the qucllimg icsr was a
suppurative arthritis and peritonitis , usually as routine IKMJHILIC Test because success of treatment
complications of pneumonia , depended on administering the specific antiserum .
ftpidtimology; Natural infection with In later stages of the disease , pneumococci arc less
pneumococci has been reported in some species of abundant.
animals such as guinea pigs but they have little The sputum , after homogenisation if necessary,
relation to Homan disease . The source ot human is iiurculated on blood agar plates and incubated at
infection is the respiratory tract of carriers and less 37 C under 5-lti per cent CO ,. Growth occurs
"
often, of patients . PnturnOcocci occurin the throat after overnight tncLihatiu-n. Where sputum is not
of approximately half die population sampled at available, as in infants, scrum- coated laryngeal swab *
any one time , They are transmitted from one to may be used for culture. Isolation from respiratory
.mother bv fingers or hi inhalation of contaminated secretions is facilitated by using blood agar
droplets or droplet nuclei . Dissemination is containing gentamicin 5 pg/ml.
facilitated bv crowding. l- rom specimens where pneumococci are
Infeciion usually leads only T O pharyngeal expected to be scanty illation may be obtained by
carriage . Disease results only when the host intraperitoneal inoculation in mice, even if cultures
resistance is lowered by contributory factors such me licgaliie . lnuculated mice die in 1-3 day ?, and
as respiratory viral injections , pulmonary pneumococci may be demonstrated in the peritoneal
congestion, stress, malnutrition , immunodeficiency exudate and heart blood . The test may be negative
or alcoholism- Splenectomy and sickle cell disease wirh occasional strains that nr avirulcnt tor mice
arc important predisposing conditions. ( type 14 strains).
Pneumococcal serotypes vary greatly in In the acute stage ot pneumonia, the organism
virulence. I hfi case totality rates it pneumonia
( mav be ubtained from blood culture in glucose
Furtlur Reading
American Academy oFPlcdiatria 1997 . Therapy for children «nrh iimrivc pneumococcal infection , fttfatrin W:289.
jvnbs MR and PC Applebaum 199S. Antibiotic resistant pneumococci , Rev Mft / Mjarbiof 6 i 77-
Turima >»rn El Ct *1 , IW 5- n <>cnoLa] iriicL tmiL Ativ ting! J McJ .3.12:12 B0.
'
Ilf|
) nfij[
Copyrighted material
in Neisseria
CM
‘JO* / .,:
I
that occur as ccunmensab m the mouth or the upper
respiratory tract .
NEISSERIA MENINGITIDIS
( Meningococcus ; Diplocdccus intraCtUaftris
meningitidis ) Meningococci^ first de -scnbfd
and isolated in 19S7 by Weichselbaum from the
-•*» .*s .a
, .2
‘
* ® l»
spinal fluid of a patient. f *
N . meningitidis causes meningococcal
meningitis [ formerly also known an cerebrospiral >
fever) which may occur sporadically, as localised
outbreaks or as epidemics, and also septicemia. Fig. £ 5.1 Merlin gecuceUs In Hold- Inset
Mnrphol y; Meningococci are Gram negative
^
oval or spherical cocci 0.6-08 pm in size ,
- enlarged view to show flat
cocci .
adfi sidn of the
the cocci arc more regular and generally 5-10 per cent CO, and high humidity.
intracellular TheJT? arc non motile . Most fresh On solid media , after int ibatfon for 24 hours ,
isolates are capsulated . rbe colonies are small (about 1 HUM in diameter )
t inltm- ul charLicteristics: Meningococci translucent , round , convex, I - M H - I grey, with
have exactinggrowth requirements and do not grow smooth glistening surface and . li entire edges.
on ordinary media. Growth occurs on media The colonies are typically lertTlCi l . r in shape ,
- r
OfJVt rh
"
d material
* Neisseria 223
rough types of colonies are found. Growth is pour siihtypeS based on outer membrane proteins and
m 1 i.c ]vimedia, producing a granular tutbidi tv with poLynaccharides.
little or no surface growth. Resistance: Memngococci arc very delicate
By .LU;HT , chocolate agar and Muelle H niton organisms , being highly susceptible to heat ,
^
starch casein hydrolysate agar ire the media -
dessication, alterations in pi I and to di aufectants.
cii mm -. m I v used for culturing men ingncocci .
-
Modified Thayer Martin { with vancomycin ,
-
They were uniformly sen .:rive to penicillin and
other antibiotics, but resistant strains have emerged
nilislir and nystatin ) i > a useful selective medium . and become common m many arexs-
Eliocheitiivd rCACtions: They arc catalase and Pathogenicity: Cerebrospinal meningitis and
oxidase positive. The prompt oxidase reaction helps nieningpcoccai septicemia are the two mu in types
the identification of ncisseria. ( both meningococcus of meningococcal disease . Meningococci are strict
and gonococcus) in mixed cultures. When freshly human parasites inhabiting the nasopharynx .
prepared 1% solution of oxidise reagent ( tetrameihyl Infection is usually asymptomatic. In some, local
pataphenylene diamine hydrochloride) is poured inflammation ensues, with rhinitis and pharyngitis.
on the cuicure media, the neisseria colonies cum Dissemination occurs only in a small proportion .
deep purple. Subcultures should be made The manner in which the cncci Spread from
immeilLately, as the organism dies on prolonged the nasopharynx tn the meninges may he directly
exposure to the reagent. The test may also be along the perineural sheath of the olfactory nerve,
performed by rubbing a little of the growth with a through the cribriform plate to the subarachnoid
loop on a strip of filter paper moistened with the space, or more probably, through the bloodstream.
oxidase reagent ( Kovach method ). A deep purple In certain cases the -. itc of entry of the
colour appears immediately. meningococcus may be the conjunctiva. Cases of
Indole and hydrogen sulphide are not produced meningococcal purulent conjunctivitis occur. On
and nil rates axe not reduced - Glucose and maltose reaching the centra! nervous system, a suppurative
are utilised, but not sucrose or lactose, producing lesion of the meninges is set up, involving the surface
acid but no gas (gonococci acidify' glucose but not of the spinal cord as well as the base and cortex of
maltose ). Arid formal ion by neisseriae is weak, the brain. The cocci are invarmhly found m the
being oxidative and therefore best tested on peptone spinal fluid, both free and within the leucocytes .
scrum agar slopes containing the sugar and Case ferity is variable but in untreated cases may
indicator. be as high as SO per cent. Survivors may have
Am iconic properi ica and DIBBM float ion: sequelae such as blindness and deafness. Some cases
Meningococci are capsulated , unlike other develop chronic or recurrent meningitis
ni ' isseriae . Rased on their capsular polysaccaride Mcningocooicinia, presents -as acute fdver wiith
antigens, meningococci are classified into at least chills, malaise and prostration. Topically a petechial
13 scrogroups, of which Groups A, B and C are
the most important. Group A is usually associated
=
rash occurs early i i the disease- Meningococci may
be isolated from the petechial lesions. Metastatic
with epidemics and Group L mostly with Idealised
outbreaks, while Group R causes both epidemics
-
involvement of the pun ears, eyes, lungs and
,
Copyrighted materia
224 * Tflsitbook of Microbiology *
intravascular coagulation. and multisystem failure. It is necessary to establish the specific etiology in
Rarely chronic meningococcemia may be seen. purulent meningitis for proper treatment . In
Meningococcal disease is favoured by deficiency' meningococcal meningitis, the cocci are present in
of the terminal complement components (C5 C9).
The pathogenic agent in meningococcal disease
- large numbers in the spinal Auid and , in the early
stage, in the blood as well. Demonstration of
appears to be the endotoxin [ LPS) released by meningococci in the nasopharynx helps in the
-
autoJy js. The vascular endothelium is particularly
-
sensilive to the endotoxin. All itt.ii' it mLl .imm iUirv
detection of carriers,
1. £ jtnnii| ] fii >fi of £ .’.S7 The fluid will be under
j
.
cascade systems as well is cytokines and nitric oxide pressure and turbid, with a large number of pus
are triggered and uprcgulated. In fulminant cases cells . For bacteriological examination, if a
adrenal hemorrhage and profound shock are sufficient | L uarttity is available , the CSF is
-
only n crvi u r of the mci i i ngococcus, Asympn ? mat ic
nasopharyngeal carriers rarely contract the illness
agglutination or counter Immunoelectrophoresis
using meningococcal antisera. Similar tests are
hut serve to infect their contacts. Transmission is also available for pneumococcus, H . influenzae
essentially hy airborne droplets or less often by type b and Group B streptococcus antigens.
fomhes. During intcrepidcmk periods, the carrier Antigen detection is particularly useful in
-
race is about 5 10 per cent . An increase in carrier parII. illy treated patients in whom smear and
rate heralds the nr Ret of an epidemic . During culture tests may be negative . The second
epidemics the carrier races in closed cummumiiL' E portion of the CSF is inoculated on blood
-
may go up co VO per cent, Meniiigi.ii is common
m children between 2 months and 5 years of age.
agar or chocolate agar plateR and incubated at
35-3fi T under 5-10%. CO{. Colonics appear
Epidemics usually occur in semidosed communities —
after IS 24 hours and may be identified by
living in crowded conditions, as in jails and ships morphology and biochemical reactions. It is
formerly, and in army camps in recent times. i mportant to remember that morphologically
Prevalance of mei:ingiii > is highest in the
' meningitis betr of Africa' stretching from Ethiopia
.
similar organismR such as iV, flavesccns JV, fljrva
and Acinetobtcter may also cause purulent
to Senegal. Frequent epidemics have occurred here . meningitis occasionally. The isolated
One of the largest was in lV9ft , when 150,000 cases meningococcus may he grouped , if rcqiiired* by
and 15,000 deaths were reported. agglutination with the appropriate sera . The
Laboratory (JUL IKKUS: The primary agents third portion of CSF is incubated overnight,
^
causing purulent haeierial meningitis are either as it in or after adding an equal volume of
meningococci , pneumococci and Haejnopfi .' fus glucose broth and then subcuitured on
influenzae type h. Other important causative agents chocolate agar . This method I N .LV sometimes
air group B streptococci, staphylococci, Escherichia succeed where direct plating fails.
coir and Listeria monocyfqgenes- 2 . Blood culture: In menulgocuCccniia and in early
Copyrighted material
* Natasaria 225
cases of meningitisP blood -culture is often arc very high in the household or close contacts of
positive Cultures should be incubated For 4^7
.. meningococcal patients, they should be provided
days , with daily subcultures. Meningococcal
. with chemoprophylaxis .
antigens can be found in che blood in active Monovalent and polyvalent vaccincs arc mMk
disease. . containing the capsular polysaccharides of groups
3 . .Vasap/isiyngBjil swuh: This is- useful for the A , L\ W-135 and V . The vaccines induce good
detection of carriers. Sampling should be done immunity after a single dose in older children and
without contamination with saliva. The swab adults hut arc of lirtLe value in children below
should be held in a suitable transport medium 3 years. The immunity is group specific . There is
( for example, Stuarts ) till it is plated . no Group B vaccine available ar present.
4 . Petechia/ lesions: Meningococci may
.
Copyrighted materia
22b 4 Textbook of Microbioiagy
or slightly umbonate, with finely granular surface in the exchange of molecules across the outer
and lobate margins. They are soft and easily membrane.
cmul ' i liable. Protein II is related to the opacity of the
Four types of colonies have been recognised - gonococcal colonies and so is called the 'opacity
Tl to 7 4.Types 1 and 2 form small brown colonies. associated' (OPA ) outer membrane protein. Strains
The cotip .LTL " jiiIi .L1 CL ] , autoaggluLinsble and virulent. with the OPA protein form opaque colonies and
Types 3 and 4 form larger, granular, nonp igrocnted those hi . king it transparent colonies. A strain may
colonies. T3 and T 4 cocci are nonpiliatcd, Form express 0 to 3 serological varieties of the OPA
smooth suspens i i ms and are avi rulcnt. Fresh i ^ oktes prorein at a rime- OPA may be responsible for
from acute cases of gonorrhea generally form Tl attachment to the host cells and also for the
and T 2 cnlnni.es. On senaf subculture, they change clumping af cocci seen in urethral exudate smears.
to T3 or T 4 colonial morphology. Tl and T2 types I’ hc outer membrane also contains
.
arc also known as P" and P" respectively, while lipopolysaccha ride (endotoxin ) which may be
1 > and 14 are known as P . "
responsible for the toxicity in gonococcal infections.
E i cwhemi L'al react inns- Gonococci resemhle Many other proteins of poorly defined roles in
meningococci except in the effect on maltose- pathogeniciry have been described. Both gonococci
Gonococci acidify only glucose and not maltose . and meningococci elaborate IgAl protease that
Antigenic properties Gonococci are splits and inactivates IgA.
antigemc& lly heterogeneousc 1 hey arc capable of RuistAAne The gonococcus is a very delicate
changing their surface structures in vitro, 7*hcy organism , readiily killed by heat , drying and
probably do so in vivo as well, to avoid host defence. antiseptics. Tt is a strict parasite and dies in 1-2
The surface structures include the following;
Pifj which are hairlike structures several
micrometres long, act as virulence factors by
promoting attachment to host cells and inhibiting
hours in exudates outside the body.
—
In cultures, the coccus dies in 3 4 days but survives
in slant cultures at 3? °C if kept under sterile paraffin
oil. Cultures may be stored for years if frozen
phagocytosis, Pili are composed of repeating peptide quickly and left at -70 °C.
subunit ?. ( pilins) consisting uf conserved (uonstanl ! lJHthojJi;n , , - iiy ? Gonorrhea is a venereal disease
and variable regions. Fill undergo antigenic and which has been known from ancient times. The
phase variation. name gonorrhea ( mcaniTig> flow of seed ) was first
The trilaminar outer membrane of gonococci employed by Galen in 130 AD.
contains many different proteins. Protein I is the The disease is acquired by sexual contact . The
major constituent ind shows antigenic diversity, first step in infection is adhesion of gonococci to
which helps in typing gonococcal strains. Protein I the urethra or other mucosal surfaces. Fill are
of a '- ingle strain is antij emcally constant, though involved iti this adhesion. Adhesion is rapid and
^
It shows considerable heterogeneiry between firm so that micturition after exposure offers no
different strains, There we two variants of protei n L, protection against infection. The cocci penetrate
called IA and IB, Any one strain carries only either through the intercellular spaces and reach the
LA or IB but not both. Using monoclonal antibodies subej4cbeiial connect i ve ti HSLLC by the th Lid day after
to protein 1 epitopes, gonococci can be classified infection, The incubation period is 2-8 days. In
Litfo several setovars, Al to 24 and Bl to 32 . men , the disease starts as an acute urethritis with a
Proteins I and III act as ligands attaching the mucopurulent discharge containing gonococci in
coccus to the host cells. They also form a
large numbers . The infection extends along the
tratsmembrane channels (porms ) which play a role urethra to the prostate , seminal vesicles and
Copyrighted material
I JF W
* NetSBflria * 227
epididymis. Chronic urethritis may lead DOSTRICTURE animals . Experimental disease may be produced in
forma r ion . The infect - on may spread to the chimpanzees by urethral inoculation . A lethal
periurethral tissues, C. IIKIIIC uhseenses and multiple infection can he produced in mice by intracerebral
dischargm sinuses ('wacercan perineum"). inoculation.
^ , the initial infection involves the
In women The only source of infection is a human carrier
J
urethra and cervix uteri , "i"hc vaginal mucosa is not or less often a patient. The existence of asymptomatic
usually affected In adults because the straiificd carriage n women makes them a reservoir serving
squamous epithelium is resistant to infection by the to perpetuate infection among their male contacts.
cocci and also because at the acid pH of vaginal The made ni infects in Is almost exdu:- ivclv venereal.
"
if
secretions. { Vulvovaginitis occurs in prepubertal Fomices do not play any significant role, as the rood
girls ). The infection may extend to Bartholin's die rapidly outside the human body. The only
glands , endometrium and fallopian tubes . Pelvic nonvenereal infection is ophthalmia neonatorum.
inflammatory disease and salpingitis mav Lead to Ort.ee very arniman , thin has been controlled by
sterility. Rarely, peri tor it is may develop with the practice of instilling 1 % silver nitrate solution
perihepatic inflammation ( Pitz — Ifugh -Curtis into the eyes of all newborn babies (Grade's method ).
syndrome ) , Clinical disease Is as a rule less severe When sulphonamides, and later penicillin, were
in women, many of wliom may cany gonococci in found very effective for the treatment of gonorrhea,
the cervix without any symptoms. Asymptomatic it was hoped that the disease could be eradicated.
carriage of gonococci is rare in men, However, after a temporary decline, its incidence
Proedv. - occurs in both sexes. It may develop has been rising steeply. In 1970, the global incidence
by direct contiguous spread in women but in men of new cases was estimated at 16 million , making
i s usually the result of anal sex . Conjunctivitis may h one of the most common infective diseases. In
occur, usually due Co aufr ijnocuf ation by the pat i ent 's same areas, gonorrhea has reached epidemic
fingers. Blood invasion may occur from the pri maty proportions , especially in adolescents and young
site of info. don and mav lead to metastatic lc-- Ii > n^
, adults . The reasons for the Increase in gonococcal
such u arthritis , ulcerative endocarditis and verv infection are largely social and cultural. In the
rarely meningitis. Occasional cases of pyemia have 19B0s, with the AIDS scare, there was a noticeable
been reported. decline in the incidence of gonorrhea, but this has
A nonvencreal infection is gonococcal not been kept up , A higher incidence of gonorrhea
ophihilmoa in che newbornr which results. from has been observed in persons belonging to blood
direct infection during passage through the birth group B. The bans for tins not known.
canal. Gonococcal bacteremia leads to skin lesions, Laboratory diagnoita In the acute stage ,
especially hemorrhagic papules and pustules on the diagnosis can he established readily bill chronic
hands, forearm , feet and legs * and to tenosynovitis cases sometimes present great difficulties , Ill acute
and suppurative arthritis, usually of the knees, ankles gonorrhea the urethral discharge contains
and wrists. gonococci in large numbers. The meatus is cleaned
Gonococci contain several plasmids . Nincty-m with & gauze soaked in saline and a sample of the
per cent of the strains haw a small cryptic plasmid discharge collected with a platinum Loop for culture ,
of unknown function . Two other transmissible or directly on slide for smears . In women, besides
plasmids contain genes that code for beta lactamase the urethral discharge, cervical swabs should also
which causes resistance to pcnicillin - be collected - This should be done carefully, using a
: Epidemiology: Gonorrhea is an exclusively speculum. High vaginal swabs arc not satisfactory
human disease , there being flu natural irtiectiort in
; In chronic infections, there may not be any
Copyrighted material
22% ^ TemtiOO* 01 Microbiology
oe .tr
I
"5?
-.*£> j.O** *
1
rapidly Again, when penicillin was introduced, all
strains were highly sensitive ( \1IC 0.005 unit/ ml ) .
From 1957, strains with decreased susceptibility
4
-
.8 ( MIC higher than 0,1 uniit/ml ) became common .
u
i As patients infected with such strains did not
m
ITJTI LJ F"
1
4 : tfV respond to the usual doses of penicillin , vciy large
.
doses of penicillin, 2.4-4 .fi million units were used .
_ From I 97fi , gonococci producing p- lactamase
-. yCr -
r
IT'
TI
( penicillinase ) have appeared, rendering penicillin
yiLr - treatment ineffective. These penicillinase- producing
IV, onrrrrllOCJe ( PPNG } have spread widely.
^Tlic Centers for Disease Control anil Prevention ,
Fig . 25.2 Gonococci In urethrai pm Inset: enlarged . USA in 1993 recommended the following schedule
view lo shew kidney Shaped cells with adjacent for uncomplicated gonoirhca: Ceftriaxone 125 mg
surfaces concave . single INI Jose or Ciprofloxacin .500 mg (or
Copyrighted materia
* Neisseria 229
Ofloxacin 400 nig ) single oral dose , plus due to gonuCOccal infection, the coed persisting us
Doxycyoline 100 mg twice daily for 7 days or L-forms and hence undetectable by routine tests.
Erythromycin 1 g single oral dose . The regimen is The majority of such cases are , however , the result
costly hut wmrltH very well against gonococci and of infection s ofdivcrsccriology. Thc most important
'
the frequently coexisting chlamydial infection. of these arc CAfam. Cndumitii , UrcapIaMma
Prophylaxis: Control of gonorrhea consist* of
early detection of cases , contact tracing , health
education and other general measures , As even
^
uia ticum and iWm /a ma bonuais. J lerpesvinis
^^
and cytomegalovirus may also account for sonic
cases . Urethritis may also be caused by other
clinical disease does not confer any immunity, bacteria ( for example , Gardncrcllt nginilis ,
vaccination has no place in prophylaxis , Ac- jnerohuL- rer IwofTi , Ac. cilccActdcitsX fungi
( Candida albicans ), pmtotna (Trichomonas
NONGONOCOCCAL {NONSPECIFIC} URETHRITIS vaginalis ), or even by mechanical OJ chemical
Along with an increase in the incidence of irritation . As etiological diagnosis is soldum
gonorrhea, there has also been an increase, in recent achieved , the management of this syndrome is
years, of eases of chronic urethritis where gonococci difficult ,
cannot be demonstrated . This has been called
lurlgOtluCoccd Or notlgpcdfr: urethritis. In some COMMENSAL NEISSEHIAE
of these , urethritis forms part of a syndrome Several species of Ncisscriac inhabit the normal
consisting of conjunctivitis and arthritis in addition respiratory tract . The characteristic features of yjroc
( Reiter's syndrome ). Some of these cases may be of the common specie: arc bated in Tabic 25, 1 -
Table 25.1 Differential characteristics of Neisseriae
Copyrighted materia
230 * Textbook of Murobiology >
Their pathogenic significance is uncertain though colonisarion by N- Jacranncs in young children may
'
some of them ( for example * N , fi& ve&cen& y N . be responsible for the presence in them of antibodies
citarrhzli * ) have been reported occasionally as protective against mcniitgococcal iitfecdon.
having caused meningitis. jVeiiieru catarrhaUy is now classified as
.V . iaetsmjL’ jf frequently isolated from the Maraxclli { BranhumcHa } catarrhxtis . It i * an
nasopharynx is closely related to meningococci , opportunistic pathogen capable of causing laryngitis *
though it is virtually aviinilcnt . It dirTers from bronchopncumoria, men i rgitis, n, nus i i is and middle
pathogenic nei &aeriar in being positive in the ear disease .
ONPG test tor beta galactosiiiase. Nasopharyngeal
Further iteailjnj
Britigan BE and PF Spading 1965. Gonococcal infection . New Engl J Med . 1 . ( <? ! . '
Rcscntstcm NC et il. 2001 . Mci in tjcwca] diseases , .’Vew tngiJ Med , 334: 1376
^
Tunlcel AR and MW Schrld 1995 . Acute bacterial meningitis. fjnerf , 346: 1675.
LJ opyrighted materia
Corynebacterium
Coryneb nutria j-rt- Gram pusitive h ornnidd fast, observed. They are Gram positive but tend to be
iqonmptilc rods with irregularly Plained. segments, decolorised easily. Granules composed ut
and somerimes granules. They frequently show dub polymctajihnsphare air seen in the cells. They air
Heaped swellings and he nee the name more strongly Gram positive than [he rest of the
coiyncbacteria ( from cvryne , meaning club) lire
, bacterial cell - Stained with LoefflptV methylene
most important member of the genus is blue, the granules take up a bluish purple colour
C- diphtheriae, the causative agent of diphtheria. and hence they are called me [ achromatic granules.
Diphtheria has been known from ancient times. They are also called vohjrijior Babes Einst granules.
Aretacus, the Cappadocian, in the second century, They 3 re often situated nr the poles of the bacilli
described the Egyptian or Syriac ulcer, which most and are called polar bodies. Special stains, such as
medical historians agree can he identified as Albert’s, Ncisscr’s and Ponder ’s have been devised
diphtheria. The disease was first recognised as a for demonstrating the granules clearly. The bacilli
clinical entity by Rretormcau who called it ate arranged in a characteristic fashion in smears .
Copyrighted material
232 4 Textbook or Microbiology *
/A mu
= f -
\ fc
\ *s c
\ <£
common in gravis , hemorrhagic complications in
gravis and intermedius, and obstructive lesions in
the air passage in mitis Infections, 111 general, mids
\ o~
/ A- ! V is the predominant strain in endemic areas, while
gravis and intermedius tend to be epidemic. The
»\ ^ l
mitis type is better able than the more virulent types
!s
^
in establish a commensal relationship with the
>
i .
host Wide variations have been noted in the
frequency of the different types in different places
& at different times. There is evidence to show that
Copyrighted material
4 Corvi ' Efbadtmum 233
Morphology Usually short rods, with Long barred forms writh Ijnng, curved ,
uniform scaining, few or no dubbed CM kb; poor pleomorphic rods wirh
granules. Some degree of granulation, very prominent granules
pleomorphism., wirh iireguladv pleomorphic
barred, snow-shoe and (ear -
drop forms
Colon v on tellurite In hours, colony is 1 2 mm * IK hour colony small* Size triable* shiny
blood Hgur in size , with greyish black
, 1mm in size* misty. Does black. In 2-3 days,
centre* paler, semitranslucent not enlarge in 48 hours, colonies become flat *
periphery and commencing dull granular centre with with a central
crcnation of edge- Tn 2-3 days* smoother, more elevation poached egg
-
3 S mm in si- sw, fUr colony
with raised dark ten ( re and
glistening periphery and
a lighter ring near The
colony
crcnared edge wirh radial
-
srriarion ‘daisy head colony 1
-
edge "frogs egg" colony
Consistence of
V
Like cold margarine * bri ( tEe,
1
„ Intermediate between Soft, buttery, easily
colonies moves AS ii whole on the plate, gravis and mirif HuibafiaUe
not easily picked out or
emultifiable
Hemolysis Variable Nonhemolvtic
t
Usually hemolytic
Growth in broth Surface pellicle, granular Turbidity In 24 hours* Diffuse TUffcidicy with
.
deposit Little or no turbidity clearing in 49 hours* soft pellicle later
with line granular
sediment
bacilli is qualitatively similar. The sfrdin almost toxin is inactivt because the active Hite on fragment
universally used for toy in production is rhe ' Mark A is masked. Activation is probably accomplished
Williams 3' straiji , which has been variously by proteases present in the culture medium and
JcM- rilKul as a initis- ( Tiipltry and. WiluKi ]
and ail infected tissues. All the enzymatic activity of the
irfermeditLs strain Cmickshank .
( ) tone in is present in. fragment A. Fragment B is
The diphtheria toxin is ; L protein and has lieen responsible for binding the toxin to the cells. The
crys- tahayecl. It has a molecular weight of about antibody to fragment B is protective by preventing
62 n 000, It is ratnemcly potent aTid the lethal dose the binding of the toxin to the cells. The toxin LH
For A 250 g guinea pt is 0.0001 mg. It consists of labile. Prolonged storage, incubation at 37 C for C’
^
wo fragments, A and B, of MW 24 n (XX> and 3? ,000, —
4-/> wceksp treatment with 0.7 0.4 per cent formalin
respectively. Both fragments are necessary for the or acid pH converts it to toxoid. Toxoid is toxin
roxie effect. When released bv rfie bacterium, the
Jr r that has lost Its toxicity but not its antigenicity, It is
Copyrighted materia
234 * Textbook al Microbiology *
concentration nfiron in the medium. The optimum occasion he as shore as one day. In carriers , the
level ol iron for toxin production is 0.1 nig per incubation period may be very prolonged. The site
litre, while a concentration of 0.5 nig per litre ol infection mav he ( 1) faucial; ( 2 ) laryngeal;
inhibits the formation of toxin . The diphtheria ( 2 ) nasal; {4 otitic; ( 5 ) conjunctival; 16) geriHxH
)
toxin act* by inhibiting protein synthesis . vulval, vaginal or prcpucial ; and { 7 ) cutaneous,
Specifically, fragment A inhibits polypeptide chain' which is usually a secondary infect ion on pre-
elongation in the presence of nicotinamide adenine existing skin lesions . Sometimes diphtheritic
dinucleotide bv inactivating the elongation factor , whitlow or ulcer mav occur. Cutaneous infections
-
/
EF 2 . It has a special affinity' for certain tissues ate commonly caused by nontoxigenic strains of
such as the myocardium, adrenals and nerve diphtheria bacilli .
cmlings - Faucial diphtheria is the commonest type and
Re*istance: Cultures may remain viable fer two mav vary from mild catarrhal inflammation to
or metre weeks it 2S-3Q "C . It is readily destroyed verv widespread involvement . According to the
by heat in 10 minutes at SK :> C and in a minute at clinical severity, diphtheria may be classified as:
r
100 ^C . It is more resistant tn the action of light, 1. Malignant nr hypertox ic i n which there is severe
desiccation and freezing than most nonsporing toxemia with marked adenitis ( buUneck). Death
bacilli . Ir has been cultured from dried bits of i « due to circulatory failure. There is high
pseudomembrane after 14 weeks. It remains fully incidence of paralytic sequelae in those who
virulent in blankets and floor dust for five weeks . Ir recover .
is easily destroyed by antiseptics. Li is susceptible 2. Septic, which leads to ulceration , cellulitis and
tn penicillin, erythromycin and broad spectrum evert gangrene around the pseudomembrane;
antihintics. and
Antigenic structure: Diphtheria bacilli arc 3. Hemorrhagic , which is characterised by
anrigeideally heterogeneous . By agglutination , bleeding from [ 3 ie edge of the membrane,
gravis strains have been classified into 13 types , cpistaxis, conjunctival hemorrhage, purpura and
intermedins into 4 types and mitis into 40 types . generalised bleeding tendency.
Gravis strains of types I and 111 have been reported The common complications are:
to be common in Great Britain , type II worldwide , I. Asphyxia due to mechanical obstruction of the
type IV mainly in Egypt and tape V in the USA . respiratory passage by the pseudomembrane for
Copyrighted material
i CoryMtiLrlLiltdi 235
which an emergency tracheostomy may become 6- clear, cloudy or bloodstained pleural exudate;
necessary.
jr
and
2, Acute circulatory failure , which may he 7. soTnetlines, pericardial effusion.
peripheral or cardiac, I nbrotatory diagnosis Laboratory
3, Pbstdiph thei me paralysis, wl ic h tyf > ic ally occurs confirmation of diphtheria is necessary for the
m the third or fourth week of the disease ; initiation of control measures and for
palatine and ciliary but not pupillary paralysis epidemiological purposes but not for the treatment
i -; characterise < , and spontaneous recovery' is the of individual cases- Specific treatment should be
rule. instituted unrnftintrefy on suspicion of diphtheria
4, Septic, such as pneumonia and of . n -j mcdin - without waiting for laboratory tests. Any delay may
Kelapse may occur in about 1% of cases. be fatal.
Diphtheria is a toxemia. The bacilli remain
contined to the site of entry', where they multiply
-
Laboratory diagnosis con i '- ts o!isolation of the
diphtheria ba . itlu -- and demonstration of its toxicity.
and form the to \ in , Thc to \ i:i causes local ncctor: c One or two swabs from the lesions are collected
changes and the resulting fibrinous exudate , under vision, using a tongue depressor. Diphtheria
-
together with the di integraring epithelial cells,
leucocytes, CTythrocytCS and bacteria, constirutc the
bacilli may not always he demonstrable in smears
from the lesion, nor car thev be confidcntlv
pseudomembrane , wh . ’ h is characteristic of
'
i different ated from some commensal coryncbactcria
diphtheritic infection. The mechanical normally found in the rhroac . Hence smear
com pile it ions of diphtheria are due to the examination alone is not sufficient for diagnosing
membrane, wh: k the system ic effects arc due to diphtheria but is important in identifying Vincent’*
the toxin. anyma. for this, a Gram or Ln- hmurt. stmned smear
Nonloxigenic str.iins of diphtheria hacilii may is examined for Vinocnl s spinochetes and fusiform
cause infection even in immunised individuals, as bacilli. Toxigenic diphtheria bacilli may be identified
immunirv. with the toxoid does not confer
Jf
in smears by immunofluorescence. For culture, tlxe
antibacterial immunity, Such infection is mild
J
swabs are inoculated uiiLoeffler 's serum slope, tellurite
though pseudomembrane formation may sometimes blood agar and a plate of ordinary blood agar, the last
occur , for differentiating streptococcal or staphylococcal
Diphtheria does not occur naturally in animals pharyngitis, which may simulate diphtheria . If the
but infection can be produced experimentally , swab cannot be i r.oculated promptly i t should kept
Susceptibility varies in different species . moistened with sterile serum so that the bacilli remain
Subcutaneous inoculation of a guinea pig with a viable. The scrum slope may show growth ML 4-B
culture of virulent diphtheria bacillus will cause hours hut if negative, will hive to be incubated for 24
—
death in 1 4 days . At autopsy, the following features
can be observed:
hours. Smears stained w: rh methylene blue or one of
the special stains ( Ncisscr or Albert stain ) will show
1. gelatinous , hemorrhagl -, edema and, often, the bacilli with metachromadc granules and typical
necrosis at the site of inoculation; arrangemeiit.TeUuriiie plates will have to be incubated
2 . swollen and congested draining lymph nudes;
3. peritoneal exudate which may be clear, cloudy as growth may sometimes be delayed. The tellurite
-
for a; least two days bd ore heii ig con i .lien d nc p'l ive,
^
or bloodstained; medium is particularly imjvirtant in the isolation of
.4 congested abdominal viscera; diphtheria bacilli from convalescents, contacts and
5- enlarged hemorrhagic adrenals, which is the earn era as in these cajscs they may he outnumbered
pathognomonic feature; bv. other bacteria,
-' n
f
opy righted material
KW.1
256 i Textbook. ot Microti ology
, »
Vlt! l I KMCR TESTti dish while the medium is still fluid. If hnree
Any isolate of the diphtheria bacillus should he scrum is not available, sheep or rabbit scrum
betted fur virulence or ttuigenicity for the may he used . When the agar has set, the surface
is dried and narrow Streaks 0-f the Strains are
bacteriological diagnosis ro be complete, Virulence
testing may be bv in vivo or in Vitro methods, the made at right angles to the filter paper strip. A
positive and negative control should be put up .
former hv. the subcutaneous or i ntradermsl test and
X
In vitro test
i . Kick !y t;vf pn iphtifum teat: A rectangular
^ FI -9 - 2E .S Elgk £ test. A - Fitter papsr
strip of filter paper impregnated with diphtheria impregnated
antitoxin (1000 -units ml ) is placed on [lie surface wliti A. ftlphihtfiw intitorin, Toxigenic sirs in ,
of a
^
normal horse serum agar in a petfi C. Nonto* igenic strain . 0 . T strain shoeing
lemlgonletly .
Coryn-^u a clorlum 237
Important pediatric di-tase all over the world but PROPH’i L wis
followi ug the development ofeffect i ve prophylat tics Diphtheria can he controlled by immunisation .
and mass Immunisation , the disease has been Three methods of immunisation are avi . liable ; , 1
virtually eradicated from most advanced countries. active, passive and combined. Of these, only active
In those dcwlo]'ing countries where childhood immunisation can provide herd immunity and lead
Limnui i isation programmes have been i nplemented
to cradic;i tion of the disease. Pass: vc and combi ned
[
effectivdy, diphtheria has become rare but in others immunisation can only provide emergency
it continues to be a serious problem. The prolonged protection to siw;ep1 ihUL i 11Llii. ILIUS!- ex loosed to risk.
'
>pyrighted material
H dr w
* Texmoo* ol Microbiology >
and was due to rbc ability.' Lit'thc toxins and antitoxins at intervals nf at least tour weeks, and preferable
to combine in vatying proportions. When the toxin six weeks or mom , followed by a fourth dose about
v/ , L >. IDELJ in instalments, the toxin added first
js a year afterwards. A further booster dose is given
combined with imnrethan i equivalent of Antitoxin , at school entrv.
laving insufficient antitoxin behind to neutralise Paative tmm uni station: This is an
[ he toxin added HuhvequeiLily. emergency measure to be employed when
Several other preparations were introduced for suHccptibles are exposed to infection, as when a case
active immunisation (for details, the third edition o ( diphtheria Is admitted to general pediatric wards.
of this textbook may be consulted ) . Only rvvu It consists of the subcutaneous administrarinn of
preparations are in Line nmv , Fonnol toxoid ( also, 500-lQQti units of antitoxin (imtidiphtheriric serum ,
known as fluid toxoid ) and adsorbed toxoid. For moi ADS), As this is a horse scrum, precaution against
toxoid is prepared by inctibating the train with hypersensitivity should be observed.
formalin at pH 7.4 7.6 for three to four weeks at < lonihincil immunisation: This consists ot
37 'C until the product is devoid of toxicity while administration of the firsr dose of adsorbed toxoid
retaining immunogcnicity- Adsorbed toxoid is on one arm , while ADS is given nn the other arm ,
purified toxoid adsorbed onto insoluble aluminium to be continued by the full course of active
—
compounds usually aluminium phosphate , lens
often the hydroxide. Adsorbed toxoid is much more
immuiLLSatioo. Ideally, all Cases that receive ADS
.
piophyl Ktically should receive combined
immunogenic than the fluid toxoid . Due to anxiety immunisation.
bout ID enhanced risk of provocative poliomyelitis,
it was replaced by fluid toxoid tar
, SLITTIC time . STAMIUKDI ^ ATIO ^ OF
A N T I T< I \
-
I NS
TOXINS A\M
Copyrighted materia
i Corynebacie'ium * 239
antitoxin unit {AU ) is defined as rhe amount of the amount of toxin which flocculates most rapidly
antitoxin that has the time mill combiningopacity with one unit of antitoxin. The Lf unit has several
far taxi n and toxoid together as one unit ofEhrEch's advantages. It is inexpensive and rapid and docs
original aniitcmn . nut need animals, Ic is also the only method available
Since toxin always contains some toxoid, two for the titration of toxoids . The amount oi toxoid
other units for measurement of toxin have been in prophylactics is expressed in Lf units. Many
introduced, the LO and L + doses. The LQ (Limes other in vitro teats have been developed for antigen
nul ) dose ul the diphtheria toxin is the largest assay. These include cell culture neutralisation tens
amount of toxin that, when mixed with one unit of using rabbit Iddncy cells, passive hemagglutination
antitoxin and injected nbcutuieously into a 250 g test with roxi n coated ta nned sheep RHC , and HI A -
guinea pig, will on the average cause no observable Treatment: Specific treatment of diphtheria
reaction. As 'no reaction is nor a definite end point, consis.rs of antitoxic and antibiotic therapy. Antitoxin
in actual practice, the end point is taker as mini mum should be given immediately when a case is
local edema.The L* (Limes tod ) dose oi diphtheria suspected as diphtheria , as the fatality rate increases
toxin is the smallest amount of toxin rhat when with delay in starting antitoxic treatment. The
mixed with one unit of antitoxin and injected dosage recommended is 20,000 to 1 ,00,000 units
subcutaneously into a 250 g guinea pig will on the for ferrous cases , half tile dose being given
average kill the animal within 96 hours. If toxin intravenously. Antitoxin treatment is generally not
combines with antitoxin in constant proportions , it indicated in cutaneous diphtheria as the causative
tvould be expected that the ditference between the strains are usually nontoxigenic.
I A d wc and the I $ d* we would be ex|ual to 1 MI ,l >,
"
.
C diphtheria? ft sensitive to penicillin and cun
Blit when the estimations arc actually made, it is be cleared from the throat within a few days by
found to wary from 10-100 MLD or more. This penicillin treatment. Diphtheria patients are given
discrepancy is due to the presence in toxin a course of penicillin though it only supplements
preparations of varying amounts of toxoid and to and docs not replace antitoxin therapy,
rhe ability of the toxin and antitoxin to combine in
J
Eiythramyrin ft more active than penicillin in rhe
varying proportions. This is known as the Ehrlich treatment of carriers-
phenomenon.
The use of death as an end point for the titration OTHER PAmOGEWC COflYNE BACTERIA
of toxin is wasteful of animals. Romer intn hduced a C. / //. CEJtA.XS
method of titration employing the erythematous This bacillus related to C. dlphr/tcnac can cause
swelling produced by the intiaJenmal injection of dLphtheriii -like lesions . It resembles the gravis type
toxin , and its neutralisation by antitoxin. The of the diphtheria bacillus but it liquefies gelatin ,
minimum reacting dote ( MRDJ is the leutuioint fcrroenis trehalose slowly and does not reduce nitrate
of toxin that when iniectrd intradermallv in a guinea
, to nitrite . It produces two types of toxins, one
pig, causes an erythematous flush 5 mm in diameter prohahly identical with the diphtheria toxin and
visible alter 36 hours. 'Hie Lr dose is the smallest the other resembling the t o x i n o f
amount of toxin which, after mixing with 1 unit of C. pscvdatubawlaiiz It is pathogenic to guinea
antitoxin , will produce & minimal skin reaction when pigs , the lesions produced resembling those caused
injected irUrndcrmally into J guinea pig. by C. diphthen«, It lias been found to cause
Ramon introduced a test tube method for in lection in cows, ami human infectw may be
titrating toxin and antitoxin based on flocculation. transmitted through cow 's milk. It is sensitive To
The flocculating or Lfunit of diphtheria toxin is .
erythromycin , Diphtheri i anritoxin is protective .
Copyrighted material
240 * Textbook of Microbiology *
IT lias been suggested that C’. ukem /ii may be and other areas.These may sometimes be mistaken
considered i subgroup of diphtheria hacilU rather for diphtheria bacilli and arc called diphtheroids.
than a separate species, In general, diphtheroids stain more uniformly than
j4ioiiafiiictBHiiii ( formerly Corynebmcteriun}} diphtheria bacilli, possess few or no mccachromatio
hairmoh-tiLunr ea.n cause phuivngitiB and skin ulcers. granules and tend E u b e arranged i n parallel rthn
C jakdinn can cause cuta neous and blood i nfeetin ins ( palisades) rather than cuneiform pattern. However,
m immunocompromised hosts. IT is usually some diphtheroids may be indistinguishable from
multi resistant, nespnncling only tci vancomycin. diphtheria bacilli microscopically. Differentiation
Cofynebacterlj of veterinary importance are rhe is by biochemical reactions and more reliably by
Preisa Notard Lae ill LIS ( C. juL' utforubervLjJosjfi ), virulence tests, The common diphtheroids are
which causes pseudettuberculosis in sheep and C. paeudodiphthcritiL-um ( C. hairnannri) found in
suppurative Lymphadenitis in horses, C. renafo the din ' ll and C. xenonJ.t found in the conjunctival
causing cystitis and pyelonephritis in cattle and sac. The former is unease positive and does not
C, equf , isolated from pneumonia in foals. forment glucose , while the latter is unease negative
and foments glucose . Both are pjTar.inamltl3. Re
Com NEB At :TERIA CAL SING positive, unlike diphtheria bacilli .
SI r t:i t E H ; ] A I . SKIN INFECTIONS
Erythra Rina , a Localised infection of the stratum OTHER CORYNEFORM UACTEHIA
corneum usually affecting the axilla and groin, is Besides genus Ctujwbflcflenuin, a number of Other
caused by C nuiuiUHnnum , This is a lipophilic genera of coryuefujuj bacteria have been defined .
coryncbactenum and can be grown readily in media Among them , the genus FroprumfMetierftJjn is of
containing 20 per cent fetal calf scrum . medical interest as three species, T! aeries. E
C. renujs has been associated with trichomycosis
'
jnauJonm and F wvixfam, are constantly present
axillaris , characterised by the formation of on human skin. Thev are anaerobic and .icrotoJeraiit,
pigmented nodules around uxitlaiy and pubic hair growing well in lipid containing media. P. £ rnes is
shafts. often isolated from acne lesions bur its pathogenic
role LR uncertain.
DtPHTHBROiD3 CrtrrrttbtfCfttrrum parvum which in frequently
CaryDcbactcria resembling C- diphtheria occur a* used as an immunomodulator is a mixture of
nnmiiil commensals in the throat, skin, conjunctiva Hropiombacterium species.
Further It ending
.
Coyle MB and BA Lipjlcy 1990 Coryneform bacteria in mfretious disease. Clin Mkmhinf iiev 3:227 .
. 9%L R (SLiTi;Knce of diphtheria in the fnDlerSHtelUnm lancet JJ 7:t 99 l
Hardy E K I i v i at| .
. .
Hotier W 1991, Cutaneous diphtheria SntJ Ctrmjml 30: fiJ 5
. .
Zainiri 1 PWN Cprynebacteri urn , 1 n Collet , JG et u ( eda ), JVactrcaJ Medical Alitmlaoingy 1^" edn. Edinburgh: Churchill -
LivifttfHfK ,
]) opyrighted material
1^ Bacillus
CM
Sporogeuous , rod-shaped bacteria are classified Anthrax was for long feared as a potential tool
into two genera , the ucruble Bacilli and the in biological warfare. This fear became an actual
anaerobic Civslriiliz. The genus Bscillus consists fact in 2001, when anthrax in the form of weapons
of aerobic hseiili forming hear resistant spores. They grade spores, having enhanced dispensability and
are Gram positive but rend to be decolourised easily virulence was seni hv mail to various destinations
P
so ns to appear G mm variable, or even frankly Gram in the USA, causing disease and death in many
negative . They are generally motile with persons.
pcritrichout flagella , the anthrax bacillus being :a Morphology: The anthrax bacillus is one of the
notable exception , Members of this group exhibit largest of pathogenic bacteria, measuring 3-10 flm
great diversity in their properties. The genus * 1-1 - b |jm . In tissues, it is found singly, in pairs or
includes psychroph ilic , mcsophilic and in short chains, the entire chain being surrounded
thermophilic species , the maximum temperatures by a capsule. The capsule is polypeptide in nature,
for vegetative growth ranging from about 25 G to being composed of a polymer ofd (-) glutamic acid.
above 75 aC and the minimum from about 5 “ C to Capsules are not formed under ordinary conditions
45 C . Tlirir salt tdlrrute varies from less than
"
of culture but only if the media contain added
2% to 25% NiCl .
Their spores are ubiquitous, being found ir ] soil,
-
bicarbonate or are incubated under 10 25% CO,.
If grown in media containing serum , athumin ,
dust, water and air and constitute the commonest charcoal or starch , capsule formation may occur in
contaminants in bacteriological culture media. the absence of CO , .
BptUftit a:J fhr.ii j.^ , the causative agent of anthrax,
'
In cultures, the bacilli are arranged end to cud
is the major pathogenic species. H . txmis can cause in long chai ns . The c nds of the bacil it arc truncated
tundhnrrLC gastroenteritis. Some species may lie or often concave and somewhat swollen So that a
responsible for opportunistic infections , that n of bacilli presents a 'bamboo stick’ appearance ,
Copyrighted material
242 * Textbook or Mtatobioko y
^
while a virulent or attenuated strmir form smooth
colon ies . OTI gelatin st:th I run a characteristic
a ‘
inverted fir tree' appearance seen * Willi slow
liquefaction commencing from ' top ( Fig , 27.3),
On blood agar , the colonies n nonhemolytic,
rhough occasional strains jimduc < a narrow zone
of hemolysis. In broth growth as floocular
ilrpoiiit , with little or no- rbiditl
When U- anthneis is grown n the surface of a
i solid medium containing "
-
5 ) units of
'
nnriaciti fast , The spores J : > nut stain by ordinary it the susceptibility of if . '
is gamma phage .
methods bur can be stained differentially by special
A selective medium ( PLET medi J l . consisting
techniques. When stained with Sudan black B, fat of polymyxin, lysozyme , lent diamine tetrn
globules may he made out within rhe bacilli . When
acetic acid ( EDTA ) and rl acetate added to
blood films containing anthrax bacilli are stained
heart irthtsion agar, h&s been to isolate iJ .
with polychrome methylene blue for a tew seconds
and examined under the microscope, an amorphous
aathneis from mixtures "tii .i ng other spore -
beating bacilli .
purplish material is noticed around tbc bacilli. This
represents the capsular material and is characteristic
of the anthrax bacillus. This is called the
M’ Fndycans reaction and is employed for the
presumptive diagnosis of anthrax in animals.
"
my righted mafern
* Bacillus * 243
_
- i ' h e 11 < ] IKIL: -. I i r i - isulion *u Glucose , maltose , L:UI
sucrose arc fermented producing acid but no gas .
Nileaiis art reduced to nitrites. Catalase is formed.
-
R t K i s l art c L : The v e ge rat E V e b a c L l 5 i ii re nor
particularly resistant and are destroyed at ftO QC LCI
30 minutes. In the carcasses of animals which have
died of anthrax, the hacilfli remain viable in the
bone marrow for a week and in the skirt for two
weeks . Normal hem fetation of smears may not kill
the bacilli in blood films. The spores arc highly
resistant to physical and chemical agents. They
have been isolated from narurally infected soil after
as. long as 60 years . They resist dry heat at 140 *C
for 1-3 hours and hoiling for 10 minutes . I’hey
survive in 5% phenol for weeks . HgC in a 1/
1000 solution may tail to kill anthrax spores in less
than 70 hours . Four per cent potassium
^
permanganate kills them in 15 minutes. Destruction
of the spores in animal products imported into
nonendemic countries is achieved by 'duckerinjr ' in
which formaldehyde is used as 2<Hi solution at .
Fig 27.3 Anthrax badbus In gelatin stab cullute
30-40 QC for 20 minutes for disinfection of wool showing- Inverted fir tree appearance
and as 0.25 per cent at 60 gC for six hours for lesion , heart blood and spleen ( more than 10* bacilli /
animal hair and bristles. The anthrax bacillus is ml). The bacilli are &een confined to the interior
susceptible to tulphotumulcs, penicillin , of the capillaries, where their numbers may be so
erythromycin , streptomycin , tetracycline and great as to obstruct the flow of blood .
chloramphenicol. Occasional strains res is ran r ro
penicillin have been met with.
Two virulence factors have been identified
the capwtfarpQjypeptkk and the infhnur roxiji, each
—
Pathogenkdty: In nature, anthrax is primarily ot which is encoded by a separate plasmid.
,
a disease of cattle and sheep, and less often of horse ? The capsular polypeptide aids virulence by
and swine hut experimentally most animals are inhibiting phagocytosis. Loss of the plasmid ( pxflJ )
susceptible to a greater or lesser degree. Rabbits, which contrail Capsule production leads to lots of
guinea pigs and mice are susceptible. Infection can virulence . This; is how rhe live attenuated anthrax
be produced with difficulty in birds. Frogs are spore vaccine ( btenie strain ) was obtained.
resistant, while toads are very susceptible , Anthrax toxin was identified bv the finding that
Following the subcutaneous inoculation of a the injection of sterile plasma of guinea pigs dvmg
culture into n guinea pig, the animal dies in 24^72 of anthrax into healthy guinea pigs killed them and
hours, showing a local , gelatinous, hemorrhagic rhit deith could be prevented by immune serum.
edenu at the site of inoculation , extensive The toxin is a complex of three fractions: tlie edema
subcutaneous congestion and characteristically, an factor (OFor Factor I), the protective antigen factor
enlarged , dark red, friable spleen. The hlood is dark ( PA or Factor 11) and the lethal factor ( LF nr Factor
red and coagulates less firmly than normally. The IJI ), llicy are not toxic individually but rhe whole
bacilli are found in large numbers in the local complex produces local edema and generalised
344 ^ Textbook ol MisrutaWOfiv
bur the mechanism of action is not known. Loss of infected wool . This is a hemorrhagic pneumonia
the plasmid (p>dl 1) wb i. l L encoder the anthrax tun in with a high fatahiy rate. Hemorrhagic meningitis
renders the strain avimlcnr. This is believed to have may occur as a complicarinn .
been the basis for the original anthrax vaccine Intestinal anthrax is rare and occurs mainly in
developed by Pasteur. The aviruleni Sterne vaccine primitive communities who eat the carcasses of
Strain is devoid of the plasmid coding for the animals dying of anthrax. A violent enteritis with
capsular polysaccharide . bloody diarrhea occurs, with high case fatality.
Hum ait anthrax mav , be industrial or non
§r
-
ANTHRAX industrial (agr ' cultural}. The former is found in
Anthrax is a 70onnsis. Animals arc infected by r
workers in industries such as meat packing or wool
ingestion of the spores present in the soil, Direct factories . Mon - industrial anthrax is often an
spread front animal to animal Is rare. 'Hie Mi - case occupational disease in those who associate
is generally a fatal septicemia but may sometimes frequently with animals , such as veterinarians *
be localised * resembling the cutaneous disease in butchers and ferment. Tl may also he found in the
human beings. Infected animals shed in the general population . Cutaneous anthrax used to be
discharges from the mouth, none and rectum, large caused by shaving brushes made with animal hair.
numbers of bacilli , which sporulstc in soil and Stomoxys cidcittans and other biting insects may
remain as the source of infection. occasionally transmit infection mechanically
Human anthrax is contracted from animals Anthrax IS enzootic in India , the number of
*
directly or indirectly The disease may be ( 1) ar. imals infected running into tens of thousand
*
cutaneous * ( 2 ) pulmonary* or ( 31 intestinal, all types annually. The disease is rare in some countries such
.
leading co fatal septicemia or meningitis. as Britain * where infection is imported through
Cutaneous anthrax follows entry of the contaminated hides, hone meal fertiliser and other
infection through the skin- The lace , neck, hands* animal products- The extent of anthrax in human
arms and back arc the usual sites . The lesion starts beings is nor dear but about 20,000 to 100 *000
as a papule 1- 3 days after infection and becomes cases are believed to occur annually throughout the
vesicular, containing fluid which may he dear nr world* mostly in rural areas- Large epidemics of
bloodstained . The whole area is congested and anthrax were reported from Russia and Zimbabwe
edematous and several satellite leniom filled with during 197S-80. An epimotic of anthrax in sheep
*
serum or yellow fluid are arranged round a central hufi been active near the Andhra-Tamil Nadu
necrotic lesion which is covered by a blaek eschar. border, causing many cutaneous and
Cropyrighted materia
i Bacillus 245
iitoifi oeficephaliLC human infection with high ganimaphage and direct fluorescent antibody tesr
[
^
mortality rate. There have heen nuthieab in ^ ID FA ) tor capsule specific staining and. for
Karnataka and Wear Bengal- Anthrax inferrinn in polysaccharide cell wall antigen are sufficient.
human being* provides permanent immunity and For a further confirm ationT PCR tor anthrax
second attack* are extremely rare. bacillus specific chromosomal marker? can he done.
Laboratory diiignosiu Anthrax may be For epidemiological studies and strain
diagnosed by micruscopy, izulfUft, animal characterisation, MLVA { multiple locus variable
inoculation and serological demonstration of the number tandem repeat analysis) or AFLP ( amplified
anthrax antigen in infected tissues . Acute and fragment length pohmorphis- m ) can be used,
convalescent phase sera should be obtained, since Frophyhxk Prevention of human anthrax is
and bodies Co the orji .iimm Can be demon-stmted hi '
mainly hy general methods such as impruverrieriC
L'LI diffusion complement fixati > jnh antigen coated
* of factory hygiene and proper sterilisation of animal
tainted red Cell agglutination and ELISA products like bides and wool. Carcasses of animals
techniques . The type of test to be employed depends suspected to have died of anthrax are buried deep
on the nature of the material available . in quicklime or ere mated to prevent soil
When an animal i -; suspected to have died of contamination ,
anthrax, autopsy is not permissible, as the spilt blood Prevention of anthrax in animal* is aided bv d
« ilJ lead Co contamination of the soil . An ear may ULtivc nnmuni ^.iLion . The original P. i ^teurV anthrax
be cut oft' from the carcass and sent to the laboratory. vaccine Is nf great historical importance. It was
Alternatively , swabs soaked in blood or several Pasteurs cocKvimnn demonstration of the protective
blood smears mav be senr. The demonstration of ^
effect of his anthrax vaccine in A public experiment
Grim positive bacilli with the morphology ot ul Pouillv - lr - Foft in lSSl that marked the
Jr
anthrax bacilli drill a. positive M'FadyeHrts RUttion beginning ol scientific icriniunu prophylaxis-.
will enahle the presumptive diagnosis to be made. Past mr 's vaccine was tbe anthrax bacillus attenuated
ImmuiiiotluoreKcenr microscopy can confirm the
identification. I solation of the hacillus is easy if gross
—
by growth at 42 43 nC-
As the spore is the common infective form in
contamination has not ouutied. The anthrax nature, vaccines eonsis-tmg of spores of attenuated
bacillus cun often be isolated from contaminated
tissues by applying them over the sh avert skin of a
-
strains were developed The Sterne vaccine
contained spores of a noncapsulated avimlmf
guinea pig. It is able to penetrate through minute mutant strain . The Mazucchi vaccine contained
abrasions and produce fatal infection , If the sample spores of stable attenuated Carbazoo strain an 2%
received is putrid so chat viable bacilli ate unlikely,
diagnosis may be established by AscoLiV
-
saponin The spore vaccines have been used
extensively in animals with good results. They give
thermoprccipitin test by demonstration of the protection for a year following a single injection.
anthrax antigen in tissue extracts. They are not considered safe for human use, though
After the biotermrism experience in the USA they have been used for human immunisation in
in 2001, the CDC (Centers for Disease Control )
have prepared guidelines for identification of anthrax
-
Russia Alum precipitated toxoid prepared from the
protective antigen has. been shown to be a safe and
bacillus.. Any large Gram positive bacillus ivith the effective vqccin-c for human use. It has been used in
-
general morphology and cultural features of
anthrax; nomnoiile, nonhemolytic on blood agar
persons occupationally exposed to anthrax infection .
Three doses intramuscularly. at intervals of six
and catalase positive can be given a presumptive weeks between first and second, and six months
report of anthrax. For initial confirmation., lysis by between second and third doses induce good
Copyrighted materia
246 * Tpjfibook of Microbiology *
rv
U opyrighted materia
Bacillus 247
*
with ihe consumption of cooked rice * usually fried POCUffnubtCT in ligated rabbit ileal loopN resembling
rice from Chinese rest u unites The illness is
r the hear labile enterotoxin of jEscfiencftia coli
characterised by acute nausea and vomiting 1-5 Strains causing the emetic type of disease produce
hours after the meal. Diarrhea is not common. a toxin which causes vomiting when fed to Rhesus
B. emeus is present in luge numbers in the cooked monkeys , resembling staphylococcal enterotoxiti.
rice and fecal samples from these patients. Both The emetic toxin was produced only when B. Cereux
types of illness are mild and self limited, requiring was grown in rice but not in other media. Two
no specific treatment . mechanisms of action have been described for the
It has been shown that the two types of disease enterotoxin of if , cereuf, one involving stimulation
are caused! by strains of J5 cereus belonging to of CAMP system and the ocher independent of it.
—
r
different serotypes. The dkldMll disease is mostly A special mannitol-egg yolk phenol red “ -
caused by serotypes 2* 6, 8 9P 10 or 127 while the
B polymyxin agar ( MYPA ) medium is useful in
,
rice associated emetic illness is caused by serotypes isolating B. ccirus from feces and other sources,. B ,
1* 3 or 5, Isolates from the diarrheal type of disease etfeu& producer leeiihiitAfit Mid lernients glucose
1' iirther R e a d i n g
Ak&aray N ec al _ 1990 Cueaneous anthrax. Trop Gtqgnph Med 42: 163
. .
Rrachman PS. 19S0. lnfadpliiHi anthrax. Ann New Yotk Acid Sd 353:83.
Dixon TC rt al. 1999. Andirax. New Eng J Med 341:815
Indira Devi K.. 2001, Anthrax ], Acad Clm Microb 3:55.
LaJicha MK and A Kumar 1996, Anthrax in South India. Lmcci 348:553.
LaforccFM. 1994. Anthrax. Clin /nTcctDrj I 9A 0O9.
MaseLson, M et ad. 1994. The Sverdlovsk anthrax outbreeJk of 1979. Science 266:1202 .
Mock M and A Foret 2001 Anthrax. Ann Rev Microtoof 55:647..
The gcntis Clostridium consists of Gram positive, 4. Spherical and Terminal , giving a drumstick
anaerobic , spore tunning bacilli . The spores art- appearance ( 07. renm).
wiiier than the bacillary bodies, nving the bacillus Closrridja aie motile with peritrichate flagella,
^
a swollen appearance , resembling a spindle. Hence with few exceptions such a*. Cl perfringens and
.
tbe name Clostridium ( AJcJ-sTer meaning a spindle ) . Q rtianr type VI which are iwnmotilccThe motility'
The genus contains bacteria tWpwiliblc tor three is slow and has- been described as ’stately ' , 0-7,
major diseases of huimr beings - gas gangrene, pertrfn erih and 07. butyricum art capsulated, while
food poisoning and tetanus. Some ot the pathogens, ^
others arc not .
for example, Cf . perfh ngrnnLand Q teram are found
'
Clostridia are easily stained . They arc Gram
normally in human and animal intestines, Mam positive hut in older cultures , cells are often Gram
species arc pathogenic hot most are saprophytes variable , m even frankly Cram negative.
found in .soil , warcr and decomposing plant and Clostridia are anaerobic. The sensitivity to
animal matter. Intestinal flfirtridia rapidly invade oxygen varies in different species. Some ( for
the blood and tissues of the host after death anil example, Cl jrovrt) are exacting anaerobes and die
initiate decomposition of die cadaver. Some (for on exposure rn oxygen , while no me others (for
example Cl acefrib( JtybYum ) are lit industrial example, Cl. Jbs'fofyrjL’ unT) are aerotoferant and may
importance, used for rhe production of chemicals even grow aerobic ally. More important than the
such as acetone and butanol. absence of oxygen is the provision of a sufficiendy
Clostridia are highly pleomorphic. Thev are rod low redox potential ( Eh ) in the medium , ITiis car
shaped, usually 3-K|lm it 0.4-1.2 pm in siie. Long be achieved by adding reducing substances such as
filaments and involution forms are common. Spore unsaturatvd fatty acids, ascorbic acid, glutathione,
formation occurs with varying frequency in different cysteine , t hi ogive cdhe acid , alkaline glucose,
sjutcies . Some [ 07. iporogej'ie.d sporulite readily sulphites or metallic irqn . A small concentration
while others ( 07. ywrfrjngejrs ] do SLJ inconstandv, ufCO , appears to enhance growth. The optimum
Spondadon takes place in the animal hody also . temperature for pathogenic Clostridia is 37 ' C .
The shape and porition of spores vary in different Some saprophytic clostridia are thermophilic and
species and these arc of use in rhe identification others psyrhmphylic. The optimum pH is 7-7.4.
and class i heat uni of clostridia , Spares may be: Growth is relatively slow on solid media .
1 . Central o r equatorial , giving the bun II Us a Colonial characteristics arc variable. Some species
spindle shape ( Cl. bifcnnctiniu), are hemolytic or blood agar. A very useful medium
2. Subrermimi, the bacillus appearing club sh aped 15 Robertson 's cooked meat broth . It contains
[Cl perfj-JJTj eJi.s ) . msaturated fatty acids which take up oxygen , the
^
3. Oval am! rerminah resembling a tenni* ran ker reaction being catalysed by hemrir in the meat ,
[Cl ferriurn ). and also sulphydril compounds which bring about
Copyrighted material
* Clostridium 249
a reduced OR potential. Clostridia grow in the and can spread along tissues and even cause
medium, rendering the broth turbid. Mott HJKM:LL"S septicemia.
produce gas . Slccharalvtic species turn the meat Many methods have been adopted for the
pink. Proteolytic species turn the meat black and classification of clostridia. These include
produce tnul and pervasive odours. In lilmLLS milk morphological features such ; LS the shape and
medium, the production of acid, clot and gas can position id spores and biochemical fortunes such
'
phenolic disinfectants Formaldehyde is not verv found in the Iteccs and it contaminates the skin of
active and spores may sometimes survive immersion the iHrrinetim. buttocks and thighs. 'tTe spores are
in 2% solution lor upfo five days. Halogens are commonly found in sod , dust and air ,
effective and 1% aqueous iodine solution kills spores Morphology: It is a plump , fJrant positive
within three hours. Clutaraldc hyde { 2% at pH 7.5- bacillus with straight, parallel sides and rounded
8.5 ) is very effective in killing spores. In general, or truncated ends , about 4- ft pm « 1 tim . usually
dns ' ridia IfC susceptible tu metronidazole , occurring singly or in cttiaius or smull bundles- . It Is
penicillin, cephalosporins and chloramphenicol ; pleomorphic, and filamentous and involution forms
lets sO to tetracycline , and resistant ro arc common - It is capsulatcd and non motile- Spores
*
aminoglycosides and quinoloncs. arc central or subicrminal but arc rarely seen in
Clostridia can produce disease onlv when the artificial culture nr in material from pathological
conditions are appropriate . Their invasive powers IcsiiOlU, and their absence is tine of the chanacteristic
are limited . Pathogenic elostridin form powerful .
morphologkal features of Cl pcrfmtgnns,
ototoxms. CL bomirnttn is virtual! v nuniuvasive and Cultural characteristics: It is an anaerobe
nonmfectiu-us. Botulism is due to the ingestion of but can also grow under mkrttttbpihUit conditions.
preformed toxin in food. Cl. ftefuirhu little tsvaffime Oxygen is not actively feme to the bacillus and
property and is confined to the primary site of cultures do not die on exposure ro air, as happens
lodgement. Tetanus results tram the .LCILOTI of the with some fastidious anaerobes. It grows over a
potent exotoxin it produces. The gas gangrene
Clostridia , besides being toxigenic, are also invasive
-
pH range of 5.5 8.0 and temperature range of
20*0-50 :: C . Though usually grown at 37 C , a "
Copyrighted material
2 SQ i Tuxtbook of Microbiology
—Central or
—
ti&
CL jineiifatis Cl pcffnntgen?
—
mcchimlyrit : proteolytic
a fiffw
stcehmrofytic
Copyrighted materia
* CloslritJium * 251
Clostridia « human palhogens yolk. The opalescence in the egg yolk media may
he produced by other lecithinase forming bacteria
A . The gti gangrene group: also (O. miiji, Cl hiJernierifans, some vibrios, some
] . Established. CLpctfhngGM aerobic spore bearers.] . In the case of these bacteria ,
pathogens Cl aep6cum the react inn is not neutralised by the CL pcrfruigens
Cl novyi
antitoxin, except with O hr fermentsn $ which
2. Los pathogenic Cl histolyricim}
CL talk* produces a serologically related ld. iTliinasc .
3 . Doubtful pathogens Cl bilesmentans The alpha toxin is hemolytic for the red cells
Cl sporogznes of most species, except horse and goat, due Dc^ its
B . Tetanus : Cl rctimi action on the phospholipids on the erythrocyte
C . Food poisoning :
,
membranes . The Ij'v.ls is of the hot-cold variety,
being best seen after incubation at 37 °C followed
1. Gastroenteritis Cl pertringens
( Type A )
by chilling at 4 JC , It is relatively hear stable and is
2 . Necrotkkig enteriti* CS. pettiringmi only partially inactivated by boiling for five minutes.
( Type C ) Beta (]3), epsilon (s) and iota (l ) toxins have
3 - BmEut CL tt 'rwJjVjMirr lethal and necrotising properties. Gamma ( y) and
D* Acute colicifi a diffkfc eta ( n ) toxins have minor lethal actions. The delta
ifi ) toxin has a lethal effect and is hemolytic for
iL ]: HL>. (a ) toxin i$ produced by all types of Ci.
perftrn rnf and most abundantly hy Type A stm i ns.
-
the red cells o-f even toed ungulates (sheep, goats,
pigs, cattle ) . The theta ( GJ H>xm is an oxygen labile
^
Thb is the most important toxin biologi«JIy and hemolysin anligenically related to streptolysin O-
is responsible for the profound toxemia of gas lr ic aliio lethal and a general cytolytic toxin . The
gangrene. It is lethal, dermonecniLi ^. and hemolytic kappa ftc ) toxin is a collagcnasc. 'the lambda ( X)
I: is a phospholipidase [lecithinase C) which, in toxin is a proteinase and gelatinasc. The mu ( ji )
the presence of Ca ” and Mg" ions, splits lecithin toxin is a hyaluronidane and the mi ( v ) toxin a
into phu & phoryl choline and d lycende. This deoxytn lM? nudcasc .
reaction is seen as an opalescence^in serum or egg .
Besides ibe toxins, Ci perttingeia also produces
yolk media and is specifically neutralised by the other soluble substances, some of which possess
antitoxin When Cl. perfringens is grown on a enzymatic properties. TTtese include the enzymes
medium containing 6% agar, 5% Filde -. pepuc which destroy the blood group substance, A and
digest of sheep blood and 20% human semen , with H, a neuraminidase which destroys myxovirus
the antitoxin spread on one half of the plate, colonies receptors on red blood cells, a substance which
on the other half without the antitoxin will he renders red blood cells panagglutinable by exposing
surrounded by a zuilc of opacity, There will be no TheirT am igena, a hemagglutinin active against the
opacity around the colonies on the half of the plate red blood cells of human beings and most animals,
with the antitoidnh due to the specific neutralisation a Hbrir.olvsin , , i hemolysin distinct from alpha ,
of the alpha toxin. Thiv specific lecirhinaae effect, theta and delta toy i ns, histari ine, a "bureti ng factor*
known as the Nagler reaction , is a useful test for which has a specific action on muscle tissue and
the rapid detection ui Cl. pcrfnngens m clinical may be responsible for the characteristic muscle
specimens ( Fig. 28 - 1 ) . The incorporation of lesions in gas gangrene and a circulating facror'
neomycin sulphate in the medium makes it more which can cause an increase m the adrenaline
selective, inhibiting coliforms and aerobic spore sensitivity of the capillary bed and also inhibit
bearers. Human serum may be replaced by 5% egg phagocytosis.
Copyrighted material
252 « Ti&xibook of Microbiology *
J \\T H O C R N r C I T Y
CL perfrifigcftS produces the following human
infections:
Gns gangrene CL perfringem Type A LS the
predominant agent causing: gas gangrene- It may
occur as the sole etiological agent but is more
commonly seen in association with other clostridia
as well as nondostridial anaerobes and even aerobes.
All clostridial wound infections do not result in
gas gangrene. More commonly, they lead only to
wound contamination , or anaerobic cellulitis, It Is
only when muscle tissues arc invaded that gas
gangrene (anaerobic ntyosicis) results.
I' OuJ poisortiftg: Some strains of Type A
(food poisoning strains) can produce ftiod poisoning.
They are characterised by the marked heat
resistance of their spores and the feeble production Ffcfl, ES-1 Nngter reaclion. Cl- perfringens colonies on
of alpha and theta toxins. They have been shown ttie right lull ol Ihe plate are surrounded fay haloes,
to produce U beat labile enteroroxin which, lib; wWle colonies on the left half ( containing srtltoeram
the enterotoMins of V choierae and enterotoxigenic to alpha to * in ) have no haloes around them,
£ . coli , leads to fluid accumulation in the rabbit Gangrenous appendicitis: Cl . perfringena
ileal Loop. Type A (and occasionally Type D ) strains have been
Food poisoning by CL peffringtns is usually isolated from gangrenous appendicitis. The
caused bv a cold or warmed up meat dish. When demonstration of antitoxin in these patients and die
contaminated meat i* cooked , the spores in rhe beneficial effects of the administration oFaniitoxin aLso
interior may survive. During storage or warming suggest the etiological role of tbe bacillus in this
they germinate and multiply in the anacrohic condition. It has been proposed that die toxemia and
environment in rhe coobd meat . Large numbers shock in some cases of intestinal obstruction and
of Clostridia may thus be consumed * which may peritonitis may bedue to the toxins of CL pertriiigEns.
pass unharmed by the gasinc acid due to the high Ntrerolising enteritis: This is a severe and
protein in the meal and reach the intestines where often fatal enteritis known by different names in
they produce the encerotoxin. After an incubation different countries: Germany ( Darmbrand) , New
period of ft-24 hours, abdominal pain , diarrhea and Guinea ( pigbel ) . East Africa, Thailand and Nepal.
—
vomiting set in - The illness in self-limited and
recovery occurs in 24 48 hours. Diagnosis is made
by isolating heat resistant CL perfringens Type A
from the feces and food . As this maybe present ut
It is caused by CL perfringens type C Strains with
heat resistant spores which germinate in the
intestine producing beta toxin causing mucosal
necrosis. The evocative name 'pigbel’ is New Guinea
normal intestines, isolation from feces, except in pidgin for abdominal pain and diarrhea following
large numbers is not meaningful. Isolation from unaccustomed feasting on pig meat along with
food has to be attempted by direct plating on trypsin iithibitors like sweet potatoes . Immunisation
selective media , as the bacillus is present in food with type C toxoid has been shown to protect
mainly as the vegetative cells. against this condition.
Copyrighted material
Table 261 : Toxins produced by Cl . parfringens. types
Typc Pathogenicity
S
a l T T\ K
* M V
-
A Ca g-infcficne: WALW infecrions, Mpritemia. + ++
* - - - -
4
B
Food poisoning
Lamb dysentery
4 ++
+ ++
-
+++ * 4
4
*
-
4
~
- 44
- 444 444
++
4+
Closndjm
P
C Enteritis Ln animals
Enteritis necroticans in. human beings
+ 4+
44 +-
44
4+
* -
=
-
4#
=
-
*4
-
# 44 4
-
-
-
-
- + 44
— — ++ ++ ++
Copyrighted
materil
bJ
a
254 i Taxlbook of M c'obioogy *
Biliary tmcsl infection: Cf p&rningens produces at least four distinct toxins and a
beer reported to produce two ore but serious fibrinolysin. The alpha toxin is hemolytic ,
i nfections ( "t the lull , iry tfact - 1CUK trilptjwemanm > dermonecrotic and lethal , the beta toxin is a
s hokCYHNtis SILLI pOStcholcCVSteCtOlTiy SCptLCCmiU.
'
leucotoxic deoxyribonuclease, rhe gamma roxin a
Bndi enous gns gangrene of tntra - hvaluroniduse and the delta toxin an oxygen labile
^
uhJmiiinal origin: Gas gangrene of the hemolysin .
J
abdominal wall has been imported as an infrequent Cl icpOcum is found in the soil or in animal
complication oi abdominal surgem The infection in res tines. It is associated wirh gas gangrene in
is endogenous, the organism being derived from human beings , usually with other clostridia . It also
the gut and contaminating the abdominal wall dU 9« " braxy ' in sheep and 'maJignanT edema' in
during surgery. Gas gangrene of the thigh as a result cattle and sheep .
of infection tracking from the abdomen 11 LIS also
been reported. CLOSTRIDIUM NOVYI
Brain abscess and meningitis: Brain ( CJ, oedemartensj
abscess and meningitis doe to Cl pcriiingcns have This is i large, stout, pleomorphic, Gram positive
been reported very rare I v. bacillus with large , oval, tubiermmal spores. It is
Panophthalmitis: Panophthalmitis DUL to Cl.
perfringen* has occasionally followed penetrating
- widely distributed in soil. It is u strict anaerobe,
readily inaL- rtvattd by exposure of cultures to air.
eye injuries. Four types ( A to D) are recognised, based oo the
Thoracic infections: Clostridial infection of production of toxins . Onh type A is of medical
the chest cavity may follow penetrating wounds of importance , as it causes gas gangrene. Gas gangrene
the thorax . This, is more often seen in batrlc caused by CJ. novyi is characterised by high
casualties than m driliui situations . mortality and large amounts of edema fluid with
Urogenital infections : Infection of the little or no observable gas in infected tissue. Other
urinary tract may occasionally follow surgical type* produce veterinary disease, There was a lethal
procedure such as nephrectomy. Clostridial outbreak of Cl. HOVIT type A inleCtion in heroin
infection tit tlie uterus in a serious and not infrequent addict* in Britain in 2000-
condition , commonly associated with septic
abortion . Septicemia i * common in this condition. CLOSTRIDIUM HI3T0 LYTIC UM
This is an actively proteolytic clostridlum, forming
CLOSTRIDIUM SEPTICJM oval , subterminal , bulging spores . This is
This bacillus was first described hv Pasteur and acroroletant and some growth may occur even in
. J
irregular and transparent initi.dlv. turning opaque Oakley ( 1 S4) defined gas gangrene as a rapidly
on continued incubation . Hemolysis occurs on
^
spreading, edematous myonecrosis* occurring
horse blood agar. Growth is promoted bv glucose . characteristically in association with severe wound*
It is saccharolytic and produces abundant gas. of extensive muscle masse * that have beer
Six groups have been recognised, based on contaminated with pathogenic clostridla ,
somatic and flagellar antigens. CJ . rcptioUB particularly with Cl /vr/rmgrns. '[Tie disease has
Copyrighted material
* Cl D51n di u m 255
been referred to in the past as 'malignant edema' . Anaerobic cellulhh in which clostridia invade
Other descriptive terms that have heen used are the fascial planes, with minimal toxin production
.macmbit fdostriLli.il) myositis’ and 'clostridial
'
and no invasion of muscle rissues . The disease is
myonecrosis' . gradual in onset and may vary from a limired Lgas
Gas gangrene is ctiaracreriaticaUy a disease of war, abscess’ to the extensive involvement of a limb.
in which extensive wounds with heaw .s
contamination Anaerobic myositis or gas gangrene, which is
re very common. In civilian life, the disease generally the mint serious, associated with clostridial invasion
follows road accidents or other types of injury of healthv muscle tissues and abundant formation
jf
involving crushing of large muscle misses. Rarely, it of ex-ntoxi ns. Gas gangrene results only if the
may even follow ^irgical operations. conditions favourable for clusterhaJ multiplication
The bacteriology of gas gangrene is varuid- exist in the wound. The most important of these is
R a rely is this due to infection with a single low oxygen tens! i to.'Hiis is achicvcLl ideally in battle
Clostridium. Generally several species of clostridia wounds in winch there are : mplanted bullets or
are found in association with anaerobic streptococci shell fragments, along with bits of clothing and
and facultative anaerobes Fnich an i . . atli , proteus soil particles. The ionised calcium salts and silicic
and staphylococci. Among the pathogenic clostridia, acid in the soil cause necrosis . Crushing tissue or
£7- yjerfh jigiens LR the mnsr frequently encountered tearing of the arteries produces anoiLp. of the
'
(approximately GQ per cent), and Cl. no and Ci . muscle. Extravasation of blood increases the
scpficum being the next common ( 20-40 per cent), pressure on the capillaries, reducing the blood
and CL histoh^L-um less often . Other clostridia supply s Iill further.The Eh and pH of tile damaged
usually found are Cl . sporogenos, CL hllax , tissues fall, and these changes along with the
.
CI. bifetmen rails Cl. aotdelh ; , CL Jtnjtivtidumaod
.
chemical changes that occur within the damaged
Q terr.' Lrm, These may not be pathogen1. ’ by and anoxic muscles, mcluHmg breakdown of
themselves . The relative incidence of the different carbohydrates and liberation of aminoacids from
species varies m dliferent Series of cases and may proteins, provide an ideal pabulum for rhe
he a reflect : an of the distribution of the species in .
proliferation of anaerobes. Ex IT uvasated hem iglobirt
different Sails. and myohe mnglohin are reduced and cease to act
Cfesp idia usually enter the wound .LIUIIL; with as oxygen carriers. As a result, aerobic oxidiLTinn is
*
implanted foreign panicle such a soil (particularly halted and anaerobic reduction of pyruvate to lactate
*
manured or culnvated soil), road dust , bus of , leads to a further fell in Eh .
clothing or shrapnel. Clostridia may also be present The clostridia multiply and elaborate toxins
on the normal ski:i, cspec i ally on the pc i i -icum and - .
wfe.Lih cau *e further ti ^-uc damage The lecithinascs
thighs. Infection may at times he endogenous. Gas damage cell membranes and increase capillary
gangrene may occasionally follow clean surgical permeability, leading to extravasation and increased
procedures (especially amputations for vascular tension in the affected muscles, causing further
disease) and even inject inns (especially adrenaline j , anoxic damage. The hemolytic anemia and
The mere presence of clostridia in wounds does hemDglohmun.L seen m CI. perinngvns infections
not constitute gas gangrene. MacEcnnan has are due to the lysi of erythrocytes by the alpha
*
distinguished three types of anaerobic wound to in. The collagenases destroy collagen barriers
infections:
*
in the tissues and hyaluronidases break down the
Simple wound contamination with no int .i >:on intercellular substances furthering invasive spread
of the underlying tissue, resulii ig in Hide more than
'
by the clostridia. The abundant produedon of gas
some delay in wound hcaliug- reduces the blood supply still further by pressure
Copyrighted material
256 4 TgKfbggk g1 Migrgfciiolggy
eBern, extending the irci ofanw damage. It thuf numbers of regularly shaped Gram positive bacilli
^
becomes possible for the infection to spread from without spores is strongly suggestive of Cf .
the original site, making the lesion a progressive perfriugtns infection. "Citron bodies" and boat or
one . leaf shaped pleomorphic bacilli with irregular
The incubation period may be as short as seven Staining suggest Cl- sepbeum. Luge bacilli with
hours or as long as six weeks after wounding , the oval , subterminal spores indicate Cl novyi. Slender
average being 1 (H-S hours with Cl , perfringtris+ hadlii with round, terminal spores maybe Cl tetani
2- 3 days with Cl scpticum and 5-6 days with or Cl tetanQmaiphunt .
Cl , novyi infection The disease develops with Aerobic and anaerobic cultures are made on
increasing pain , tenderness and edema of the fresh and heated blood agar, preferably on 5-6 per
affected part along with systemic signs of toxemia. cem agar to prevent swarming , A plate of serum
There is a thin watery discharge from the wound , or egg yolk agar , with Cl periiingens antitoxin
which later becomes profuse and serosanguinous. spread on one half is used for the 'Nagler reaction'.
Accumulation of gas makes the tissues crepitant . Four tubes of Robertson's cooked meat broth are
In untreated cases, the disease process extends inoculated and heated it 100 ’C for 5, 1C, 15 and
rapidly and inexorably. Profound toxemia and 20 minutes , incubated and subculturcd on blood
prostration develop and death occurs due to agar plates after 24-48 hours, to differentiate the
circulatory failure .
r
organisms wi th heat resistant spores. Blood cultures
Laboratory diagnosis: The diagnonis of gas are often positive, especially in Cl pcriUngcni and
gangrene must be made primarily on clinical CL sfpacwn infections. However Cl . perfringent
grounds, and the function of the laboratoiy is only bacteremia may occur without gas gangrene. The
to provide confirmation of the clinical diagnose as isolates are identified baaed on their morphological ,
well as identification and enumeration of the cultural biochemical and toxigenic characters.
infecting organisms. Bacteriological examination Prophylaxis find therapy: Surgery is the
also helps to differentiate gas gangrene from most important prophylactic and therapeutic
anaerobic streptococcal myositis, which may be measure in gas gangrene . All damaged tissues
indistinguishable from it clinically in the early should be removed promptly and the wounds
stages. In the latter, Gram stained films show large irrigated to remove blood dots, necronc tissue and
numbers of streptococci and pus ceils , but noc foreign materials. In established gas gangrene,
butiHi , contrasting with the scanty pus cells and uncompromising excision of all affected parts may
diverse bacterial flora seen in films from gas be life- saving . Where facilities exist , hyperbaric
gangrene. oxygen may be beneficial in treatment.
The specimens to be collected arc: ( 1} Alms Antibiotics are effective in prophylaxis, in
from the muscles at the edge of the affected area, combination with surgical methods. The drug of
from the tissue in the necrotic area and from choice is metronidazole given IV before surgery
the exudate in the deeper parts of the wound; and repeated S hourly for 24 hours. As mixed
( 2) exudates from the parts where infection appears aerobic and anaerobic infections arc usual , a more
to be most active and from the depths of the wound, broadspectrum antibiotic prophylaxis, such as a
to be collected with a capillary pipene or a swab; combination of metronidazole , gentamicin and
and ( 3) necrotic tissue and muscle fragments. amoxycillin is advisable .
Gram stained films give presumptive Passive immunisation with 'anti -gas gangrene
information about the species of dostridia present serum' ( equine polyvalent antitoxin in a dose of
and their relative numbers. The presence of large 10,000 IU Cl- per&ingcns, 10,0001U Cl norland
GODvrianift - - nateria l
ru
< C iQstridiuFri E 257
—
"logins
: Cl tefsni produces at least two distinct animals do not germinate and arc destroyed by
tQxins a hemolysin ( tctxnotrw'.t ) and a powerful phagocytes. Germination and toxin production
neurotoxin ( feranojpas /njn ) . The two are occur only if favourable conditions exist, such as
antigenically and pharmacologically distinct and reduced O-R potent id, devitalised tissues* foreign
their production is mutually independent. A third bodies or concurrent infection. The toxin produced
toxin , a nonspasmogenic , peripherally active locally is absorbed by the motor nerve endings and
neurotoxin , has been identified. It is not known transported ro the central nervous system
whether this plays any role in the pathogenesis of i ntraxonally.The toxin is specifically and avidlyfixed
Copyrighted materia
* ClDSU dium r 2Sy
cause of death , particularly in the newborn , But strai ns are s tab- inoculated on each half of the plate,
immunisation of infants and expectant mothers hits which is then incubated anaerobically for two days.
reduced the incidence tr> 5 large L^ vtcnt . Toxigcuic Cl. reraru strains show hemolysis around
Laboratory diagnosis: The diagnosis of tbe colonies, only OIL the half without the antitoxin,
tetanus should always be made on cluneal grounds. l .vsih is inhibited by the antitoxin on the other half.
Laboratory tears only hel[i in confirmation. Not This* may help in identification of the culture as
inficquciitfy ir may not IK possible to establish a Cl. tetani but is unreliable as a test of toxigenic in '
laboratory diagnosis at alf since it indicates the production only of tetaiudysin
Laboratory Lliagirosis may he made by and not necessarily of teranospastuin, which is the
demonstration of Q, refartr by micrwcopy, culturv pathogeniv toxin .
or by animal inocnlation. Microscopy is unreliable
and the demonstration oi the typical drumstick '
'
Toxigenkiiy is best rested in animals. A two to
four -day-old cooked men culture (0- 2 ml ) is
-
bacilli in wounds in itself is not diagnostic of inoculated into rhe root of the rail of a mouse. A
tetanus, The bacilli may be presem in some wounds second mouse that has received the tetanus
without leranui developing, i t may not also be antitoxin ( 1000 units ) an hour earlier serves a> the
possible to distinguish by microscDpv between Cl. control. Symptoms develop in the test animal in
tcttinr and morphologically si mi I nr bacilli such as 12-2-1 hours , tiegi ruling with stiffness in the tail
Cl. aanosnotphum and LV. ohouicb Diagnosis
^ . Rigidity proceeds to the leg on the inoculated side ,
by culture is more dependable. Isolation i $ more the oppos i re- leg, tr u ok a nd forelimbs, in that order .
likeEv from excised hits ot tissue from the neurotic
r
The animal dies within TWO days r
Inlt may be killed
r
depths of wounds than from wound swabs, The earlier as the appearance of ascending rctanus is
material is inoculated on one half of a blood agar diagnostic.
.
plate. Cl tetwiproduces a swarming growth which Prophylaxis: Tetanus is a preventable disease ,
may be detected on the opposite half of the plate As the spores are ubiquirons , wound contamination
after 1 -2 days incuh- utioo anaerobically . Tlie is unavoidable. The disease is due To the action of
material is also inoculated into three rubes or rhe toxin . Therefore the obvious and most
et>okrd meat broth, one of which is heated to dependable method of prevention is to build up
SO cC tor lo minutes, the second tor ri miitutts, antitoxic immunity bv active immunisation by
and the thud left unhaUetL The purpose of heating routine Immunisation of children and booster doses
tor different periods is to kill vegetative bacteria, where appropriate.
while leaving undamaged tetanus sjwires, which vary The nature of prophylaxis depends Largely on
widely in heat resistance . The cooked meat tubes rbe type of the wound and the immune status
urt iucubaled at 37 "C and SubcullUted On one half of the patient . Tlie available methods of
ot blood agar plates daily for Uplo four Javs. Cl. prophylaxis axe (1) surgical attention; ( 2 ) antibiotics;
reran / may be isolated in pure culture by
subculturing from the swarming edge of the
—
and (3) immunisation passive, active or combined.
burgical prophylaxis aims at removal of foreign
growth. The incorporation of polymyxin li , to bodies , necrotic tissue and blood dots, to prevent
which clostridiu are resistant, makes the medium an anaerobic environment favourable for the
more selective. tetanus bacillus. The extent of surgical treatment
For iide ntification and Iwatiptmirity testing , blood
'
may vary from simple cleansing to radical excision,
agar plates ( with 4 agar to inhibit swarming), depending on the type of wound.
^
having tetanus antitoxin (1500 units per ml ) spread Antibiotic prophylaxis aims at destroying or
over one half of the plate are used. The Cl . tetani inbihiting tetanus bacilli and pyogenic bacteria in
Copyrighted material
4 Clostridium » 261
wcmnds so thiLt the production of toxin is prevented. kept ready. In persons- with i history ot any allergy,
In experimentally infected animals tetanus can be the trial dose should be 0.05 ml of a 1/ 10 dilution
prevented by antibiotic:*- when adminis- Eered four of ATS.
hours after infection but not alter eight hours . This
, Bovine and ovine ATS were introduced to
emphasises the need for prompt administration ot overcome reactions to horse serum but these in turn
antibiotics. Long- acting penicillin injection ib the nan also produce hypersensitivity.. Passive immunity
drug of choke. An alternative: is ciythrumydri 500 without risk of hypersensitivity can be obtained by
mg b.dL for five dap, Antibiotics are to be starred rhe use of human antitetanus: immunoglobulin
before wound toilet* Baoifucin or neomycin may (TIG). This is effective in smaller doses (210 units)
be applied locally also. Antibiotics have no action and. has a longer half- life (3“ 5 weeks ). As TIG in
on the toxin . Hence , antibiotic prophylaxis does
,
prepared by immunisation of human volunteers, its
not replace immunisation but serves as 3 useful n.vai I ability" is limited.
adjunct. FHHVC immunisation is an emergency procedure
Passive immunisation is by injection of tetanus to be used only once. The former practice of persons
antitoxin, Anti tec anus serum ( ATS ) Irom receiving ATS every time thev arc wounded was
hyperimmune horses was the preparation originally" not only useless and wasteful hut alsro positively
used . The dose employed was 1500 1LJ given dangerous . It is better to eliminate the use of ATS
subcutaneously or inTramtiscularly in nonimmune altogether, tetanus being controlled by active
persons MKWI after receiving any" tetanus prgne injury immunisation * with human ITG being moved for
ATS was useful not only in reducing the incidence emergency use In the nonimmune.
of tetanus but also in prolonging the incubation Active immunisation is not onlv the most IF
period and reducing the mortality when it did not effective nuthod of' prophylaxis bur also rhe only
prevent the disease. However, equine ATS carried
, means whereby tetanus following unnoticed injuries
rwo disadvantages implicit in the use of any can be prevented . Th : $ is achieved by spaced
heterologous: serum "immune elimination and 1
injtetioivs of fanned hm>idh which is available cither
-
hypervciuithity. The half life of ATS in human as ' plain tONo d ' , nr adsorbed nr aluminium
'
beings is normally about seven days bur in persons hydroxide or phosphate . 1 In adsorbed 10X 0 id is a
who have had prior injections of horse serum , it is better antigen . The tetanus toxoid is j- iven either
eliminated much more quickly by combination with alone or along wirh the diphtheria tiwcoid and the
pre -existing antibodies. Prior sensitisation also leads pertussis vaccine as the ‘triple vaccine', in which
to hypersensitivity reactions which may range from pertussis vaccine acre as art adjuvant also. A course
mild local reactions To serum sickness , and even uf immunisation consists of three doses of tetanus
fata] anaphylaxis. It is* therefore, obligatory that a tfJXQi . 1 given intramuscularly, with an interval of
rest for hyperaemitiviiy should invariably be made 4-6 weeks between the first two injections and the
before administration of ATS The intraderma] tc^t
, third dose 6 months later [or as per
for hypcrsensirivicyp which m in common use -, has recommendations of the National ITOTTH] nisarion
been reported to be unreliable, A "trial dost given
1
Programme ). A full course of immunisation confers
subcutaneously would be a belter index of : nainu - 11 ry for a pc i i.od of at least ten yean, A "booster
hypersensitivity A dose of 0.5 ml of ATS is given dose * of toKoi d i R recommended after ten years. ATS
subcutaneously and the patient observed for at least nr TIG Rhould not be given to an immuni -ed
half an hour for general reactions . As even this individual. Instead, a booster dose of toxoid i - given
dose may precipitate anaphylaxis in some cases., a if wounding occurs three years or more after the
Anion loaded with adrenaline ( 1 /1000 ) must be full course cif immumsation. Too frequent injection
of toxoid shoti [d he avoided as hypersensitivity However , because the patients are isolated, there is
rcucticmii ]ruy occur occasionally. a common impression that they arc highly
An illustration ol' the efficacy of active infectious. This is not true. TelirtUs- patienis ate
immunisation that in World War LI , onlv 12 eases hardly ever infectious , and person to person
of rotaries occurred in 2 , 7J? 4 , K 1 hospital transmission does not occur at all.
admissions for wounds or injuries , among the
^ Tncatmen t cons isrs of ensuring quiet, control Ling
American soldiers who had been previously spasms, maintaining airway by tracheostomy with
immunised . intermittent positive pre -Saure respiration and
Combined immunisation consists of attention to feeding . Human TIG 10,000 LU
administering to a rnnimmure person exposed 10 suitably diluted may be given by slow IV infusion,
the risk of tetanus TIG injection at one site, along tollowed , if needed, by another 5,000 IU later. Ever
with the first dose of toxoid at the contralateral though TIG may not neutralise the toxin already
site , followed Hy the second and third. JTNKCIH of bound to the nervous tissue, it can inactivate the
toxoid ac monthly intervals. It is important to use unbound toxin and any further toxin that may he
adsorbed toxoid as the immune response to plain produced. Antibacterial therapy with penicillin or
toxoid maybe inhibited by TIG . Ideally, combined metronidazole should he started at once and
immunisation should be employed whenever ootitinued for a week or more. ATS used to he given
passive immunisation is called for. intravenously in massive doses as part of the
Table 2? , 3 shows the recommended integrated treatment . Several controlled trials have been
prophylaxis of tetanus following injuty. undertaken to assess the value of antitoxin and the
Trealiment: Tetanus patients should he treated Optimum dose. The results indicate that antitoxin
in hospitals, preferably in special units . The reason is of value in treatment but that 10 ,000 1U
for isolating them is to protect them from noise intravenously gives as good replies as much higher
and light which may provoke rhe convulsions. dciSCE.
Mote : Immune - Patient has had a full course of three injections of toxoid.
Fortuity immune
- - Patient has had iwy injections of toxoid.
.
NorLi. n : rmint: Patient bjs had on** cr m: irtjKtidit of fcHtiwd , (if irtirtULnisarion status is rutlotown.
TIC - Tpianui Immune Globulin .
* The toxoid needs to be pwen only if three years or mure have elapsed after active immunisation or
Copyrighted materia
I
- Clostridium * 263
Patients recovering from tetanus should receive that is responsible for its pathogenicity. The toxin
u fiill course of active immunisation , as an attack of differs from other exotoxins in that it is not
the disease docs not confer immunity. Second released during the life of [ lie organism . It is
attacks of tetanus have been recorded. produced mtTacellnlarlv and appear; in the medium
only on the death and autolysis of the cell . It is
CLOSTRIDIUM BOTULINUM believed to be synthesised initially as a nontoxic
Cl. fjoniffnuru causes botulism, a paralytic disease protoxin or progenitor toxin. Trypsin and other
usually presenting as a form of food poisoning . proteolytic enzymes activate progenitor toxin to
The name botulism is derived from LH a usage" active Toxin .
( hofu/ us, Latin for sausage ) formerly associated with The toxin has been isolated as a pure crystalline
this type of food poisoning. C7. botufoiuin was first protein which is probably the most toxic substance
isolated by van Ermertgem ( 1896 ) from a piece of known lr has a MW 70.000 and a lethal dose for
,
ham that caused an outbreak of botulism . The mice of 0,000,000,033 mg. The lethal dose for
bacillus LS a widely distributed saprophyte, occurring human beings is probably 1-2 |ig. It is a neurotoxin
.
in vitgin sojln vegetables, hay silage, animal manure and acts slowly, taking several hours bo kill.
and sea mud.
Morphology : It is a Gr ^ m positive bacillus
about 3 p x 1 Jim , noncapsulated , motile by
.
pcritrkhiBe flagelL producing subierminal,, oval,
bulging spores .
—
The toxin is relatively stable, being inactivated
only after 30 40 minutes at SO and 10 minutes
at 100 ° G. Food suspected to be contaminated with
botulinum toxin can be rendered completely safe
bv pressure cooking nr boiling for 20 minutes- It
Cultural characteristic!: It is a strict resists digestion and is absorbed through the small
anaerobe. Optimum temperature is 35 but some intestines in active form. It acts bv blocking the
strains may grow even at 1-5 °C . Good growth production or release of acetylcholine at the synapses
occurs on ordinary media . Surface colonics arc large, and neuromuscular junctions . The onset is marked
irregular, semitransparent, with fimbriate border. by diplopia, dvspllagia and dysarthria due to cranial
Biochemical rcactionsvaiyin different types, Spores nerve involvement - A symmetric descending
are produced consistently when grown in alkaline paralysis is the characteristic pattern, ending in
glucose gelatin media at 20-25 ^ C. They art not death by respiratory paiuhnis.
usually produced at higher tcmper^ turcs- A small quantity of Cl- botulinum type A toxin
Resistance: Spores are heat and radiation injected into a muscle selectively weakens it by
resistant, surviving several hours at 100 C and for blocking the release of acetylcholine at the
upto 10 minutes at 120 ^ C. Spores of nonproieolvtic neuromuscular junction. Muscles so injected atrophy
types of B , E and F are much less resistant to heat .
.
Classification: Eight types of Cl botulinum
—
hut recover in 2 4 months as new terminal axon
sprouts form and restore transmission .
.
have been identified (Types A* 13, Cl C2 , D, E, E, Intramuscular injection of the toxin, first used to
G ) based on the immunological difference in the treat strabismus , is now recognised as a safe and
toxins produced by [Item. The toxins produced by effective symptomatic therapy for many
the different types arc identical in their neuromuscular diseases.
pharmacological activity but arc neutralised only The botulinum toxin can he toixoided . It is
by the homologous antiserum. An exception is C 2 specifically neutralised by its antitoxin and is a good
toxin , which shown cntcmtoxLC activity, while all antigen . The- toxins produced by the different types
the others are neurotoxins . a
of botulinum appear to be identical , except for
Toxin: C7, botulinum produces a powerful exotoxin immunological differences . Toxin production
Copyrighted material
264 Of WiCfObiQ
1
^y *
appears to be dcremaned by the presence of manifestations are constipar .on, poor feeding,
bacteriophages, at least ir types C arid D. Icthaigy, weakness, pooled oral secretions, weak or
Pathogenicity: O. ht Ir :JJ' IV ? -: JM is noninvasive and altered cry, fioppiness and loss of head control.
virtually nonrtfectinus, Its pathogenicity is -due to
is not generally demonstrable in blood .
-
Pati i;nt& excrete t n and spores i n th i : r feces. Toxin
the acrion of its toxin , the manifestations of which
arc collectively called botulism , Botulism is of three Management consists of supportive care and asnstml
-
types foodborne botulism, wound botulism and
infant botulism.
feedingi - Am itoxiita and antibiotics ins not inch cared.
Degrees of severity vary horn very mi lid illness to
FfMxJhttrnc botulism in due t[ j tht
indention ' if fatal disease. Some cases u1 sudden infant death
preformed toxin The types ot the bacillus and the
. svndrome
.
1
have been found to be due to infant
nature of rbe food responsible var\ in different botulism. Honey has been incriminated as a likely
regions. Human disease is usually caused by type* food item through which the hacillus enters the
A, B, E and very rarely F. Types C and D arc usually
associated with outbreaks in eatde and wild fowl . Laboratory diagnosis: Diagnosis may be
Type C has been assoc iated with sudden death in a ennfirmed by demonstration of the bacillus or the
—
few patients. The source of botulism is usually
preserved lotaJ meat and meat products in Europe,
canned vegetables in America and fish in ^ apan.
Type !; is iHsociatcd with fish and other seafoods-
toxin In food or feces. Gram positive spuring bacilli
may be demonstrable in smears made from the (bod .
Cf tafvhnuFTT mav be isolated from the food or
the patients feces. The food is macerared in sterile
.
Proteolylic varieties of Cl bondmuzn can digest .
sisJir&e, and the fi.lrrite inoculated info mice or guinea
food, which then appears spoiled . The cans are pigs inCraperLtoneally. Control animals protected bv
often inflated and show bubbles on opening. polyvalent mtiioxiti remain healthy. Typing it done
Nonprorcolytic varieties leave food unehanged. by passive protection with type- specific antitoxin .
-
Symptoms begin usually 12 36 hours after The toxin may occasionally be demonstrable in the
ingestion of food. Vomiring, thirst , consr - pahon, patients blood, or in the liver postmortem.
ocular paresi >, difficulty in swallowing, speaking A retrospective diagnosis may be made hy
anti brtathing constitute the common fcatuneFC Coma detection ofnttkoDon in the patients scrum but it
or delirium may supervene . Death is due to may not be seen in. all cases.
respiratory f nlure and incurs 1-2 days after onset. Control ? As most cases of botulism follow
Case fatality varies from 25— 70 per cent. consumption of inadequately canned or preserved
Wound bofu /rsm is a very rare condition tood , control can be achieved by proper canning
resulting from wound infection with C7. fwtuirmtm. and preservation. When an outbreak occurs, a
[ ; : xicl i & produced at the site of infection and is prophylactic dose of antitoxin should be given
absorbed. The symptoms are those of foodbome Lntnmutmkffy to all who consumed the article of
botulism except tor the gaNtnunteNtiiLxl components food.
which are absent. Type A has been responsible for Active immunisation has been shown to be
most of the cases studied . effective . It immunisation is needed, as in laboratory
-
Infant botulism was recogm ed as a clinical workers exposed to the risk, two injections of
entity in 1976. This is a toxico-infection. C’J. aluminium sulphate adsorbed toxoid may be givers
bomlirtutn spores arc ingested in food , get at an interval of ten weeks, followed by a booster a
established in the gut and there produce the to \ i.n . year later Antitoxin may be cried for treatment.
Cases occur :n infants helow six months. Older Polyvalcnt antiserum to types A, B and E may be
children and adults are not susceptible . The administered as soon as a clinical diagnosis is madcr
Supportive therapy with rn^irtTETianOt Cit ropilflti<Wl antibiotic therapy. Many antibiotics have been
is of equal or greater importance- incriminated including ampicillin, tetracycline and
chloramphenicol but linaunYviii ml cfodamyrin arc
CLOSTfllDlUM DIFFICILE AND ANTIBIOTIC puiticuliiily prone to cause pseiLtlnmerribFjnous colitis.
ASSOCIATED COLITIS It has now been shown that antibiotic associated
CL ( Hfficik wus first isolated in ly.i.S frym the fcL es'
colitis is due to the active mu triplication of Q
of newborn infant*- . It was HO named because of the dtfBa'fe and lies production of an cnteioroxin as well
unusual difficulty in isolating it. It is a long, skndcr as a cytotoxm . Diagnosis can be made by
C rrajn positive h .uillus with a pronounced tendency demonstrating the toxin in the feces of patients by
to lose its Gram reaction . Spores are large , oval itscharacteristic effect on Hcp-2 and human diploid
and terminal. It is mm hemolytic, sticcbarolytic and cell cultures nit by ELISA . The toxin is specifically
weakly proteolytic , It was not considered neutralised bv the C7, sordelii antitoxin. CL difficile
X
pathogenic till iy ?7 when it was found ro he can aJso be prown Jrom the teces o-f patients.
responsible tor antibiotic nissociaccd colitis. CL difficile strains are usually sncsista.nl to most
Acute colitis, with ur without me mb t .me antibiotics Metronidazole is the dru ot choice .
formation , is an important complication of oral
*
Further Keiidiin
Bartlett JG . 1994. Clostridium difficile . Clin Infect Dis, 18;S265 ..
^
Brazier JS. 1995. Laboratory diagnosis of CL difficile associated disease . Rev Med AHcrohial fr:236.
L uerdcn BI and BS DraBar {ede ) - 199 L Amkansbes jji Airman disease. London; Arnold
^ .
Sanford JP. 1995 . Tetanus - forgotten, bur not pone . Sew Entf J Med , 332:812,
Copyrighted material
o> Nonsporing Anaerobes
CM
-
present have been estimated to be 10* llGVml in
the small intestine. ItfVm! in saliva and 10 IL /g in
e. Bifidobacterium
f Aetirtomyeeii
the colon - B. Gram negative
Afuerobk bacteria differ widely in the degree a. Haeteroida:
of anaerobiosis required for their growth, borne b. Prevotcllla
species fail to grow if the atmosphere contains as c . Pbrphyromonas
little as 0.03 per cent oxygen, while at the other d. Fusobacterium
extreme , some arc aerotolerant and may grow e. Lcptotrichia
sparsely on [ Ire surface of aerobic places . III . Spirochetes
Consequently the techniques employed for the a - Treponema
propagation and study of anaerobes vary in b. Borrelia
complexity. Besides the medically important species listed
Eatly methods of classification used such above, there ate several anaerobes that occur in soil
unstable criteria as cell and colony morphology, and water and which may be of industrial and
biochemical reactions and antibiotic sensitivity agricultural importance (for example ,
patterns. Current (dassifLcatitm is based on DNA methamobacterLa , butyrivibrios).
base composition and analysis pf the fatty arid end
products of metabolism . Medically important ANAEROBIC COCCI
anaerobes may be broadly classified as fotlowsr ANAEROBIC cocci represent a heterogeneous
Copyrighted material
s Nonsporipg Anaerobes 2fi 7
factors, and antibodies to them can he detected in nods, often with pointed ends. They are part of the
patients . Thev are normal inhabitants of the normal oral flora and arc seen in acure necroticng
intestinal, respiratory and female genial tracts- le ^ iMftR in the mouth.
Bactcroides species have been classified based
on their u ace ha roly tic effects. Asiceharoly nc
-
A common condi t ion i Vincent 's angi:ia, wli i ch
may resemble diphtheria , with the inflamed
pigmented specicH have been separated IS the genus
lsii|
_
ph>rnomcinasn containing Pgingivalis reRp u > il le
pharyngeal mucosa showing a greyish membrane
which peeb easily. Stained aments show large
for periodontal disease , ft cndodontulis causing fusildrm and spiral bacilli .
denta ] root canal infections and other species.
Moderately Hacchaioiytic speties inliibiTed by 20% ANAEROBIC INFECTIONS
bile are placed in the genus Prcvotella. containing There has been a reawakening of interest m
F. mehninogetiica, F buccafis, F. dentieok and anaerobic infections during recent years . This is
Others. The genus TtatrernideR proper now includes due to the availability of improved and simplified
the important species ft- foigifts, and others Ruch techniques for the isolation and identification of
as fl , vutfiaitit , B . chs ra si on j( and B. anaerobes.
theta / otaom.vron. Anaerobe infections are usually endogenous and
ft . fragi / iR is the most frequent of the are caused by tissue invasion by bacteria normally
nonsporing anaerobes isolated from clinical resident Oft the respect ive body surfaces. Anaerobic
specimens. It is often recovered from blood, bacteria arc normally present on the skin, mouth,
pleural and peritoneal fluids, CSF, brain abscesses, nasopharynx and upper respiratory tract, intestines
wounds and urogen ::al infections - P and vagina {Table 29.1) . Anaerobic infections
mtkimiotfemci is easy to recognise because of generally follow some precipitating factor such as
the black or brown colour of the colonies.. The trauma , tissue necrosis , impaired circulation ,
colour is not due to the melanin pigment as was hematoma formation or the presence of foreign
once thought but to a hem - it derivative, It has been bodies . Diabetes, malnutrition, malignancy or
- _
i IJL .H > . I from various infections luludimt lung or
( ,
prolonged treatment w i l h aminoglycoside
-
liver abscess, mastoiditis , intestinal le mns and
lesions of the mouth and gums. Cultures of ft
antibiotics may act as predisposing factors .
Anaerobic infections are typically polymicrobial,
jne/ jmrto enK'a and even dressings from wounds mote than one anaerobe being responsible besides
^
infected with the bacillus give a characteristic red aerobic bacteria. While the infection is usually
fluorescence when exposed to ultraviolet light. localised, general ms- semi nation may occur bv
The genus FusofwcfejTum contains fong, thin bacrerem ::i . Tabic 29.2 lists the common sines and
or spindle shaped bacilli with pointed ends . E type of anaerobe infections and the bacteria
nurfeatum is a normal inhabitant of the mouth and responsible.
is found in oral infection and pleuropulmonary Iherc are some clinical features which suggest
sepsis . F, rtcei'ophtJfMim produces a wide range of the presence of anaerobie infection. Pus produced
exotoAins and has been responsible lot liver abscess by anaerobes is characteristically putrid, with a
Copyrighted materia
a Non &poting Anaerobes ^ 269
Table 29,1 N&nnal anaerobic Hare ri the human body
Anaerofcf
CIosmdiujYi
Siin —
JVfou th njtsophMiynx Intestine ViginM
+4
Actinomyces * >
Bifidobacterium 4 +4 +
lJropionibacterium
ff&yvnoidcs trwg&i 44
R inchninagenrcu 4 4 4 +4
FuBob&c Ecrin m 4 4 4
Spirochetes 4
pcriltivc, nauseating odour. However, there roav be used , they should be sent in Stuart’s transport
he exceptions; infections solely due ro B tragi!is medium -
may he free of this imelL Pronounced cellulitis is a ln the laboratory, exposure should be Limited
common feature of anaerobic wound infections;. to the minimum. Examination oh a Cram stained
Toxemia and fever are not marked. smear is useful. Pus in anaerobic infection usually
Lrtbornlory diagnosis: As anaerobes form shows a large variety of different organisms and
pan of the normal flora of the skin atid mucous numerous pus cells . Rarely, as in brain abscess, H
surfaces, their isolation from specimens has lii be single type of organism alone may he seen .
interpreted cautiously. The mere presence of an Examination of the specimen under ultraviolet
anaerobe does not prove 1 causal role - Specimens light may show the bright red fluorescence of
should be collected in such a manner as to avoid
"
R mehninngcniai. ( las liquid chromatography of
resident flora . For example , the sputum is the specimen rriav vie Id presumptive information
unsatisfactory' for culture from a suspected case of on the types of anaerobes present .
lung abscess; only material collected by aspiration Several special media have been described for
would be acceptable. anaerobes but for routine diagnostic work, freEblv
As some anaerobes die on exposure to oxygen, prepared blood agar with neomycin , yeast extract,
earn should be exercised to minimise contact with heroin and vitamin K is adequate . Plates arc
air during collection , transport ami handling of incubated at n 7 ' CT in an anaerobic jar , with 10%
specimens. A satisfactory method of collection is CO ,. 'Ehc Caspak system provides a convenient
ro aspirate the specimen into an airtight syringe, method nf routine anaerobic cultures . Plates are
plunge the needle into a sterile robber cork to seal examined after 24 or hours . Some anaerobes, inch
it and send ir immediately to the laboratory. E\is :LS fusobucteria , require longer periods of incubation.
and other fluids may be collected in small bottles Raralld aerobic cultures should alwavTi be set up. This
with airt ight caps and transported qu ickly, cn suri ng is ncccsaxy as a control for the growth on anaerobic
that the specimens fill the bottles completely, bwabs plates and also because in most anaerobic infections
are generally unsatisfactory hut where they arc to aerobic bacteria are also invoked.
appendicitis, peritonitis;
ischiorectal abscess; , JJ. fragiUs
wound infection after
cnlorecraJ surgery
Femaie genitalia:
Wound! infection Mowing
genital surgery;
Puerperal sepsis; P mehminogemca ,
tubo-ovarian abscess, anaerobic cocci; K fragitis
Bartholin's abscess,
septic abortion Genital aruuernbes and Cl perfringens
Sirin and raft timm
Infected sebacious cyst. Anaerobic cocci
Brcast abscess, axillary abscess Anaerobic cocci; jRme /aninogertj-ca ( Staph , sureu#
' '
commonest cause}
Cdlulitkj. diabetic ulcer , gangrene B. rragjJjs and others.
'
Further Rending
Duerden DL and BS Dnumr ( eds ), 1991. Ajisfcirohttr in Hiimitn Disease- London: Arnold.
,
Finenld M . 1995.. Overview of clinically important anaerobes. Clin Jm/ect Dis. 30: S2QS,
KiiMf DL. 1993. InJfeetLoisa due in mixed anawebie organisms. In Hamsons Principles of Internal Modidnc^ 14 edn. New
H
Copyrighted material
8 Enterobacteriaceae I: Conforms - Proteus
'
-
111 r L i mes of human be i ngs .11 nl animals is composed
uf nonspuring, noma .- bl fast , (. iram nej lLve bacilli
III . NortlactoSe fermenters (fur example
Salmonella ,
rhry exhibit general morphalugi cal and ^ ,
Shigella )
biochemical similarities and me grouped together This method of classification was derived from
in the large and complex family Enrero'iMCfcrjJce*?. the use of lactose in MACCDHKEY's medium the ,
Members of this family may or may nor be most popular medium for the isolation uf fecal
capsulared and are morile by peritrichale flagella * bacilli . Though taxononucally unacceptable, this
nr ate non motile - They arc acmh is - and facultatively scheme had practical value in diagnostic
anaerobic, grow readily in ordinary' media, ferment bacteriology. The majority of the commensal
glucose producing avid and gas or acid only, reduce intestinal bacilli arc lactose fermenting ( LF). As
nitrates to nitrites and form catalase but not oxidase. the most common member of this group Is the colon
Within the family * they exhibit very wide bacillus* or Escherichia coh. all lactose fermerumg,
biochemical and antigenic heterogeneity. T ltougb enteric bacilli were called coliform bacilli . The
the family i ^ s ibdivided into groups or tribes, genera, major intestinal pathogens, Salmonella and Shu^rl/a
subgenera , species and types* many strains are met are nonlactose fermenters ( NLF), and hence readily
with that possess every conceivable combination detectahlr by the colourless colonies they form on
of characters and do not fall into any such arbitrary MacConkey's medium. There remained a small
taxonomic category . The frequency of genetic group which showed delayed fermentation of
mechanisms fluch as conjugation and transduction I .iceo^' and with the creep t ion o t Shigella sonnei,
in these bacteria contributes to their infinite variety . they were all commcnsLta. This heterogenous group
Classification of these bacteria into well demarcated of late lactose fermenters was called panaco/on
compartments, though necessary for their systematic bacilli.
study would therefore be artificial . The Classification based on a single property, such
*
-
classification of Entcmhacti riaceae has hecn
controversial and there hive been successive changes
as lactose fermentation ., is contrary to modern
taxunoimdal concepts. The current practice is to
in their grouping and nomendaturc. group together bacteria that possess a number of
The oldest method was to class ' ty these bacteria common morphological and biochemical
into three groups based on their action on lactose. properties, and similar DNA base compositions,
T. Lactose fermenters (for example While the three widely used systems for the
Kschcrichia, classification of Entewhacteriaceac ( Bergtys
Klebsiella ) manual* Kauffmann, Edwards-Ewing) have certain
II . Late Lactose fermenters (for example differences, the general approach is the same. The
Shigella sonnei family is fust classified into its major subdivision -
Copyrighted materia
272 i TgxIbOOk 01 Mizrahitjlogy *
group or
-
h trill* cnn iisth gf One or MOT*
tribe, Rjs
genera and each genus one or more suhgenen and
ftQgUfnnrV, F.. hcrmznii and F.. whOUtthkh have
been isolated infrequently from clinical specimens ,
Copyrighted material
4 Erigrobacienacoae lr Colilorma - Proteus 273
nnir I , maltose and many other sugars arc The somatic lipopolysiicchiridc surface Q
fermented with the production M acid jnd gSt$, antigen, besides exerting crtdiUtode activity, also
Typical strains do not ferment sucrose. The trior protects the bacillus from phagocytosis and the
biochemical tests widely employed in rite bactericidal effects of complement The envelope
classification of enterobacteria are tile indole , or K antigens also afford protection against
methyl red ( MR ) t Voges-Froskauer ( VP ) and phagocytosis and antibacterial factors in normal
citrate utilisation test's, generally referred to hy the serum, though it is not effective in the presence id
.
mnemonic ' IMViC , E coli ! H indole and MR antibody to Q or R antigen . Most strains iff E. coii
positive, and VP and citrate negative (IMViC + 4 responsible for neomtil meningitis and septicemia
). Gelatin is not liquified * H ,S is not formed, carry the Kl envelope antigen which is a virulence
unci is not split and growth does not occur in KCN factor resembling the group B antigen ot
medium. meningococci.
Antigenic stcwCturtj berotyping of E coli is . Fimbriae also promote virulence. 'Tire common
based on three antigens - the somatic antigen O, fimbriae seen in most enterobacteria , which arc
the capsular antigen K and the flagellar antigen H . chtomosnmally determined , present in large
So tar some 170 tvpes ofO antigens, IDO K antigens numbers and causing mannose sensitive
acid 75 11 antigens have been recognised . The hemugglulination, in probably not relevant in
antigenic pattern oi a strain is recorded as the pathogenesis. A different kind of fimbriae which
number of the particular antigen it carries., as for are plasmid coded, found only in send! number -'
example 0111 : K5S: H2- and mediating mannose resistant hemagglutinins
Tlic K antigen is the acidic polysaccharide have been shown to act as virulence factors. Some
antigen located in tile ‘envelope’ or ToicTCHiapsute. oi them may no? occur as morphologically separate
( K for Kup^ cl, German for capsule ). It endows the structures but only as surface antigens - tor example,
O antigen and renders the strain insgglunnable by K 33 and K 99 antigens in strains causing diarrhea
the O antiserum . It may also Contribute to virulence in animals, or the Eoloniaxiion Fattur Aotjjgem
by inhibiting phagocytosis . Formerly K antigens .
( CFA ) in enterotoxigenic E coli causing human
were subdivided into three kinds - the tlienookIdle diarrhea. Fimbriae arc also nfimporrarice in urinary
L antigens, the thermostable A antigens ami the K tract infection , as for example the P fimbria which
antigens found on enteropithogenic strains binds specifically ro the F blood group substance
associated with infantile diarrhea. Later it was or human erythrocytes and uiocpitheliid cells.
shown that the B antigen was not a separate entity'. Er coli produce two kinds of ercotoxina -
K intigens are therefore currently classitied into Aeznofyiov and enteranudns. Hemolysins do not
two groups , 1 and 1!, generally corresponding to appear to be relevant in pathogenesis though they
the former A and L antigens. arc produced more commonly by virulent strains
Several different serotypes ot E. ctili are found tli .LI 1 by svirulentnnidt Fflterotauuiih are inifjortanl
in the normal intestine Most of them do not have in the pathogenesis of diarrhea. I'hrce distinct types
K antigens . The normal colon strains belong to the of E. coli cntcrotoMins have been identified - lie ^ f
'early ' O groups (1, 2 , 3 , 4 etc . ) , while the labile toxin ( LT ) , lie at stable toxin ( ST) and
entcropathogenic strains belong to the ’ later ' O verotox. in ( VT ) also kn0wn an Shiga-like toxin
groups ( 26, 55, 86, 111 , etc}. ( SLT).
Virukrtue factors: Two types of virulence .
Thc E coii cnterotoKin LT was discovered in
Copyrighted material
274 < T& x.tCiook of Mcrobicnogy
Table 30 1 Enterabacteriacefie ; Important di & tsng rushing leaturEs ol ihe dillerenl genera
I i 1
'
a
| 1a I|
*
I s I
G
45
LU * w I l £
— —
Motility 4 4 —
»
4 4 4 4 4 4 + 4
Gas from glucose- + * 4 + 4 + + d d + +
Acid from lactose 4 4 — “ 4 4
Add from sucrose d d 4 4 d ^ d
Growth in KCN - + d - + 4 + 4 4 4 4
Indole + 4 d - d - d + 4
MR + 4 -
1 f f “ 4 + +
VP + 4 +
Citrate
H2S
Urease
Phenylalanine
+
+
-
1
4 -
— 4
+
4
4
d
4 4 d.
+
4
d
+
d
d
deaminase ( PPA )
Arginine
- — — - “ 4 + 4
dehydro! ase d d + - d .
Lvsine
Jr
decarboxylase
Ornithine
+ + — + - d d 4 4 - - -
decarboxylase d + d t d - 4 4 4 d + -
(d
-. pewits
different in different specie* nr strain )- )
Jinporunr exceptions:
lS cyphi do< ¥ not produce g» from sugm,
’Sh . sonncL ferments Eactose and sucrose lat-c
method which they lud earlier used for identifying leading to increased outflow of water and
the cholera enremtnxin ( CD , vtr injection of E.
% electrolytes into the gut lumen, with consequent
entf culture filtrates into dosed ligated loops of rabbit diarrhea. Though the mechanism of action of LT
ileum induced outpouring of fluid and ballooning and CT is the same „ she Latter is about a hundred
of the ]oops, E, toll IT rrsemMc & the chotera tostin times more potent than the former. LT is a powerful
, .
in its structure , antigenie propetficn and mode of antigen and can therefore,, be detected by a number
,
Copyrighted material
i Entprabactermceao I : Cdlprurms - ProlfluS t 275
intestines of infant mice within four hours of a time* though recurrences may be due to different
intragastric administration . This mLinl mouse test serotypes.
LR the standard method fur demonstration of ST , . Infection may be precipitated by urinary
It also induces fluid accumulation in the intestinal obstruction due to prostatic enlargement , calculi
loops of neonatal hut rot weaned piglets. ST , is or pregnancy. About 5-7 per cent of pregnant
methanol soluble . ST,, causes fluid accumulation women have been reported to haw urinary infection
in young y inlets ( uptn nine weeks) but not in infant without any symptoms. Such .!> vmpttur.' .rt re
mice . The mode of action of ST„ is not known but bdcftmiru undetected and untreated may lead to
: it is not through cAMP or cGMP. It is not symptomatic infection later in pregnancy,
methanol soluble. SI genes are earned on plasct i ids pyelonephritis and hypertension in the pregnant
which may also cany other genes, such as for IT woman, as well as to prematurity and perinatal
and drug res stance . However, ST . and ST & genes death of the fetus.
are not seen to be carried on the same plasmid. Whik infections of the lower urinary tract seem
E , colt Vc rocyiotoxin or Veroroxln ( VT ) was to be 'ascending Infection caused by fecal conforms,
so named because H was first detected (1977) by Us pyelonephritis is probably due CO hematogenous
cytuUixit: effect on Veto cells , a cell line derived infection . Strains canning K antigens are more
from African green monkey kidney cells. It is also
known as Shiga-like toxin ( $ LT ) because ir is
-
commonly rcspon ible for pyelonephritis, while
most isolates from cystitis lack K. anrji cns ,
similar to the Shigella dt-senreriae type 1 toxin in ^
Bacteriological diagnosis of urinary tracr
its physical, antigenic and biological properties . infection has undergone a marked change following
Besides cytotoxicity in Vero or HeLa cells , VT also the development by Kass of the concept of
shows cnterotmricitv in rabbit ileal loopc-- and mouse 'significant bacteriuri . L . Normal urine is sterile but
1
paralytic-lethality as does the Shiga toxin . VT is during voiding may become contaminated with
also composed of A and B subunits. VT genes genital commensals . In order to avoid such
appear to be phage encoded . An antigenically con rami nation , urine used to be collected by
different VT, called VT . has been identified, which catheterisation for culture. Any bacterial growth
is not neutralised bv the Shiga antitoxin, unlike from cathetcri ^cd urine was considered to denote
VTr infection . Even under ideal Conditions ,
catheterisation leads to urinary infection in at least
CLINICAI INFECTIONS two percent , and when pTecaulumK are inadequate ,
Four main types of clinical syndromes are caused the risk is much higher. Hence catheterisaLion is
by E. coll : 1) urinary tract infection , 2 ) di.irthcA, no longer considered justifiable for diagnostic
3) pyogenic infections, and 4) -septicemia. purposes. Instead, clean-voided midstream samples
I I r t n n r y tr -act infection :. E . troti and o t h e r H >f]r 11
' .sre employed I > r eulI :irL . S . H 11 -.- ic .
L' ' '
eol [forms account for the large majority of naturally sbould be collected carefully to reduce
acquired urinary tract infections. Those acquired contamination to the minimum. In men, lr is
in the hospital, following iiiRmnnentarion , are more sufficient if midstream urine is collected after the
often caused bv other bacteria such as Pseudomonas
.a
prepuce i :i retracted and the glans pcnb cleaned
asid. PfuTcui. with wet cotton . Tn women, anogenital toilet is more
The E. coh\ $emtypet commonly reipooiible For Important and should consist of careful cleaning
urinary tract infections lire chose normally found in
BJ ‘l1
with soap and water. Noniiriiant antiseptics such
the feces, O groups I , 2, 4, G , 7 h etc. Only one as chid r ho ! dine have been recommended for vulval
scrofvpe is generally isolated from infected urme at cleaning. Uni re should be passed keeping the labia
Copyrighted material
276 * Textbook of Microbiology
A& SRjr LT ST
In rests
Ligated ttbbiT ileal h>op
Read at ft hours # +
Read it IS hours 4
ON A probes 4
- .^
s- L p ^i r L
^L^ cJ bv tm
Tbc fiisl pnrtiijn of urine that
rK. voiding and are of no significance. Counts between
flushes out commensal bacteria from the anterior the two levels are infrequent when the sample is
urethra is discarded. The next portion of the urine collected properly and processed promptly. Such
( midstream sample;J i -s Collected directly in CO a. results should he considered equivocal and the
Sterile wide mouthed container and transported to culture repeated. Needless to say, interpretation of
the laboratory without delay. Urine is a good b’acteriuria, should always he with reference to the
medium for the growth of coliforms and other condition of the patient. In patients on antibacterial
urinary pathogens, and hence delay in processing or diuretic drugs and with some bacteria like Staph ,
will vitiate the results of quantitative Culture. If aureus, even low counts may be significant -
delay of morn than 1-2 hours is unavoidable , the For quantitative culture, serial ten fold dilutions
specimen should be refrigeruted - of urine are tested hv the pour plate or surface
In quantitative cultures , midstream unite culture methods. This, however, is too complicated
samples w i U give a biphaslc diatribu Lit m of colon i L :H , for routine diagnostic work, for which
most specimens containing either less than 10,000 semiquandradve techniques are more convenient.
or more than 100,000 bacteria per ml. Kass and The most widely used techn ique employs a standard
other investigators have established that in the loop which transfers a fixed, small volume of urine.
presence of active infection in the urinary tract the One loopful of urine is placed on a non inhibitory
urine will contain 104,004 bacteria or more per medium (blood agar] and another loopful on an
ml. This level is, therefore, considered to represent indicator medium ( MacConkey ) . The former
significant bacterinria. Counts of 10,004 bacteria medium gives a quantitative measurement of
or Cess per ml are due to contamination dunmg bacteriitria* while the lattet enables a presumptive
~
y\
Lj opyrighted materia
• Efiierobactenaceae I : CoiiEorms ' Protejs 277
diagnosis o- f the bacterium . The isolates ire in the urine only when the kidneys are infected
identified hy their properties. and not when the infection i * confined to the
Bacteriological investigation of urinary tract bladder. Antibody coated bacteria are detected by
infection is not complete without an antibiotic immunofluorescence lining fluorescent tagged
sensitivity test of the isolate . E. coii and other antihuman globulin or by staphylococcal
common urinary pathogens develop drug resistance coagglutmlTiim .
so frequently that no antihactcm therapy can be Uiorrbeat Right from 1885 when Escherich
instituted meaningfully without testing individual fust isolated the bacillus from the feces of infants
strains. Resistance is often to multiple drugs and is with enteririis, E . coii had been suspected to be a
of the transferable variety. Antibiotic sensitivity tests causative agent of diarrhea. However, as there was
may be done directly using the uri :ie samples as no way then of differentiatingdiirrheagenic E . coii
inocula and the results confirmed by repeating the strains from the welter of commensal strains
test with individual isolates. Invariably present in normal feces, it remained
Because urinary Luc L injection i £ £Uth a common unconfirmed till serotyping schemes for E . coii
problem and bacteriological facilities are not always were developed. It was only in 1945 that Bray
availableh several screening techniques hive been established the etiological role of a specific type of
introduced for the presumptive diagnosis of E.culi subsequently recognised as type Cl 11) during
significant bacterium . These include the following: ^
a hospital outbreak of childhood diarrhea in
1 ) Gricss nitrite test - based on the absence of nitrite London. Soon many other emeropathogemc
in normal urine . The presence of nitrite, detectable serotypes of E , coii came to be recognised as
by a simple test , indicates the presence of mtiate - responsible for infantile diarrhea. Subsequently,
reducing bacteria in urine, 2) catalase test — the other varieties of E . coii diarrhea came to be
presence of catalase as evidenced by frothing on identified in children as well as in adults. At least
addition of hydrogen peroxide indicatesbaclcriuria, five different types of Jiarriieagemc E . coli are now
though a positive result is obtained also in recognised - entcropathogcnic, enterotoxigenic,
hematuria, 3) tri jihcnyltctraioliijni chloride { ETC ) entero invasive , entero - hemorrhag ic
test - bised on the production of a pink - red (Shigatovigeuic or Verotoxigenic ) and entero-
precipitate in the reagent caused by the respiratory aggregarive E , coti-
activity of growing batteria + 4) microscopic Entcropathogen ic Ei . cnli ( EPEGfc These
demonstration of bacteria in Gram stained films of have been associated m & i nly wn h di ,irrhea m i nfants
urine, 5 ) glucose tesr paper - based on the utilisation and children usually occurring as institutional
of the minute amounts of glucose present L:I normal outbreaks but they can also cause sporadic diarrhea
urine, by bacteria causing the infection, and 6) dip in children and less often iii adults , EPEC diarrhea
slide culture methods - ig*r coated slides are was common worldwide from the late 1940* to the
immersed in urine or even exposed to the stream of 1960a . Afterwards, it has become less common,
urine during voiding, incubated and the growth EPEC were identified by serotyping, initially
estimated by colony counting or by colour change by their O and B antigens (for example , 026:B6,
of indicators. None of the screening methods is as 055:B5 t 0I 11:B4 and so on ). After the existence of
sensitive or reliable as a culture . the B antigens became suspect , only Q typing i =
The antibody coated bacteria test has been practised.
employed for the localisation of the sice af urinary The diagnosis of EPEC diarrhea is relalively
infection . This is based on the assumption that easy during outbreaks but very difficult in sporadic
bacteria coated with specific antibod ies are present cases. Fresh diarrheal feces is plated on blood agar
Copyrighted material
278 i Tejcibook of Microbio- logy »
qnd MacC-grikey media. After ( mrnight incubation, ticiihn.il ur culonisaliun factor aTiligertii, of which a
K oIi colonics arc emulsified in saline on a slide number have been identified (CFA I, II , ITI ,[V ).
and tested for agglutination hy polyvalent and Diagnosis of El EC diarrhea depends on the
monovalent EPEC O antisera . At least ten colonics demonstration of entcrotctvin in E . coli isolates by
per plate should be tested as many serotypes are anv of the methods listed in Table 30.2 . A strain of
jf
present in a single culture. If isolated colonies are ETEC may produce either IT or ST or boThB
negative,, the confluent growth is emulsified and Defection of LT is easy as many in vitro methods
tested. During outbreaks, if the causative serotype arc available, such as tissue culture tests ( rounding
is- known , cultures need be tested only with the
j , of Yj mouse adrenal cells and elongation of CHO
particular antiserum. EPEC antisera are now cells due to intracellular increase ol cAMF
difficult to obtain and so specific diagnosis is concentration ), and serological tests, ( ELISA ,
available only in few laboratories, When the passive agglutination and Immunofysin tests ). In
outbreak is caused by a strain with some readily
,
a1
vivo tests such as rabbit loop or intradermal tests
demonstrable feature such as ' ANUT to ferment may be used when in vitro tests are not available.
sorbitol, rapid identification is possible by using The deceedon of ST is more difficult. Tine infant
appropriate culture media. mouse test is still widely employed - The poor
The pathogenesis of EPEC di .urhca is not fully antigenicity of ST has prevented the development
understood. EPEC do not ordinarily produce ol serological cescs ,, chough ST ELISA using
enterotoxins, nor arc they invasive. In infantile mnnndnnal antibody has been introduced. Genetic
enterids, the bacilli are seen ro be adherenr ro the probes art available for detection of ST and LT in
mucosa of the upper small intestine , intimately .
Ecoli culture, or directly i.n feces food or water.
attached to cup-like projections (' pedestals 1 ot the Enterdiivitivt E. coli ( ElEG): These
enterocyte membrane, causing disruption of the resemble shigcllac in many respects . Many of these
brush border microvilli . The name enfcncwdfwrenf strains are nonmotile, do not ferment lactose or
E. coli has been proposed for these strains, which fiernienr 3 r late with acid, but without producing
can be identified by their adhesion ro HEp-2 cells. any gas and do not form lysine decarboxylase. Many
Enterotoxigenic H . cnli ( ETBC ): of these show O antigen cross reaction with
Diarrhea caused bv £TEC is endemic in the shigellae. These ‘atypical’ E . coli strains had earlier
•li
OCClUtt in children as well M adults . Foi laboratory which the bacteria were found beneath the skin
(li nosis of FI EC, the Screny lest used to be and in the deeper tissues . Washing would not
^
employed ( that is , instillation of a suspension of remove the bacteria from such vegetables and
freshly isolated EIEC or shigella into the eyes of cooking alone may ensure safety Thin finding
guinea pigs leads TO mucopurulent conjunctivitis extends rhe scope of EHEC food poisoning to
and severe keratitis). Mice may be used instead of vegetarians also,
guinea pigs-. Cell penetration of Hel .a or HEP-2 Laboratory diagnosis of VTEC diarrhea can he
cells in culture is a more humane diagnostic test. made by demonstration of rhe havilEi nr VT in frees
Tliin ahijitv to penetrate- cells in- determined hv a diltCtly or in culture. As VTEC forms only a
large plasmid , detection of which can also he a minority of focal conforms in infected cases, testing
diagnostic tent. The plasmid to ties for outer individual colonies may not be successful. The
membrane antigens called the 'virulence marker sensitivity can be considerably inert- used by using
antigens’ { VMA ) which can be delected bv the DNA probes tor VT1 and VT . genes. VT can be
EUSA ( VMA ELISA ) tear detected by its cytotoxic effects on Venn or HeLa
Flnl crnlicmc )Trhii (ji [: Ft .vtjli ( EHEC ) cells. Demonstration of VT neutralising antibodies
S bigamy!genic K , coli ( STEC ) dr in convalescent sera may help in retrospective
Verotoxigcnic K . coli ( VTBC ): E. coJi diagnosis.
strains producing vcrocvloroxm ( VT) or Sliiga - like Most VTEC strains belong to the serotype
toxin (SIT ) can give rise Co diarrheal disease ranging 0I57:H7 which does not lemient sorbitol , unlike
in severity from mild diarrhea to fatal hemorrhagic the majority ot E. CQI L SO the use of sorbitol
'
colitis and hemorrhagic uremic syndrome ( HUS) MieConkey ntedium helps in screening for 0:157
particularly in young children and the elderly. The VTEC.
primary target for VT appears to be the vascular Em ernuggregutix e E. mill ( EAEC ):
endothelial cells.This may explain rhe pathogenesis These strain? MC SO named because they appear
of HUS, in which a characterisuc renal lesion is aggregated in a Stacked brick’ formation on Hcp-
capillary microangiopatbv. VTEC also produces 2 cells or glass . They have been associated with
diarrhea in cattle and pigs. The typical EHEC is persistent diarrhea* especially in developing
serotype Ol 57: H 7 hut a few others such as 02 b:
? countries. Most of the are 0- untypahte, but many
Hi also belong to this category. arc H-tvpable. Tn animal experiments they cause
The disease may occur sporadic ally or as shortening of villi , he morrhagic necrosis and mild
outbreaks of food poisonong . The source of edema with mononuclear infiltration of the
infection is contamination by human or animal gubimicHL They form a low molecular weight heat
feces, directly or indirectly. Changing life stv1es and stable enterotoxin called EASTl
citing habits, with growing popularity of fust thud* ( rrtcrciajrjTFTcjriifjvc bvni- *tnbie enttr taxin - l )
"
^
have led to a remarkable increase in EHEC food l*yo enic infections:E. coir form the most
poisomng. Jn the USA, about 20,000 cases of 0:157 ^
common cause of intra -abdominal infections, such
food poisoning occur every year, many with as peritonitis and abscesses resulting from Spillage
hcinorrliagic complications . A Large outbreak of ot bowel contents . They also cause pyogenic
food poisoning caused by E. troli 0 :1 57 „ with QVW infections in the perianal area. They are an
10,000 cases occurred in japan in mostly important cause of neonatal meningitis.
affecting school children . Investigation of this SepticeTiiia: Blood Stream invasion by £ coli r
epidemic revealed the source of infection to he salad mav lead to fatal conditions like septic shock and
vegetables such as radish and alfalfa sprouts, in 'systemic inflammatory response syndrome’ (.SlRJj),
Copyrighted material
280 * Textbook gi Microbiology *
AR R. coll commonly show multiple drug antigen serologically identical the antigen nt S.
Co
resistance, antibiotic sensitivity testing of strains is jyphi and 5* paratyphi C- These may he used for
important in Treatment. the estimation of VI antibodies or for raising Vi
antisera.
EDWARDSIELLA
The genus contain! the species £dwareJsi>Ua Cirrobactcr is a normal intestinal inhabitant.
fands which is a nnnc .LpHulatcd, motile bacillus with Many strains formerly called paracolons belong ro
weak fermentative powers , The name tarda refers this group . It has been isolated from a few eases of
to its lardy or weak fermentation of sugars. Of the enteric fever hut its etiological role is not established.
sugars commonly used , only glucose and maltose It may cause infections of the urinary tract, gall
are fermented. It forms irdnle and HjS, utilises bladder, mitldle ear and meninges.
citrate and decarbaxylates lysine and ornithine.
.
E tarda is a normal intestinal inhabitant of KLEBSIELLA
snakes and other cold blooded animals. It has been The genus Klebsiella consists of nonmotile ,
cultured from normal tfld diarrheiiic human teccs. capsulatcd rods that grow well on ordinary media
Its pathogenic role is uncertain but it has been forming large , dome shaped, mucoid colonies of
isolated from wounds, urine, blood, and from CSF varying degrees of stickiness.They are short, plump,
in cases of fatal meningitis. straight rods, about 1-2 K 0.5-0.8 mm in size. The
capsule is often prominent and can be made out
CITRQBACTEfl even in dram stained smears as haloes around the
,
These arc motile bacilli which utilise citrate, grow bacilli. KlrhsieUae are widely distributed in nature,
in KCN medium , produce H3S and ferment lactose occurring both as commensals in the intestines and
late or not at all. Three species are recognised, Citiv, as saprophytes in soil and water. Their classification
frrundii which gives typical reactions and Citru, has undergone various modifications- They have
ioseri ( formerly Cirro. diverses ) and Cifro. been classified i n t o three species based on
amaJonaticirs which do not form H S- Cltrp. biochemical reactions and into over 80 serotypes
^
freundii strains were formerly classified as the based on the capsular ( K) antigens (Table 30.3).
-
'Ballerup Bethesda group . They exhibit extensive
’
KLEBSIELLA PNEUMONIAE
antigenic sharing with satmoncllac- This may cause
confusion in the diagnostic laboratory Some strains ( Friedlanders bacUhrs , Bacillus mucufus otpsuiatus)
(for exmiple, the Bhainagar strain ) have a Vi This bacillus was first isolated by Fried Under
+
- d
d
——
MR
VP
—
+
+
— ——
Citrate 4- d
Urease
Malonate
+
4
d
— +
—
Lysine 4 d —
Copyrighted materia
* Fnterptjgdefiacaae l : Conforms Proteus » 283
persons who have medical problems such as and belongs to capsular type 3.
aJcoholism , chronic bronchopulmonary disease or The species A'. o Tnca may he rarely isolated
diabetes meliitus. The disease is characterised by from clinical specimens. ^
massive mucoid inflammatory exudate of lobar or
m
E. cloacae E . acrogcncs
Gas from gjyceml - +
Aesculin hydrolysis - +
] .yimc decarboxylase
Arginine dihydiulaK
-
+
i
Copyrighted materia
282 * Tgxlboofc 01 Micmtuoiogy *
positive , Biochemical rca *cti < > n ^ arc evident best ro their pleomorphism, after the Greek god EYoteui
at 22 LC ; n 37 C they may be negative or irregular. who could assume any shape.
Only one genus is recognised , tl , aim lr is '
The tribe Profcrac is classified into three genera
found in human and animal feces, sewage, soil and
water .
- Pro re us, Moig-AncUa and Provkfcnria . Most of
them except for cume Pruvindencia strain --... produce
a powerful urease which rapidly hydrolyses UTea
SERHAT1A fo ammonia and carbon dioxide. A characteristic
This ditfcis from Hatnia in farming a pint, red or feature which distinguishes Pro ferae from Other
1
magenta , nondifllfoiblc pigment called prodigies in enterobacteria is the presence, in all members of
which is formed optimally at room temperature. the tribe, <> t the nuitnc phenyl alanine deaminase
Only one specks is of medical importance - 5, which converts phenyl danine to phenyl pyruvic
/nareescej] .? ( £# iic > //trs prodigiosus' ) , Jr is acid (PPA reaction ). All of them with few
l
pleomorphic, with minute coccobicillaiy and exceptions show the following features:
normal haciliury forms-. It i ^ a saprophyte found in Gram negative , noncapsulated, pleomorphic,
water , soil and find . Ir may grow in sputum utter
motile rods,
collection and may suggest hemoptysis because of * resistant to KCNt
the pigment formed Cpseuduhetnupivsis ' ). * degrade tyrosine;
Nosocomial infections due to S, rthucescttis are * tail to acidify lactose, dulcitol or malonate;
heing reported with increasing frequency, The ' do not form arginine Or lvsirte Jec.Lrhox vlasc or
bacillus lias been associated with meningitis * beta galactosidasc;
endocarditis, septicemia , peritonitis , respiratory MR positive, VP negative .
.
infection and many oilier conditions Multiple drug
*
included in this chapter. The name ' Proteus refers film of breath ) . Nonflagellaled Variants grew as
Ornithine 4 - + - - -
decukiyliie
Indole - + # 4 4-
PermencaEi ^ n. of adi*iiiTi>] - - - + ± ±
Fermeniarion of frehaloi* 4 t t 4 -
Copyrighted material
^ EnlerobacifiriaCEfEie I : Colilorms- - Froteun r 283
non motile strains of Pt. vulgarcalled rhe lX infrequently. Nosocomial wound infections also
-trains ' , wtre agglutinated by sera from typhus fever occur .
parienCS- This heternphilic agglutination due to the The genus Providencia ( formerly known as
sharing of a carbohydrate hipten by certain strains Aofetrs inconstant} con tains three species seen in
of Proteus and iiJcettsLae forms the basis of the clinical infeiTioiu. PPyv akalifacieos is some times
Wei I -Felix reaction for the diagnosis of some seen in normal human feces but far more frequently
TickertsiaJ infections . Three nonmotile Proteus in rHirrhftdl stools though its etiological role is
strains 0X 2 , OXIV anti OXK are used in the uncertain. Prov. sEtWtji is a common cause c4 mitUUV
agglutination tost . infection and if infection in burn. Prov. rettgerii is
i
Proteus baLilli are widely distributed in nature part of the normal fecal flora of reptiles and
as saprophyte*, being found in dccompotmg animal amphibians, and sometimes causes nosocomial
matter, in sewage, in manured soil and in human infection of the urinary tract, wounds, bums and
and animal feces. They are frequently present on blood,
the moist areas of the skin . They are opportunistic Proteus bacilli arc resistant to many of rhe
pathogens, commonly responsible lor urinary and common antibiotics. An exception is Pr. mimhilis
Septic infections , often nO£tic0fnul which is sensitive to anupLdilijrt Lind cephalosporins.
]
The genus ADMIX contains rwo species of Provident ia are the most resistant, particularly Am
medical importance - Pr. mirabilix , which is an stiwrtii which is also resistant to disinfectants such
important urinary and nosocomial pathogen , and as ehlorhexkline, Cctrimide, hcnyMlkonium chlun.de
Pr. vulgaris which is found much less commonly and heavy metal compounds such as silver
in human infections. Pr. mfrabjJrs ! H indole negative sulphonarnide., making it a major pathogen in bum ®
and Pr. vulgnrrt. nidufo positive. units. Jt is sensitive to phenol and gluiaraldehyde-
Cultures of proteus bacilli have a characteristic Amikacin and dprofioxodn arc generally effective
putrefactive HMJOUT described as 'fishy ' Of 'senutuT in tmtnwnt
Pr. mirtbiln aod / V. vujgarirswarm on solid culture
media . Discrete colonics are seen in young cultures ERWINIA
but thereafter actively motile cells spread on tlie These ire anaerogenic bacilli forming a yellowish
surface of die plate in successive waves to font ] a pigment, usually found in soil and causing plant
thin filmy layer in concentric circles. Swarming infections E , hcrhk' O'b has occasionally been
*
growth is a problem in the laboratory when mixed isolated from respiratory and urinary infections in
growth is obtained in which proteus bacilli are predisposed or hospitalised patients.
Copyrighted material
284 4 Tftxtboofc of Microbiology
Further U . Gliding
BlIMlllJf 1990. The pathogenicity ofenteropathogcnic E#cAmcAia coL ] ModMiaobioL 47:383.
Barnwell JG . 19901. PadiiDphy5®log> of Larrhed. disorders . Her Jn/iet DJS. l 2 S.3fl.
^
'
s “
Cooke EM. 1985. EsrAfriciji coli, an overview. J Hjg (Cmb) 95: 523 .
Ewing WH , l 9B6, Bdmtxb and &mng $ Modfiotioa o f E r-r e; 4^ sdn New York Bbener,
.
GIOR Hj . 1991. The puhogeneti* ef Eicheridtii coir diarrhea- RewMed MksebkL 237 .
Kawr* P and JB K-aper . 1998. Diarfhjjgenic E .ccli. Qnn Microbial Reis 1 L742.
uo
y py righted materi 3
CO Emerobacteriaceae II: Shigella
the clinical picture of dysenterv. is fermented with the production of acid, without
Morphology : ShigeElac are short, Crain gas, except for the Newcastle and Manchester
-
negative rods, about 0, 5 pm * 1 3 pm in size.They biotypes of Sh. flexnen type 6, and some strains of
are nonmofile, nnnsporing and noncapsulated. Sh . bovJii tvpes 13 and 14, which form gas.
Fimbriae may be present . Fermentation of mannitol is of importance in
(lulturAl characteristics: They are aerobes classification and shigellae have traditionally been
and facultative anaerobes, wh a growth tempe - divided into mannitol fermenting And
rature range of 10- 40 °C and optima of 37 <S. and
r
nonfermenring species . .Sh, flexneri , Sh , bovdii and
pH 7.4 . They grow on ordinary media bur less .
Sh Monnei ferment mannitol, while Sh , djventciiat
readilv than other enterobacteria. After overnight does not . Exceptions are not infrequent. Lactate
incubation, colonies are small , ahout 2 mm in and sucrose are not fermented, except hy LSJJ. smaa
diameter, circular, convex, smooth and translucent . which ferments them late . Adonitol, inositol and
Occasionally on primary' isolation and I reqoent Iv salicin are not leriUentod-
in subcultures, a proportion of the colonies may be Antigenic structure: Shigcllae possess ore
of the rough type. Colonies on MacConltcv agar or more Lmaior ' antigens and a large number of
arc colourless due to the absence of Lactose ‘ .
minor" somatic O antigens Some strains possess
ferment attorn An exception is Sh, sonner which K antigens. These arc not relevant in typing but
ferments lactose late and forms pale pink colonies. may sometimes interfere with agglutination by O
Deoxycholate citrate agar ( DCA) i $ a useful Antisera . Fimbria! antigens arc also present. In
selective medium. Growth is inhibited on Wilson general, the antigenic structure of shigellae is
and blairs hismuth sulphite medium. simple, compared to the complex structure of
iCcBtNtnnee Shigelkc arc not specially resistant. salmoncllae.There 15 considerable antigenic sharing
They arc killed it 56 in one hour and by 1% between some members of rhe genus as well as
phenol in 30 minutes. ! n ice they last tor 1-6 between shigellae and £. coii. Common fimbria!
Copyrighted material
2&> * Textbook off Microbiology t
an ripens may also occur, particularly in Sh. ScxnCii- A2 . Fragment Al appears to inactivate host ceil tiO
li is , therefore, important that: the identification b ribosome , interfering with protein synthesis .
of shlgcllae should he made by a combination ot -
Sh thsevjHTFje type 2 (5b, schmils. i) forms
antigenic and biochemical properties and nor by indole and ferments sorbitol and rhamnnsc .
slide agglurbiiiNDn alone. Serotypes 3-7 were described by Large and Sachs
in India and hence used to be known as the l .aige-
CLASSIFICATION Sachs group . Three fiirther serotypes have been
Shigcllac arc classified into lour species or dusi- ribud making a total of ten.
subgroups based on a combination of biochemical Sh . Ilexntri ( subgroup H ) i This gioup is
and serological characteristic? Serotypes are
r named after Flexner, who described the first of the
distinguished within the species , -Sir . aonnri is mannitol fermenting shigellae from Philippines
serologically homogeneous and is danificd by ( 19 D0 ). This group is hiocheinically heterogeneous
Cclicin typing. find antigen ic ally the most complex among
Sh . dyscnterinc ( subgroup M i This species shigcllac. Based on type specific and group specific
of mannitol nontermenting bacilli consists ot ten antigens , they have been classified into six serotypes
serotypes . Type 1 is the bacillus originally ( 1 -6 ) and several subtypes ( la; lb; 2a , 2b; 3a , 3b.
described by Shiga ( ifi . sfrfgar}, Jr is indole negative 3c, 4a , 4bn 5 a , 5b). In addition , two antigenic
and is the only member ot the family that is always "
variants' culled X and Y are recognised , which lack
catalase negative .. (Si. tehmitzi and Sh. so/incr arc the type specific antigens. Serotype t> is always indole
invariably catalase positive , while among other negative and occurs in three biotypes, some ot which
shigella species , some strains may be catalase form gas from sugars.
negative, ) Sh . Itovdii ( subgroup Cfc 'Hiis group consists
Sh . Jyrejifrriiir type 1 forms a toxin (Shiga of dvsenterv bacilli that resemble Sh . tlexneri
toxin ), the earliest example of an exo toxin produced biochemically but not antigenieally. Tlie group in
by a Gram negative bacillus. Three types of toxic named after Boyd , who first described these strains
activity have been demonstrated in shigella culm re from India ( 1931 ) , Fifteen serotypes have been
filtrates : ( 1 ) neurotoxicity, demonstrable by identified . X!J . boydii are isolated least frequently
paralysis and death on injection into mice or from cases of bucillarv dysentery.
rabbi ra. JI Tintigh known as ‘henrotovi n', the primary Sh . sonnei ( subgroup Dfc litis bacillus , first
site of its action appears to be not the nervous described by Sonne ( 1915 ) in Denmark, ferments
tissue hut the blood vessels, iniainlv of the central lactose and sucrose late . It is indole negative. It is
nervous system, with the neurological effects being antigcnically homogeneous bait may occur in two
ton » ndar v ; (2 ) eilTcruUlxicity, with induction < 11 tluid forms - phase J and phase II - the latter forming
accumulation in ligated rabbit ileal loop . Two colonies that are larger, flatter and more irregular
new shigella enterotoxins have hern identified , On subculture , phase I produces both types of
designated as Sh- ET- 1 and 2, the former colonies but phase II is considered to be a Joss
confuted to Sh . flexneri 2a and the latter more variation. Organisms in phase 11 maybe isolated
widespread; and ( J ) cytotoxicity, causing cytopathic from patient^ bait are more common in convalescents
changes in cultured Vero cells . This appears to be and carriers.
the same as Verotoxin 1 ( or Shiga -likc toxin ) Sh . Mtnnci causes the mildest form of bacillary
produced bv certain strains of E cali ( VTEC) . The dysentery, In many cases the disease may only be a
toxi n consists of binding ( fl ) and acti ve { A subun i is.
' mild diarrhea. 1 lowever, Sh . sojinei infecrion persists
'
Subunit A is divided into two fragments Al and as the most common shigellosis in the advanced
Copyrighted materia
* Emerobactefiac*ae II . Shigella » 207
eoimtries. For epidemiologici! purposes, Sh.. sonjwj Congo red binding rest. Invasive property is related
has been classified into many colicin types, to the presence in the bacillus of large plasmids
( M.W. 140 « 10") coding for the outer membrane
P \ THOGBPJIC 1 T"i protein responsible for cell penetration , These
fihigcllac cause bacillary dysentery. Infection occurs proteins are called 'virulence marker antigens'
by ingestion. The minimum infective dose is ] < iwL ( VMA ) . Detection of VMA by ELISA serves as a
OS few its Li I 100 Willi being Capable of iiii dating virulence test for Shigellaef as for ctuerouwosive
die disease, probably lwvjusr they survive gastric fcall .
acidity better than -other enterohxLttria. Their Baci 11 ;in (JysciUUJ h as A sh ort i m Lthat N m period
" '
rot noninvasive ones . The invasive property' of the whoit spectrum of disease caused by sbigellae.
bacillus can be demonstrated by its ability to \ [ uni an beings arc the only natural hosts for
penetrate cultured I IcLa or Hcp-2 cells or by the shigellie . Captive monkeys have been found
Subgroup A B c D
Species Sh_ dywentcrixe Sh >
ffcxncri Sh. baydn Sh. sonnei
Mann Lhi ]
Lactose
Sucrose
—— ——
A
——
A A
A Late
A Late
Dulcitol
1 rvdole
=
d
—A
d
d
*
-
Ornithine decarboxylase - -
Serotypes lfi 6 + variants 15 Only one
A - Acid
-
d Variable
Copyrighted material
m < TextbwK of Microbiology
Table 31.2 Blotypes cl Sh. Itexneri Type 6 Shigellosis is worldwide in distribution hut
Biotypt Frmacii fa troii of .
epidenuolpgicaUy there arc a lumber nfdifferences
herwren the nature and extent of the infection in
Ghicote Mi nnitoi the affluent and in rhe poor countries . Where
RU>yd RK
environmental sanitation is good , as in Britain,
A A
Manchester shigellosis is mainly seen in young children and in
AG AG
Newcastle A or AG special situation* like mental hospitals (asylum
dysentery ). Sh. SOJinej' it the predominant infecting
A = Avici
-
AG Acid and GM
infected but such infections may h.ivc hecn of
agent. In the USA , Sh. sonnei is the main type in
the north , while Sh. Hanoi is more common in
hudlixi origin , Experimentally, dysentery cm be the south . In countries where environmental
produard only ]]i monkeys. Human volmweer studies mutation is poor, endemic shigellosis is found in
huve clarified the spectrum of shigellosis . Of H group all age groups and is caused by all species. In India,
of volunteers ingestin 10 , 000 Sh . tlexneri 2a Sh - tfexnerr has been the predominant species,
^
bacilli , a quarter remained ibymptqattttic, another having formed .50-B5 percent of isolates in different
quarter had transient fever a day or two Later, a series. Sh. dysenteriae ( 8-25 per cent ) and Sh.
quarter developed fever with watery diarrhea, while sonnet ( 2 ~ 24
per cent ) are the next common specicS-
tuiJv OrtC quarter developed topical dvscntcrv. Sh . toidii (0-8 per cent ) has been isolated least
Evpldemiulngy : Epidemics of bacillary frequently.
dysenrery have always accompanied wars and ufreu I he picture baa changed in recent years. After
influenced [ fieii outcome. In several campaigns, a long period of quiescence, Sh . dysenticrj je type 1
'
more men have died of dysentery than were killed suddenly appeared in an extensive and virulent
LO bardc. A recent instance was the major epidemic epidemic form in Central America in 1968, In 1973,
affecting many thousands , with high case fatal itv, ;L similar outbreak started in Bangladesh and later
which occurred during the Rwandan civil war Jit in Sri Lanlca. Several localised outbreaks were
1994, Epidemics in civilian communities are observed In India from 1974, followed by extensive
associated with poverty and Each of sanitation epidemics in various Stiles from the early 19ft0?.
The only source of infection are human bongs I he epidemic strains showed plasmidbornc
- cases , ( if less ^ftctl carriers . Chronic carriage is
rare , the bacilli disappearing from feces within a
multiple drug resistance -
Laboratory diagnosis: Diagnosis is made by
few weeks* except in some malnourished vhidren isolating the bacillus from feces. Fresh feces should
or AIDS patient*. ishigcllac exhibit a high rate of be inoculated without delay or transported in a
secondary household transmission . The modes of sulmhle medium such as Sachs' buffered glycerol
transmission may he as fellows : (1) direct, ill rough
-
contaminated fingers : hand -to mouth' infection;
-
saline, pi i 1.0 7 . A. Highly alkaline transport media
used for vibrios are inhibitory for shigcllac . Rectal
( 2) through fomibes such as door handles , water swabs are not satisfactory.
taps, lavatory seats; (3) through water; ( 4 ) through For Inoculation i r i s best to use mucus flakes if
contaminated fiw > d or drink. Skigelhisw , cspcciallv they (ire present in the sample. MacConkcy and
Sh. sonnei infection , may occur as food poisoning: DC A plates are inoculated. A f t e r overnight
,
and ( 5) through flies which may transmit the incubation at 37 "C, the plates ate inspected for
infection as mechanical vectors ; (6) Shigellosis may nonlactose fermenting colonies, which are tested
occur in young mate homosexuals ns part of the for motility and b i o c h e m i c a l reactions, Anv
gay bowd syndrome. nonmotile bacillus that is urettSC, citrate, H S and
^
Copyrighted material
4 Eriterabacianacgae ( I: Shig&lla h 289
KCN negative should be curt her i nvest i gated by widely prevalent in shigellae . Indiscriminate
biochemical tests (Table 31.1). Identification is antibiotic treatment will only worsen the problem
confirmed by slide agglutinsbon with polyvalent
. of drag resistance in intestinal bacteria. Antibiotics
and monovalent sera - Demonstm tion of antibodies should therefore be 1 i mited to severe or toy i q gases,
in sera is not usefiil.
, and in the very young, debilitated and the aged.
Trcntmcntz Uncomplicated shigellosis self is a The choice of antibiotic should be based on the
limited condition in which the patient usually sensitivity of the prcvailitig stTain. Many strs i ns are
recovers spontaneously in a few days. However, in still susceptible to nalidixic acid or norfloxacin and
acute cases , particularly in infants and young other fluoroquinoloncs-
children, dehydration has to be corrected promptly. Conlrol; Control consists essentially in improving
Oral rehydration is adequate in most cases. persona] and environmental sanitation. Antibiotics
Routine antibacterial treatment is nor indicated have no place in prophylaxis. No effective vaccine
in dysentery. Multiple drug resistance plasmids axe is available .
LCWIF MJ - 1997 - Sh igella - In Greenwood D {cd }, JWedica/ AfifiubioJcKy, 15* edn , Edinburgh: Church ill - L -'iugrtDfK -
The b cmis Sitrn&neilx couii -sts ot bacilli that with perilrichate flagella , except lor one type,
^
parasitise the iurevrincs nf a large numhcr ol- .
,Y giiltin iriim - pultorum , which is always
ve rtrbratc species and inlcct hum in beings, leading non motile. Non motile mutants of other types mav
to enteric fever , gastroenteritis, septicemia with or sometimes be found . They do not Form capsules or
without focal sjppLiriiiiiii, and the carrier state . spores but may possess fimbriae.
The most important member ot the genus is Cultural characteristics: Salmonellae are
S .-iirrutneHa lyphi , the can native agent ot typhoid ae n > bi £ and fitultf tively arise n itaic, growing ie adi ly
Fever. The typhoid bacillus was first observed by on simple media over a range of pH 6-S and
Ebertb ( IfitfO) in the mesenteric nudes and spleen
oF fatal cases of typhoid fever and was Isolated by
—
temperature 15 41 ' C {optimum 37 ' C ) .
C olonies are large, 2-3 mm in dianteter, circulai,
tJafFky ( 1884 ) . It came to be known as the Ebcrth- low convex and smooth. Thev are more translucent
I
tiaffky bacillus or Ehcrthelh lyphi. Salmon and tfian coliForm colonics . On MicConlcey and
Smith ( 1K 85 ) described a bacillus which was deoxycholate citrate media , colonies are colourless
believed to cause hog cholera ( mistakenly, as It is a due to the .absence of lactose Fermentatiou. On
-
virus di.Hcahc ) . nu: bac illus, later called Y choScrac -
"
group, consisting of the typhoid and paratyphoid positive, VP negative and citrate positive. Y lyphi
bacilli that are exclusively or primarily human and a lew other sal monel lac do not grow in
parasites and f 2) the food poisoning group, which Simmons' citrate medium as they need tryptophan
^
are essentially animal parasites but which can also as the growth factor. Urea is riot hydrolysed. H S
-
infect human being :, producing patrocntcritis,
septicemia or localised infection!.
is produced , except bv Y paratyphi A, S. cholene
stris and some other stjjecics.
^-
Morplmli kgy:5almoncliae ait Cram negative rods, The enteric fever group may he separated
about 1-3 pm * 0-5 pm in size . They arc motile biochemically ('fable 32.1 ) ,
Copyrighted material
Entenobacieriaceae III : Salmonella t 291
Rcsiyfcmce: The bacilli are killed at 5 ? ‘C in The O antigen is unaffected by boiling, alcohol
one hour or at 60 " U in 1 ? minutes, Boiling fir or weak acids . When mixed with antbcru , O
chlorination of water and pvteurisation o f milk antigen suspensions Form compact, chalky, granular
destroy die bacilli . In polluted water and soil, LILCV clumps . O agghitination takes place more slowly
survive for weeks anti in ire tor months. Cultures and at a higher temperature optimum (St >— 5.S ^C)
may he viable for years if pCCKnul from fining. than H agglutination ( H 7 *C)- The O antigen is
They sue killed within five minutes, by mercuric less immunogenic than the IT antigen and the litre
chloride ( 1 : 500) or 5% phenol. uf the O antibody induced after infection or
Antigenia structure : Salmoncllnc possess the immunisation is generally lower than that of the
following antigens based on hi eh they arc classified ft antibodyr
- .
and identified: (1) flagellar antigen 11, (2) somatic The O antigen is not a single factor but a mosaic
antigen O, and (1) a surface antigen Vi, found in of two or more antigenic factoid. Sulmonelllae aie
some species Several strains carry fimbriae . classified into a number of groups based on the
Fimbrial antigens are nut important in identification 1 n e sei ice of duncteristic O ai i rigens on the banerial
but may cause confusion due tu their nonspecific surface.
nature and widespread sharing among \ i urUigcTi: Many strains of $. typfii tail ro
enterobacteria. agglutinate with the O anti serum when freshly
LI Ulltj £ L:li: This antigen present on the flagella isolated. This is due to the presence of a. surface
is a heat labile protein. Ir is destroyed by boiling <ir poly saccharide antijtcn enveloping the O antigen .
by treatment with alcohol but not by formaldehyde . Felix and Pitt, who first described this antigen ,
When mixed with antisera , H suspensions believed that it was related to virulence anti gave it
agglutinate rapidly, producing large , loose , fluffs the name ' Vi antigen'. It is analogous to the K
clumps. The H antigen is strongly immImogenic antigens ot cdliiorms. It is heat labile . Bacilli
and induces antibody formation rapidly and in high inagglu finable with the O antiserum become
litres following infection or immunisation . The igglutinible after boiling or heating at 60 "C for
flagellar antigen is of a duul nature, oerurring in one hwr. It is also destmved liy N 11C l and O.Ji N
one of two phases. NaOH It is unaffected by alcohol of 0.2% formal.
p
S, typhi A d A d
Sr purmphi A AG — —
S. paratyphi B AC AG — AG
5- paratyphi C AG AG AC
Copyrighted material
292 TRxtbook or M exobiology
*
In human volunteer experiments, steal ns possessing
m
the VI antigen were found to cause clinical disease
more consistently than those lacking it.
The Vi antigen L ? poorly immunogenic and only
low rirres of antibody are produced following
infection . No Vi antibody s induced by the
]
S population of motile cells rich in H antigen , from cL , d , etc. , and after I, as ill , z2 , Ctt . Fhasr 2 antigens
*
such cultures, selection may he carried nut by using art far fewer and urc termed 1 , 2 etc. In some
*
Craigic ' s rube . This cunsisti of a wide tube species, antigens belonging to phase 1 mav occur
containing soft agar (0.2*Hi) at the centre of which .
as the phase 2 antigens ( for example, e n, zlS ) .
is embedded a short, narrow tube open at both ends, Strains thar possess both phases are called dipiuiyii.-.
in such a way that it projects above the agar. The Some, tike & typhi, occur in phase 1 only ami art
strain is inoculated carefully into the inner tube . called morlOphahiu.
Copyrighted material
1 Entorobaetenaeeae ill Salmonella * 293
A (.illume will contain. LL'IIS with the flagellar morphology and loss of the O antigen and of
antigens of both phases but generally One Or the virulence. The colony becomes large , rough and
other phase wi]] predominate so that the culture is irregular. Suspensions in saline arc
agglutinated only by one of the phase antisera . For auto aggluti nable. Conversion into R forms occurs
serotyping of Salmonella isolates , it may be by mutation. R forms may be common in laboratory
necessary to identity the flagellar antigens of both strains maintained by serial subcultivation. S - R
phases. A culture in phase 1 can be converted to variation may be prevented to some extent by
phase 2 by passing it through a Craigic's rube maintaining cultures on Dorset 's egg media in the
containing specific phase 1 antiserum, and the cold , ot ideally by lyophilisition.
reverse conversion achieved by using phase 2 Mucoid colonies, associated with the
antiserum . development of a new mucoid or LM’ antigen , have
\ variation; Fresh isolates of S, typhi been described with S , paratyphi B and some other
generally carry a surface layer of Vi antigen that species.
completely musks the O antigen . Such bacilli arc Variations in O antigen: Changes in rhe
agglutinable with the Vi antiserum but not with Structural formulae of the Q antigen may be 1nductd
the O antiserum. This is called the V form. After a by lysogcnisatEon with some converting phages,
number of subcultures, the V] antigen is completely resulting in the alteration of serotypes . Thus, $,
Lost. Such cultures arc inagglutiiiable with the Vi anatum is converted into S. newington by one
antiserum but readily agglutinable with rhe O phage and the latter into S, jiajjmeapoLs by another
antiserum . This is called the Wform. Intermediate phage ( Fig, 32,2).
stages during the loss of the Vi antigen , when the Classification and nomenclature: The
bacillus is aggluti nable wi r b both Vi and O antisera, classification and nomenclature of salmnnellac have
arc called ' V\V forms’, undergone several modifications over the years.
Ocher Vi-conraining bacilli such as 5. paratyphi Inclusion in the genus is based on common
C anti S . duhlin seldom have the O antigen biochemical properties. Classification within the
completely masked by the Vi antigen . genus is on antigenic characterisation based on the
S R variation: The smooth -to- rmjgh variation Kauffmajin-White scheme. This scheme depends
is associated with the change in the colony on the identification, by agglutination , of the
Copyrighted material
294 * Te* ttHH3 k ot Microt^ ology *
1.2
1.4, .5 , 12 1.2
S, cAesctr J , 5, 12 e, h E n, x
7 Cl
- S' paratyphi C
, &, 7 , ( Vi) c 1.5
S. choleric-sun 6s 7 c 1.5
S - C2 S' rrruejfcifwn 6, 8 d 1.2
-
9 D 4, 12 , ( Vi ) d -
S. trtKfitidis
Sr gpHmamm
1, 9, 12
1, 9, 12 - ——
10 - £1 S' amtum
,
1 10 e, h 1.6
structural formulae of the G and H antigen* ofthe inn > four subgcnera (Table 32.3):
strains {Tabic 32,2). ubgrjjtrs /, the largest and medically the most
Salmonellae at Initially classified into ^
important group contain* all the species which
ierob icil groups , based on the presence of commonly cause human and animal infections.
^
distinctive O antigen factots, which ire designated hah U contains mostly specie
; isolated
^ 3
enuf
4
— #
-- - —
many but now with some 2400 serotypes of
salmoncllae, giving individual names isuoi realistic.
On the basis of biochemical reactions ,
KaufFmann proposed that salmoncllae be classified
Milonare
Sol Lein
KCN — —— # 4'
-
+
+
Copyrighted material
* EntiefDtiaciBriaD V
' '
n III: Salmonella 295
Modern tajconnm ical techniques, especially abortus-nvis in sheep and S.gulWnanjir; in pouitr v.
DNA studies, have shown that all the members of Others such as S. typhimurium, have a wide hcM
tin genus Salmuneifae and of the funner genus range affect ing animals, I 'i rds and humans. lniL L [ iofl
Arisema are so closely related that they should all in animals may vary from an asymptamit ic
be considered as belonging to a single species, in a conditi on to fatal, and someti mes cpi ^ootic di seasc.
genetic , phylogenetic and evolutionary sense. ,\ typhimurrum and 5. cnKtiridis cause a fatal
Variations in properties such as antigenic structure, septu emi .i in rats and mice. S- putfanim causes
'
biochemical reactinns and host preferences 'white diauhea in chicks and 5- galiinarvm fowl
'
-—
salmonellae. S. enterica i classified into seven
subspecies based on DNA reussut latum tests.
( 2) septicemia, with or without local suppurative
-
Ir ionsj and (3 ) gastroenteritis or food poisoning.
These subspecies are named enrerica , naiamac,
a.r / zonae. didrizotae* houtcnac, bongori and indict ENTERIC FEVER
Subspecies ertrenca corresponds to the former The term enteric fever Includes Typhoid lever
submenus T. caused by ft. Typh; and paratyphoid fever caused by
Such classification and nomenclature , while V p.ojrcphi A, B and C.
being taxonomic ally correct , would be loo Typhoid fever was once prevalent aii er the H
complicated for use in clinical bacteriology. For world and was not well demarcated fr other
example, the taxonomically correct name for the prolonged fevers. A detailed study of the disease
typhoid bacillus would he * Salrrumella enterjca, was presented by Bretnnneau (1 j who Jn m 11 tied
subspecies enteric a , serotype typin . Therefore, the the intestinal lesions. The name typhoid wa > u cn
^
'
old pracucc of referring to clinically imports or by Louis (1829) to distinguish it from typhus fewer.
salmoncllae serotypes by the species name continues Budd (1856 } pointed out that the dtsejic was
m clinical bacteriology. transmitted through the excreta of patients, Ebetth
Sometimes serotype* may have to be further (i 860) described the typhoid bacillus and Gafftcy
differentiated. Thus, S. gallinanun and S. puiloruiA
cannot be distinguished serologically but they can
-
( 1884 ) i olated ii in pure culture. Its causative role
was confirmed by Mete bn i kntff and Besredka (1900 )
be idenrified by biochemical reactions ( S , . hy infecting apes experimentally, S- paratyphi A was
galiinanim is anaerogenic and ferments dtdciiol isolated by Gwyn (1898) , S, paratyphi B ( £.
unlike ft . pul/firum]. Important pathogens such as .schoftmuMcn) by Achard and Rcnsaude (189fi ) and
S. lyphif S. pafxtyphi A and B, and S . typhimurium S. paratyphi C ( S. frrahfcjtfjf ) by Uhlenhuth and
can be further typed for cp^ icrniolog ^ cal purposes Hubcncr (1908 ) from cases resembling typhoid
by phage susceptibility, biochemical properties , fever.
bacterociti production and antibiogiam, Thc infection is acquired by ingesnon . In
I Vti hogtnicityt Salmonella* are strict parasites human volunteer experiments, the ID50 was found
of animals or humans. S. typh\ t S , para pfu A and be about Iff to 10" bacilli . On reaching the gut ,
^
usually, hut not invariably S . paratyphi B are
to
the bacilli attach themselves to microvilli of the
confined to human beings. Other salmoncllae are ileal mucosa and penetrate to the lamina propria
parasitic in various animals - domestic animals, and submucosa - They are phagd rtosed there by
^
—
rodents, repifles - and birds. Some species axe host
adapted S. abuttus-rqiir found only in horses, Sr
polymorphs and macrophages ^ The alulitv to resist
intracellular killing and to multiply within these
Copyrighted material
II JR 'ligR
296 i Twcttxnfc of Microbiology *
cells is a measure of theb virulence. 'I'hey enter the thromboses and peripheral neuritis are other
mesenteric lymph nudes, where chey multiply and , complications found . Osteomyelitis is a rare sequel.
via the thoracic duct , enter the bloodstream. A Convalescence is slow. In about .5-10 per cent
transient bacteremia follows , during which the of cases, relapse OCCUR during convalescence - The
h.icilh are seeded in the Liver , bladder spleen,
, relapse rate is higher in patterns treated early u i.ch
bone marrow, lymph nodes, lungs and kidneys, chloramphenicol ( 15-50 per cent ).
where further multiplication takes place . Towards S . paratyphi A and B cause paratyphoid fever
the end of the incubation period, there occurs which resembles typhoid fever bui is generally
massive bacteremia from these sites of milder. 5. paratyphi C may also cause paratyphoid
multiplication, heralding the onset of clinical fever but more often it leads to a fbmk septicemia
disease . with suppuradve complications. Other salmonellae
A & hilt ii a
"
good culture medium fur the have on occasion been repotted to cause enteric
bacillus , it multiplies abundantly in the gall bladder fever. These have included S. dublint S. baricUy^ S.
and is discharged continuously info the intestine sendai . 5. enfe .nr.'dis, £ typhimvrium, S- eastfcnume ,
where it involves rhe Feyer’s patches and lymphoid S . saintpaul , S . oranienburg and S . panama .
follicles uf the lie urn . These become inflamed, Infection with Alkaligenes fiecalit also may
undergo necrosis and slough off. leaving behind sometimes cause a similar clinical picture .
the characteristic typhoid ulcers. Ulceration of the Epidemiology: Typhoid fever has been virtually
bowel leads Co the two major complications of the eliminated from the advanced countries during the
disease - intestinal perforation and hemorrhage, last several decades mainly as a result of
DLLIMG the 5-4 weeks that normally constitute the improvement in water supply and sanitation hut it
,
course of the disease , [ he intestinal lesions undergo continues to be endemic in the poor nations of
healing- the world . The control of paratyphoid fever has
The incubation pcr: id is usually 7-14 days but not been so successful . The distribution of
may range from 3-56 days and appears to be related paratyphoid bacilli shows marked geographical
to rhe dose of infection . The clinical course may differences. £ paratyphi A is prevalent 11 L India and
vary from a mildundiflfercnt iated pYrw i a (ambulant other Asian countries, Eastern Europe and South
typhoid ) to a rapidly fatal disease. The onset is America, £ paratyphi B in Wfestcrn Europe, Britain
usually gradual , with headache, malaise, anorexia , and Notch America; and S. paratyphi C in Eastern
a coated tongue and abdominal discomfort mth Europe and Guyana.
cither constipation nr diarrhea. The typical features Enteric fever is endemic in all parts of India.
are a step-ladder pyrexia , with reladve bradycardia An incidence of 930 per 100,000 was recorded in
and toxemia. A soft, palpable spleen is a constant the late 199Gs in a 5-year community based study
finding. J Jepatomegaly is also common. 'Rose spots of children in Delhi . Worldwide, 16 million cases
that fade on pressure appear on the skin during the are estimated to occur annually with 600 ,000
second or third week but are seldom noticeable in deaths! The proportion of typhoid to paratyphoid
dark skinned patients. A is about 10: 1 . Paratyphoid B is rare and C very
The most important complications are intestinal rare . The disease occurs at all ages but is probably
perforation, hemorrhage and circulatory collapsc. most common in the 5-20 years age group. The
Some degree of bronchitis or bronchopneumonia age incidence is related to the eadecnidty of the
\ < always found. Some develop psychoses, deafness disease and the Level of sanitation.
or meningitis. Cholecystitis, arthritis , abscesses, The source ol infection is a patient , or far mote
peri osteitis, nephritis, hemolytic anemia , venous frequently, a carrier Patients who continue to shed
Copyrighted materia
< EntoiotacteriKWB III: Salmonella * 297
typhniJ hacilli in fcccs far three weeks To three the demonstration of typhoid bacillus antigen in
month* after clinical cure are called conwafescMf hiood or urine.
current Those who shed the bacilli for more than Dkiid culture: Bacteremia occurs early in the
three months but less than a year are called disease and blood cultures are positive in
' temporary' carrion' and those who shed the bacilli approximately 90 pci cent of cases in the first week
for over a year are called 'chronic Cartiers ', About of fever. The popular belief that blood culture for
-
2 4 per cent of patients become chronic carriers.
The development of ( he carrier State is more
diagnosis of typhoid fever is useful only in the first
week i * erroneous . Blood culture is positive in
common in women and in the older age groups approximately 75 per cent of cases in the second
(over 40 years ). Some persons may become earners week, 60 per cem in. the third week and 25 per
following inapparent infection (syinptamleSs cent thereafter t i 111 the subsidence of pyrexia. Blood
eret < ] r ) . 1 he shedding ( bacilli is usually cultures rapidly became negative an treatment with
intermittent. The hacilij persist in. the gall bladder antibiotics.
or kidney and arc clin iinatcd in the feces ( fecal
'
About 5-10 m! of blood is collected by
carrier ) or urine ( urinary carrier ), respectively. venepuncture and inoculated into a culture bottle
Urinary eairiage is less frequent and iv generally
associated with some urinary lesion such as calculi
—
Containing 50 100 ml of 0.5 per cent bile hnotb.
Blood contains substances that inhibit the growth
or schistosomiasis of the bacilli and hence it is essential that the broth
Food handlers or cooks who become carrieth be taken in sufficient quantity to provide at least
ate parti iTularly dangerous, The best known of such fourfold dilution of blood. The addition ofliquoid
typhoid carriers was Mary Mai Ion {'Typhoid ( sodium polyanethul sulphunale ) counteracts the
Mary „ a New York cook who, over a pc ri ; >d of 15 bactericidal action of blood.
years, caused at least seven outbreaks affecting over After incubation overnight at 37 BC , the bik
200 persons. broth is subculturcd on MacConkey agar. Fak
Carriers occur with paratyphoid bacilli also. nonlactose fermenting colonies that may appear on
While & paratyphi A occurs only in human beings, this medium arc picked out for biochemical tests
S . pimrypbi B can infect animals such as dogs or and motility, Salmonellae will be motile , indole and
cow*, which may act as source* of human disease. urease negative and ferment glucose, mannitol and
Typhoid fever occurs in two epidemiological maltose but not lactose or sucrose. The typhoid
types. The first is endemic or residua] typhoid that bacillus will be anaerogenic, while paratyphoid
occurs throughout the year though seasonal bacilli will form acid and gas from sugar*.
variations may sometimes be apparent. The second Identifier ion of the isolate is by slide agglutination.
is epidemic typhoid , which may occur in endemic A loopful of the growth from an agar slope is
or nonendemic areas. Typhoid epidemics are water, emulsified in two drops of saline on a slide. One
milk or foodborne . emulsion acta as a control to show that the strain is
Laboratory d inruns is : Bacteriological nor aucoagglutinable. If S. lyphl is suspected ( that
diagnosis of enters fever consists of the isolation is, when no gas is farmed from glucose ), :L loopful
of the bacilli from the pat i LLnt And the demonstration of typhoid O antiserum (factor 9/group D ) is added
of antibodies in his scrum , A positive blood culture to one drop of bacterial emulsion on the slide, and
is diagnostic, while the same significance cannot agglutination looked for after rocking the slide
be attached to isolation from feces or urine . gently. Prompt agglutination indicates that the
Demonstration of antibodies is not conclusive isolate belongs to Salmonella group D. Its identity
evidence of current infection - A thud method is a$ $. ryphi is established by agglutination with the
Copyrighted materia
296 Textbook of Microbiology *
*
fffgpgap antiserum (ami-d scrum). Quite often , fresh Another advantage is that a sample of scrum also
isolates of S, tvphi lire in rhe V k >nn and do not becomes available. Even though agglutinins may
agglutinate with the O antiserum. Such strains may be absent in the early stages of the disease, a Widal
he tested for agglutination against anti-Vi scrum. tesr provides a bascl i nc ri r re against wh ich the results
Alternatively, growth h ttl ed off in :L mull of tes.CS performed Idler may i;: - " evaluated.
*
^
amount of saline boiled tor 20 minutes and tested Feoes culture: Salmondlac are shed in feces
for agglutination with the O unrihenLcn . Where the throughout die course of the disease andl even in
isolate is a noatyphoid Salmonella (producing g.is convalescence with varying frequency. Hence fecal
*
from sugars}, it is rested for agglutination with O cultures: are almost as valuable as blood cultures in
:md H antisera for groups A , B and C. For diagnosis. A positive fecal culliire , hiTwever* nwv
occur in carriers as well as in patients, d bc use ot
^
i dentilration of unusual serotypes, the help of the
National Salmonella Reference Centre should be enrichment and selective media and repeated
sought . The National Salmonella Reference Centre sampling increase rhe rare of isolation Fecal culture
In India is located at The Central Research Institute, is particularly valuable In patients on antibiotic as
,
KasauEi . The reference centre tor salmoncll ie of . the drug LWS nor eliminate the hacilEi from the
*
.MUII.II origin is at rhe Indue Veterinary Research guc as rapidly as it does from the blood.
Institute, Ir..irnagar. Fecal -samples arc plated directly on. MacConkev,
If salmorcllae ate not obtained from the first DCA and WUson-Blair media . The last is highly
subculture from bile broth subcultures should be selective and should be placed heavily. On
*
repeated every other day rill growth is obtained. MarConkev anil DCA media, nalinorirllaje appear
Cultures should He deelured negative only after -
as pair Color i L H. OO the Wilhon-Rlair medium,
incubation fur leu days. To etiinmate the risk of 5. rty Ju forms large black colonies, ivith a metallic
introducing contamination during repeated .
sheen . S. ffcirjfyphj A producer green colonies- due
subcultures, mid also tnr economy and safety, tothe absence of H ,S production.
Castaneda's method of culture may be adopted, In For enrichment, specimens Are inoculated into
tIns, a double medium is used. The bottle of bile one tube each of selenite and recratliionute broth
*
.
broth bus ,m igur slant on one side . After and incubated tnr 12-1 H Knurs before subculture
inoculation of blood, the bottle is incubated in the ontoplates.
upright position.For subculture, the bottle is merely Urine culture: SalmonetIae are sEied in the
tiEted HO that rhe broth runs over the surface of the urine irregularly ini lntre< ] Lientlv. Hence urine
agar, Ir is reincubated in the upright position- If culture is less useful than the culture of blood
salmoncllac ire present, colonies will appear on or feces. Cultures are generally positive only in
the slant. the second and third weeks and then only in
An altemuri vt ro blrrod tuI ti i re is rhe dot < i 11 ture. ab^ut .
yer cent of cases Repeated sampling
Here , 5 ml of blood is withdrawn from rhe patient improves the rate of isolation. Clean voided urine
into a sterile test tube and allowed to dot. The samples are centrifuged and the deposit inoculated
senam is. pipetted oft ami used for the Widal rest . into enrichment and selective media as for fecal
The clot is broken up with a sterile lass rod ami
^ culture .
added In a hottle of bile broth. The incorporation Other materials for Cullure: Isolation may
of streptokinase ( 100 units per ml) in rhe broth he obtained from several other sources but they are
facilitates lysis of rhe clot. Clot cultures yield a not usually employed. Bone marrow culture is
higher rate of isolation than blood cultures as the valuable as it is positive in most cases even when
bactericidal action of [ he serum is obviated . blood cultures are negative. Culture of hi Le obtained
Copyrighted materia
* Entergbacleriaceae III : Salmonella 299
by duodenal aspiration is usually positive and may may be added as a preservative. It is important to
be employed lor the detection of carriers . Other use standard smooth strains for antigen prejtararicm .
materials which may yield isolation at times are The strains used usually are the 5. iyphr 901 , 'O'
nose spots, pus from suppurative lesions, CSF and and ' If strains . Karh batch of antigen should he
sputum. At autopsy, cultures may be obtained from compared with a standard . Readymade Widal kits
[ he gall bladder, livef , spleen acid mesenteric lymph .iI stained antigens available commercially are now
nodes. widely used ..
r
\S itliil reaction: This is a tent for the The results of the Widal tost should he
measurement of H and O agglutinins for typhoid interpreted taking into account the following:
and paratyphoid bacilli in the patient 's sera. Two 1 . The agglutination titre will depend on the stage
types of Cubes are generally used for the tesfr"^ of the disc asc. Aggluti nins usually appear by the
narrow tube with a conical bottom ( Drcycris end of the find week, so thnt blood taken earlier
agglutination tulie ) for the ]]
agglutination , and a may give a negative result. The litre increases
short round bottomed tube ( KclLx tube ) for the O steadily rill the third or the fourth week, after
ugglutination . Ecjual volumes ( ( 1.4 m 3 ) of serial which if declines gradually.
dilutions of the scrum (from 1/ 10 to 1 /640} and 2. Demonstration of a rise in titre of antibodies, by
the H and Q antigens art mixed in Dreyer 's and testing two or more serum samples, is more
Felix agglutination tuhes , respectively and incubated meaningful than a single test , If the first sample
in a water bath at 37 *C overnight. Some workers is taken laic in the disease , a rise may not be
recommend incubation at 50-55 C tor two hours , demonstrable. Instead , a fall in titre may he seen
followed by overnight incubation at room in some cases.
temperature. Control tubes containing the antigen 3. The results of n single test should be interpreted
and normal saline arc set to check for with caution . It is difficult toby down levels of
autoagglutination . The agglutination litres of [ he significance though it Is generally seated that
serum are read. II agglutination Leads to the tit res of 1/10Q or more for O agglutinins and 1 /
formation of loose , cotton woolly chimps, while O 200 or mom for H agglutinin? are significant.
agglutination is seen as a disclike pattern at [ fie IL is necessary co obtain information on the
bottom of [ he tube . In both , the sujiernatant fluid distribution of agglutinin levels in ‘normal sera'
is rendered clear. in different areas,
The antigens used in the test are the H and O 4 . Agglutinins may be present OIL account of prior
antigens of 5. and [he H antigens of .Si parafyphr disease , in apparent infection or immunisation.
A and H . The paratyphoid ( f antigens are not Therefore, the mere presence of agglutinin in
employed as they CTOSS-icact with the typhoid O flic Widal test should not be taken as proof of
antigen due In their sharing of factor 12. The II typhoid fever.
agglutinable suspension is prepared by adding 0.1 % H agglutinins persist longer than O agglutinins .
formalin -
to a 24 hour hmth culture or saline Semm from an individual pmiOUiuwd with TAB
suspension of an agar culture- fr> r preparing the O vaccine will generally have antibodies to -5’,
suspension , the bacillus is cultured on phenol agai typhi, S. paratyphi A and B, while in case of
( 1:1100 ) and the growth Scraped off in a small infection antibodies will be seen only against
volume of saline. It is mixed with 20 times its the iniecting species.
volume of absolute alcohol, heated at 40-50 aC for 5. Persons who have bad prior infection or
20 minutes, centrifuged and the deposit resuspended immunisation may develop an anamnestic
in salhie TO the appropriate density. Chloroform response during an unrelated fever. This may
Copyrighted material
300 ^ Textbook d UiorabiDbgy
bcditferciriated by repetition of the test after a widely and if is essential , therefore, to test repeated
wreck. The anamnestic response shows only a sample^. . Cholagogue purgatives increase the chance
transient ri '-e, while in enter 1 , - fever the rise is of isolation. For the detection of urinary carriers ,
sustained. repeated urine cultures should he carried out.
6. Bacterial suspensions used as amigens should be The Widal reaction is of no value in the
free from fimbria. False positive results may detection of carriers in endemic countries . The
otherwise occur . demonstration of VI agglutinins has been claimed
7. Cases treated early with chloramphenicol may to indicate the carrier state. While this is useful as
show a poor agglutinin response. a screen ing test , confirmation should he made bv
Other serological met hods of diagnosis include culture.
indirect hemagglutination , CIE and ELISA , The tracing of carriers in cities may be
accomplished by the 'sewer-swab1 technique. Gauze
i JnHar^ s r R A T i o N OF CIRCULATING pads left in sewers and drains are cultured , and by
ANTIGEN tracing positive swabs, one may be led to the house
Typhoid bacillus an Ligetis are consistently present harbouring a carrier. Another technique of isolating
in the blood In the early phase of the disease, and salmonellae from sewage is filtration through
also in the urine of patients. The antigen can be millipore membranes and culturing die membranes
demonstrated by sensitised Staphylococcal on highly selective media such as Wilson and Blair
coagglutination test. Staph surtus (Cowan I strain )
, media.
which contains protein A , i * stabilised with
formaldehyde and coated with 5- typhi anrihody. B M :TBRROPH *GB TVPING
When a 1% suspension of such sensitised Intraspecies classification of S, typhi fot
staphylococcal cells is mixed on a slide with serum epidemiillogical purposes was made pOStihle by
from patients in the first week of typhoid fever, the bacteriophage typing, first developed bv C raigic and
typhoid antigen present in the scram combi nes wi rh Yen (1937 ). They found that a bacteriophage acting
the antibody attached to staphylococcal cells on the Vi antigen of the typhoid bacillus [Vi phage
produc i ng vi sible agglut i nation wi:h i n two m L uutes. II ) irn highly adaptable. The parent phage is called
The test is rapid, sensitive and specific but is not phage A It could be made specific for a particular
positive after the first week of the disease. strain of typhoid bacillus by serial propagation in
Other laboratory tests: A white cell count the strain . Such adaptation was obtained by
is useful Lcticopenia wirh a relative lymphocytosis phenotypic or genotypic variation. At present, 97
is seen . Eosinuphili are said to be absent bolt in. the Vi II phage types of S. rtp/ti are recognised . As
tropics., with a high incidence of helminthic phage typing of S . typhi depends on the presence
infestation, eosinophils are usually present . of Vi antigens, a proportion of strains ( Vi negative )
will be untypable . The phage type is stable. Apart
D l x i i N O S l S O F l A u u i l- ltS from helping in tracing the source of epidemics,
The detection of carriers is important for phage typing also provider information on the
epidemiological and public health purposes. trends and patterns in the epidemiology of typhoid
Laboratory tests are also useful in screening food at the local , national and international levels. Phage
handlers and cooka to detect earner state. typing is carried out at the National Phage Typing
The identification of fecal carriers i -. by isolation Centres and is coordinated by the International
of the bacillus from feces or from bile. The Reference Centre . The National Salmonella Phage
fretpency and intensity' of bacillary shedding vary Typing Centre fur India is located at the Lady
Copyrighted materia
H Enterabartariacflae III . Salmonella » 301
Harding Medical Collegeh New Uclhi , Phage various parts of Europe, .Africa and Asia where
types A and El are the most common and are typhoid and paratyphoid fevers were endemic. No
present throughout India. However* rhe relative controlled held trial has been conducted with the
prevalence in different regions* in subiect to change TAB vaccine . In civilian practice , protection is
from time to limic. mainly required against typhoid fever. If paratyphoid
The preponderance of one or two phage types components are considered necessary either A or
in a region limits the utility of phage typing as H may be added, but not both, as only one of them
.
an epidemiological tool Additional markers is found in any one area , 'nicrcfbrc, in India , instead
have, therefore, been employed for the -subdivision of the TAB vaccine , a divalent typhoid-paratyphoid
of strain H belonging It ) a phage type . These include A vaccine {eliminating paratyphoid B which is very
(1) Ni.CoU.cS complementary phage tvping of rare in the country ) or the monovalent typhoid
type A strains into 10 Types; ( 2 ) Krisfenscns vaccine is prefemed -
biotyping based on fermentation of xylose and The vaccine is given in two doses of 0.5 mJ
arabinose; (3) production oftctrithiomte reductase; subcutaneously at an internal of 4-6 weeks. Local
( 4) bacteriocin production; and ( 5 ) antibiogram. and general reactions lasting for one or two days
Currently , more discriminating genotyping are |L ui [e frequent . Such reactions may be avoided
methods like phasmid finger printing, muhiLocu ? if the vaccine is administered in a dose of 0-1 ml
enzyme electrophoresis , 1S-20Q profiling and intradermally. In nonendcmic areas, vaccination is
random amplified polymorphic DNA analysis haw recommended foe troops, medical and paramedical
been employed lor epidemiological characterisation personnel . In endemic areas vaccination is
in advanced centres . recommended for ail children , in whom a single
Phage typing has been applied also tn S, paratyphi dose might give adequate protection , which may
. .
A and B, S typhimurium , 5 enteritidis and 5,
1001
Dublin , Among the S. pa ^ iryphr A isolated from
India, phage types 1 and 2 are the most common * t T
A
Copyrighted material
302 * Textbook oJ Microbiology »
mutant ). On
- - —
if a stable imitanc of 5, typht strain Ty2 la, lacking
the enzyme UTVP gaJsLctote 4 ejiimtfvire (Gal F.
ingestion , it initiates infection but 'self
destructs" after four or five cell divisions , and
of typhoid ftver.
While antibacterial therapy has been so effective
in the treatment of cases , it has been disappointing
LII the treatment of carriers . A combination of
therefore cannot induce any illness. The vaccine LH antihacterial therapy along with the vaccine has
^
an enteric centred capsu containing 10’ viable
lyophilised mutant bacilli. The course ctinsists of
been tried in the eradication of carrier state , litis
combination has also been used to prevent relapses .
one capsule orally, taken an hour before food, with Elimination of the carrier state muv require hemic
a glass ol water or mi ]];, on days ], 3 and S . Wo measures such a* cholecystectomy, pyelolithotomy
antibiotic should be taken during tins period . or nephrectomy.
The injectable vaccine ( typhiai-Vi) contains Drug resistance: Though occasional resistant
purified Vi polysaccharide antigen ( 25 pg per dose ) strains had been identified in the laboratory,
from S, jyphj Strain Ty 2. It is given as a 'ingle resistance to chloramphenicol dial not pose any
subcutaneous or intramuscular injection, which problem in typhoid fever till 1972 , when resistant
causes only minimal local reaction. strains emerged in Mexico ami in Kerala {India).
Both these vaccines are recommended only in Tr Mexico, the resistant strain caused an explosive
chose over five tcatv of age, the same dose being epidemic, with high mortality. Traveller* who got
used IDT children and adults . In both case; infected in Mexico bad, on occasion , conveyed the
protection is stated to commence 2-3 weeks after mutant strain to Worth America and Eurofie but
adminictation and lasts for at least three ye a rs , after Lt did not get established in these areas.
which a booster may be given . Roth the vaccines Chloramphenicol resistant typhoid fever has
are effective and only their relatively high cost stands become a problem in many countries in Asia,
in the WuV . of their wider use.
J
In India, chloramphenicol resistant typhoid fever
Typhoid bacilli arc primarily intracellular appeared in epidemic form first in Calicut ( Kerala )
penuites, and cell mediated immunity rather than in early 1972 . Jr became endemic and was confined
humoral antibodies may he more relevant in to Kerala till 197fl . Subsequently such strains
protection against die disease. Cell mediated carrying drug resistance plasmids appeared in many
immunity develops during the course of thedisease. Other parts of India . Though resistant to
Cellular i mm u n i tv to the typhoid bacillus is chloramphenicol , such strains were initially
common in population* in endemic :Lrrjie. Absence sensitive to amjncillin, amoxycillin , cotriUMBMik
of CM I ha* been claimed to indicate susceptibility. and furazolidone, which were HucccssfiiUv used for
r
The killed vaccines currently used do not stimulate treatment . Rv late 11990* , typhoid bacillus Strains
CMI. resistant to many or all of these drugs began to
Treatment : Specific antibacterial therapy for spread m most parts of India. Ar present, the drugs
enteric fever became available only in 1948 with useful in treat ment of such multi resistant typhoid
the introduction of chloramphenicol , which cases arc the later fluoroquinolones ( such as
continued as the sheer anchor against the disease ciprofloxacin , pefloxacin, oflaxacin ) and the third
till the 1970s when resistance became common . generation cephalosporin:, (such as ceftazidime .
Copyrighted material
1 Erlerobacteriacaae III : Salmon alia > 303
ceftrioxune* cefotaxime}. Furazolidone is still active animats . Gastroenteritis may occur without food
against most isolates but its action is too slow for it poisoning as in cross infectlor in hospitals.
to be used alone in treatment. Rccendy many strains Clinically, the disease develops after a short
have become res in taut to fluoroquinolones* but incubation period of 2*1 hours or less. with diarrhea *
,
several isolates of typhoid bacilli are now sensitive vomiting, abdominal pun and fever . It may vary in
chJonmpbenicol severity from the passage of one or two loose stools
"
to
to an acme cholera -like disease. Ic usuallv
ir
subsides
SALMONELLA GASTROENTERITIS in 2 4 days but in some cases a more prolonged
~
Salmonella gastroenteritis ( more appropriately enteritis develops, with passage of mucus and pus
enterocolitis) or rood poisoning is generally a in tcces, resembling dysentery. In a few, typhnidal
zoonotic disease rhe source or Infection being or septicemic type of fever may develop ,
'
.
anuxul products It: maybe caused by any salmonella Laboratory diagnosis is made by isolating the
except SI typhi The first instance of salmonella food BihCK)Della from the feces. In outbreaks of food
poisoning to have been identified was in 18S 8, poisoning, the causative article of food CM often
when ( juftner in Germany isolated , L bacillus ( .S'. be identified by taking a proper history. Isolation
enter! tidir ) from the meat of an emergency
,
Copyrighted material
304 * TsxUnoh of Wicroo dog * *
endocardins, pneumoniaand memngjitii, Antecedent Srt} j?himunum phage type 29 in England in the
gastroentcrifis may or may not he present . The ease lVfitls. Human infections were initially gastrn
fatality may be as Ki urh as 25 per cert.
Salmonellae maybe isolated from the blood or
-
crteriti - due to spread ftorr- infected animafe, through
food. Subsequently some salmonellae appear to have
from the pus from the suppurative lesion . Feces changed their ecology In some ways . From being
culture may also sometimes be positive. Septicemic responsible for zoonotic infections only as in the past ,
salmonellosis should be treated with some multiresisiant salmonellae have now become
diluramphcnitol < jr other appropriate antibiotics
as determined by sensitivity tests.
important agents of hospital LTOSS injection . Such
nosocomial salmonellosis manifests particularly in
-
neonates as septicemia meningitis and suppurative
MULTI RESISTANT SALMON ELL AE lemons, Diiintiea may not always be present.
R factors conferring multiple drug re stance have In India * several hospital outbreaks of neonatal
become widely disseminated among salmonellae . septicemia caused by multi resistant salmonellae
The clinical significance of thus phenomenon was have occurred in recent years. Mortality in neonates
first observed during the studies of human and is veTy high unless early treatment Is started with
veterinary infections with drug resisranr antibiotics to which the infecting strain is sensitive.
Further Reading
^ ^ ^ .
Chriarie AB. I 9 H7. Inf&xious Diseases. Epidemiology atid Cfinih Pri- ciire , 411 edsi. Friinhut h: Ch Lfchill-LfonEsftme,
itnd Paratyphoid . In Topicr and Wilson s Mktobiolffgy' and JWi refflJsra Jji&.'rint wh 4* cdri - vul -3.
'
LunJuri: Arnold.
Ivanort B c[ aL 1994. Vaccination ajjiinsi ryphoid fever: Fraeflt Bull Wlci Hkh Ota: 72^957-
Mindd BK.. 1994. SalfflHKfll cyphi iusil other KaLmuiiidcDyj. Gut 3- 5:726.
Mina SH cr JL 199ft . Midcaiim rnAtani Sdiunetli typhi: A kabnJ pfo koL JModMkriobioL 44:317.
^
WHO, 1W. C ?4 wifml nf Sal me HIE]la infer turns in
1 ^
jminuli, fluSt WIrf I f l t h ^ .
Orgn 12 S!M ,
vJ opyrighted materia
[
CO
CO Vibrio
. ir i
^
due to two nr more cells lying end To end .
Pleomorphism is frequent in old cultures. In stained
films of mucous flakes from acute cholera cases, ’
the vibrios are seen arranged in parallel rows ,
described by Koch as the 'fish in stream appearance.
1
Dvrraniei cl :C
3D6 * Textbook uT
which later becomes clear due ut hemndigCEti < ui . Vibrio colonics maybe identified by the 'string
In gckrin stab culture, mfiindibuliform ( funnel- test’. A loopful of the growthis mined with a drop
for tile cultivation of cholera vibrios They may he maltose , mannose , md sucrose bur nor inositol,
classified as follows: strabitiosc, or lactose, though lactose may be split
Holding nr transport media: ( 1 ) Vrnkat - very lilowly. lndnh: is formed and nitrates are
nunarrRfifn &krighiifla ( VR) medium: A simple .
reduced to nitrites These two properties contribute
modified form of this medium is prepared by lo the VI iultra red reaction ' which JH tested by
dissrilvirig 2i ) g oudt sea Halt and 5 g peptone in adding a few drops of concentrated sulphuric acid
Otic litre of distilled water and adjusting the pH to to a 24- hour pepronc water culture. With cholera
K />-S.S. Jt is dispensed ID serewgapped bottles in vibrios, a reddish pink colour is developed due to
10-15 ml amounts . About 1-3 ml stool is to be the formation of nicroso- indole . Catalase anti
added to each bottle - In this medium vibrios do caudate ten It are positive. Methyl red and urease
(lot muhlplv but remain viable for Hcv-eial weeks. tests are negative. Vibrios dccarboxvlate lysine and
(.2 ) Cary-Rlair medium:1his is a buffered solution ornithine but do not utilise arginine. Gelatin is
of Hodium chloride , sodium rhioglvcollate, liquefied. Vibrios elaborate several enzyme!;
disodium phosphate and calcium chloride at pH including L -OILAGTNASE , cLiittte, chitinasc,
8,4 . It is .t suitable nanapmt medium for Salmonella nucleotidase, decathoirylaEC, lipas<\ mucinasc and
and Shigella an well as tor vibrios . (.1) Autoclaved neuraminidase (receptor destroying enzyme).
sea water also serves as ji holding medium. Resistance: Cholera vibrios are susceptible to
Enrichment mediiu (1) Alkaline peptone water hear, drying ur.d acids, hut resist high alkalinity.
at pH £.6; [ 2 ) Mfjn$ ur't tau. ro / holate tellurite They arc destroyed at 55 UC in 15 minutes . Dried
peptone water at pH . 2 . Both these arc good
^ on linen or thread , they survive for 1-3 days but
transport JIS well as enrichment media . die 1(1 about three hours on cover slips. Survival in
IJlilting nttdin: (1 ) Alkaline bile salt agar ( RSA) water is Influenced by its pHh temperature, salinity,
pH S.2: This simple medilirci lias HEKKKJ ihe test of presence of organic pollution and other factors. In
rime and is still widely used - The colonics ate simitar geiLLTol , the El Tor vibrio survives longer rhan rhe
to those on nutrient agar. ( 2 ) Monsur 's gelatin c lassical cholera vibrio. In the laboratory vibrios
timiDcbalajte trypticase teflurrtsogir (GTDV) medium: survive for months in sterile sea water, and this has
Cholera vibriiw product small, rr .inslucent colonies been suggested us ;i method for the survival of vibrios
with a greyrih black centre and a turbid halo. 'I'hc in nature hi grossly contaminated writer, such as the
,
1
colonics become 3-4 mm in size in 48 liourn. [3 ) Ganges water of India, the vibrios do not survive for
rCIIS medium:Thin medium , « Jiitnini ug tiliosulfiltc , any length of rime* due to die apparently Jaige amounts
citrate, bile saits and sucrose, is available commcmoHy of vibriophflges present. They survive in clean tap
and is v«y widely used at preset ]!. Cholera vibrios water for thirty dap. In untreated night soil, they may
,
produce large yellow convex colonies which may sumvx for several days. Vibrios are susceptible to the
become green O i l continued incubation. common disinfectants.
Copyrighted materia
4 Vibnc » 307
On fruits, they survive for 1 - 5 days at room indistinguishable from cholera . However, by and
temperature and for a week in the refrigerator. In large , N’ AC vibrios were nor pathogenic and
general , IOIHI materials left a.t room temperature do commonly isolated from envi momenta! sources and
not act as m important swnxofinftttinn for longer healthy human intesrircs.
than a day or two blit those stored in [ lie cold may While all isolates from epidemic cholera
harbour vibrios for more than two weeks. belonged to group 0 - 1 , not all tnemben ol the
They are killed in a few minutes in the group were capable ol causing L [ i 11 LL . iI dioleft, The
'
gastric juice ot’ normal acidity bur they may survive first such members which acquired prominence
for 24 hours in achlorhydric gastric juice. were the vibrios isolated by CrOHschlich { 190?)
Clat &ificfltitm: Jn the past* many oxidase from sis llaj pilgrims who died at the Tor
positive, motile, curved rods were rather loosely i| uafajuiut station on the Si mu Pen insula. They had
grouped as vibrios . Precise criteria have been laid died not from tholera but from dysentery or
down for differentiating vibrios from related genera
'
gangrene of the colon. These came to be ealled the
( Table 33.1). lil Tor vibrios. They were identical to cholera
Hribcrg ( 1934) classified vibrios into six group vibrios in all laboratory tents except that they were
based on the forme ntution of mannose, sucrose and hemolytic to sheep erythrocytes and gave a positive
atabincise . Two more groups were added later . \ bgcs-Proskaner reaction . As the El lor vibrios
Cholera vibrios belong to Croup I (Table 33.2). were subsequently isolated from water sources and
A serological classification WH introduced by normal human intestines , they were considered to
Gardner and Vcnkatraman (1935), Cholera vibrios be nnnparhogenic , In 1937 , El Tor vibrios were
and biochemically similar vibrios , possessing , L recognised as endemic in Celebes (Sulawesi ) ,
common fliLgellar ( H ) antigen were classified as Indonesia, causing a choleraic disease ( paracbalera ).
< Jroup A vibrios, and the rest as Group B vibrios I lowevcr , outside thfo endemic area. El lor vibrios
comprising a heterogeneous collection. Based on were considered nonpathogenic .
IILL: major somatic (Q ) antigen, Group A vibrio* The situation dunged in 1961 when El Tor
we re classified subgroups' ( now called ( 1
into " vibrios gave rise to the seventh pandemic -of
berOgrOUpfi Or tCfOVUl ), 1 39 of which are currently cl Lid era . Besides increased virulence and
known ( Table 33.3) . All isolates from epidemic invasiveness, the pandemic El Tor strains showed
cholera ( till 1992 ) belonged to serogroup 0 - 1 . altered laboratory reactions . The new El Tor strains
Therefore in the diagnostic laboratory group 0- 1 were often nonhemolytic and V— P negative . Mew
antiserum (commonly called ' cholera differentiatm properties were defined such as chick
nondifferentifll serum ) Lime to be used fur
1 ^
celt SLgglutIllation , polymyxin sensitivity arid phage
identifying pathogenic cholera vibrios ( which ate susceptibility tents ( Table 3.3.4) . It wan accepted
referred- » ‘igghltfortblc vibrios' ) . Other vibrio tbut El TOT vibrios were indeed cholera vibrios,
isolates which were not agglutinated by the 0- 1 which contained two bfon jet , the old or 'classical '
antiserum. came to be called norragjf / urjNjMe or ^
cholera vibrios, and the El Tot vibrios.
!M AG wbflM. Frhcy were considered nonpathogenic Bused on minor surface antigenic characteristics
and hence also called nmcfeafeii vibrio* ( NCV ) , both classical and El Tor biotypes of cholera vibrios
Both these terms are not strictly' appropriate . were classified into three serotypes, Ogiwa , Inaba,
Though SAG vibrios are not agglutlnablie by the and Hikojima ( Table 33.5 ). The Ogawa And Inaba
G-l antiserum , they arc readilyagglutinated by their Strains- are agglutinated by their own respective
own antiserii. The term noncholera vibrio is uol specific sere only, while die Hikojima strains are
correct as some of them can cause a disease clinically' agglutinated hv both Ogswa and Iniba antisera .
Copyrighted material
303 * Textbook ;: F Micre- biology
+
+
—
+
V
+
V
+
V
+
—
~
Note : 1 * no gas produced; 2 - gas mayor may nor be produced; V = [motion virisble ,
There ts. no difference in pathogenic itv between Further classification car he made hy phage
the three serotypes . Scrotypinj; is only of typing. Phage typing schemes have been
efi ide miulogical signi fieanct . standardised for classical and El Tor bioiypet an an
The non-Gl vibrios ( the so tilled NAG vibrios)
have been classified into many serogroupt, currently
-
well us for 0 139 vibrios. New molecular methods
like ri ho typing have added further rctmements to
uplo 139. The latent Hemgrnup 0-139, identified strain typing.
in 1992 causes epidemics of cholera, emphasising
that they tan no Longer be considered as nunchokn CHOLERA
vibrios. Cholera > an acute diarrheal disease caused by
Modern taxonomies! criteria , particularly , V. cholcrsc , In its most severe form, cholera is a
UNA studies , have led to the recognition that all dramatic and terrifying illness in which profuse
the cholera vibrios that belong to Gardner and painless watery diarrhea and copious effortless
Venlsatraman 's group A and share simdar vomiting may lead to hypovolemic shock and death
biochemical properties and a common H antigen m less than 24 hours. In treated canes , the disease
arc so closely related that they constitute a single -
may last 4 6 dpys, during which period the patient
species Vibrio cbolenc , which can be classified may pass a total volume of liquid stool equal iu
into serogmups (or scrovars ) , bintypes and scrotypes. twice his body weight. All the clinical features of
Accordingly the present nomenclature will be severe cholera result from this massive loss of fluid
indicative of all these features, as lor example, .
and electrolyte* The cholera stool is typically a
VT cho/crae scrovar 01, biotype El Tor, serotype colnrlcw witerr fluid with flecks of mucus, said
Ogawa, to resemble water in which rice has been washed
Table 33 2 Heiberg grouping of vibrios
Group Fermentation of mannose Sucrose AraWncic
T A A
11
111
-A A
A A
IV -A A A
-— -
V
VI —
-—
=
Vll A A
vm — A
Copyrighted materia
* Vibrio M9
VIBRIO
Croup A Group B
Cholera vibrios Biochemically and
Biochemical M mllaiiricG; anrigenically
Common U aorigen hererojfe ntnui
( Vihrift fhalet it )-
O SUBGROUPS
( SEROGROUPS ) :
SEROVARS )
01 Mon -01 ( cujicA[]y up( G (M 3)
BIOTYPES ’
I
[Criteria Listed \
in Table 33.4 ) j
f
'
C1?aSri Es1,S
E3. Ttrf
l 1
f \
SEROTYPES Ogawa Inaba Hikciima
Copyrighted material
310 * Tgxlbaok GJ Microti ulOQy *
introduced the canine model infected with able 33.4 DttteraflMitlon between classical
cholera vfbrios through a stomach tube, after cholera and El Tor vibrio*
administration of sodium bicarbonate developed Twt CAurieii cAflipra El Tor
diarrhea and vomiting, usually ending in Liexrh.
—
v
In human infection, the vihrias enter orally fbmolpis - +
cholera toxin, CT, or CTX) which is very similar into the small intestinal lumen , oflarge quantities
to the heat labile toxin ( LT ) of !' coif In structural, of water and electrolytes and the consequent warcry
chemical , biological nod antigenic properties , diarrhea. The fluid secreted is isotonic with plasma
though CT is far more potent than 11 in biological but contain * much more of potassium and
activity CT production is determined hy a bicarbonate. The rflgjn also inhibits intestinal
filamentous phage integrated with the bacterial absorption of idmm and chloride. All clinical
chromosome . It can also replicate ns a plasmid manifestations and complications in cholera result
which ] can be transmitted to n ^ ntoxigenk strains, from the massive water and electrolyte depletion
rendering [ hern toxigenic. Cl , TCP and Other thus caused.
virulence factors arc regulated by the TodtR gene CT also exhibits other biological effects which
product, ToxR protein. can be used for its detection and estimation. These
Copyrighted material
i Vibrio * 311
indudc activation nnipolysis in rat tCttiCulU' tissue, spread steadily westwards , invading India in 1964 .
elongation of Chinese hamster ovary (CHO ) ccll-s By 196 b , it had spread throughout the Indian
in culture and histological changes in adrenal subcontinent and West ASM . In the 1970s the
Tumour (Y ) cell culture and vero ull | , JT also pandemic extended to Africa and parts of Southern
increases skin capillary permeability; and so has been Europe.
called the Lpemieiab Llity factor ( PF). It ran be During the course of the pandemic the vibrios
"
,
the [ hinges and Brahmaputra in Bengal , is its The seventh pandemic of cholera lias heen
homeland, where it has heen known from very different from nil the others It is the first to have
*
ancient Times. Till early in the nineteenth century', originated from outside the Indian subcontinent h
,
cholera was virtually confined to India, periodically is alio the first to have been caused by the El Tar
r
causing large epidemics in different pans of the biotype, in contrast to all the earlier ones causerl by
country. the classical cholera vibrios. The severity of illness
From tS17 to 1523 cholera vibrios had spread was much less, with a large proportion of mild and
from Bengal , in sis separate pandemic waves , asymptomatic ink'd ions. Mortality was low and the
Involving most parts of the world. It was largely carrier rate high. El Tor vibrios tended to remain
due to the threat of pandemic cholera char endemic in many new geographic areas , causing
international health organisations came into being. pcri*> div epidemic hursts. The El 'for vibrio has
After the cm! of the 6th pandemic in 1523* till proved to he much hardier than the classical vibrios ,
1961 tine disease remained confined to its endemic capable of surviving in the environment: much
ureas, except far an isolated epidemic in Egypt in longer . A peculiar phenomenon has been rhe
1947 , replacement nt the classical hintype by the El Tor
Theseventh pandemic originated from Sulawesi vibrios following the pandemic spread. Thus, in
(Celel >es) * Indonesia, in 1961 when the El Tot India the classical vibrio is hardly ever encountered
vibrios which hid been smouldering there tor many after the El Tor epidemic took root , though in
decades suddenly became more virulent. After Bangladesh , the classical vibrio had staged a
spreading to Hongkong anil the Philippines , it rundudL
Copyrighted material
312 4 Tiaytbook of Microbiology *
An event of gneat WftnifkartCt wa* the sudden may initiate the next tandemit of choiera , The new
emergence of non-0l V! fMfiatf ( former NAG strain continued spreading, eastward'; to tile South
vibrio) as the enust of epidemic cholera, In October East Asian countries, and westwards to Pakistan,
1992, a new non-01 vibrio was isolated from China and some parts of Europe. Bui surprising)), 1
*
cholera outtrrea.lt in Madras { Chennai). Similar bv 1994 the FI Tor strain regained its dominance
outbreaks soon followed in different parts of India. and the threat ot an 0-139 pandemic diminished.
By January, 1993, the tiew strain had become -
Both 0 -1 ElTarud0 139 strains began tacoaast
epidemic in Bangladesh also. In Lite affected Ateis , in endemic areas.
this strain replaced the EL1 lor vibrios as the
"
Cholera is an exclusively human disease .
.
epidemic and environmental serovar Jt also showed Infection originates from die patient or the carrier.
a tendency to be more invasive, causing bacreremic Carriers may be incubatory convalescent, healthy
illness in some. The new epidemic strain was or chronic. Incubatory carriers shed vibrios only
r '
Jr
designated Serovar 0- 139 ( or 0- 139 Bengal) . during the brief incubation period of 1 > days .
Unlike the 0 -1 cholera vibrio, the 0- 139 vibrio is -
Convalescents may LXCICLC them for 1 3 week The
*
capsulated. As it possessed novel surface antigens, healthy or contact carrier who has had subclinical
the 0- 1 atnun vaccines could not protect agajnst infecrion usually sheds the vibrios tor les- s than 1(1
0“ 139 infection. There was nn natural antibody days. The chronic carrier continues to he active tor
against the strain in any human population then. It months or years^ rhe longest duration recorded
was therefore considered likely that the 0-139 strain -
hc mg 1(1 years . Chronic Curriers were rure in
ft
*
to
966
961 19B 1
*
1971
1934
h 19B1
7%
1991
.
Fig 43.2 Spread . -
^
( ) Unique onvironmenfal reservoirs
El Tor Cholera 1961 lW, Fltwnll) WtflUlted $p«lal environ rtfttntal esmoltfi of toxigenic
El Tor Cholera vibrio 01 are also indicaled. (Source; WHO ] .
Copyrighted material
A Vibrio 313
*
classical cholera but with El Tor infection they sue As cholera vibrios may die in a few hours at
seen more often . Persistent infection of the gall tropical temperatures , it is necessary to preserve
bladder accounts for chronic carriage . the specimen at 4 °C or in some appropriate
Infection it acquired through fecally holding medium . Stool samples may be preserved
contaminated water 01 food . Direct person- to - in VR fluid or Caty-Blair medium for long periods.
person spread by contact may not be common but If the specimen can reach the laboratory in a few
hand contamination of stored drinking water has hours, it may be transported in enrichment media
been shown to be an important method of domestic such as alkaline peptone water or Monsur ' s
spread of infection . Large scale movement of medium, thus saving the time required fbr isolation .
persons, as occurs during fairs and festivals , has If transport media are not available, strips of
traditionally beet ] associated with the spread of blotting paper may be soaked in the watery stool
cholera . and sent to the laboratory packed in plastic
The persistence of the vibrio during the envelopes . Whenever possible, specimens should
intenepitleiTiLc periiHls was a matter of controversy. be plated at the bedside and the inoculated plates
In the endemic areas it may be maintained by sent to the laboratory.
continuous transmission of subchnical or mild Diagnosis by direct microscopic examination of
infection . It is now known that the natural habitat cholera stool is not recommended as the results
of cholera vibrios is the saline waters of coastal arc rot rclrahlc - For rapid diagnosis, the
seas and brackish estuaries, where they can persist
for long periods , particularly in association with
small crustaceans such as copepods , crabs or
plankton.
A significant difference in susceptibility to cholera
has been reported in relation to blood groups, group —*
O persons being the most susceptible and group AB - 0 ' r
f
Copyrighted material
314 « Textbook gl Microbpoloqy »
employed usually are bile salt agar , Maction key considered to be unn -Ol chulera vibrio. Specific
agar for nunselective and TCB 5 agar fur selective antisctum against 0-139 is available. In the fully
plates. The plates should not be older than 3-5 equipped laboratory* diagnostic tests in cholera and
dap and should be dried well beforc streaking [t , other diarrheal diseases should consist of a batterv 'J
is possible to identify vibrio colonies on nonseledivc of tesrs designed to isolate other known pathogens
media after incuhatn m lor 4-5 hours by examina!ioU also.
under a stereoscope with oblique illumination . FCKI isolation ul vibrio# from Carri e rs* essen: iiallvrf
"
Generally* the plates arc exan tied after nwernight the Bune techniques are to be followed, except that
incubation at 37 Colonies suggestive of vibrios more than one cycle of enrichment may be
should be picked w ith a straight wire and tested by necessary. As vibrio excretion is intermittent *
,
Jr "
slide agglufina : ii ' ii w : rh cholera O subgroup J repeated stool cxarc : nation will y: dd better results.
scrum (cholera ' nondiflerenri - d' serum ) , If positive* Examination of stools after a purgarive f magnesium
agglutination may be repeated using speciftu Ogjwa
and 1 naba sera for sen ttvp i ng. Hikoj im a sir J 11 is w 111
—
sulphate 15 30 g or Mannitol 30 g ) * or of Kile
after duodenal intubation is of special value .
agglutinate equally well with Ogawa and 1 naba sera. Serological examination is of litrle use in the
If agglurination is negative with one colony it is , diagnosis of cases though : t may be helpful in
essentLiil to repeat the test with at least five more assessing the prevalence of cholera I n an area. 'The
colonies* as ‘agglul i lable and nun -01 vibrius may tests avi dabte are agglutination using live or killed
1
coexist in the name specimen. J: slide agglutination vibrio suspensions , indirect hemagglutination ,
i -i positive* the isolate is tested (or ehi ik red cell vibrint idal test and antitoxin assay. Of these, the
agglutination. This is employed for presumptive complement dependent vibriocidal antibody test is
differentiation between El Tor and classical cholera the most useful.
1
vibnoS. A report cat ] be sen! al ill is stage * usually Fur examination of water samples fur vibrios *
the day after the specimen is received , If no vibrios clit ' hmentor filtration methods may be employed ,
'
are isolated * a second cycle of en richment and In the former , 900 ml of water arc added to 100 ml
plating may succeed in some cases. tenfold concentrated peptone water at pH 9.2,
u opyrighted
y materia
* V brio » MS
incubated at 37 ‘-'CJ! for 6-8 hours and a second numbers of Ogawa and Inaba serotypes* given by
ennchincnt done before plating on selective media. subcutaneous or ini ram uvular injccidoo. Many
For the filtration technique the water to be tested laboratories employ classical cholera and El Tor
should be filtered through the Mi11ipore menil e vibrio in equal numbers in rhe vaccine . Strain
filter, which is then placed directly on rhe surface 0* 139 vaccine has diso beco prepared. The
of a selective medium and incubated . Colonics concentration of the vaccine has been increased to
ap}>ear after overnight incuturion . Sewage should 12,000 million per ml ,, in order to improve the
he diluted in saline , filtered through gauze and anrigcnic stimulus,
treated US for water . Several eoncrolled field trials in endemic areas
Immunity: Jn cholera, the vibrios remain with wiooa types of injected vaccines have shown
uuitfioed to the mteseme, where they multiply and that the protection atfbrded hv them dod not exceed
elaborate the enterutoxii ] which is. responsible for -
5Q tifl per cent; the duration of protection m only
the disease . Immunity therefore, mzy be directed 3 6 months the rate ®f protection in endemic ITeii
“ '
against the bacterium or against the toxin — increases with age; i single dose of vaccine i $
antibacterial or antitoxic. Natural infection confers ineffective in children below five years of age while
some amount of immunity but it docs not seem to two doses at 1-4 week intervals mto ptoreeiire; a
last for more chan 6^12 months and rcinfcctinns single dose confers good protection in adults- due
are known after tills period. to its acting m a booster on top of prior naturd
Immunisation with killed vactines induces only immunisation ; cell - free somatic antigen
antibacterial immunity. The protector effect of these preparations are as- effective as whole cell vaccine;
vaccines , especially puimed somatic antigens used chcrc is good cross- protection between classical and
a* vuciines , though shor [ - Lived , proves that El Tor vibrios ; rhe crotfs- protection between Ogawa
antibacterial immuniry can protect agamsi intecrion. and Inaba serotypes is doubtful and requires further
The protection appears to be serotype specific hut study , pending which ViCcirte Containing the
not hiorype specific . homologous- serotype is to be employed. .Aluminium
Immunity may be local , in the intestine, or hydroxide and phosphate adjuvant vaccines
SVStcmk . The appearance of local antibodies in the
'
produced berrer inumuilly, particularly in young
nrestinc has been known for a long rime. These .
children* TOKOid vaccln ee have not been been
are known as ‘coproamibodies' as they appear in successful. Injectable vaccines do not provide any
the feces . Tliey consist of IgG , IgM and IgA . local immunity. in the mtcttiiul mucosa. They are
at f*
Prophylaxis The prevention of cholera requires also unacceptably feactogenic. I lenee attention has
essentially general measures such as provision of been directed to oral vaccines .
protected water supply and improvement of Two tvpe-s of oral vaccines have been tried
environmental sanitation - As these arc noT easily recently; MEW oral whole cell vaccines with and
_ :
attainable , vaccination con nucs fo be the most without the inclusion of the B subumr of CT„ and
v. i dely used method of prevc nrion in enden iic areas. /eve ora/ vsiconeii with classical , El Tor and 0- 13^
Cholera vaccines wete introduced bi krran
, strains, with their toxin genes deleted. While the
within a vear of the discovery of the vibrio. The
r /
results have been promising, problems- remain to
origin id vaccinffl were live sus- pen -sions of vibrius. be solved before they are cleared fcf genets! m
As they gav* rise to adverse reactions, they were An Ideal cholera vaccine is yet to be found .
-
replaced by killed vaccines The vaccines used
iradiri -DnaHy are killed suspensions containing 3000
Cholera vaccination was- a compulsory requirement
for International travel , but now very few countries
'
Copyrighted material
316 i Texltiook of Miaobiology »
Trout morn : The treatment of cholera consists countries and it is now considered an important
essentially of the prompt and adequate replacement cause of food poisoning throughout the world. It
of lost fluid and electrolytes . Oral administration inhabits the coastal seas, where ir is found in fishes,
of fluid containing glucose and electrolytes, either arthropods such as shrimps and crahs, and molluscs
alone or supplemented by intravenous fluids is a such as oysters . In Calcutta , it has also been found
highly successful and freely available method of in small pond fishes.
treal mg cholera . Cereal hafied preparations are- In morphology, it resembles the cholera
equally effective and usually more acceptable . vibrio, except that it is capsulatcd , shows b .|x>lar
'
AmibacCen .il therapy is of secondary importance . sta:iling and has a tendency to pleomorphism,
Oral tetracycline was recommended for reducing espec ially when grown on 3% salt agar and in old
the period of vibrio excretion and the need for cultures . Unlike other vibrios, it produces
parenteral fluids. Initially cholera vibrio* were pen trie hous flagella when grown on solid
in Liifbnnly suscept i ble to ail ant i him ics acti ve agai nst media. Pillar flagella are formed in liquid
Gram negative bacilli , but since 1979, multiple cultures .
drug resistant strains have become increasingly Ir grows only in media containing NaCl. It can
common. tolerate salt concentration upto 8 per cent but not
10 per cent. The optimum salt concentration is 2-
VIBRIO MIMICUS 4 per cent. On 1 CBS agar, the colonies an; green
So named because it closely resembles cholera with an opaque, raised centre and flat translucent
.
vibrios m bioclientical features, V jujinfcu^ can be periphery. The siring test is positive .
differentiated by its failure to ferment sucrose. Like It is oxidase, catalase, niirate, indole and citrate
V, cholerae, it grows best at low salt concentrations positive. Glucose, maltose, mannitol, mannose and
( 0..5-l % ). Tt has been responsible for many sporadic arah mose ate fermented producing acid only.
'
eases of diarrheal disease on the Gulf Coast of the Lactose, sucrose, salicin, xylose, adonitol, inositol
USA, Infection is acquired from eating seafood, and sorbitol are HOE fermented .
,
-
especially oysters i :ic di easc is self 1 incited.
Clinical manifestations resemble those caused by
- It is killed at aC in 1,5 minutes. It docs not
grow at 4 "C but can survive refrigeration and
V , panthacmolyticus. freeing. Dtving destroys it. It dies in distil led water
or vinegar in a few minutes.
HALOPHIUC VIBRIOS "
I hrcc antigenic components have been
Vibrios that have a high requirement of sodium recognised somatic O, capsular K and flagellar
chloride arc known as halophilic vibrios. Their H antigens . Scrotyping Is based on O and K
natural habitat is sea water and marine life. Some antigens, 12 O groups have been recognised and
halophilic vibi ios have been shown to cause human -
59 di ; inct K antigens-
— .
disease V pnrahicniultrticuii V. a mofyr / cijs and
V. vulnificus. ^ Not all strains of V , parahacmolyticus are
pathogenic for human beings, Jt has been found
that strains isolated from environmental sources
VIBRIO PAR AH \ EMOLI TICUB (such as water, fish, crabs or oysters } are nearly
V. patahaeinofyticus is an enternpathogcnlc always nonhemolytic when grown on a special high
halophilic vibrio originally i -^olited in 1951 in Japan salt blood agar { Wagauuma agar) , whi c strains
'
as the Causative agent of an outbreak of food from human patients arc almost always hcmoiytjc.
poisoning due to sea fish - Gastroenteric due to This is called the Kanagawa phenomenon and is
this vibri ' i has since been identified in several due to a heat stable hemnl >sin. The significance of
Copyrighted materia
4 Vibrio 317
Indole
V.P.
Nirrafc rcductLan
—
4 +
+
Urease
Sy cru e Imne ill aciufl
^
—-
Swanning »
laboratory test for pathogen icitv; Kan ugawa posi t ivc V vulnificus, previously known as L* vibrio or
Strains hiring considered pathogenic ioi human JJentvAcir vulnifici , is a marine vibrio of medical
beings and negative a trains nonpathogcnic . No importance . It if VP negative and ferments lactose
enterotoxin has been identified - The vibrio is but not sucrose . IT has a salt tolerance of less than
believed to cause en.teritii by invasion of the eight per cent . It causes two Types of illness. The
intestinal epithelium. V. parjhacmohtLu^ causes first is wound infection following contact of open
food poisoning ass wilted with marine food. It also wounds with seawater. The second type occurs in
causes acute diarrhea, unutoditcd with food compromised hosts particularly those with liver
poisonirg. .Abdominal pain, diarrhea , vomiting and disease. Following ingestion of the vibrio, usually
fever arc the usual signs. Feces contains cellular in oysters, it penetrates the gut mucosa without
exudate and often also blond . Dehydration is of causing gastrointestinal manifestations and cliters
moderate degree and recovery occurs in 1-d days . the bloodstream, rapidly leading to septicemia with
Cases ate more common in summer, and in adults high mortality.
than in children . In Calcutta, V partihacmo/yTrcuff
-
could tic isolated from 5 10 per cent of diarrhea AKHOMUNAM P l. hKltlMUN AS
AM>
'
cases admitted ro the Infectious Diseases Hospital . Besides the genu*. Vibrio, the family Vibrionaceac
.
V puahMcmofyticusis common in sea fish in some also contains the genera Aeromorias and
other parts of India but human cases are much less Pfei;it> jTionas, some members of which have been
frequent. associated with human lesions.
AtromooMt hydtophiliL, originally isolated from
VlBHIfl Al lilMil YTICJI * frogs, in which it causes the "red icy disease', has
This halophilic vibrio resembles V piTth^ cmo- been reported from many cases ot diarrhea and from
Jjtkut in many respects and was tormerlv considered some pyogenic lesions in human beings .
a biotype of rhe latter , It has a higher sail tolerance, PlesmmoBMM thigdfaidcs also has been reported
Is VP positive and ferments sucrose ( Tahle .33.6). from diarrhea ] disease . Both these arc oxidase
It i« frequently found in sea fish. Its status as a positive , polar flugclluted, Crum negative rods and
human pathogen is uncertain - It has been associated may be mistaken for vibrios . They may be
with infections of eyes, cars ml wuunde in human differentiated horn vibrios by bioohem ical test* such
beings exposed to sea water. as utilisation of aminoacids.
Copyrighted material
318 * Texlooo* ol MicrobiolDBV *
further Reading,
Rjiruu. D mi WB G ret:no ugh 1992, CiWffa. Maw York:Plenum.
Collier L ec al teds ) - 199H. Toptcy and Williams MknMology andMicrobial Infections S'1 , edn. LoreJon : Arnold Vok . 2
*
and 3 L
Cofatelt RR iirhJ Hut ] 1994 . Environment } reservoir of Vibrio choleric Annair Acad So 740:44.
* ,
FaruK] SM ec al. 200.1!. Kcnecgecu.i: and evoJuii^n of V, eholerjc 0- 139. Proc Nit ALSIJ Sci (USA } 100: 004.
Honda T nni.iT lida L 99 j. The (whitgen iciiy of Vainnio /idriharjiwdfrTkus. Rev Med Microbiol 4:106.
Kjper JIB el a.1. 1995, Chcdera. Oifi MicrohM Ret. S:4H.
1
.
Lacey SWr 1995 , Chdten CM Infect /3UL 20:1409,
MctaL-anus JJ .Tii -ij.:! |L S^dcd-T 1994. ChnJE-r-j. v-jeciiics. Sere-aiL-e 265: 0 7.
^
Naif GB CT al. 199 b. Vrrtno ohtu1*rae G 139 -\Rengd. RevM td Aficra6io/ 7:43.
,
Copyrighted material
Pseudomonas
Pseudomonas are a large group of aerobic , on MacCohkey and DCA media , forming non-
nonsporing Gram negative bacilli , motile by polar lactose- fermenting colonies. Many strains arc
flagella , They are ubiquitous, mostly saprophytic, hemolytic on biood agar . In broth, it forms a dense
being found in water , soil or other moist turbidity with a surface pellicle.
environments Some of them are pathogenic to Pi. &cfuginoi& produces a number of pigments,
plants* insects arid reptiles . A few cause human the best known being pyocyanin and fluotescin .
infection , typically opportunistic . Pyocyanin is a bluish green phcnazinc pigment
based on molecular analysis, pseudomonads have soluble in wuer and chloroform, Fluorescin
been reclassified and many former Pseudomonas ( pyoverdin ) is a greenish yellow pigment soluble
species reallocated to new genera such as in water but not in chloroform , In old cultures it
13urkhoIderiiit Stenotrophomonas and others. may be oxidised to a yellowish brown pigment .
PyocvanLi} is produced only by Pi . aerujfrrtfMa but
PSEUDOMONAS AERUGINOSA fluorescin may be produced by many other species
Ps- pyucyanca: BaaHus pyocysncus also. Other pigment produced are pyarubin {red)
Morphology: It in a HLENDER Gram negative
. and pyomelanin ( brown ) in various combinations.
bacillus, 1.5-3 pm * 0 5 pm, actively motile by a
, Some strains may be nonpigmented. It is Tint known
polar flagellum. Occasional strains have two or whether the pigments have any role in
three flagella . Clinical isolates ace often pillared . It pathogenesis. Some of the pigments particularly
is noncapsulated hut many strains have a mucoid pyocyanin . Inhibit the growth of many other
slime layer . Mucoid strains, particularly isolates bacteria and may therefore contribute to Ps.
from cystic fibrosis patients have an abundance of aeruginosa emerging as the dominant bacterium in
extracellular polysaccharides composed of alginate mixed injections.
polymers. This forms a loose capsule (glycocalyx ) Biochemical reactions : The metabolism is
in which microcolonies of the bacillus ate OMidatiue and nonfermentative . Peptone water sugars
enmeshed and protected front host defences. are unsuitable for detecting acid production, since
Cultural characteristics; It is an obligate this i:- weak and gets neutralised by alkali produced
aerobe, but can grow anaerobically if nitrate is from peptone. An ammonium salts medium in
available - Growth occurs at a wide range of which the sugar is the only carbon source is the
temperatures, 6-42 "C, the optimum being 3? “ C- best . Glucose is Utilised oxidatively, forming acid
lt grows well on ordinary media , producing large, only Indole , MR , VP and H ,S tests sue negative .
opaque , irregular colonies, with a distinctive, musty, Nitrates arc reduced to nitrites and further to
mawkish or earthy smell. Iridescent patches with gaseous nitrogen . Catalase , oxidase and arginine
A metallic sheen are seen in cultures on nutrient dibydrolase tests arc positive .
agar. C rystals are seen beneath the patches. It glows ClyssilloilLont As Ps . aeruginosa has become
Copyrighted material
330 i Taxttnofc of Microbiology
a very important cause of hospital infections , ICS community outside the hospital, the most common
classification is essential hir epidemiological infection caused by Pi. aeruginosa is suppurauve
purposes . Serotyping , bacteriocin ( pyocin , otitis, which is chronic though not disabling. In
acruginusin ) typing and bactenopli . ige typing have the hospital, it may cause localised or generalised
been used but are not entirely sati ^factory. infections. Localised lesions are cn mm only
Restriction endonuclease typing with pulsed-field infections of wounds and bedsores, eye infections
gel electrophoresis is the most reliable method and urinary infections following catheterisation. Fs .
avaUable- aeruginosa is the most common and most serious
Rm ^ unce: The bacillus Is not particularly heat cause of infection in bums . It is also one of the
resistantj being killed at 55 nC in one hour but
, agents responsible for iatrogenic meningitis
exhibits a high degree of re-.istsuicc to chemical following lumbar puncture. It frequently causes
agents. It is resistant to the common antiseptics and post - tracheostomy pulmonary infection. Septicemia
disinfectant* such an quaternary ammonium and endocarditis may occur in patients who are
compounds, chloroxylenol and hexachlorcphane debilitated due to concomitant infection, malignancy
and may even grow profusely in battles of such or immunosuppressive therapy. Ecthyma gangreno-
antiseptic lotions kept for use in hospitals- Indeed, sum and many other types of skin lesions have
selective media hive been devised for ft, aeruginosa been described occurring either alone or as
incorporating dettol or cetrimide. It is sensitive to part of generalised infection , mainly in patients
acids, beta ghiraraldehyde, silver salts and strong with leukemia and other types of malignancy.
phenolic Jisirifectants. Its suscept i billty to silver has Infection of the nail bed is not uncommon
been applied clinically in the us-e of a Liver following excessive exposure of hands to detergents
sulphonair.i. de compounds as topical cream in and water.
bairns. ft. afitigjjTUiia 1 ns been described as one ot the
Psr aentgr /ioM possesses a considerable degree .
agents responsible for infantile Hi urfiea and sepsis .
of natural resistance to antibiotics. Examples of Strains isolated from outbreaks of diarrhea may
din i tally effect ice antibiotics axe amlnoglycoKi des form a heat labile enterotoxi n and give a pnsirive
(gentamicin, amikacin ), cephalosporins (edotsedme, rabbit ileal loop reaction, ft , acrugincuu has been
ceftazidime , cefopemone) , fluoroquinolones reported to cause a self-limited febrile illness
(ciprofloxacin , ofloxacin , pcfloxacln ), penicillins { Shanghai Aver) resembling typhoid fever In some
( piperacillin , ticarcillin , azlocillin ) . For localised tropical areas.
infetions, topical colistin, polymyxin B or 1% acetic The pre-eminent role of Pi. aeruginosa in
acid may be useful. hosp i tal i nfccti on is. due ti > i rs resi stance to common
Palhogenrctt ) : ' Blue pus was known as a
1
antibioticsand antiseptics, and its ability to establish
surgic al enrity long before Gcasvd (1SS3) isolated itself widely in hospitals. Being an extremely
/V. aeruginosa from such cases. Both the specific adaptable organism it can sut vive and multiply even
names of the bacillus refer to its capacity to cause with minimal nutrients, if moisture is available.
“ blue
pufi' the term aeruginosa , meaning verdigris
T Equipment such as respirators and endoscopes,
which is bluish green in colour and pyocyana , articles such as bed pans and medicines such as
being a literal translation of ' blue pus . 1
loLions, ointments and eye drops and even stocks of
The pathogenic importance of the bacillus was distilled water or plants and flowers may be
not adequately recognised till recently, when It haa frequently contaminated - ft aeruginosa is present
established itself as one of the most troublesome on the skin of the axilla and perineum in some
agents causing nosocomial infections . In the persons. Fecal carriage is not common but may be
-
jr"*
1
opyrig hted _ n-
n atari
L I IL L
< Pseudomonas > 321
frequent following oral antibiotic; treatment or caused by a few other species , such as Ps.
hospitalisation . fhioicsrejis, ft puiidi and ft. sfurzeri.
The mechanisms of pathogenesis are not
clearly understood . Several toxic extracellular STENOTRGPHO MONAS M ALTO PHI LA
products have been identified in the culture -
( VO n Ml M I rY PSBUDOMONAS
MALTOPHILA )
liltnttSn such as exotoxins A and S. Enotoxin
A acts as in NADase , resembling the This i* a saprophyte and opportunistic pathogen ,
diphtheria toxin . Good antibody response to causing wound infection , utinaiy tract infection and
exotoxin A is considered a favourable sign in septicemia . It is usually oxidase negative and
severe infections xvitli ft. aeruginosa, Orher acidifies maltose in addition to glucose , lactose and
toxic products include proteases , elastises , sucrose. Infections usually respond to cotrimoxazole
hemolysins and enterotoxin. The slime layer and chloramphenicol .
acts as a capsule in enhancing virulence.
Laboratory diagnosis The bacterium grows BURKH OLDEST A CEPACIA ( FORMERLY
readily on most media,The identification of PSEL OOMONA S Ch' PA t . fA J
pigmented strains of the bacillus from clinical I’ his is a plant pathogen causing onion rot ( ccpu,
specimens is easy. But about 10 per cent of isolates I .arin for onion}. It is increasingly being recognised
may be nonpigmented . Prompt oxidase reaction and as an opportunist environmental pathogen ,
arginine hydrolysis help in their identification . It particularly in those with cystic fibrosis or chronic
mi; be necessary to use selective media such as granulomatous disease, in whom it causes fatal
-
ci irimide agar for isolation from feces or other necrotising pneumonia , It is nutritionally very
versatile. It can grow in many common disinfectants
samples with mixed flora . As Ps. a&Vginot& is a
frequent contaminant, isolation of the bacillus from and can ever use penicillin G as a sole source of
a specimen should not always be taken as proof of carbon I It n oxidase positive and acidifies mannitol,
its etiological role. Repeated Isolations help to sorbitol and sucrose. It can cause urinary, respiratory
confirm the diagnosi -s. and wound in (ectlnns, peritonitis, endocarditis and
Control; Prevention of ft aerugiaota cross
infection in hospitals requires constant vigilance
- septicemia . It is inherently resistant to mnst
antibiotics .
and strict attention to asepsis. Antibiotic treat merit
is not jdwavs- satisfactory. Animals with eicjWri- GLANDERS
mentally infected hums have been protected by prior BLRKHOLDBRIA MALLEI ( FORHELY
immunisation with the homologous strains. PSEUDOMONAS MALLBI )
Immunotherapy in human hunts cases with Tile bacillus had also been classified variously
antiserum to Ps, aeruginosa may be useful . as Locfficrdh , Pfeiffcrdh , Milteomym ,
Pseudomonas vaccines are being tried in Cystic Actinubacillus and Acjjrefrtiiacfer. It is the
flbtosis patients who arc highly vulnerable to causative agent of glanders ( mxfledjs, in Latin}, a
pseudomonas infection . disease primarily of equine animals - horses,
Specific antibacterial therapy constitutes only mules and asses - but capable of heing
one aspect of the management of serious transmitted to other animals and to human
pseudomonas infections . Treatment of the beings, The bacillus was discovered by Loeffkr
underlying diseases, correction of granulopenia and and Schutz (1882J .
appropriate supfjortive therapy need attention . .
ft. malfe; is a slender, nonmotile Gram negative
bacillus, 2-5 pm * 0.5 pm staining irregularly and
Occasional opportunist infections may be
Copyrighted materia
322 ^ TexlOnok ql MiorObiulOQv *
diagnostic of glanders, as ir may also be produced rnav be an acute septicemia , a Subacute Cyphoid -
"
by inoculation of Other bacteria such as Brucella likc disease , or pneumonia and hemoptysis
species , Preisz- NocarJ bacillus, Actinobadllus resemhlingtulierLuldsis. In clrrotliC form , there mac
ligmcmi and Pa . pseudomallui . be multiple caseous or suppurative fiici , with absces- s
Human infection is usually occupational , found formation in the skin and subcutaneous tissues,
in ostler?, grooms and veterinarians. It may be acute bones and internal organs. Acute melioidosis has a
or chronic and is protean in character, with high case faijJity rate. Serological evidence indicares
locali &arion in the respiratory tract , shin or that inappareut infection is common in endemic
subcutaneous tissues. In acute glanders, there is areas. Long latency and reactivation may occur as
fever, mucopurulent nasal discharge and severe rhe bacillus can survive intracellularlv in the
proftration. The fatality rate is high. While htiman reticuloendothelial system. The bacillus has been
infection is acquired only rarely from infected isolated from water and soil in endemic areas, Ir is
animals, Laboratory cultures are highly infectious a soil saprophyte that cause? infection in rodent ?
and PSL mailer is one of the most dangerous bacteria and humans accidentally. Human infection occurs
to work with. commonly through skin abrasions or by
Animals suffering from glanders develop a inhalation.
delayed hypersensitivity to rhe bacterial protein. Diagnosis may be made by demonstration of the
This is the basis of the miUtiii test used for bacillus in exudates by microscopy (small irregularly
diagnosing glanders. This is analogous to the staining Gram negative bacilli , showing typical
tuberculin test and may be performed by tile bipolar 'safety pin appearance with methylene blue
subcutaneous, intracutfmttius or conjunctival stain ), isolation hv culture from sputum, pus, blood
methods . oi unne , or bv serology { F1LJ 5 A for IgM and lgG
Copyrighted materia
« PHudomonHi > 323
Further Reeding
.
Biltch AL and Smith RP ( tds ). 1994. Pseudomonas teruginoa infections NrwYcrk] Marcel Dekker.
Copyrighted material
K?
CO
Yersinia, Pasteurella, Francisella
The plague bacillus and many other Gram negative* sire , with rounded ends and convex sides* arranged
shore bacit!] that are primary pathogens of rodents singly, in short chains Dr in small groups. In smears
were grouped together in the genus Pasteuretla. stained with Giernsa or methylene blue, it shows
Based on cultural and biochemical differences* this bipolar staining ( safety pin appearance ) with [ he
.
-
gnou p has been divided ] i no [] Ltee genera Yet surra *
PasteurcH i and Francisella. The genus Yersinia ,
two ends densely stained and rhe central area clear
{Fig. .55.1). Plegmorphism is very conmon and in
containing the radical ly important species Y.patis
{ the causative agent ot plague ) * Y. pyeudo
'
club -shaped, filamentous and glam forms.
—
old cultures, involution forms are seen coccoid ,
fizbtn. rdnsr:i ( a primary pathogen of rodents) and Pleomorphiiun i & ohanicterirally enhanced in media
Y.cntcrocoHticM ( which causes enteric and systemic containing 3% NaCl.
diseases in animals and human beings) was so The bacillus is surrounded by a slime layer
named after Alexandic Yerstn * who discovered rhe (envelope or capsule ). Jt is nonmotile * nnrtsporing
^
plague bacillus. The genus tjsfnni is now assigned
to the family Enterobjctcriaceae . The genus
and non acid fast .
Cultural rharaderistics: The plague
PasreuneiJa contains several related bacteria causing bacillus LH aerobic and facultatively anaerobic ,
hemorrhagic septicemia in different species of timwth occurs over a wule range of pi I (pi I 5-9, 6,
animals and occasionally producing local and optimum pi I 7.2) and temperature ( range 2~45 “ C ) .
systemic infection * in human beings, grouped under The optimum temperature for growth ( unlike most
it common species named f ? rntdrochfa. One of these * pathogens) is 27 ‘G but the envelope develops best
P. Aviacptkt is the chicken cholera bacillus used at 37 ^C-
by Pasteur for the development of the first attenuated 1 T is not nutritionally exacting and grows on
bacterial vaccine . Hence the name PdsCeurella. The ordinary media. On nutrient agar, colonies arc small,
genus Fnndtttla , co mailing of F. rrWarensis* is delicate, transparent discs, becoming opatjue on
named after Francis for bis pioneering studies on continued incubation. Colonics on blood agar or
tularemia, caused by this bacillus . other heolui containing media are dark brawn due
to the absorption of the hemin pigment . Colourless
Y K H S I M A PtiSTis ( K O k A t K i i i .Y colonics arc formed on MacGonkjey's agar, In broth ,
P A H i ' l - I I k l - l . l A PKS 'I IS ) a flocmlent growth occurs at the bottom anti along
The plague bacillus was discovered independently the sides i > t the tube, with little or no turbidity. A
and simultaneous I v hv Yersin ANLI Kitasato ( 1 S 44 ) delicate pellicle may form later. If grown in a flask
in Hong Kong at the beginning of the last of broth with oil or ghee (clarified butter ) floated
pandemic of the disease. on tup (ghee broth ) a characteristic growth occurs
Morphology: Yi peVtiY is a short , plump, ovoid, which bangs down into the broth hum the surface,
Gram negative bacillus, about 1.5 im * 0.7 pm in resembling stalactites ( stxbctiCe growth ) ( Fig* 35.2).
^
Copyrighted materia
* Yersi.- na . Pasleurella , Ftaivci Etll .a 325
^ *
1: nmii ^ niJii . jl rcueliotlNi Glucose * maltose and Aniigcnsir Inxios nnd other virulence
nmnriito] but not lactose* sucrose or rhamnosc arc laclors: PI ag\ic bacilli arc antigcnically
k'nnenfet! with the rmrl rtirFfl afudd hut no homogeneous and serotypes da not exist . The
^
fndolc U not produced , IT is MR positive und VP antigenic structure is complex. At least 20 antigens
and citrate negative catalase positive and acseulin have been detected by gd diffusion and biochemical
positive and mxtdnse iud urease negative . Gelatin is analysis. Many ui them have been claimed to be
not Lt[ uefied * Based on the fermentation of glycerol virulence factors. They include the following:
and reduction r,fnitrate * Devignat hsf iListirL ULshed 1, A heat labile protein envelope antigen ( Fraction
ihnrophysiological varieties of Y , /wstjs. This typing
'
^ 1 or FT) best formed in cultures incubated at
appeaih rn he o1 epldemioLogicd significance 37 aG, Tr inhibits phagocytosis and LK generally
because of the different geographical distribution present only in virulent strains. This plasmid
of the types (Table 35.1 ), encoded antigen has been considered! a virulence
Ik' si ^ . nu L- : The plague bacillus is easily determinant but (KTaiianal strains deficient in
destroyed by exposure to heat, sura light , drying and Fraction 1 antigen have been isolated from fatal
chemical disinfectant. IT is destroyed by heat at human cases. The antibody to this antigen is
,
55 JC nr by phenol in 15 minutes. It remains protective in mice,
vi able for tong peri ods i n cold, moist ctjvi ran ments . 2, Two antigens designated V and W and always
It can survive for several months* and cwn multiply , produced together have been considered to be
in the soil ot rodent burrows. All strains; are lysed the virulence (actors as they inhibit phagocytosis
.
by a 5-pccitic Jintipl Tguc bacteriophage at 22 “ t . and intracellular killing of the bacillus .
Production of V and W antigens is plasmid
mediated.
I
©0
Copyrighted material
336 * TB* Ebook of M crobioiogy *
Copyrighted materia
* Ystrinla Paateuralla, FrandsaHa * 327
Historians of plague identify 41 epidemics remains localised at the site of flea bite, with only
before the birth of Christ and 109 epidemics in minor constitutional symptoms ( pcstis minor). As
ihe next 15 centuries. There are records of 45 the plague bacillus usually enter* through flea bites
pandemics between AD 1500 and 1720, The disease on the legs, the inguinal nodes are involved acid
was quiescent in the eighteenth and nineteenth hence the name 'bubonic’ : bubon meaning groin ) .
centuries and confined to endemic foci . The last The gland* become enlarged and suppurate. The
pandemic started in Hong Kong in 1S94 and spread hadlli enter the bloodstream and produce
throughout tine world , (caused by y.pcsfss van septicemia - Sometimes there are hemorrhage:- in. to
of /cntaJis ) . Indi :t was one of the countries worst the skin and mucosa. Disseminated intravascular
hit by this pandemii Plague reached Bombay in
P
.
days The lymph nodes draining the site of entry of
the bacillus become Infected. In some, the infection
in blood because the bacterial mass blocks the
passage mechanically The blond, mixed with the
Copyrighted materia
I
l* CA^l
'7
.
'
*
*
- :
jpr
#
*3
I >
-
\ -
H
1
1 4
(?
P
/
Fig. 35.3 Natural foci ef plague, known and suspected jKnown areas - dark; Suspecled - shadedl
bacteria is regurgitated into the bite, transmitting the twentieth century, helped to clarify the
the infection. Infection may also be transferred by epidemiology of plague. It ivss (bund thar plague
contamination c »f the bite wound with the feces of produced epizootics First in Rattus oorvegicua
infected flenH . When a diseased r:tt ( lies ( rat fall ), ( sewer rut ). When their number dwindled, the
the fleas leave the carcass and in the absence of disease passed to the domestic rat, R, It was
another rut , may hire human beings , Causing from the domestic ru that the infection Spread to
bubonic plague . human beings.
Several species of fleas may act as vectors, the Two natural cycles of plague CKJSI, the domestic
most important being Xcnnpsvlh chtopi* , X . astia and the wild . The tenni htrban or domestic plague'
and C’erutnphyfhifr fa^qintus . X . ohttopis , the refers In plague that is intimately associated with
predominant species in north Jndia is a more human beings And rodents living with them ,
efficient vector than the south Indian species X . possessing a definite potential for producing
Utk. This has contributed to the more extensive epidemics. "Wild nr sylvatic plague ' occurs in nature
nature of plague outbreaks in the north a* compared and in wild rodents, independent ot human beings.
to south India . Plague epidemics generally occur I “ he rodents involved vary in di ffenmt regions, Over
"
Copyrighted material
* Yersinia. Pasteurelist , FranciseHa 129
{ Tatcra indies) and the bandicoot are infected . During epizootics, it is easy to diagnose plague
Human infection may occur during skiruling And itl ran. Buboes are usually present NL the CffViCll
handling of carcases of infected wild animals , region . Tticv arc hard and can bo moved uitder the
Carnivores, including cats and dogs can fret infected skin t ) n section , rhe bubo may show congestion,
,
by eating infected rodents or through their fleas. hemorrhagic points or grey necrosis . Smcare from
Clinical plague isseldom seen in dogs, but may desktop the bubo stained with methylene blue show the
in call. Human infection frfntii[ili.dat ion < H RSpinloty bipolar stained bacilli . The fluorescent antibody
droplets from infected cans has been repotted . technique may be of use in identifying plague bacilli
In enzootic foci, plague may persist for long in the impression films of the tissues. Bacilli in
periods. Infected fleas may survive lor ewer a year. bubo show considerable pleortiorphism. The liver
The bacilli can remain alive and even multiply in is mottled, with red, yellow or grey stippling. The
the soil of abandoned rodent burrows. They can spleen is enlarged, and moulded over the stomach,
infect new rodents rhat may reoccupy such burrows. with granules or nodules on the surface . A
This Itay account for the 1* nag period of (JuitHCOtC characteristic feature is pleural effusion which may
acid subsequent re - emergence charActerivtic of he clear, abundant and straw coloured nr, less often ,
plague. Attenuated strains of plague bacilli have bloodstained , Bacilli may he demonstrated
been isolated from natural foci. They may regain microscopically in spleen smear* and heart bfood.
virulence when plague becomes active. Eradication Cultures tuny be made from the buboes , spleen ,
of plague is an unlikely prospect as it is a disease of heart blood acid particularly, from bone marrow in
—
the earth of rodents that live in burrows and of
the fleas that live oa them . Only when hu man be i ugs'
decomposed carcasses.
In badly puoiikd carcasses , microscopy and
or domestic animals trespass on these natural t < >ci culture may not be successful. The putrificd tissue
do human infectious set in . rubbed on the shaven abdomen of a guinea pig can
In the 1990s , there Iras been a n -emergence of infect the animal. Diagnosis may also be established
plague in countries where it had ceased to be
noticed for many years . This has happened in the fhiimcscenf staining.
-
by demonstrating the F I antigen by immno-
-
China in Asia, Malawi and Zimbabwe in Africa,
the erstwhile USSR in Europe and in the USA.
—
developing and the developed countries India and In human bubonic plague , the bacilli may be
readily demonstrated in buboes by microscopy,
culture or animal inoculation . Blond cultures ate
Plague bacillus strains carrying plasmid h -nrne often positive .
resistance to mi il ri pic a ntihiotirs wr re re|iorted hunt In pneumonic plague , the bacilli can be
Madagascar jn 19^5, These have the potential to demonstrated in the sputum by micmscopv, culture
spread and pose a great threat. or animal inoculation.
Laboratory diagnosis: The laboratory should Serological tests are sometimes useful in
lie able to diagnose plague not only in Immins, but diagnosis . Antibodies to the F-I antigen may be
in rodents also , as timely detection ul infection in detected bv passive hemsgglutination. Rise in titre
rats may help to prevent epidemic spread . of antibodies in paired sera m titre of 128 or above
A rat which died of plague may carry infected in a single scram sample can be considered positive.
ileus and should be handled with care. Pouring IgG and IgM ELISA test -s have been developed.
kerosene oil over the carcass is a simple method of PCk is a rapid and sensitive method for presumptive
eliminating the fleas. In the laboratory, the carcass diagnosis of plague in clinical material and fleas,
should be dipped In 1% lysol to destroy 1'rophyLftxis:; In the prevention of domestic
ectoparasites. plague, general measures such as control of fleas
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H Yersinia, Pas1auf & lta. Ranchsella 351
Motility at 22 ®
C +
*
Growth on MacCfonl^ jfs agar f
Acid from sucrose *
* + *
Add from nulbK t +
Indole + +
Oxidase s-
Urease 1
— + r
*
Ofnithine decarboxylase
- - * 4-
Y, enterocolitis^ h.i - been isolated from a wide and sheep . It may some times occur
cats, rats, cattle
range of domestic and wild animals and , in recent ns icorrmiensal in the human respiratory tract also.
years, LS increasingly being reported from human Human infection is rare hut may occur following
clinkaJ material. It produces Three types of disease nnimdl bites or trauma I'he clinical tnInifctUMni
,
in human beings. The first Type occurs in young may be local suppuration following animal bites
children ns self - limited gastroenteritis or { wound infection , cellulitis, abscess, osteomyelitis),
enterocolitis which may be either inflammatory or meningitis following bead injury, respiratory tract
noninflammatory. The second is mesenteric adenitis infect LOO {pneumonia , bronchitis , sinusms ) or
and inflammatory terminal ileitis in older children appendicitis and appendicial ahseefis.
that may mi mb appendicitis . The third category LH The bacillus is sensitive rn tetracycline and
i systemic disease typically in adults , often streptomycin And most strains to penicillin SLS well.
characterised by bacteremia, meningitis, arthralgia
or erythema nodosum . Persons belonging to HI . A FkAMCIStiLLA TIFLAKKNSIS
- B 27 group are prone to develop reactive arthritis. (TASTEL' flELJ.A TVLAREN5i$t BflULkLLA
TULARESSIS )
PASTKI KEIXA M I i . m c m . v This is the causative agent of tularemia, a disease
( formerly FastcurclU Scptica ) of rahhitj anil other rodents, originally described
A group of related bacteria isolated from
'
mTulare county, California. Infection is transmitted
hemorrhagic septicemia in a variety of animals and hy ticks and several other arthropod vectors. Human
birds had, in the paat, been named according TO infection may occur by direct contact with infected
their species of origin— P. hftvincptLii , rodents such a * rabhits or through rich hires. It can
HVWeplica, etc. Though rhev show some degree of also be acquired hv ingestion of contaminated meat
host specificity, they arc so alike in other respects or water and in halation of infective aerosols,
that rhev are now considered strams of a single It is a minute , capsulaTed , non motile Gram
species designated P. muhocidx . negative bacillus, about 0.5-0.7 pm * 0.2 pm in
P. multcifhia is a non motile . Gram negative sire. It resemhle-s mycoplasma in being filterable
bacillus generally resembling YendrUa but differing and in multiplying by filament formation and
In being osidase positive , producing indole and budding, besides binary fission- In infected animals,
failing TO grow on MacConkcyr’s agar. It acts as an intracellular parasite, being found in
The bacillus is often carried in the upper Urge masses inside the liver and spleen cells- Jt has
respiratory tract of a variety of animals such as dogs, fastidious growth requirements and special media
Copyrighted material
332 Textbook of Microbiol
*
^
such AS Francis blood dcxrrosc cystine agar have ro like respiratory infection. The disease may also he
l:c employed for its isolation . Minute ttartSp.Lrent waterborne, as a result of water pollution by the
colonies appear after incubation for 3-5 days- \ LTL 1 .I ot infected rodents. The bacillus is highly
'
Srrainsof S. fu/arenfrjs have been subdivided i nto infectious and laboratory infection has been quite
'
biotypes based on their virulence and common. Diagnosis may be made by culture or by
epidemiolugical beliiviour. Highly virulent strains inoculation into guinea pigs or mice. Agglutinating
arc found only in N. America, while strains of low aniiliodLcE mav. be demonstrated in sera from
Jr
-
virulence arc seen in Europe and A i ^ also.
In human beings, tularemia may present as a
patients
An attenuated vaccine is available- which cin
local ulceration with lymphadenitis, a typhoid like be administered by scarification to pamnt who sure
fever with glandular enlargement or an influenza subject TO hiph risk of infection.
Further Rmding
Brubaker BR . 1991 . Factors ptfomoring infecriMs. caused by j-^rsiniae. Clin Microbiol Rev: 4:309.
Butler X 1994 . Yersinia infecnons. Clin injfikf Dis. 19:655,
Campbell and JM Hughes. 1995, Plague in India. Ann Inf Marat 122155.
Cover TL and RC Aber 1939. Yersinia entcr&calihca. New Engl J Med 321:16*
Gage KL 1993. Plague . In Topley amJ WJfson- l Microbiology and Mhcmbml Infections , 9 , K «3n. London: Ainold.
IWBtzcr R . 1954. Pb uc WHO Monograph scrie-s No. 22.
^
u opyrighted
y materi 3
CO Haemophilus
CO i
i
i
Thegemis Haemophilus contain snull^ nonmotile , Cultural cheracterittioE The bacillu* Lai;
nonsporing, oxidase positive, pleomorphic, Gram fastidious growth requirements . The accessory
negative bacilli thut ire parasitic on hurmn being* growth factors, named X and V , present in blond
or animal *. They arc characterised by their are essential for growth . The heat stable X factor
requirement of One or both of two accessory gn^wth is hem in or other porphyrin* requited for the
factors ( X and V } present in blood ( ffoemapWirj;, synthesis of cytochrome ,md other heme enzymes
meaning blood Loving ). such as catalase ,ind peroxidase involved in acrohic
Pfeiffer (1892} observed that a small, Cram respiration . The X factor is not required for
negative bacillus was "constantly present ' in the anaerobic growth . The V factor was so named
sputum of patient* from the influenza pandemic of because it was originally thought to be a bacterial
18 B9-92 and mistakenly proposed this as the vitamin. It is heat labile being destroyed at 120 °C
causative agent of human influenza . Tbia amt to in a few minutes. It i* present in red blood cells
be known as the ’ influenza bacillus’ ( Pfeiffer's and in many other animal and plant cells lit is ,
bacillus),. liter renamed i iaanophilus influenzae . synthesised by some fungi and bacteria ( forexample ,
The causal relationship between this bacillus and Staph , aunnts) in. excess of their requirements and
human in flucnza could not he Fubs-tanliated and Wit released into the surrounding medium. The V factor
finally disproved when Smith , Andrcwes and i K a cocnzymo, nicotinamide adenine di nucleotide
Laidlaw (1933) isolated the influenza virus . ( NAI 3) nr NAD phosphate ( NADP) which act*
as [i hydrogen acceptor in the metabolism of the
^.M O P H I I U S h l l I 1-. \ Z ,M-
I\ cell .
'
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334 s TexItKHiK 01 Microbiology
The major antigenic determinant of capsulated pathogen, causing acute invasive infections. The
strains is the capsular polysaccharide based on bacilli spread through blood, being protected from
which H . jjiij'Juc'rjjiae strains have been classified phagocytes by their capsule . Haem up Inins
—
hv Pittman into six capsuLr types types a to f.
Typing was originally done by agglutination but
meningitis is ihe mwt important infection in this
group , others being laryngocpiglottitii,
other methods such as Quellung reaction , conjunctivitis, bacteremia, pneumonia , arthritis ,
|mn iptlaliun, caflgglutiufllion , Cl E and ELISA may endocarditis and pericarditis. These infections are
also he used . Capsular typing in of m e d i c a l usually seen in children and arc caused bv the
r r
importance as about 95 per cent of H . influenzae eapsulared strains, type b accounting for nnxsi cases.
isolates from acute invasive infections such as In the second group, the bacillus spreads by local
meningitis belong to type b. Diagnostic kits for the invasion along mucosal surfaces and causes
identification of H . influenzae type b ( Hit ) arc secondary or superadded infections, usually of the
commercially available. respiratory tract. These include otitis media, sinusitis
The type b capsular polysaccharide has a unique and exacerbations of chronic bronchitis and
chemical structure , containing the pentose sugars
iibos- e and rihitnt instead of the hexoses and
-
bronchiectasis These are usually seen in adults and
are often caused by rhe noncapsulated strains.
hexosamines JS in the other five serotvpes. The Meningitis: Thi* is the most serious disease
capsular polyribosyl ribitol phosphate ( PRP) ati digen produced by H . influenzae with case fatality rates
of Hit induces IgG , IgM and IgA antibodies winch up to 90 per cent m the uncreated. The bacilli reach
are hactericidsd, opsonic and protective. Hib PRP the meninges from the nasopharynx through the
is therefore employed for immunisation . Hib bloodstream , The disease is more common in
capsular anCigen shows cros-s reaction with the children between two months and three years of
capsular an r igens of some frram positive and ( i ram age. Thi -; age incidence has been correlated with
negative bacteria. the absence of bactericidal anti - PRJ 3 antihodies .
H . influenzae strains lacking a capsule can not Older children develop immunity as a result of
he typed and are called ' nontypahle strains . Next
1
subclinical infection. It has been reported that in
ro JIib- the nontypablc strains arc the most relevant
i the tropics , non -type b strains may be responsible
in clinical infections, for meningitis more often than in the temperate
The outer membrane prorein antigens show zones.
considerable variation. OMP antigens of Hib Laryn &oep gLottiti * ( croup): This is an
htive been classified into at least Id subtypes. acute
'
inflammation of the epiglottis , with
H , influenzae lipooligosaochandcs are antigenicaily obstructive laryngitis , seen in children above two
complex . OMP and LOS subtyping may be of years . Untreated cases may be fatal within hours.
eyidemn iloyical value. Tracheostomy is often necessary to relieve
hi. influenzae is the first free I K i ng organLin respiratory obstruction caused by the grossly
whose complete genome has been sequenoed. enlarged uvula. Thi -. cond irion is always associated
Pathogenicity: hi . influence is an exclusively with bacteremia and blood cultures are usually r
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t H a &m Dphi I u s * 337
the upper respiratory tract . Infection is transmitted rreognsed in Brazil in J 9?4, EPF is now endemic
by the respiratory route . Carriage in the upper in South America.
respiratory tract is common parliculariy in young
children but such strains arc usually noncapsulated HAEMOPHILUS DUCREYI
and not responsible for acute invasive infection, Ducrey ( 1890 ) demonstrated this bacillus in
Immuniiy in type specific. As the large majority chancroid lesions and Liv. inoculation into tile skill
Jr
of serious infectionn are caused by type l> strains, on the firearm , was able to transmit the lei- 1 on
active immunisation wiih Hib PEtP vaccine is through several generations.
indicated . Purified PRP ii immunogenic in older Chancroid or soft sore is a venereal disease
children and adults. However , in Common with characterised by tender n-onmdurated irregular ulcers
other polysaccharide antigens, PRP is poorly on the genitalia - 'Phis infection remains localised ,
immunogenic In children below two years - Its spreading only to the regional lymph nodes which
immunogen^ icy can be jnpnoved by coupling with arc enlarged and painful , Autoinoculation lesions
protein carriers like diphtheria and tetanus toxoids may be produced by contact . There is no immunity
or meningococcus outer membrane protein. Such following infection but a hypersensitivity results,
conjugate Hib PRP are available for use in young which can be demonstrated bv Imradermal Jl
Young household contacts of patients. with ff , ducreyi is a short, ov. : id bacillus ( 1 — 1.5 pm
svatemic H . jnriuenjfiite infection have increased risk
If
a 0.6 mm ) with a tendency to occur in end to end
of getting intccted - llifampicin given lor four diyS pairs tir short chains . It is Gram negative but often
prevents secondary infection in contacts and also may appear Gram positive and frequently shows
eradicates carrier stare. bipolar staining . The bacilli may be arranged in
small groups or whorls or in parallel chains giving
HASMOPHJLUS AEOYPTIU 3 a school ill fish or 'rail road track' appearance.
-
(AocA Wfeefa bacillus, formerly H, SQjprjcus) '
Primacy isolation is difficult . It can be growtl
Even before Pfeiffer described the 'influenza OTl fresh clotted rabbi [ blond. Smears made after
hacitluP, Koch ( 1 SS3) bad observed a small bacillus 24-46 hours incubation show tangled chains of
in conjunctivitis cases in Egypt . It was first cultivated harilli. It may also be grown on the chuinnJJanioic
by Weeks ( 18® 7 ) in Mew York and came to be membrane of the chick embryo. On chocolate agar,
known as Kuch-Weeks bacillus . Recent DNA enriched with isovitalex and fetal calf serum , and
studio have shown that the baci '.lus is identical containing vancomycin as a selective agent , H „
with nou-capsulated H . influenzae. Therefore, the ducreyi forms small , gray translucent colonics alter
former H . aegrptimx has lost its separate species incubation at 35 °C under 10 per cent CO . and
status and is now comr . lered as a biotype ofri . high humidity in 2-E days.
i .' M I
- I t is worldwide in distribution and causes The species is antigenit ally homogeneous and
'
a highly contagious form of corjunci iv iris (' pink cultures mav be i den rifled by agglutination with
eye' ) . Jr is especially common in the tropics and the antiserum . Intradermal ! - looilation of the culture
subtropics and may occur in epidemic forms . It into rabbits produces a local ulcerative lesion .
responds to Irmai sulphunainideB or ge ft : 11 Lei i L . H . ducreyi is susceptible to sulphomunidcs and
It has also been identified as the causative agent many antibiotics. Cases resistant to sulphonamidrs
of Brazilian purpuric fever ( BPF }, in which and tetracyclines haw been reported . Etythro'
conjunctivitis proceeds to a fulminant septicemia myein , corrimoxazole, ciprofloxacin or ceftriaxone
in in bin H and children with high fatality. First may be used for treatment .
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co Bordetella
The genus BordtteUi is named after Jules It is Gram negative. Bipolar me Achromatic
Bordet, who along with Gcngou identified lilt granules may be demonstrated on staining with
itmal ] ovoid baeillus cawing whoopunt cough * in tnluidine blue.
the sputum of children suffering from the disease Cultural characlorisirios: It is aerobic No
(1900) and succeeded in cultivating ii in a growth occurs anaerobically It grown best at 35-
crjmplex medium (1906 ) . The bacillus is now 36 C
known as Bofdctclin pertuisii ( jWTUSSrs meaning Complex media are necessary tor primary
.
intense cough ) A related blcillut, Bore ).
ia was isolated from mild cases nt
paftpertvs&
whooping cough ( 1937) . Bt>rd , branchixepticn
orjginallv isolated from dogs with broncho-
—
isolation - The msilium in common use is the
Bordet-Gengou glycerine |iotat ( i - blood agar . Blood
is required not to provide additional nutritive factors
but raTher to neutralise inhibitory materials formed
pneumonia ( 11911) may occasionally mfect human during bacterial growth. Charcoal or ioit exchange
beings- , producing a condition resembling pcrtussis. resiilS incorporated in culture media may serve the
It has been suggested that BvfiL bronchiseptia same purpose . Charcoal blood agar U a useful
represents the ancestral form from which [ fie Other medium. I [ does not grow on simple media like
UVHH species hiive evolved - The ( mirth member oJ nutrient agar.
the genua is Bond avium which causes respiratory
, Gmwtb is slow. After incubation for 2
disease in tuikcys. , -
hours colonics on Bordet Gengou medium are
RoKiisiThii. A FHRTIJSSIS
-
small , dome .haped, nontb, opaque , vise]d. greyish
white , retractile and glistening, resembling ' bisected
( Bordet ( iengou bacillus: pearls' or 'mercury drops'. Colonies are surrounded
formerly I hemophilus pertussis) by a hazy zone of hemolysis . Contluem growth
Morphology: Word- pertUKK is a small , ovoid presents an 'aluminium paint appearance.
'
-
coccobacULus ( mean length 0- i pm) In primary
cultures, ceils are of uniturnn > ize and shape but on
ItiuchuTnicul reactions: It is biochemically
inactive. It docs not ferment sugars , form indole,
subculture they may become longer and thread lik^ L reduce nitrates , utilise citrate or split urea . It
It is noncnoiile and nonsporing. It is capsuUred but produces oxidase and usually catalase also.
rends to lose the capsule or repeated cultivation. Resistance: It is a delicate organism , heing
The capsule can he demonstrated bv special stains killed readily by heat ( 55 QC (or 30 minutes.), drying
bu t docs not swell i n the|neHc i LLC of the ant i scrum . and disinfectants. Rut unlike H . ui/Pirenfirc LI retains
-
In culture him the bacilli tend to hf arranged in
,
Copyrighted material
340 < Texlbaak of Microbiology »
Antigenic constituents and ^ irul ^ nc ^ by the pancreatic islet cells. It is now known that
factors: Several antigenic fractions and putative all these are manifestations of the pertussis toxin,
virulence factors have been described bur their role PT is a 117 ,000 molecular weight hexamtr
in the pathogenesis of pertussis remains to be protein composed of six subunits with an A - 11
clarified They include the following: structure ( the A portion heing the enzymatically
l .Aggiutinagcn -i -. 15 ofd : * tullie possess genus active moiety and If the binding component) . It
specific and species specific surface agglutinogens con he toxoided. PI’ toxoid is the major component
associated with the capsular K antigens or or acellular pertussis vaccina. Antibody to PT
'
fimbriae . Gy agglutinin absorption tests , 14 can protect mice agaln &t intraaanl, intruperitoneid
agglutinating factors have been identified Factor or intracerebral challenge .
7 is common to all three mammalian species of 3. Filamentous hemagglutinin (FHA)\ Thin is one
bordetrlhe, Factor 1 ? is specific for Bord . of the three hemagglutinins produced by Bord .
btOitchiscptics and Factor 14 for Bord. pejTu.sSj.s, rhe otbtrS being PT and a lipid factor.
parapertussis . Factors 1 to 6 arc found only in Purified FHA appears as a filamentous structure
Strain!of BOfdt pCtttiS$Bt all gfwhich carry' Factor in the electron mienusuope and hence the name.
1 and one or more of the other factors . Bordetellae If is present on the badlluy surface and is readily
are cIn -bs- IFLCD into Various types based or the shed , ll adheres to rhe cilia of respiratory
agglutinogens thev carry. AH strains causing epithelium anti to erythrocytes. Resides facilitating
infections belong to types 1 , 2 and 3 , it is essential adhesion of Bord- pertussis to respiratory
chat pertussis vaccine strains should have factors epithelium, FMA and PT hemagglutinins also
1 , 2 and 3 . Factor specific antibodies arc present promote secondary infection bv coating other
in the Hera oi convalescent and immunised bacteria such as hfocinuphilos influenzae am!
persons. Agglutinogens promote virulence by pneumococci and assisting their binding to
helping bacteria to attach to respiratory epithelial respi ratory epithelium. This phenomena has been
cells. They are useful in serotyping strains and in termed piracy of adhauu.
epidemiological studies. Antibody to FHA can protect mice against aerosol
2. Pertussin toxin (PT}- This is present only it ] challenge . FHA is used in acellular pertussis
.
Bord , pertussis It plays an important role In the vaccines along with PT toxoid,
pathogenesis of whooping cough . PI’ is expressed 4. AdenvJjtr cyctsse ( AC ): All mammalian
or the surface of the bacillus and secreted into bordctcUae but not Bord - tvinm produce adenylate
the surrounding medium. The toxin exhibits cyclase. At least two types of AC arc known, only
diverse biological and biochemical activities, one of which has the ability to enter target cells
which formerly had been believed to be caused and act as a toxin . This is known as AC toxin
bv different substances that had been named ( ACT ). It act > by Catalysing the production of
accordingly. Examples are the LYJNPHOCJ-TOSIS CAMP bv various types of cells-.
producing Victor or LPF causing profound .
5 jffe. jr labile toxin (HLT ): It is a cytoplasmic
lymphocytosis in pertussis patients as well as an protein present in all bordetcllae. It is in activated
experimental animals; and two effect? seen only in 30 minutes at 56 °C . It is dermoneerotk: and
it ] experimental anini . LLH , hut not in patients, such lethal in cuice. Its pathogenic role is not known,
as the histamine seif ft rifting fitefor or HSF ft - cjtofariri ( TCT ): It is a low molecular
responsible for heightened sensitivity to histamine weight peptidoglyean produced by oil bordetcllae.
In experimental animals, and the islet activating It induces ciliary damage in hamster tracheal ring
prtftein nr 1AP induong excessive i nsul ] n secreti on cultures and inhibition of DNA synthesis in
I
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* Bordetella 341
epithelial cell cultures, Its rule in disease is nut In hum ,m beings, alter an incubation period of
known.
7. LJ pop*1h ^Jediunde ( LPS ) or the heat ATa dj/er
"
-
about 1 2 weeks, the disease takes a protracted
course comprising three stages - the catarrhal ,
toxin in present in at! hordetetlae and exhihirs paroxysmal anrl convalescent - each Lasting
features of Gram negative bacterial endotoxins, It approximately two week * . The onset is insidious,
is present in the whole cell pertussis vaccine but wirh Low grade fever , catarrhal symptoms and a dry
is not considered to be a protective antigen. irritating cough Clinical diagnosis in the catarrhal
8. Ptmctiii : Pertactin is an outer membrane stage is difficult. This is unfortunate as this is rhe
protein (0MP) antigen present in all virulent stage at which the disease can be arrested by
strains ot B . pertussis . Mice i mm united with antibiotic treatment. This is also the stage of
pertactir resist npiratoiy challenge wirh rhe maximum infectivity. As the catarrhal stage
bacillus. AnlilnK.lv EL» pertactin can he seen in the advances to the paroxysmal stage, the cough
blood of children after infection or immunization. increases in intensity and comes on in distinctive
I
PenaCtiii is included in acellular jsertussis vaccines. bouts. During the paroxysm , the patient is subjected
Variation: tford. perrus.sjs undergoes a smooth
'
to violent spasms of continuous coughing, followed
to rough variation. All fresh isolates are in the hv a long inrush ot air into the almost empty lungs ,
smooth form ( Phase 1 ) . On -iuh culture , they wirh a characteristic whoop ( hence the name ). The
undergo progressive loss of surface antigen* , and paroxysmal stage is followed by convalescence,
pass through phases II and III , finally becoming during which the frequency and severity of
phase IV which is the rough, avirulcnt form, coughing gradually decrease .
A reversible change in the capsular antigen has
been described as VnodulttHu'- The bacillus may
-
The disease usually lasts 6 8 weeks though in
some it may he very protracted . Complications may
occur in one of three potential 'modes', X, 1 and C, be 1} due to pressure eifeers duri ng the violent bouts
each of which has a characteristic surface antigen . of toughing (subconjunctival hemorrhage*
X, I am ) C refer to the colour of the confluent subcutaneous emphysema ) , 2 ) respiratory
colonies on rhe Bordet-Gengou medium - X for ( broncho - pneumonia , lung collapse ) , or 3)
Xnuhic ( yellow), C for cyanic ( blue ) and 1 for neurological (convulsions , coma). Respiratory
intermedia te. Modulation is influenced by the
'
corn plications are self limited, the atelectasis-
nature oH the culture medium. On the flordet- resolving spontaneously hut the neurologic a I
GcngOU medium, fresh isolates always occur in the complications may resulr in permanent sequelae
X mode. such as epilepsy, paralysis, retardation, blindness or
Patlmgoiriciiyi fiord , pertussis is an obligate deafness.
human parasite but infect ion can he produced The inflation is limited to the respiratory tract
experimentally in several species of animals, the and the bacilli do not invade the bloodstream. In
white mouse being most often employed. hitiarusal the initial stages, the bacilli arc confined to rhe
inoculation in mice induces a characteristic patchy nasopharynx, trachea and bronchi. Clumps ot bacilli
interstitial pneumonia, histologically resembling the may be see n c nmes hed in the cilia of the respiratory
human disease. Intraperitorteml inoculation of large epithelium. As the disease progresses, inflammation
doses is fatal due to ttueonii. Intracerebral extends into the lungs , producing a diffuse
inoculation causes a fatal infection. Immunised mice bronchopneumonia with desquamation of the
are protected - This forms the basis for the alveolar epithelium.
intracerebral mouse potency utay for pert uuL$ Blood changes in the disease are distinctive and
vaccines . helpflit in diagnosis . A marked IcucocvTosis occurs,
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Hidden page
- i Bondetelte 343
Innimnufluonescenct: is useful in identifying the produces better and more sustained protection and
bacillus in direct smem of clicuCAl specimens and less reaction than the plain vaccines. Pertussis vaccine
Ilf cultures . The differentiating features of is usually
a
administered in combination with
bordetelkt are listed in Table 37.1 . diphtheria and tetanus toxoid { triple vaccine ) . Not
Serologicil diagnosis is not helpful. Klsc in only is this more convenient, but Bord. pertussis
antibody hire may be demonstrated in paired serum also acts as an adjuvant for the toxoids, producing
samples by agglutination , gel precipitation or belter antibody response.
complement fixation tests. As antibodies appear Late, In view of the high incidence and severity ot
the second sample of serum should he collected the disease in the newborn, it is advisable to start
some weeks after the onset of the discasc.
r emunstration of spc-L-ifLU *ecTctOrv IgA antibody
^
in nasopharyngeal secretions by ELISA has been
proposed as ;L diagnostic method in culture negative
—
immunisation as early as possible . Three injections
at intervals of 4 6 weeks are to be given before the
age of six months, followed by a booster at the end
of the first vear of life,
- r
,
infectivity is highest in the earliest Stage of the receive chcmnprnphvlaxi * with erythromycin .
disease when clinical diagnosis is nor easy. Non immunised contacts should receive
Specific immunisation with killed Boni. pertussis erythromycin prophylaxis for ten days after contact
vaccine has bee n found very effective. 1 r is of u rmosr
m with the patient has ceased . Pertussis vaccination
importance to use a smooth phase I strain tor may induce reactions ranging from local soreness
wane production. The method of inactivation and fever , to shock, convulsions and encephalopathy.
should be such rhat antigen ic potency is unaffected. Provocation poUomyclmv is , L rare complication.
Detoxication with 0.2% merchaobte during several Factors contributing to toxicity or pOitYlcdna]
months,' storage at 4 C has been recommended as encephalopathy have not hecn defined - The latter
.
a satisfactory procedure The alum absorbed vaccine compticition is estimited ro occur in one in 15 10 —
Table 17- 1 Differentiating feature* of Enrdetella species
Bord . pertuswj Band. parapertussis Bord. bnonehisepica Bord , arium
MdCilitj'
Gruwth on nutrient agar
—— —
+
+
*
4
—
Growrh on Bordet Geng-Qu 3-6 1-2 1 1
medium {days )
Urease
Nitrate Cu nitrife ——
*
—
4 f
*
- ——
Citrate uliliwitwjn V + V
Oxidase
Toxins:
+
— --
I 4
ACT 4 4 4
PI 4
- -
V Variable
Copyrighted material
344 * TexltiooK ol Microbiology
million inject ion R . Eslinutcd NEUROLOGICJI Ire fitment: fiord pctmu^is is- susceptible
, tn
complications of natural disease have ranged tnont several antibiotics ( except penicillin ) blit
1.5-14 per cent in hospitalised cases; $. third of antimicrobial therapy i;; beneficial only if initiated
-
these recover, a third Live mirhc tiild , L third die
or have severe detects .
within the first ten days of the disease.
Erythromycin or one of rbe newer macrolides is
[ fsvvcrc complications such &s enccjihalopathy, the drug of choice. Chloramphenicol and
seizures, shook or hyperpyrexia develop following
the vaccine, subsequent doses of the vaccine are
-
cotrimanaoU an also useful .
now used in some other countries also : LS they cause ( Bond , bronchiCam's)
far fewer reactions, particularly lit older children. Tli i T- is motile hy peri I rich ate flagella . It is
Both whole celt and acellular vaccines llave a antigenicaUy related to Bond, pertussis and firucelb
protection rate of about % ]ter cent . With wltole abortus. It occurs naturally in the respiratory tract
cell WfljflBt the protection dedincR to 50 per cent in of several apedes of animals. It has been found to
about five years and is absent after 12 years, The Liiose . L very small proportion ( O. t per «nt) of cases
-I U II ' L T h e a d i n g
Cheny JDtral. l 9Siit. ftepn ^ i of the usk !»(t on hrtllHii, ftdrj jr K 1 : 93£J .
EiiiTflrLaJ . 1992. E rtUllii: idulls, infant and herds. Lancer , 359:526,
^
Fritdmin RJ .. 193?. FtrtuisluThe IUSL-JW sod new diagnostic irischmL'- . { Vin JWnTwfrkJ Krv t ; 165.
Gustaffson I . et aJ. 19%. A connclled tnal of acellular pertussis vaccines. .A’etv tng! JXted. 333:3+9.
Tinman M . 19 H 4. The concept of perruasic as a rotm mediated disease. Pediair Infccr Die. .1:4t > 7.
WHM AA and EL f lewlrtt 1986. Virulence factors ofAnfaklla pemissjs. Arm Re v Micnibiot +0.661 .
Hewlett EL 1997. Pertussis: Currenr rancepTF of pathogenesiF . Fkdiatric Infect UJS. J 16:5
Copyrighted material
Brucella
The genus Efircelii consists of very small , from desert wood fats. Br. canis may occasionally
nomnotile , aerobic, Gram negative Coceobacilh that cause a mild human disease , but [ he other two are
grow poorly on ordinary media anti have little or not pathogenic for humans .
no fermentative powers , ITcy arc strict parasites of Morphology: Bruccllae arc cotcobacllli or short
uni trials and may also infect humans.
Brucellosis is a zoonosis, primarily affecting
—
rods 0.5-0-7 pm * T6 1.5 pm in size, arranged
singly nr in short chains. The cells are &o small
goats , sheep, cattle. bu It nines pipt and Other
, that they may be mistaken lor cocci . as was done
animals and transmitted to humans by contact wifh by Bruc e who called them jMtCAKDCOU ItKllteJU.
infected animals or through their products . The In older cultures, irregular forms appear. They arc
human d is ease was recognised along the non motile, noncapsulated and nnmpnring. Thry are
Mediterranean littoral from very early times and Cram negative and nonaeid fa*t , Bipolar sraining
has been known under various names such as is not uncommon.
Mediterranean fever , Miltn fever and undul.inr fever . ( ltd!Lind characteristics:. Bruce line are strict
A British army doctor, David Bruce { 138 b) aerobes and do not grow anaerobically. Br. abortus
isolated a small microorganism from the spleen of
fatal cases in Malta and transmitted the disease to tor growth . The optimum temperature is 37
—
is capnophilic, many strains requiring 5 109 CO .
*
monkeys experimentally- This was named BnincfJa ( range 20-40 "C) and pH &, h 7.4. They may grow
“
meJitenm ( Brucella after Bruce, tnctianixr after on simple media, though growth is slow and scanty'.
Melita, the Roman name for Malta) . A Maltese Growth is improved by the addition of scrum or
bacteriologist Zartmiit ( 19051 showed that Bf . liver extract . Liver infusion media were widely used
mcffticn.sMwas transmitted to humans by goat 's milk . tor the cultivation ofbnacellae. The media employed
Following this, the disease was eliminated from currently ire serum dextrose agar, scrum potato
British soldiers by prohibiting them from using jnfus]on agar, trypt lease soy agar, or tryptose agar.
gnat s milk and milk products, while the disense The addition of bacitracin , polymyxin and
remained undiminished in the civilian population , cycluheximiide to the above media makes them
which continued to use them . Bang ( 1397 ) selective.
.
described Hr. aimrftis [ be cause of contagious
abortion In cattle , The third major species in the
In liquid media , growth is uniform, and a
powdery or viscous deposit in lormcd in ddctiljtUftt.
genus, Br. mix was isolated by Tnum ( 19 M ) from On solid media, colonics arc small , rnoiit,
pigs in the USA. These three species cause human translucent and glistening. Mucoid, smooth and
bnicellorrs. rough types of colonies appear , associared with
Other Species causing animal in lections include changes in antigenic structure and virulence.
fir: cam .?, isolated from cases of canine abortion ,
"
Erytliritol has a specially stimulating effect on
Br, avis from abortion in sheep and Hr, neotomac the growth of bruccllac.
Copyrighted materia
34* * Texltwok ol Microbiology *
ordinarily fermented, though th rponn uxid^itivt antigen resembling Salmonella Vi antigen has been
capacity. Btucdke ^
catalase positive . oxidase
ate described .
positive ( except tor Hr. rjeoJTnmaeand Br. Otis which Bructllfl bflCttrinphflgtt Several bacterid
are negative ) and urease positive . NitrsUc^ arc phnigC' that Iy^c the Brucella strains have been
reduced Do nitrites. Citrate is not uuliscd . Indole is isolated . All these phases are serologically similar.
not produced aid MR and VP tenth are negative . The Thlist (Tb ) phage has been designated as the
K exist mice: Bmcelke ate destroyed by heat at reference pha e and at RTD Lyses only Br. abortus.
60 ' C in 10 minutes and by 1 % phenol in 15 ^
Hr. sms is lysed at 10,000 KTD, while Br mditensis
minutes. They arc killed bv pasteurisation . I hey is not Lysed at all.
mav survive in soil ind m .irmrc tor hever .il weeks .
1
ClflSNtficnlidn; Brucellac may be classified into
They remain viable tor 10 days in refrigerated milk , different species, b^sed on CO . requirements , H ,S
one month in 111- cream , four months ii butter and production, sensitivity to dyes {basic fuchsin and
for varying periods in cheese depending on its pH . lliiomn ), agglutination fo monospecific sera , pliugc
'fhev may also survive for raanv weeks in meat . Ivsis and oxidative metabolic tests with amino acids
r
They ate sensitive to direct sunlight and acid, and and carbohydrates. The three major species are Br
fend tn die in buttermilk. Hr. mttitensis mac remain me / rJcn.sb, ffr. u / jorrus , infecting primarily goat* or
alive for sis days in urine, six weeks in dust and ten sheep, cattle and swine, respectively. Many hiotypes
weeks in water. have been recognised, in these species.
Antigenic structures The somatic antigens of Br. itlia strains that produce H ,S are known 4s
hrueeDae cdnciLLii two main antigenic determinants , 'American' strains and those that do not a ' Danish'
*
A ami M which are present in dificrent iimiunls in strains.
the three major species. Hr. abortus contains about Pathogenicity: All three major species of
2U times A as M : Ih. mclitaua about 2D times M bruccllac are pathogenic to human beings . Br.
as A - Kr. sib* has an intermediate antigenic pattern. rncMtnsps is the must pathogenic , Hr. nhtyrtii. and f
Absorption of the minor antigenic component from Br . suis of Intermediate pathogenicity. The
an antiserum wjH teave most ot LILL major afttilsody incubation fjenod is usually about 10— 30 days, but
component and such absorbed A and M may *i > meti mes be very prolonged - Hu man i nfisrrinn
monospecific ser .i arc useful for species may be of three types :
identification hy the agglutination tc^t . The species 1 . latent infection with only serological hut no
identification of brucella strains is not , however , so clinical evidence;
straightforward and strains are often seen t h a t 2 . acute or subacute brucellosis; and
behave biochemically as abortus and serologically 3. chronic brucellosis .
as meUtensis and vice versa . Species and biotype Acute brucellosis is moctiy due to Br ineliiensia .
identification depend:, on a varietv of other factors ( It is usual!v known undulant ftwr, but this is
beside* antigenic structure ( Table 3S . 1 ) . misleading as only some cases show the undulont
Antigenic cross -reactions exi ^ c between pattern . ) It is associated with prolonged bacteremia
brucellac and V. c holeme and persons receiving and irregular fever . The symptomatology is varied,
rhe cholera vaccine may develop brucella agglutinins consisting of muscular and articular pan is , asthmatic
Lasting tor about three years . Antigenic cross - attacks , nocturnal drenching sweats, exhaustion,
re actio ns also exist with F . coii 0 : 11 b ; 0: 1.5 7 , anorexia , constipation, nervous irritability and chills.
Sal monel La serotypes group N ( 0:30 antigen The usual turnplication* are articular, osseous,
Kauffman anti White ) , JV nulrophila , Y . visceral or neurological .
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Hidden page
Hidden page
i Bmcellh 34y
Laboratory methods for diagnosis include culture, killed suspension of a standard stm H of Br- abortus]
'
serology and hypersensitivity tests . are rinsed and incubated at 37 for 24 hours or
Blood culture is the most definitive method for 50 C for IS hours , A ti ’ re of 160 or more is
3
the diagnosis of brucellosis- Blood is inoculated considered significant - Most patn - nfs with acute
. ufo 1 hnttfe of trypr i case soy broth and. incubated
at 37 °C under 5-10% CO . As bacteria in blood
brucellosis develop ri tries of 640 nr more by 3 4
weeks of illness. Trines tend to decline after the
—
^
ire usually scanty, large volumes of blood (5 nil.) acute phase of the illness.
should be inoculated . Subcultures are made on solid Several sources of error have to he guarded
-
media every 3 5 days, beginning on the fourth day , against . Sera often contain "blocking or
Growth mav often be delayed and cultures should nonagglutinating' antibodies . The blocking effect
not he declared negative in less than 6-8 weeks . may somet mes be removed by prior hcaf ng of the
-
BACTEC cultures may become po itive in 5 to 6 serum ar 55 QC for 30 minutes or by using 4% saline
as the di I uent for the test.The most reliable meth nd
days.
The Castaneda method of blood culture has for obviating the blocking effect and detecting the
several advantages and is recommended. Here , both '
incomplete' anti I >oi lies i - the an r i irlobnli n {Coombs)
n
liquid and solid media are available in the same test. As the prozone phenomenon to high titres
bottle. The blood is inoculated into the broth and ( upto 1/640) is very frequent in brucellosis d : s
the bottle incubated in the upright position. For csscnfiij that several scrum dilutions he tested - A
subculture , it is sufficient if the bottle is tilted so positive agglutination test may be produced by
that the broth flows over the surface of the agar cholera , tularemia nr yersinia infection , or
slant. It is again incubated in an upright position . immunisation. Cholera induced agglutinins mav he
Colonies appear on the slant . This method differentiated by the agglutinin absorption test and
minimises materials and manipulation, reducing also as they are removed by treatment with 2 -
chances of contamination and risk of infection to mercapto-ethanol. In order that results from
laboratory workers . different laboratory arc comparable , it is the
Blood cultures arc positive only in about 3CH50 practice to express agglutinin titles in International
per cent of cases, even when repeated samples are l 1 n : 75- This done by using a standard reference
rested ifr. meljfensjs and Sr mis ire isolated more
,
'
-
serum for compa i'- on .
readily than Br.abortus Bone mannow cultures y ield In brucellosis, borh IgM and IgG antibodies
a higher rate of isolation and remain positive long appear in 7-10 days after the onset of clinical
after the blood culture has become negative , infection , As thedisease progresses, IgMnnt bodies
Cultures may also be obtained from lymph nodes, decline, while rhe IgG antibodies persist oi increase
cerebrospinal fluid , urine and abscesses, i: present, in ritre. In chronic infections, IgM may often be
and, on occasion , also from sputum, brrusl milk, absent and only IgG can be demonstrated . The
vaginal discharges and seminal fluid . agglutination rest idenffies mainly the IgM
As cultures are often unsuccessful, serological antibody, while both IgM and IgG fix complement .
methods are important in diagnosis. Several The IgG and IgA antibodies may act as ' block : ng’
serological tests have been developed , including or 'nonagglcitmuling ' antibodies . It is thus evident
agglutination , complement fixation and ELISA. that the agglut inatk m test is usually posirivr in acute
The standard agglutination tes- t ( SA 1 ' iS infection but may be only weakly positive or even
performed most often . Th .* is a tube agglutination negative in chronic cases . The results of the
-
test ill which L L: U.II volumes of serial dilutions of agglutination tests therefore have lo be evaluated
the patient 's serum and the standardised antigen (a carefully. While a high titre of agglutinins, and
Copyrighted material
350 * Texlbook pF Micrnbiaioriy
especially demonstration of a rise in tirre, ran be the milk ring test a sample of whole milk is mixed
taken .LK diagnostic, even i negative agglutination well with a drop of the stained brucella antigen (a
test may not exclude the possibility of brucellosis. concentrated suspension of killed Br,abortus stained
The complement fixation test is more useful in with hematoxylin ) and incubated in a water bath
chronic cates detects IgG antibody also.
as it -
at 70 T for 40 50 minu tes, If antibodies are present
ELISA is sensitive, specific and can detect IgM in the milk, the bacilli are agglutinated and rise
and IgG antibody separately l f i s therefore useful with the cream to form 1 blue ring at the top .
fur differentiation between the acute and chronic leaving the milk unstained. If antibodies are absent,
phases of brucellosi s and also for screcr.ing large no coloured ring is formed and the milk remains
numbers of sera. uniformly blue . The whey agglutination test is
Delayed hypersensitivity type skin tests with another useful method for detecting the antiisodics
brucella antigens (‘brucellins’) ate not useful in in milk _
diagnosing acute brucellosis. They parallel the Prophylaxis: As the majority of human
tuberculin test in indicating only prior sensiiisanon infections ire acquired by consumption of
with the amigertSp and may remain positive lor years.
Brucellin testing may lead to a rise in litre of antibodies.
.
contaminated m iIk prevention con ^ i *t5 of checking
brucellosis in dairy animals. In many advanced
The methods used for the laboratoiy diagnosis countries, this1 iis achieved by the detection of
of human brucellosis may also be employed for the infected animals, their ehmina Non by slaughter and
diagnosis ofar.imal infections. In addition , bnicellae -
the development of certified brucella free berds-
P 3Ceuri >-ati < m of milk is an additional safeguard.
may be demonstrated microscopically in
pathological specimens by suitable staining or by
^ Vaccines have been developed for use in am mils.
immunofluorescence- Several rapid methods have Br. abortus strain vacrine is protective in, cattle.
been employed for the detection of brucellosis in No suitable vaccine is available for human use.
herds of cattle. These include the rapid plate Treatment: The usual regimen is a combination
agglutination resi and the Rose Bengal card test. of doxytydine for 45 days with streptomycin IM
For the detection of infected animals in dairies, daily for the first 2 weeks in adults., and in
pooled milk samples may be tested for bacilli by children cotrimoxazole along with rifampin n or
Culture and In: antibodies by several techniques. In £ LL:iUrn win .
Further It Lading
Corbel MJ. 1997. Brucellosis, an overview. Emeig. ng Infccf Off. 3: No. 2.
Corbel MJ , 199?. Vaccine* ajjaiurt bacterial J Afled jVJjrrobrof 46:267.
Expen Committee on hi ucthusii . 1936. Stub Report, Technical Report Series bin . 74. ). GUKVIIWHO.
Young J .' l . 1995. An overview of human ttfueellasu. Clin lnitci Lhi. 21:233.
:>pyrianiea materia i
L
Mycobacterium I: Tuberculosis
Mycobacteria are slender rods that sometimes cold blooded and warm blooded animals, from skin
show branching filamentous forms resembling ulcers, and from soil, water and o-ther environmental
fungal mycelium. In liquid cultures they form a sources . They were initially called atypical
mold-like pellicle. Hence the name 'mycobacteria , mycobareria and broadly categorised into hour
meaning fungusdike bacteria . They do not stair categories: photochromogens , scotochromogens,
readily, but once stained , resist decolourisatlon with non-photochromogens and rapid growens, based on
dilute mineral acids . Mycobacteria are therefore their growth rates and pigmentation . They are
called 'acid fast bacilli’ or AFB . They are aerobic, opportunistic pathogens and can lead to many types
non motile, noncapsulated and noisporing. Growth of diseases; they arc described in Chapter 40,
is generally slow. The genus includes obligate Saprophytic mycobacteria were isolated from a
parasites , opportunistic pathogens and saprophytes. number of sources. These included M . butyrieum
The Unit member of this genus fn be identified from butter, M. phlei from grass, M, stcrcoris from
was the lepra bacillus discovered by\ Lansen in 1963. dung and M. smegmarif from smegma . {Contrary
Koch ( 1882 ) isolated the mammalian tubercle to common belief,. M . ftnegmam is seldom found
bacillus and proved its causative role in tuberculosis in smegma , though other rapidly growing
by satisfying Koch 's postulates , Tuberculosis in mycobacteria occur frequenly there, contaminating
humans was subsequently shown to be caused by- urine cultures ) .
—
two types of the bacillus the human and bovine
types, designated Mveotacreifiim firberoifos!? and MYCOBACTERIUM TUBERCULOSIS
Mr iwvjs, respectively. The term M . fubejTii /osj's
'
Morphology: Mr tuberculosis is a straight or
comple* includes, besides the hunuin and bovine types, slightly curved md, about 3 pm x 0.3 pm , occulting
two other mammalian types also : M. ifrjcatium ringlv, in pains or as small clumps. The size depends
causing human tuberculosis in on conditions of growth, and long filamentous, cluh
possessing properties intermediate hetween human shaped and branching forms may he sometimes seen.
and bovine types, and M. microti ( the vole bacillus M . fid-vis is usually straighber, shorter and stouter.
pathogenic for votes and other small mammals but Tubercle bacilli have been described as Gram
not for humans)., positive, though strictly speaking this is not correct,
The second human pathogenic mycobacterium as after staining with basic dyes they resist
is the lepra bacillus causing leprosy. Though this dccolourisatlon by alcohol even without the
was the mycobacterium first described , it is the least mordanting effect of iodine. When stained with
understood because it has not been possible to carboi fuchsin by the Zichl-Neebcn method or by
convincingly culture it in vitro so far . fluorescent dyes Uuramine O, rhodamine), they
The third group of mycobacterium it a mixed resiat decofourisatian by 20 per cent sulphuric acid
group of isolates from diverse sources; from birds, and absolute alcohol for 10 minutes (acid and
Copyrighted material
352 * TcirtHck DI M c - obio 'ogy »
aleoho ] fast } . A -rid fastness has been ascribed employed for non fine culture i> Lowcnstcirv-Jcnscn
variously to the presence in [ he bacillus of an i. l J) medium without starch, as recommended hy
uniaporufiiblc wa* ( mycoloic acid ) or to a the Interna n ' nal Union Against Tuberculosis
'
semi perm cable membrane around the cell . It is CIUAT ) . This consists of coagulated hens’ egg,
i el ,mi to the integrity of the cell and appears to be mineral salt solution, asparagine and malachite
a property of the lipitl- rich waxy cell wall . Stsiioing greenh the last acting as a selective agent inhibiting
maybe on iform or granular. Beaded or barred forms other bacteria. Among the several liquid media
are frequently seen inM. rubmido - -srs', bur M . bovi $ described, Dubos ', Middlebrouk's, Proskauer and
stains more uniformly J
Beck ' s , Sula’s and SautorV media are the more
Eleetf on micrographs of tl 11n sec ^ons show that common. 1 dquid. media are not generally employed
the thick cell wall is Composed of three layers for routine cultivation , but arc used for strainviry
,
Culturfll characteristic^: The bacilli grow rough, raised, irregular colonies with a wrinkled
slowly, the generation time in vitro being 14-15 surface. They are creamy whi rch bccom ingyellowish
honrs. Colonies appear in abraLt two weeks and may or buff coloured on further incubation. They are
sometimes- take up to eight weeks- Optimum tenacious and not easily emulsified . M. bovis
temperature : s 37 UC and growth docs nor occur colonies, in comparison are flat, smooth, moist,
below 25 °C or above 40 't . Optimum pH is 6.4- white and break up easily when touched.
7.0. AI. ru6en. ulo.sjs i - an obligate aemhe, while In liquid media without dispersing agents rhe
j\f. bavin is mieroaemphilic: on
primary isolation , growth begins at the bottom, creeps up the sides
becoming aerobic on subculture, A1 tirbercir/osis and forms a prominent surface pellide which may
grows luxui ianily it ] culture as compared to M. bovi* extend alon r the rides above the medium. Diffuse
which grows sparsely. They are therefore termed ^
growth is obtained in Duhos' medium containing
eugenic and dvsgonic respo^ ri \ ely, The addition of Tween- 00 ( sorKtun monooleate). Virulent strums
0.5 4 glycerol improves the growth of AJ.
^ tend to form long serpentine cords in liquid media,
lubcrCtllptis, hut lias no effect on or may even while aviruient strains grow in a more dispersed
impair the growth of AJ. fwvjs. Sodium pyruvate manner. The cord factor itself is not a virulence
helps the gmwih of both types. Human tubercle factor as ir is present in some aviruient strains as
bacilli do not grow in presence of F- nirrobenzoic well.
acid, unlike orher slow growing nonchrumugenii. Resistance: Mycobacteria are not specially heat
Tnhercle bacilli do not have exacting growth resistant, being killed at 60 in 15— 70 minutes.
requirements but arc highly susceptible even to
^
Survival i- influenced by the material in which
traces of toxic substances Like fatty achk in culture the bacteria are present. Cultures may be hilled by
media. The toxicitj is neutraiIscd bysemm albnmin exposure to direct sunlight for two hours, hut bacilli
or charcoal , Koch originally grew the bacillus on in sputum may remal n alivc for 20— 30 hours. Bacilli
hear coagulated bovine serum. Several media, both in droplet nticle i may refcf n viability for 3— 10 days
'
solid and liquid Have been described for the under Suitable conditions. Cultures remain viable
cultivation of tubercle bacilli . The solid media ar room temperature for 6— S months and may be
conrain egg (LowcnsteLn"-Jensen , Petragnini , stored for up to two yearn at -20 "C .
Dorset), blood Harsh is), scrum ( LoeffW) or potato Tubercle bacilli are relatively resistant to
(Pawlowsky ) . The solid medium most widely' chemical disinfectants, surviving exposure to 5%
Copyrighted materia
4 Mycobacterium I . Tuberculosis * 353
phenol , 15% sulphuric acid, 3% nitric acid * 5 % and G.2% catechol in distilled water U added ro
oxalic wid and 4% sodium hydroxide - They are 5 ml of the test culture and allowed to Stand for
sensitive to formaldehyde and gluicraldthyde. 'fhey few minures . Effervescence indicates catalase
arc destroyed by tincture of iodine in five minutes production and browning indicates peroxidase
and bv 80% ethanol in 2-10 minutes. Ethanol is a
I!
activity.
suitable d is in tecta nr tor skin , gloves and clinical Amid tr sc lusts: The ability ro split amides has
thermometers. heen used id differentiate mycobacteria. A useful
m
with atypical inycobacieria.The bacilli are grown bacilli, delayed hypersensilivty is developed to die
in ; L medium containing 0.001 M fri potassium bacillary protein (tuberculin). Tuberculins from
phcnolphchalcin disulphate. 2 N . NiOH is added human , bovine and murine bacilli appear to be
drop by drop to the culture. A pink colour indicates indistinguishable - Some degree of antigenic
j positive reaction . relationship exists between tubercle bacilli and
Neutral red test: Virulent strains of tubercle atypical mycobacteria * as shown bv weak cross
hiK illi are able to bind neutral red in alkaline buffer
'
reactions in skin tearing with different tubercLilins .
solution, while avirtilent strains are unable to do Time is also some antigenic relationship between
so. lepra and tuliercie bacilli.
-
Catalase Peroxidase tests: These help in By various serological tests ir has been
established that JW. fuberctiiosj s strains arc
'
comparison . On the other hand, tubercle bacilli been demonstrated in sera of pwients, bur they have
are peroxidase positive , but not atypical not been found useful in diagnosis or relevant in
mycobacteria. Catalase and peroxidase activities are miiy
lost when tubercle bacilli become INli resistant. Bacteriophage: Many mycobacteriophdges
Catalase negative strains of tubercle bacilli are not have been isolated from soil, water and other
virulent fur guinea pigs. environmental sources as well As from lysogenic
A mixture of equal volumes of 30 vol. H ,0 , strains. Many toycob*cteria infected with temperate
Copyrighted material
354 * Textbaofc ol Microbiology *
phagw are not truly lysogenic, instead of being produces tuberculosis in cattle, humans, other
integrated with the bacterial chromosome, the primates , carnivores including dogs and cats ,
phage germane appears free, like a plasmid . This is badgers, swine , parrots and some birds of prey.
called p&tudoljwjgeny. Experimentally it is highly pathogenic for guinea
Tubercle bacilli have been classified into font pigs and calves , moderately pathogenic for dogs,
—
phage types A , B, C , and a type intermediate
between A and B, and therefore designated I ( for
cats, horses and rats , and nonpathogenie for fowl.
BCG, the tuberculous vaccine, is an attenuated strain
'intermediate'). Phage type A is the commonest and Of M fan .
*
is present worldwide . Type B is common in Europe Strains of tubercle hacilli isolated from parts of
and N . America. Type C is seen rarely. Type 1 is Africa, that show properties intermediate between
common in India and neighbouring countries. human and bovine types haw been called African
Phage 33 D isolated from an environmental strains* or Af. aBicaaiun . The name Mdsn type 'has
mycobacterium lyses all variants ofM. tubcjT[i/osj^ h beer given to strains of tubercle bacilli originally
but not BCG . isolated from south India, which are of low virulence
Bftcleriocdns: AJ. fuberru/osf .t is divisible into tor guinea pigs, susceptible to hydrogen peroxide,
two types by means of bacceriocins produced by lsoruazid sensitive and usually of phage type L Such
rapidly growing mycobaetcria. strains have also been isolated from some other
Molecular typing: DNA fingerprinting Asian countries and from Asian expatriates abroad.
provides a method tor di fife re flt i ati n g between Though species names are sometimes used for
strains of tubercle bacilli. Restriction endonulease different varieties of tubercle bacilli infecting
treatment yields nucleic acid fragments of varying humans, they are not considered independent
lengths, the patterns af which arc strain Specific . entities, but only variants of the M. fufwjrti/oris
1
This 'restriction fragment length polymorphism complex.
( RFI.P} enables strain typing for epidemiological M. microti is a natural pathogen of voles. It
purposes. Fingerprinting can also be done with a does not cause natural infection in humans, but can
chromosomal insertion sequence, IS Slid, which cause ulcers after experimental infection . Because
K present in most strains of tubercle bacilli. it is antigemcaliy identical with human Tubercle
As the entire genome of tubercle hacillus has bacilli , it was tried as a substitute for BCG for
been sequenced, more molecular typing methods human vaccination . It was given up because of
may become available . serious Inca! reactions.
Host ranfjt: M. rubercufosrir causes natural rmhngcnesis: The source of infection is usually
infection in humans, other primates, dogs and some an open case of pulmonary tuberculosis. Ir is
Other animals which have close contact with estimated that an open case of tuberculosis in India
humans. Experimentally it is highly infectious for may infect cm an average some 25 contacts before
guinea pigs and hamsters , but virtually death or cure . Other forms of tuberculosis are of
nonpathogenie for rabbits , cats, goats, bovines and
, much less importance in public health . The mode
fowl. Mice are moderately susceptible and develop of infection is by direct inhalation of KWtdlittd
progressive infection following intraperiruneal, bacilli Contained in droplet nuclei of expectorated
intravenous or intracerebral inoculation. Variation sputum. Coughing, sneezing and speaking release
in virulence among strains is frequent . Isolates from
cutaneous and urogenital tuberculosis are often of
—
numerous droplets- as many an 3000 infectious
nuclei per cough. Dried bacilli in dust arc much
low virulence for experimental animals . less infectious. Spread occurs most often among
Af - bends is more pathogenic for animals . It household or other close and prolonged contacts
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Hidden page
356 * Textbook ot Microbiology *
in Home Egyptian mummies. Tubercle bacillus common worldwide . In many advanced countries ,
I >NA has been detected by molecular uyih/n in a an in the UK it has been almost eliminated bv
—
J
three million persons die from the disease . The pasteurised or heated milk . Person - to person
transmission of of A f. hovrs Is very rare .
-
large majority of the cases and deaths are from the '
-
B
Copyrighted materia
* MyMfcact & nLim l : Tubereulatia * 357
smtsuhs and they should tioi be reused, as acid fasr auramine rhodamine fluorescent dyes and when
bacilli always be rtnuwcd
Itfly nut (rum
slides hy , examined under ultraviolet illumination, the bacilli
cleaning. Smears should be prepared from the thick will appear as bright rods against a dark
purulent part of the sputum. Smears are dried, heat background . Beeause of rhe contrast; the bacilli can
fixed and stained by the Zichl-Ncclscn tec hnique. be seen ever under the high drv objective, cnahling
The smear is covered with strong cartel futhsin large areas of the smear to be screened rapidly.
and gently heated to steaming for 5-7 minutes, Microscopic demonstration of acid fast bacilli
without letting the stain boil and become dry. The provides only presumptive evidence ot tuberculous
slide then washed with water and decolourised
is infection , as even saprophytic mycobacteria may
with 20% sulphuric acid till no more stain comes present a similar appearance - However most
off and then with 95% ethanol tor two minutes . saprophytic species srain uniformly without
Decolourindon may be carried out as a single step appearing barred or beaded and arc usually only
with acid alcohol {3% HC1 in 95% ethanol ). After acid fist without being alcohol fast. As saprophytic
washing, the smear is coumtTsraincd with Loetflcr’s mycobacteria may be present in tap watcrh rubber
Tnethvlene blue , 1% picric acid or 0.2% malachite tubes, cork or bark, they can gel into clinical
green fix one minute . Under the nil immersion materials unless scrupulous care is taker in their
objective, acid fast bacilli arc seen as bright red collection - Saprophytic bacilli are not ordinarily
rods while the background is blue, yellow or green present in respiratory secretions, hue they may be a
depe ndi ng on the conn rerstai n used - At least \ (1 ,000 problem with gastric aspirates, feces and urogenital
acid fast bacilli should be present pet ml of sputum specimens .
for them to he readily demonstrablc in direct smears. Cnncentration method! ! Several methods
A negative report should not be given till at least have been described far the homogenisation and
300 fields have been examined , taking about 10 concentration of sputum and other specimens. They
minutes. A positive report can be given only if two can be classified as methods useful far microscopy
or more typical bacilli have been seen. Smears arc only and those useful for culture .md animal
graded depending on the number of biL- illi seen: inoculation as well. To rhe former group belong
(Table 39.1) treatment with and form!nT sodium ciubonare or
When several smears arc to be examined daily, hypochlorite, detergents like tcrgitol, flotation
it is more convenient to use fluorescent microscopy. methods using hydrocarbons, and [ he autoclave
Smears are stained with aunmme phenol or method . These methods kill the hucilli without
Table 39.1 ZN smear evaluation and APS report altering their morphology or staining reaction.
Concentration methods that do not kill the baLLlli
JVrJ. of SWJT in Report and so can be used for culture and animal inoculation
AFB ( m! immersion field )
include the following'
PetrofFs method: This simple method is
0 300 r AFB rvot seen widely used. Sputum is incubated with an equal
1-2 300 F Doubtful,
repoiT smear
volume of 4% sodium hydroxide solution at 37 C "
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359 i Texioook oA MicrotiiDiogy
A - IITIP ] cr method has been described results to be given in 2- 3 weeks . But these are
eliminating ccntiifugatii m and neutral is? t ion. available only in advanced laboratnrics-
Sputum is treated with an approximately equal Semitivity testa: As drug resistance is an
volume of a sterile sol Lit:on containing 20 g important problem in tuberculosis, it is deniable
cetrimonium bromide and 40 g of NaOH per to have sensitivity of : solatcs tested as an aid to
!: rrc of d i -- tilled water The contents are mi xed w: i h
. treatment. Sensitivity tests for tubercle bacilli are
a co[coo swab and let stand for five minutes . About ma nly of three types. The first is the absolute
0.2 ml of the inoculum i - smeared firmly with the
.
B
concentration method in which a number of media
swab over the entire surfaceof acid buffered medium containing serial concentrations of the drugs arc
( IUAT- LJ medium with 20.5 g KH FO, per litre ). inoculated and the minimum inhibitory
The same nwab is used I LIT inoculating^ a second concentrations calculated . The second is the
jf
slope after stirring the contents ag. iin. The results res i -- Cance rat i o method in wh i ch two sets of media
arc as good as wuh Fetroff 's method . containing graded concentrations of the drugs arc
Instead of alkali , homogenisation can be i noculatcd, one set wi ;h the test stm i n and the ot her
achieved by treatment with dilute acids ( 6% with a standard strain of known sensitivity, The
sulphuiic acid, 3% hydrochloric acid or 5% oxalic third is the proportion method which indicates the
acid ), N acetyl cysteine with NaOH ., pancreallit , average sensitivity of the strain, taking into account
desogen , zephiran and cetrimide . Flocculation [ he face thac any population will contain cells with
a direct drug sensitivity test may also he set up. The animals arc weighed before inoculation and
Cultures arc examined for growth after incubation at intervals thereafter . Progressive loss of weight is
at 37 UC for four days { for rapid growing an indication o| infection. Infected animals show a
mycobacteria, fungi and contaminant bacteria ) and positive tuberculin skin reaction . One animal is
at least twice weekly thereafter. A negative report killed after four weeks and autnpsied . it it shows
is given if no growth Occurs after 9- 12 weeks . Any no evidence of tuberculous , the other is autopMcd
growth seen :. s smeared and tested by ZN stain : ng . after eight weeks.
For routine purposes , a slow growing At autopsv a positive animal will show a
nonpLginemed , niacin positive acid fast bacillus is caseous legion at the site of inoculation .
taken as AJ. ruhejTufosj s. Confirmation is bv.
'
Copyrighted materia
< Mycobacterium I : Tuberculosis » 359
+
M . fortuitum complex Tdluntc reduction Type of growth M. tuberculosis
+
M. M, pWir figment Scanty, smooth,
flar colonies
Rabbit [mthogcRLcirv
In light In dark No pigment
—
B.tC ,
+
M. bans
the lesions of tuberculosis , but will be smear based DNA amplification, "iTnc use of Rf’LP and
IS fingerprinting for epidemiological typing of
Guinea pig inoculation, once *0 commonly used , strains has been referred to . Demonstration of
is row seldom resorted to because it is cumbersome, mutation in specific drug sensitivity genes is a useful
Costly ami less sensitive than culture, particularly indicator of drug resistance . Such tests for
with catalase negative , 1 NH resistant strains isolated rlfampicin resistance are already available ,
mediated amplification, targeting- rihosomal RNA mycobacterial lipo - arahinomannan has been
has been introduced at an improvement on PCR reported to he of some value.
Copyrighted materia
360 * Texitook oi Microbiology *
Table 39.3
Type Niacin Nitrate reduction Oxygen Growth in TCH' Phzge type
Preferen ce
HununCdaiuial ) + * Aerobic -
i A -B . C
Asian type * * Aerobic 1
Afcan r^TK' variable MicrMetophilk A
Vole * }- variable Mkrtaftiophilit J
B
of which maybe more successful th vn of the fluid . tubercle hat-ilk ]* is injected in a guinea pig which
I'he use of PCR and DNA probes may help detect had received a prior injection of the tubercle bacillus
the bacilli speedily a ]uf enure often . 4-6 WOeltfi earlier, an indurated lesion appear* ir
Bone marrow and liver biopsy specimens from the sire in a day or two. TTiis undergoes necrosis in
miliary tuberculosis and blood from those with another day or so to form a shallow ulcer that heals
HIV coinfection arc useful for culture. Pus from rapidly without involving the draining nodes or
tuberculous abscesses often yields positive results other tissues , This is known as the Koch
in smear and culture. phenomenon and is a combination of hyper-
Pleural effusion and other exudates mav be sensitivity and immunity. The Koch phenomenon
—
j
collected with citrate to prevent coagulation. If free has three components a ‘local reaction, a < (bcaT
1
from other bacteria, they mav be used tor culture response manifested as congestion and even
afrer centrifugation. If other bacteria are present, hemorrhage around the tuberculous foci in rismesj
prior concentration Is necessity and a constitutional' or 'systemic' response of fever
Urinary excretion of bacilli in renal tuberculosis which may sometimes He fatal.
in intermittent , licncc it is advisable to test 3 6 - Allergy can he induced by infection with
morning samples of urme . Each sample is virulent as well as uvimlent tubercle bacilli , but
Centrifuged for 3UU0 rpm for 30 minute* and the not ordinarily by injection of killed bacilli or
sediment used for culture after concentration . bacterial proteins. Howevetf for demonstrating
Allergy find immunity: Infection with allergy, live or killed bacilli or ruberculopiotein can
tubercle bacillus induces cell mediated immunity 1
be employed.
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Hidden page
\bl * Texioook oi Mi^biaiogy *
of early detection and treilment of eases , since been enforced in [ lie manufacture of BCG
chcmoprophyla ^ h and immunoprophylaxis. vaccine., The recognised complications of BGG
Immunoprophylaxis is :T . intrude min] injection vaCcirte are the following:
of the live attenuated vaccine developed by Calmette Local Abscess , indolent ulcer, keloid, tuberculides,
. I I I > I Guerin (1921), the Bacille Calmette Guerin confluent lesions, lupoid lesions, lupus vulgaris,
-
or BCG. Thi H a strain of iVf. hovix attenuated by
229 serial subcultures in a glycerine-bilc-potato
fltyior .'flJ Enlargement and suppuration of dram:] i ;-:
lymph nodes.
medium over u period of 12 years. Injection of BCG Gejertk Fever , mediasn . nal adenitis, erythema
in animals induces self limited infection , with nodosum , tendency to keloid formation after
multiplication and dissemination of the bacillus ill wounding at other sites, and very rarely nonfatal
different organs and production of small tubercles . meningitis . The very few cases of progressive
Within a few weelcs the bacilli stop multiplying tuberculosis reported are believed to have been in
although they survive in the tissues for long periods. i m munodefic lent subjects.
This gives fine to delayed hypersensitivity and The consensus opinion is that BCG may not
immunity. Following BCG vaccination, a tuberculin protect from the risk of tuberculosis infection , but
negative recipient is converted to a positive reactor . gi '.^s protection to infants and young children
The : mm unity may last for 10-15 years and is against the more serious types of the disease , such
similar to the immunity following natural infec cion, as meningitis and disseminated tuberculosis. The
except that it does not carry' any risk of disease due recominendalkin therefore is that in endemic
to reactivation, as in the latter case- countries such as India , BCG vaccine be
Several In' Id ITI.LIH hive Keen conducted to assess jdinn iKtered to babies by mtraderma] injection on
'
rhe efficacy of the BCG vaccine. The results have the deltoid immediately after birth, or as early as
varied widely, from fiO per cent protection to a total possible thereatter, before the age nf 12 months.
absence of protection . In south Indi .i., a trial The WK , i nc need not be admi n i ^ tcred after the age
conducted at Madanapalle showed 60 per cent of two years, BCG should not be given to infants
efficacy while a later large trial in Chingalpattu and children with active HIV disease, though it
did not reveal any protection in adults, though a may be g: vcn with benefit to asymptomatic HIV
15-year follow up showed some protection in young positives. Babies bom to mothers wilh ATB positive
persons ! he reasons for such w ide disparity are s p u t u m should not be gr ^en BCG at birth , but only
not clear but have been attributed to several factors after a course of preventive chemotherapy.
such as the differences in the prevalence and BCG induces a nonspecific stimulation of the
virulence of tubercle bacilli in various communities, immune system providing some protection against
the type and potency of vaccines used and the leprosy and Icukcmi .i - Multiple injection of BCG
presence of ’atypical mycobacteria in the areas. has been tried as adjunctive therapy in some
The BCG vaccine aroused much criticism and malignancies. Some workers have reported that
it has been suggested that it may regain its virulence, BCG is superior to PPD for tuberculin testing.
though there has rot been any evidence of it so far. Chemoprophylaxis or preventive chemotherapy
The Lubeck disaster in which several children is the administration of antituberculous drugs
developed fatal tuberculosis following oral ( usually only isoniazid ) to persons with latent
immunisation earned the vaccine much notoriety' tuberculosis (asymptomatic tuberculin positive ) and
ui rhe early days of the vaccine. This was due to a a high risk of developing active tuberculosis, or to
-
mix up by which ivc virulent tubercle was given
"
*“ *u
opyrighted materia
j
* Mycobacterium \ : Tuberculosis 363
active tuberculosis and in children living with a 1 ntr< id need fo r this very purpose. ( 1 nfortu n ately this
case of arrive tuboculow in the house . bonis / id S was not implemented properly. Due tn a
mg/ kg daily tot 6-12 months is the usual course, combination of lapses in prescribing practices, drug
Trial* have shown that this reduces the risk of del ivery a nd patient compliance, res istance h as built
developing active disease by 90 per ccnr. HIV up in tubercle bacilli over the years, reducing the
infected coni acts of active tuberculosis also benefit efficacy
ar
of [ reatnienr.
from tills prophylaxis. Drug resistance map be 'primary' ( pretieatment,
initial ), when the patient is infected with a strain
THERAPY of tubercle bacillus which is already resistant, or
Cbcmothei ipv bus revolutionised the OU|Qj| fl1tilt acquired’ (secondary pout -treatment ) , when the
ot tuberculosis , It has been established that ^ infecting strain initially sensitive becomes resistant,
sanatorium regimens, bed rest, fresh air and rich usually as a result of impiopet or inadequate
fbodf as well as operative interventions, such as treatment. This is the more common type of
artificial ptieuntothnrut and thoracopluty arc not resistance . When Acquired resistant strains become
essential for cure it domiciliary treatment with increasingly common in an area, the chance of new
effective antituberculous drugs is given in optimal patients presenting with primary resistance
dose and duration . increases. When an infecting strain acquires
Antituberculous drugs are of two typesf resistance to one drug, the chance of its becoming
bactericidal and bacteri static. Of the bactericidal resistant to other drugs increases , unless the
drugs , rifamplciii ( R ) and pyrazinatnidc ( Z) are treatment schedule contains an adequate number
called sterilising drugs because they are able to ofdfective drug . At present the spectrum ofsirams
effectively kill the bacilli til the lesions. Ot the Other ^
prevalent in the community conn resistance to all
bactericidal drugs, uoniaad (H) is effective only available antituberculous drugs .
against replicating bacilli and streptomycin ( S ) only A very serious consequence of unchecked drug
against extracellular bacilli and so are not bv resistance has been the emergence and spread of
themselves able to sterilise the lesions. The multidrug resistant tuberculosis ( MDK -TH ) .
bactericidal drugs , along witli the hucteristatic drug Though the term multidrug resistance means only
etbamhutot ( E ) constitute the flrnt line drugs in resistance to two nr more drugs, in the context of
-
antituberculous fbenpy. ' 1 V old practice of daily tuberculosis , it specifically refers to resistance to
adminis [ ration of drugs for two years or SO bus been rifumpiuin And isonisaid , with or without reuis-tanue
replaced by short course regimens of 6-7 months, to One or more other drugs. This is because R ami
which are effective ind convenient A typical H form the sheet anchor of short - term
example of such a schedule for a new smear po > iTivc chemotherapy and any strain resistant to both three
ease is a combination o f four drugs ( HRZD- ) given drugs is unLikelv to respond to treatment.
three times a week during an in it ill intensive phase MDR -TB is a global problem , menacing the
-
of two months , followed by 4 5 months of |xxir and tOie rich nations alike . It may lie primary
continuing phase with only two dings ( HR ) three or acquired - Its presence in those with concomitant
times a week. HI v infection make* it more dangerous. When first
The major problem in chemotherapy is drug line drugs become ineffective , second! line drugs
tc si stance , which in tubercle bacilli is due To have to he tried. I.argc numbers of old and new
mutation, with an approximate rate of once in 10* drugs ,trc being used - tiiiinoloncs , uniiuglyooeides,
cell divisions. This could have been effectively macroltdcs, para amino salicylic acid, thtacetazonc,
checked bv multiple drug therapy, which was cycloserine , capreonTycin and others. They are
Copyrighted material
Hidden page
Mycobacterium II: Non-Tuberculous
Mycobacteria (NTM)
Mvcobicteria other than manmiilian tubercle rh;m that [ if tubercle bacilli. The important species
bacilli, which may ocusknal aiiKhuiniii disease in rhis group are M. Aaiwasjj, M . mttnnam and
^
resembling tuhercuLosis„have been tilled 'atypical', Mr strniae.
anonymous' or WJuified' mycobacteria, MIL M . kastsii ciuset chronic pulmonary disease
"
such as .U. phlei which are incapable of infecting resembles JVf. ^^sif but can be differentiated bv
a
human beings or animals. While human infection its poor growth at 37 aC,negative nitratase, positive
with them is common in some areas, disease is rare.
, pynzimiiudc hydrolase and 1.- fbooHidue aL- tiviricR.
They are unable ro cause progressive disease when Several photochromogen LC mycobacteria were
injected into guinea pigs. isolated in llJ6J from monkeys exported from India.
Non - tuberculous mycobacteria have been They have been classified into two species: niacin
classitied into lour groups hy RuuVOit ( 1959) huicd positiveM, sjNiijL' and niacin negative M. rafarieiuzL
cm pigment production and rate of growth: Group They have subsequently been associated with
1 photochromogeitif Group II scotoehmmpgens, pulmonary disease in human beings.
Group Ill no nphocochro Imogens and Group IV Group II — fwOtochromogens: These strains
rapid growers. Though other methods of
, form pigmented colonies (yellow-onfige-red) even
classification have been described, Runyon's in the dark. They are widely distributed in rhe
dUtiffcttiOA has found universal acLeptailce. enviremnient and sometimes contaminate cultures
Species identification depends on several additional .
of tubercle bacilli. M SLToiulaceum may cause
characteristic;; (Table dthl). scrofula (cervical oriemris) in children .
Cmup I phntochreImogens: 1 hese strains M, gunJonae, [iften found in Cap water (hence
form colonics that produce no pigment in the dark called ‘ the tap water sCOtochromogtn ' h is a
bur when rhe young culture is exposed to light for common contaminant in clinic ,i[ specimens and a
one hour in die presence of air , and reineubated for rare cause nf pulmonary disease, Ir differs From Af .
-
2 4 2 8 hours , a yellow orange pigment appears . scrofulxceum in failing to hydrolyse urea,
They are slow growing, [ bough growth LS faster nicotinamide and pyraainamide .
Copyrighted material
366 * TexIbtiOfe. of MiCfObiOMpgy
Tablte 40.1 DiHerenfliationi belween tubercle bacilli and so species of atypical mycobacteria
1
Tbr
'w
.
J
j
5
E
_
_£_
:r
! Si t
i i 1 I 5
I t :f
Z^=
P" '
3 i i £ 8
E
£
"
j
«r* c
Growth in 7 days 4 4 4 4
Growth sit 25 "C + * * ± 4 4 4 4
Growth at 3-7 + + + 4 t 4 4 4 4 4 +
Growth at 45 , lC 4 4
Pigment in dark 4 4
Pigment in light t 4 4 4
Urease 4 + 4- 4 4 4 4 4 4 4
Niacin 4 ±
Nitrate reduction 4 4 4 4 4
bacillus' beeaiiie it was fm> r identified as a human M . .tejTopj, originally isolated from toads, may
Table *>0.2 Dltfersnlkation between V, trfeerens and W. nwrinum
Chmrwcter Af. ufrera /tr M , marinum
Distribution Tropics Temperate zone
Clinical Course Chronic progressive ulcer Self -limited ulcer
Bacilli in ulcer Abundant Scanty
Rate of growth
GrowTh ar 2$
Slower, 4 8 weeks
-
—
l ister, 1 2 weeks
4
Growth at 37 - +
Culture him Bacilli in ititfds No cord formation
Pigment in tighr -
Mouse footpad legion Edema, rarely ulcer
j —
Marked inflammation purulent
ulcer
Copyrighted materia
* Mycobacterium II : S Dir - Tuberculosis Mycobacteria ( NTMj » 167
abscesses following injection of vaccines and other usually seen on the legs or arms and arc believed ro
preparations contaminated by these mycobacteria follow Infection through minor injuries;.. After an
have been reported on a number of occasions . The incubation period oh a few weeks, indurated nodules
bacilli arc found in the soil, and infection usually appear, which break down forming indolent ulcers
follows some injury; which slowly extend under the skin ,
AJ , fbrtrrri m and AJ , dieiond do not produce
^ Initially, smears from the edge of the ulcer ^ how
any pigment . Pulmonary lesions caused by Af, large clumps of bacilli which are add list and
'
be distinguished radio logically
/ortm frrjTr cannot alcohol fast. Cater, the irnmunoreactivc phase sets
from typical tuberculosis. No effective in and the bacilli disappear, " [‘he ulcers then heal
chemotherapy is available. Af . smcgtnttii , with disfiguring scans.
commonly considered as saprophyte in smegma, i ? The bacillus grows on LoWClUttin-Jensen
seldom seen in thar location . It is a frequent isolate
from soft tissue lesions following trauma or surgery.
-
medium slowly, in 4 8 weeks . The temperature
of incubation is critical; growth occurs between
Some noncultivable or poorly growing .10 ^Cand . So
’
hut not at 25 °C or 17 °C . Inocu
lation into the foot pad of mice leads to edema of
-
mycobacteria identified from the blood of AH b
patients have been characterised bv their IfiS RNA
^ the limb though ulceration is infrequent. A toxin
base sequences lliey grow sparsely in some liquid
, is produced by Af , ulccmns that causes
media. Tramples are Af genevente , Af. ConBtittitia inflammation and necrosis when injected into the
,
and At intamedium. skin of guinea pigs. "J ' bis is the only known instance
A rapid grower, Af, vatxze is reported [ < be an of rosin produced by any mycobacterium species.
immunomoJulalert capable ui inhibiting tissue M , mannum: This LV a natural pathogen of
destroying hypersensitivity responses and coldblooded animals, causing tuberculosis- in llsh
stimulating protective immune processes in and amphibia. It may also occur as a saprophyte in
tuberculosis. Clinical trials of Af, vacat vaccine fresh or salt water. I Inman infection originates from
Copyrighted materia
365 s Texlftook d Microbiology *
Contaminated swimming pools or fish tanki-. The M mirimim is not pathogenic for guinea pigs but
lesion, beginning .is a papule and breaking down in trade mud inoculation in rabbits leads to a
to form an indolent nuvlcar, usually follows superficial granulomatous lesion - Footpad
ibrasicins ]id therefore DLLWE on [ he prominence*
j inoculation in mice leads to a more severe lesion
—was first described from Sweden under the
elbows, knees, ankles nose, fingers or toes . It
name
than with M. idcemv, local inflammation being
followed by a purulent ulcer formation .
SWirnrning pool granuloma ' , and flit hfltiHus was
‘
Infection with Af. marinuni , hut not M.
named At bidnei ( fmm baJneum meaning barb) It . ulcaans, may cause a low - grade tuberculin reaction.
lias since been reported from other European M hiemophihim , fir* t described in 197S ,
countries and from North America - Its distribution causes skin lesions . It requires henlin for gtowth.
is in temperate areas in contrast to Af , tdeerans, Tt grows af .12 ¥ C! in 2-4 weeks but not ;U 17 DC -
which lias ,a tropical prevalence. EI uni an infection Tahle 40.3 shows rhe range of human infections
niav occur in epidemic form . The LLLLTIS are self - produced by ditferenr species -of atypical
limited and undergo spontaneous healing- mycohactcriiL.
HaciEli are scanty in smears from ulcers .
Growth occurs io uboul two weeks it TO ''C [range
1’icnlngy and epidemiology: As environ
mental bacteria are widely distributed io nature,
-
-
25 35 rC) and primary cultures do nof grow ^ t itifectiun with them is quite common, from soil,
37 <C but they do after adaptation. Colonies arc
'
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Mycobacterium III: M. Leprae
Leprosy is a disease of great antiquity,having been The bacilli are seen singly and in groups,
recognised from Vcdic rimes in India anil from .
ii it race!lul Lib or lying fate outside the cells. TlLey
'
Biblical times in the Middle Last . [ r probably frequently appear as aggloincrates, the bacilli heing
originated in the tropics nod spread to the rest of -
bound together hy a lipid likc substance, the glia.
the world. Leprosy has always been held in These masses are known as 'glohi '. The parallel
superstitious dread and the person suffering from rows ol bacilli in the gtobi present a "cigar hundlt '
leprosy considered "unclean and a social outcasts . appeamticc . In tissue sections, the clumps of bacilli
Tire lejura bacillus was first observed by Hansen in resemble cigarette ends . The globi appear in
1A 69. Though this wan the first baeteriai pathogen Virchow's lepra cclh' or Toacry cells' which are
"
Copyrighted material
« Mycobacterium III - M Lsprae » 371
produced, simulating lepromacous leprosy; The lepromatous type is seen where the host resistance
nine- banded armadillo { Dasypus novemcioctus) is is Low. The bacilli are seen in large numbers or as
highly susceptible to Infection with lepra bacilli , globl inside lepra cells or extracellular!)'- To is is
Following inoculation into armadilloes , a known as 'multibaci 1.1 ary disease . Superficial
1
generalised infection occurs with extensive nodular lesions ( lepromata ) develop which consist
mutt [ plication of the bacilli and production of of granulation tissue containing a dense collection
le"-- i ' ins typical of lepromatous leprosy. Some wild of vacuolated cells in different stages of
armsud Hoes in captivity have beer observed ro he development from mononuclear cells to lepra cells .
naturally infected with a mycobacterium resembling The nodules ulcerate, become secondarily infected
the lepra bacillus. " Natural disease' has also been and cause distortion and n :i:illation . Bacilh invade
identified in chimpanzees and mangabey monkeys the mucosa of the nose, mouth and upper respiratory
from West Africa but ir is nor known whether they. tract and are shed in large numbers in nasal and
have any relevance to human infection. oral secretions.The reticuloendothelial system, eves,
In animal experiments, rhe generation time of testes, kidneys and bones are also involved .
the lepra bacillus has beer found to be excepl i -unally
long, 12-13 days on the average but may vary from
"
-
ILu iLcima L common. The lepromatous type LS
more infective than the other types. The prognosis
-
£ 42 days, in comparison with about 14 hours in Is poor. Cell mediated immunity' is deficient and
the lepromin test is negative MI leproittaiOUS leprosy.
the case of rhe tubercle bacillus and ahuut 2Q minutes
in the case of coliform bacilli On the other hard, there exaggerated and
in an
Af . leprae genome has been mapped and the broad humoral immune response . Antibodies in
genes for its major protein antigens cloned and high litres- are seen against mycohacCen.il as well
scqucnced - as several other an! igens . Autoan I ibodies ate
Resi stance: Lepra bacilli have hcen found to common. Most cases show biologitaJ false positive
remain viable in a warm hum .d environment for reaction in standard serological tests for syphilis.
'
9- 16 days and in moist soil for 4b days.‘iTacy surv ivc At the other end of the spectrum Ls tuberculoid
exposure to direct sunlight for two hours and leprosy, which is seen in patients with a high degree
ultraviolet light for 30 minutes . of resistance. The skin lesions are few and sharply
demarcated , consisting of macular anesthetic
LEPROSY patches. Neural involvement occurs early and may
Leprosy is a chrome granulomatous disease of be pronounced . leading to deformities, particularly
humans primarily inyoking [ he skin, peripheral in the hands and feet. Bacilli are scanty in the
nerves and nasal mucosa but capable of affecting lesions and infectivity is minimal. This is known as
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Hidden page
H Mycobacterium III: M. L &prao » 373
The deficiency in cell mediated immunity is occurring spontaneously or more often during
-
specific ro the lepra bacillus aittignu, 1 cpromafous
patients a« not more susceptible to viruses, parasites
cbe mother spy . It is a cell mediated immune
reaction , with an influx of lymphocytes into Lesions,
acid Other pathUgertii against which CM! is and a shift to tuberculoid morphology. The lesions
important Tuberculin reactivity may be suppressed develop ervthenia and swelling, along with pain
in untreated Icprnmatous patients hut it becomes and tenderness- A similar clinical picture is seen in
positive following treatment , unlike the lepromin the ’downgrading reaction' which occurs usually
response which remains negative Lepromaious in untreated or pregnant patients , Here, biopsy of
^
patients hair large numbers. of C K * (suppressor)
lymphocytes in circulation , which can he
the lesions shows a shift to the lepromatous pattern,
reflecting a decrease of CML
specifically activated by the lepra bacillus antigens . Type 2 reaction or Erythema Nodosum
The lymphocytes present in skin granulomas are Leprosum ( ENL) occurs in the LL and lfL types,
altruist exclusively CDS + cells, in contrast to usually a tew months after institution of
tuberculoid lesions which contain pcedocniniiitly
, chemotherapy Crops of tender, inflamed
CD 4+ cells . In lepromatous leprosy, there is subcutaneous nodules appear, with fever ,
extensive polyclonal B cell activation with large lymphudenopathv and arthralgia. This i & an Arthur
amounts of antibodies being produced, hnth type response to antigens released from dear! lepra
anti mycobacterial and autoimmune . The cells and is characterised by neutrophil infiltration
ilbuminigkibuliti ratio is reversed . The antimyeo- and IgC and complement deposition in the lesiors-
bacterial antihodics ate nor beneficial. On the other
hand, they may have an enhancing effect. L E P H O M I N TEST
There is evidence of genetic effect in the pattern Till nece n[ I y, the only method fur studying imiriLiii ][ y
of response to lepra bacillus infection , HLA DR2
is seen preponderantly in persons with the
- in leprosy was a skin test for delayed hypersensitivity,
the lepromin test first described by Mitsuda in 1914.
Lulterculoid type uf reactiuith while HLA- MT1 and The original aotiger ] (lepromin ) was, boiled ,
-
HI J\ DQ]arc associated with leproma tons d i sease.
Though leprosy is a chronic disease, its course
emulsified, lepromatous tissue rich in Lepra bacilli -
Tire response to the intradermil injection of
may ho intenvperaed with acute evacerbations which lepromin is typically hip ha si;: , -consisting of two
a re of an allergic rub Lre - Two types of such reactions separate events - The first is the early reaction of
occur. Fernandez, which consists of erythema and
Type 1 or the ' reversal reaction’ or the 'lepra
reaction ' is seen mostly in borderline leprosy,
-
induration developing in 24 4& hours and usually
remaining lor 3-5 davs. This is analogous to the
Tdbte 41 ,1 Characterisation Di
^ diHerem types or leprosy
Jradeferminafe
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374 i TfeVfbOOfc d Microbiology
tuberculin reaction, This is usually poorly defined negative pensons are mote prone to develop the
and carries little significance, The second and more disease.
meaningful l:- the late reaction ot Mitsui La, starting 4. lb verify the identity of caiHlidatE lepra bacilli ,
in 1-2 weeks, reaching a peak In four weeks and Cultivablc add fast bacilli claimed to be lepra
gradually subsiding in rbe next few weeks. The bacilli should give match mg results when tested
reaction consists of an induiared skin nodulE , which ML parallel with standard lepromin.
may ulcerate, Histologically, there is infiltration with
lymphocytes, epithelioid cells and giant cells. The I laboratory diagnosis: Bacteriological
Mitnuda btc reaction docs not indicate pre-existing diagnosis is easy in the leprcmnacous but may be
DTI L tn.it i s a measure of the CMI induced by the difficult in the tuherculoltl ease?. The diagnosis
injected lepromin itself. Tt thus disti ilguishcs consists of demonstration of acid fast bacilli in the
between persons who can mount a CMI response lesions. For routine examination, specimens are
collected from the nasal mucosa, skin lesions and
against the lepra bacillus antigens and those who
cannot .
ear lobules. A blunt , narrow scalpel is introduced
into the rose and the internal septum scraped
The original crude Mitsuda antigens extracted sufficiently to remove a piece of mucus membrane,
-
from skin le ions of IcprorruitoLLb patients ( integral
lepromins ) were standardised on the basis of tissue
which is transferred to a slide and teamed out into a
uni form smear. Samples from the skin should be
content . Modern antigens are standardised obtained from the edges of the lesion rather than
according to their lepra bacillus content ( 4 * 10T from the centre. The skin ti pinched up tight to
lepra bacilli per ml ). Standard lepromins arc be mg minimise bleeding and a cut about 5 mm long made
.
ITLI .IRL . I Liwrcj -
ingly from armadillo derived lepra with a scalpel, deep enough to get into the infiltrated
bacilli (lepromin-A ), reflating human derived layers. After wiping off blood or lymph that may
lepromin - H. have exuded, the scalpel blade is turned transversely
The lepromin test is not used to diagnose to scrape the hides and bottom of the cut so as to
leprosy, nor does it indicate prior contact with the
lepra bacillus. Healthy person ^ in noncndcmiL; areas
with no chance of contact with the bacillus may
give a positive lepromin test. The test is employed
—
obtain a .tittle tissue pulp which is smeared
uniformly on a slide. About 5 6 different areas of
the skin should be sampled, including the skin over
the buttocks, forehead, chin, cheek and ears. The
for the following purposes! smears are stained by the ZiehTIVeelsen technique
1. To classify the Ichors of leprosy patients. The using ?% instead of 20% sulphuric arid for
lepromin test is positive in tuberculoid , negative decolourisation. Biopsy of the nodukr lesions and
m leprOittatous and variable in dimorphous and thickened nerves, and lymph node puncture may
indeterminate types of dtica$C - be necessary in some eases.
The smear; are graded, based cm the number of
-
2.Tu assess the prognnsi . and response to treatment.
A pos i r ive reaction indicates good prognosis and
bacilli as follows:
1-10 bacilli in 100 fields - 1+
a negative one bad prognosis, Conversion to
lepromin positivity during treatment is evidence
of improvement.
-
1 10 bacilli in 10 fields
-
1 10 bacilli per field
2+
3+
--
3. lo assess the resistance ol individuals to leprosy.
It is dcurable to recruit only lepromin positive
persons for work in leprosaria as lepromin
10-100 bacilli per field
100-1000 bacilli per field
The bacteriological ( bacterial ) index ( hi ) is added to this negimen or substituted lor clofazimine,
calculated by totalimg fbe number of pluses ( +s ! A minimum follow' up of four years for
scored in all the smears and divided by the number pauciba . illary and eight years for multibacdiary
of smears, Thus, if eight smears examined have a cases would be necessary to detect any relapse .
rota! ( ] f sixteen pluses , the RJ will be 2. For An immunoihenapeu:ic vaccine ( Mycobacterium
calculating BI , a minimum of four skin lesions, a W ) developed at the National Institute of
nasal swab and both the car lobes have to be Immunology New Delhi is cl.umrd to enhance the
examined. effect uf MDT.
The morpliological index ( MI ) is expressed . L > Prophylaxis: Case finding and adequate therapy
the percentage of uniformly st .lined bacilli out of have been the methods employed for prophylaxis.
the coral number of bacilli counted. Long - term chemoprophylaxis has given
Mouse foor pad inoculation has hern reported encouraging results in child contacts of infectious
to be more sensitive than skin slit smears for cases in India and the Philippines.
detect ion of lepra bacilli in tissues . Bur this is There is some degree of antigenic relationship
unsuitable for routine diagnosis and feasible only between the lepra and tubercle bacilli . It is an old
for drug potency or resistance testing and research clinical observation that leprosy and tuberculosis
studies. do not usually coexist. BCG vSc/ ine- was observed
Detection of antibody against M . ieprae to induce lepromin positivity and hence its use in
phenohe glycollpid anh en has beer clumed to be the prevention of leprosy was suggested by
^
a specific diagnostic test. Attempts to develop Fernandez as- early as in 1939, Shepard found that
molecular diagnostic methods arc in progress. lepra baciili did not multiply in the footpads of
Meanwhile, microscopic demonstration of lepra mice immunised with BCG, Controlled trials gave
baulli and histology remain the most useful divergent results, from high to no protection. Field
diiigriosric procedures. trills with different leprn vaccines ( BCG + lulled
Treatment: Dapsonc was the first effective ^
lepra baciili ; 1CRC bacillus ) have nor given
chemotherapeutic agent against leprosy. Its use as conclusive results so far.
a monotherapy for several years led to the
-
development of re istunt strains o1 Lepra bacilli . In
view of this, mulriple drag therapy ( MOT ) is now
MYCOBACTERIUM LEPRAE MURIUM
This is the causative agent of rat leprosy. It was
recommended in leprosy, as in tuberculosis, The first described by Stefansky in 1901 at Odes&a - It
currenr recommendations for patients with has beer subsequently reported from several
pautibacillary lesinns ( T, II , BT) is the Concurrent Countries , Rat leprosy ii characterised by
administration of rifampicln 600 mg once a month subcutaneous indurations , lymphadenopathy,
and dnpsofld (K) mg duly for tix inourhs . For emaciation, ulcerations and lots of hair. Acid fast
multib a ciliary lesions ( BB , BL , l . l . i , the bacilli resembling lepra bacilli are found in the
recommendation is tifampicin 600 mg once a lesions in large numbers . However, tbc disease
month, dapsone 100 mg daily and clofazimine 50 differs from human leprosy in rhat the nerves are
mg daily for two years nr until slki. n smears are not affected. M. leprae and Af. itprjt murium are
negative. Ethionamide or prothjonamide may be not closely related by DNA studies.
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Hidden page
Spirochetes
Elongated, motile, usable bacteria twitted ipiraDy successfully cultivated in cell free media though
along the long Ji\ is are Termed ‘Spirochetes ' ( from the commensals may be grown in artificial media.
Span, meaning coil .md tVjjjfe, meaning hair).They Treponemes cause the following diseases in
are structurally more complex than other bacteria. humans:
A characteristic feature is die presence of varying L Venereal syphilis caused by T. pallidum
no tubers oh enJoffagtHif which are polur flagella 2 . Endemic syphilis caused hv X ptfSiiUtm
wound along the helical protoplasmic cylinder, and [T. cndcaiicum )
situated between the outer membrane and t.ie- 11 wall. T . Yaws OJUSCLI bv F. pertenae
Endofogclte arc believed to be responsible for 4, Pints caused by 7! canrcum
motility hut the enact mechanisms of locomotion
i
Ihcy art almost identical in their morphology,
are nor understood . antigenic structure and other features, though there
Spirochetes vary- widely in size, some being as are differences in the clinical features and natural
Icing a * 5(H) pm and others as short as 5 pm. They history' of the diseases rhey produce. If has been
are f ’rran negative , Many are free - living suggested that the pathogenic treponemes represent
saprophytes , while some are obligate parasites . only evolutionary variation! of a single species and
They muv be ,LC tabic, anaerobic or facultative . that the diseases caused by them, though deferent
Reproduction is by transverse fission . clinically and epidemiologically, should he
Spirochetes belong TO the order Spirocherales, considered pares of a continuous spectrum of
comprising two families— S piroebetaccae treponemaJVises- Accordingly, the species 71
containing the genera Spirocticts , Cristapirs , pallidum is now considered to include three
Trepoaemt i\ud Eorrelit; and Leptncpinoeie subspecies— subspecies pallidum causing venereal
containing the genus Leptoajwta. Human jiathogens syphilis, endemicurn causing endemic syphilis and
arc found in the genera Treponema, honvli .i -.ind pertenue causing yJws.
Leptospira . Members of the genus jfunxhiea :mL
s;ij>ri 5|Thvrys found in water and lew » jjTn while TRKPONEMA PAU.mirM
Cnstzspira are found in molluscs. Treponema palEdum, the causative agent of syphilis
was discovered by Schauiiinn and! Hoffmann ( 190 )
TREPONEMA in the chancres and! inguinal lymph nodes of
^
Treponemes ffcnrpoe, meaning to turn, and nema, syphilitic patients. The name paii/ dum refers to its
meaning thread ) are relatively short slender pale staining.
spiroc hetes wi fh fi nc spj nils and poi riled nr mu nded Morphology: It is a thin., delicate spirochete
ends. Some of them arc pathogenic, while othcra with (apering ends, about 10 |im long (range 4^14
occur US commensals in rbc mouth , inrestines, and pm} and 0.1-0.2 pm wide lit has about ten regular
,
genitalia . Pathogenic treponemes have n< n been spirals, which arc sharp and angular, at regular
Copyrighted materia
37 K * Texltraqk n1 MicTobiglpgy >
microscope in wer films hut ran be made out by treponemal tests for the 1
of syphilis. The
relative staining with Indian ink. Its morphology Keitei trepnneme grows well thioglycollatV
and motility on be seen under the dark ground or medium containing serum . The Reiter treponeme
phase contrast microscope. It does not take ordinary is now classified as 7? ; i/rnis
bacteria ] stains bur stains light rose red wirh KCMVLLIIICC : 31 pallidum ii i L delicate, being
prolonged Giemsa staining. It can be Stained bv readily inactivated by drying or i beat ( 41-42
silver impregnation method?- Fontana 's method is in one hour ), Hence fomires are ! i importance
useful for staining films and Levadiri's method for in the transmission of infection. S isvcpfit ) lit) of T.
tissue HCtlrUms. paiiiduin to heat was the basiv fever therapy'
"
andantibiotics.
and outer membrane layer, to interdigitate at its Viili iiiL' sir'incuirtC The unt gttnk structure
centre. Unlike die flagella of other bacteria, these
^
cndofUgella do nut protrude outside,, but remain
within the outer membrane layer. w
nnaiGNa
Hidden page
m * Textbook ol Microbiology *
primary Icsfon heals , During this in he mil ( he patient two years duration (.15 per cent ) . The Lesions of
i & asymptomatic . The secondary Lesions arc due ro congenital syphilis usually develop only after the
wideypre .n] multiplication of llie fipiruchetea and fourth month of gestation , the time when fetal
their dissemination through rhe blond . Roseolar -
immune competence starts ajqK aring . This suggests
or papular skin rashes * mucous patches in the that the pathogenesis ncquireH immune response
oropharynx and condylomata at the mucocutaneous from the fetus. Congenital syphilis can be prevented
junctions arc the characteristic lesions. Spirochetes if the mother is given adequate treatment before
arc abundant in the lesions and consequently the the fourth month ot pregnancy.The ohstetrie history
patient is most infectious during the second ire in an untreated syphilitic woman is typically one
stage , 'Hiere may also be ophthalmic * osseous and of abortions and stillbirths followed by live hirth &
m
meningeal involvement. Secondary lesions are of infants with stigmata of syphilis and finally of
highly variable in distribution , intensity and healthv infants.
duration but they usually undergo spontaneous Laboratory diagnosis: As the manifestations
healing, in some instances taking as long as four or of syphilis are protean and as there are
five years
j
. asymptomatic periods during the natural course of
After the secondary lesions disappear, there is a the disease , Laboratory aid is essential for rhe
period of quiescence known as ' Latent syphilis". diagnosis of the disease . It is also Important in
Diagnosis during this period is possible only by assessing the cure alter treatment, Because of the
serological tests. Jn many eases , this is followed by social and emotional overtones of the disease , the
natural cure but in others , after several rears , diagnosis ot stplulis should impose a great sense
manifestation !; of tertiary syphilis appear. These of responsibility' on the laboratory'. Laboratory
consist of cardiovascular lesions including diagnosis consists of demonstration of the
aneutySms , c humic gmauLOttttia (gURimtia ) :md fpifOCheteti under the micft > S £Ope and of ^rttihiklie*
meningovascular manifestations. tertiary lesions in serum or L!SF.
contain few spirochetes and may represent Microscopy ; Diagnosis by microscopy is
manifestations ot delayed liypeirensiTivity. In ; L few applicable m primary ami secondary stages ami in
eases , neurological m anilestations such as tabes eases of congenital syphilis with superficial lesions.
dorsalis or general paralysis of the insane develop Specimens should be collected with care JS the
several decades after the initial infection . These are lesions arc highly infectious. The lesion is cleaned
known as late tertiary' or quaternary syphilis. with a gauze soaked in warm saline and the margins
In syphilis acquired nonvenerratly ( as gentlv scraped so that the superficial epithelium is
occupationally in doctors or nurses ), the natural abraded . Gentle pressure is applied to the base of
evolution is as in venereal syphilis, except that the
, the lesion and the scrum that exudes is collected
primary' chancre is extrageoital , usually <1 0 the preventing admixture with blood . Wet film ate
fingers . In the rare instances where syphilis is prepared with the exudate and after applying thin
transmitted! by blood tfUlifutioil * the primary covurs- lips , examined under the dark ground
chancre docs not occur . In congenita! syphilis , microscope . 7? paffidum is identified by its slender
where infection is transmitted from mother to fetus spiral structure and slow movement. Differentiation
transplacentally, the manifestations and course are from saprophitic spirochetes commonly present in
different . Transplacental transmission can rake place the genital area needs experience .
at any stage ui pregnancy. A woman with early Dark ground examination is useful but negative
syphilis can infect her fetus much mote commonly results do not exclude the diagnosis of syphilis *
-
( 75 95 per cent ) tlnm nnc with syphilis of osier because of m low sensitivity. A treponemal
Copyrighted material
< Sp*roc*iete& 381
concentration of lfl + per ml in the exudates h cardiolipin arnigen, The Wassermann test is no
required for the test to he positive. longer in use now.
The direct fluorescent antibody test for i . pallidum The first flocculation test used widely was rhe
( UFA -TP ) ia a better and. safer method for tube flocculation rest of Kahn. The Kahn test lias
microscopic diagnosis, Smears of the exudate are been replaced by the simpler and mote rapid VDRL
fixed with acretone and sent lo the Laboratory; where test , which ghres more quantirati .re results ( VDRL ,
1
the DFA -TP test is done using fluorescent tagged for Veneral Disease Research Laboratory, (JSPHS,
anti - T! im anti > erum . The use uf specific New Turk, where the test was developed ) . In the
monoclonal antibody has made the test more VDRL test, the inactivated serum ( that is, serum
reliable - heated at 56 UC for 30 minuta ) is mixed u rh
Serological tests: These tests form the cardiolipin antigen on a special slide and located
ma -. iistay of laboratory diagnosis A large number for four minutes. Cardiolipin remains as uniform
of Tests have- been described ., of which only a few crystals in normal serum hut forms visible dumps
am now employed - on combining with reagin antibody, The reaction
Serological tests for syphilis may he classified is read under a low power micmscnpe . By testing
as follows : serial dilutions, foe antibody titre can be determined .
1 . tests for anribodies reacting with cardiolipin The results arc reported qualitatively as 'reactive',
antigen ncagin tests; standard tests for syphilis; 'weak reactive' or 'nor rcacrivc’ . For quantitative
STS i*e|jcirtmgj the reciprocal of the end point is giver
2. tests for antibodies reacting with group specific as the litre, for example 'reactive 4 dilution or 1
tests for syphilis’ or STS . (The antibody reacting have been developed, of which the Rapid Plasma
with cardiolipin is known as mgin. This can he Reagin ( RPR ) test is the most popular. The RPR
misleading, as the 7gF antibody in atopy is also test uses VDRL antigen containing fine carbon
called reagi :i , though there is no connection particles, which make foe result more clear cut and
between the two . ) evident to the naked eve . . RRR test can be done
The first of the reagh antibody tests was the with unheated scrum or plasma * but is not suitable
Wassermann complement fixation test ( 1906 ), for testing CSF. Automated RPR test ( ART ) is
which ori i natly used a watciy extract of the liver ava i labtc for large scale tests. An automated VDR\r
^
of a syphilitic ferns AS the antigen . This was Inter ELISA rest has been devebsped which can measure
substituted by an alcoholic extract of ox heart tissue, IgC and lgM antibodies separately and is suitable
to which Lecithin and cholesterol were added . This for large scale resting of seta .
crude extract was subsequently replaced by a As candioii ] iin antigen is present both in T paltdfon
purified lipid extract -of beef heart (called and in mammalian tissues- , reagin anl iboiLies may
furdiolipin) , with added lecithin and cholesterol , be induced by treponemal or host ti -isue antigens.
as standardised by Pangborn ( 1945 ) . The This aecnu nts fnr foe bi ilogic a I false positive ( BI V !
complement fixation test remained the principal reactions, which constitute foe major di -iadvantage
serological test for syphilis, tLIL i : was replaced by of STS. BIT reaction are defined as positive
^
the simpler flocculation tests which also use the reactions obtained in cardiolipin tests , w . th ncgaf .ve
'
opyri <
B dr \
hted material
382 * Tfrultiook ol Microbiology *
results in specific treponemal tests, in the absence employed in this group was the keircr protein
—
ot past or present treponemal infections anti not
caused by technical faults . They represent
complement fixation ( RPCF) test , using a
UpopolyBicehimde-protem complex antigen derived
nontiepaneaul cardinKpin antibody responses, from the treponeme . Its sensitivity and specificity
BFP reactions may occur in about one percent were lower th:in those of Tests nsing T. p.-iUrdiim.
of normal sera . BFP antibody is usually IgM, while Though RPCF was. generally free from BFP
tragi n antibody insvphilii. is mainly IJ J , Clinically , reactions, it still gave some false positive reactions.
^
HI'"!3 reactions may be classified as acute nr chronic.
Acute BFP reactions last only for a few weeks or
RPCF and Other Reifer tfEpOnaiK tests are not n -mv
in general use.
months and are usu & Uv associated with acute 3. Specific T puSbdutn rests. These rests use the
infections , injuries or inflammatory conditions . virulent: Nichols strain of T. pallidum maintained
Chronic BFP reactions persist lor longer than si* by serial inoculation in rabbit testes.
months and arc typically seen in SJ,F, and other The first in this group is the Treponema
.
coDagen diseases Leprosy, malaria, relapsing fever, pnHkiun ] immobilisation (TPI ) test introduced in
infections mononucleosis, hepatitis and tropical 1949 . The test serum is incubated with complement
cnsiuophilia examples of Other conditions
arc .
and T p illiilnm maintained in a complex medicn:
associated with BFP reactions. anactobicalllv. It ' antibodies are present , the
Rcagin antibody becomes detectable 7-10 days ireponemes arc immobilised , that is, rendered
after the appearance of primary chancre (or 3-5 ronmotile, whfcn examined under dark ground ,
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i Spirochete* * 2E 1
frepoTicmc, rabbi t testis a nd sheep cryth nocytes- Sen all - Treatment in the primary jtage leads to
am screened in an initial dilution of 1:$0 but tines scrorcvcreal in about lour months; in the seconds ry
of 5120 or more are common Ml the secondary stage.
TPHA is junt as specific as FTA AfiS nml almost
-
and early lament stages , it takes 12 IS months-: in
later stages, it may take five yean- or more. In some
as sensitive, except in the primary stage. Jt is also cases low rltrc reactivity may persist indefinitely in
much simpler and more economical. No special spite of effective treatment Specific ii*E >ui ]eT[]al tests
equipment is needed . Kits are available are of little value as indicators of clinical cure, as
commercially. These advantages have made TPHA they tend to remain positive in spite of treatment -
a standard confirmatory t« t - TPHA titres may foil rapidly following treatment
Tablc 42.1 shows the relative sensitivities or '
in secondary syphilis bn remain positive lor life in
the serological tests in common use. low litres ,
Enzyme immunoassays ( ElA ) have been TPIIA and FTA-ABS .ire helpful in excluding
developed using T. pallidum antigens and are or confirming the diagnosis of syphilis and for
available conn merci ally ( Bio - Enza Bead test; Captia identi lying BFP reactions. Though false positive
Syphil is - CJ tost) . A rapid aggluti nation Lest J i as been reactions were believed u > have been eliminated
developed , using latex particles coated with three with the introduction of these specific tests, it is
immunodominant proteins of T. pallidum , obtained not truly so. Both TPHA and FTA - ABS can give
by recombinant technology'. It is claimed to He as false positive results , though verv rarely. All
specific as TPHA* and more sensitive . serologic ,il rests for syphilis may he positive in
The practice tor uero logical sere en I rig for nonvenureal trepone mitoses , and some in a lew-
syphilis varies in different countries. In the UK , a other spirochcatal infections as well . In Lyme
combination ot VDRI , and TPHA tesla LS Lurid . disease, VDRL rest is negative, but FTA- ABS may
"
primary stage. A repeat test 1- 1 months later will diagnosis- of syphilis, past or present, except in the
bring even this no light . In the USA, screening is very early sriges . In ncurosyphilis, a negative CSF
by VDRL or RPR test alone. This may fail ro detect VDRL test may not be conclusive but a negative
about one per cent of secondary syphilis due to the ITJH .A test eliminates the possibility of
proaone effect and about 30 per cent of latent or ncurosyphilis.
hue syphilis. Detection of specific IgM antibody may he
Table 42. T Frequency ol reactive serological tests helpful in sume situations. Being the initial type of
in untreated syphilis [percentage ) antibody to appear, IgM is detectable by the second
week of infection . IgM mti body production ceras
Siage VDRL / RPH FTA -ABS TPHA
soon after elimination of inferrion by treatment.
Primary 70-80 H 5 -10 Q 65 -Si Persistence of IgM antibody indicates continuing
Secondary 100 100 100 active disease and the need for treatment. As IgM
Latent/ late 60- 70 93- 100 95 -100 does riiit cross the placenta its presence in neonatal
,
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184 H Te tboo-k al Microbiology *
*
confirms
^ ^ . syphilis and helps predominant in early syphilitic lesions.
scrum cisn iiir il
d] fferenrLa1 e ] tfrom surdpoSrlnvctv due W passively Specifically sensitised Thl cells secrete cytokines
transferred maternal antibody (lyphilo toxemia) , favouring clearance of spirochetes bv activated
Many techniques have been developed tor the macrophages.
selective detection ot IgM antibodies, These include Reinfections do not appear to occur in a person
modifications of rhe FTA-AES, TPHA,E1A and already having active infection , Tl was believed that
VDRL using whole sera or separated IgM
rests, prut ? ] iminon or infection immunity, as seen in some
fractions. When such te>- ts are not avai 'able-, parallel parasitic infections , holds good in syphilis also and
tests of maternal and neonatal sen may settle the that a patient becomes susceptible to reinfection
diagnosis of congenital syphilis „ in which the -
only vvliL rt his original intention is cured. However*
neonatal serum ma y show LL IIL LICT Li tie of antibody it has been shown that some degree of immunity
^
than the maternal scnim. Serial tearing is also useful to reinfection occurs in experimental animals and
because the litre of passively transferred antibody persons whose infection has been completely
decreases rapidly, rhe VDRL test becoming eliminated by treatment .
negative by three months. ProphyIjixiii As transmission is by direct
Epidemiology; Venereal syphilis is worldwide contact, it is possible to protect against syphilis hy
in distribution. During the five centuries that it: has avoidance of sexual contact with an infected
been recorded and studied, the disease has individual. When this- is not complied with, the
undergone much variation in its natural history and use of physical barriers ( such ns condoms) ,
.
clinical features As originally described it was a
, antiseptics (potassium permanganate ] or antibiotics
highly virulent disease with tlnrid cutaneous may minimise the risk . The use of prophylactic
.
manifestations With the discovery of the dramatic penicillin carries the danger that it may suppress
therapeutic response to pcnidllin, it was hoped that the printin' Lesion without eliminating the infection
it may even be possible to eradicate syphilis, as the so rhar recognition ami treatment of the disease
di >ease has no extra human reservoir. However, not .
may become more difficult No vaccine is available .
on I y has it not been pnss iblc to el [minatc the d i sease Treatment: Penicillin is uniformly effective in
but an mcrease has occurred in its incidence, due syphilis hut it is necessary to give an adequate dose
ro the changing Liisroms, habits and values in and maintain the drug Level for a sufficiently long
ancicty. period to establish cure . A single infection of 2 A
The advent of the AIDS pandemic has had an million units of he n/ athine penicillin G Is adequate
impact on syphilis, in most places, fear of AIDS -
in L urly cases . For Late syphilis, this amount may be
and safer sex practices led to ;L tall in rhe inc idence
,
repeated weekly for three weeks.In pa bents allergic
of syphilis and all ST t init i ,illy, but this trend did 10 penicillin , doxvcvcline mavbc usedl. Ceftriaxone
m >r continue everywhere. Concurrent infection with is effective, particularly in ncumsyphllis Penicillin
syphilis and HIV is common andmay lead to earlier treatment in syphilis sometimes induces a reaction,
evolution of neurosyphilis. the Jarisch- licrxheimer reaction, consisting of
Immunity: The immune mechanisms in syphilis fever, [il.Liaise and exacerbation of symptom!. It is
arc not adequately understood. Humoral immune frequent, but harmless, in primary and secondary
response against the Crepontmc docs not appeal to syphilis , and can be managed with bed rest and
be effective rhe disease progresses unhindered aspirin. It is rare in late syphilis but can be
for decades even in the presence of a vigorous dangerous in some cases of gummatous ,
antibody response , Cell mediated immunity mav be Cardiovascular or ncurosyphihs It is believed to be
more relevant . T lymphocytes and macrophages are doc ro the liberation of trade products like tumor
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336 * Tox ( book of Microbiology *
grown anil mistakenly Called 7] jwlhtlunt — for iiorrelliae have been assigned to various species
example, the Reiter and Kazan strains which hive based on the Iccki that CArty them. Over ten species
been identified as 7 ] phagcd&lis anil the avirulent of borreliae are known to infect human beings and
Nichols and Noguchii strains which have been cause RF ( 13. duttemii, B. hcrsnuii, B. purhcri , etc . )
recognised as T. refringen^ . They are generally confined to certain geographic
Jn experimental work on T. pallidum in areas. There is evidence from DNA homology
rabbits , T, parmtuiscunkuii (formerly T. curuciJi), studies to indicate that ail of them may belong to a
which has a very similar appearance and causes single specie;;, with separate host adaptation. The
natural venereal infection in rabbits , mar pose descriptions that follow apply to all of them, unless
problem H. stated otherwise.
Mi irpluNlo|jy; B . recurrent }* i* an irregular spiral
BOftREUA
with one or both ends pointed. Jt is ft-20 pm long
Borrcliae are Imge, motile, retractile spirochete and 11.2-0- 4 pm wide. It possesses 5-10 loose spiral
with irfcguLir* wide, open coils. They arc usually colls at intervals of about 2 mm. It stains well with
-
5 30 pm long and 0.3 0.7 pm wide . They ate
^
Giemn and Wterial trains and is Gram negative,
readily stained by ordinary methods and arc Gram Cultural characteristics: Borrelia are
negative . Several species of Uorrelia occur as micTuaeropbiJic. Optimum temperature for growth
commensals on the buccal and genital mucosa. is 2 K-30T, Cultivation is difficult but has been
Borreliae of medical importance are those causing successful in complex media containing serous
relapsing fever, B. vinctxrh which sometimes causes fluids. Growth occurs on the chorioallantoic
fusospirochccosis and 13, burgdorferi , the causative membrane ofchkk embryos. For primary isolation ,
agpor of l .ymc disease . the best method is to inoculate mice ur rats
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« Spirocnmes p
387
antigens.
PathogeniciLy; Afrer an incubation period of
2-10 days relapsing fever Sets in as fever of sudden
Fig. 42.2 Borrrito rEcurrentii in peripheral blood smear
onset. During this period, borreliae are ahundanr
in the patient 's blood. The fever subsides in in Hume outbreaks, a high rate of fatality. tr lift;,,
-
days. After an afebrile period of 4 10 days during
which borreliae arc not demonstrable in blood,
the horrrlia is, confined tn the hemotymph and 15
not shed in saliva or excreta . So the infection is
another bout of lever sets in.The borreliae reappear transmitted not by the bite of lice but by their being
in blood during the relapses of fever, The disease curbed and rubbed into abraded *kiq, fi , rKvrrentis
ultimately subsides after 3-10 relapses. is not transmitted transovariallv m lice .
Jr
Experimentally, rodents such as rats, mice and, Tickltome relapsing fever occurs an sporadic
less readily, guinea pigs may be infected by cases in endemic areas . It is a ‘place disease' and is
intrape riloneal iniection. Borreliae may survive in frequently associated with certain dwellings or other
the brains id infected animals after they haver
locations rhur are inhabited by infected ticks. The
disappeared from the blood. disease is milder but relapses are more frequent
Epi (itimiulu | y: Louscbarnc relapsing fever than in Lousebome fever. The borrelni persists in
tends to occur as epidemics whenever poverty, the body of infected ticks throughout their life and
overcrowding and lack ot personal hygiene is also transmitted transovarially so that the ticks
encourage louse infestation. Epidemic relapsing act as reservoir mi well as vectors. The borrelia
^
fever used IO be very common during wans and in invades all pam of the body of the rick and is shed
jails of former days but with improverneinls in in its saliva and feces, So the infection is transmitted
hygiene and the discovery of insecticides., it has to humans Through the hire of ticks or their
now become rare-. It survives in some areas, as in discharges. Several species of soft ticks hclonging
pans of Africa and appears as outbreaks whenever to the genus QiPithodorus act as vectors , different
civil strike and famine encmarage large scale louse spcciea being responsible in different regions. In
infestation. The louscbnrnc disease presents a more India , the vector species are O, cholozoiu ,O, cro&si,
severe clinical picture than the tickbome variety O. hhorensa and the fowl tick, Aign persicus.
and is associated with jaundice, hemorrhages and . These *oft ticks can live for ten years or more with
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4 Spirochetes * 391
and truss absorption reactions usingtmnumf rabbit Aseptic meningitis is common in canlcoht infection
sera or more recentlyJr
with monoclonal antibodies . and abdominal symptoms in grippotvphosa
( icuetic methods , such as restriction endonuclease infections However, clinical syndrome* are not
analysis and 1 > NA pairing arc i ] *ed tor further semtvpe specific and any type of ilines* can he
classification into serotypes. produced by any serotype.
Pathogenicity: In natural reservoir Hosts , Laboratory diagnosis : Diagnosis may He
Icptospiral infection is asymptomatic:. However, made by demonstration of rbc 1 ept £« pircs
when infect ion is transmitted to other animals , microscopically in blood or urine, by isolating them
dlinica] disease may result. Human * are Inferred in culture or by inoculation of guinea pigs or by
when the leptospires in water contaminated by the serological tests,
urine of carrier animals enwrs the body through l , Examination of blood: A* leptospines disappear
cuts or abrasions on the skin nr through intact from the blood after the first week , blond
mucosa of mouth , nose or conjunctiva. The examination is helpful only in the curly stages of
incubation period is usually about JO davs ( range the disease , he fore antibiotics are given .
.
2-26 days) . The clinic il picture varies from mild 1 cptospircs may be demonstrated by examination
/
undifferentiated pyrexia to severe or fata ] illness of the blood under the dark field microscope or
with hepatorenal damage (Weds disease ). Ln severe
, by 1 mmunofUior'cscL- nce but this is of lit; k practical
cases, the onset La acute , with rigor, vomiciog, value.
head .ichcand intense injection of the eye*.The fever Three or four drops of blood arc inoculated into
is irregular and usually subsides in about ten days. each of several bijou bottles containing EMJH
Jaundice occurs in about 10-20 pci cent ol ases '
I or similar medium. The bottles are incubated at
by the sccnnd or third day. I 'urpuric hemorrhages 37 V . for two days and left thereafter at room
sometimes occur on the shin and mucosa . temperature in the dark for two weeks. Samples
Albuminuria is a constant feature. from the cultures am: examined cvciy third day
This typical presentation is unusual . for the presence of leptospires under dark ground
Leptospirosis is now classified into two clinical types illumination. Primary isolation may be delayed
— jcfcrj e and nevuVrenV- Many eases present as
'
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392 i Textbook of Micrctnoiagy »
withdrawn by cardiac puncture is inoculated into formal] nised suspensions of prevalent leptospira
culture media. serovars are tested for macroscopic agglutination
%
^ t urine Leptospira appear in with serial dilutions of the test scrum . The
the urine in the second week of the disease and microscopic agglutination test (MAT) generally
intermittently thereafter for A— 6 weeks . The urine uses live cultures of different serotypes and
should He examined imincdiafely after voiding as agglutination is observed under the low power
leprospires readily undergo lysis in acid urine . dark field microscope . This tcsi is more specific
Centrifuged deposit of the urine may lie examined and is usuallv done only in reference laboratories.
under dark gnuind illumination . Direct culture of Due to the presence of some degree of cross
urine is seldom successful because of reaction between different serovars, agglutinin
Lontamination but isolation is usually possible bv absorption tests may sometimes become necessary
inoculation into guinea pigs . for accurate diagnosis .
The identification of the isolates of kptnspircs J. /)r: mwiv (if Jepiainimijs rn :INJJDJ /S: Infection
itf made by agglutination with type specific sera. ^
in rodents and ocher animals may be diagnosed
Due to the large number of serotypes and the high by serological rests or by culturing pieces of
degree of antigenic cross, reactions between them , kidneys.
identification of isolates is a complicated procedure 5. F y j j t n n.j r p f J D of u-. j f L T for patliafienii
and is generally confirmed hy one of the WHO/ leptospin * ( f a shaved and scarified area of the
FAQ Reference I .aboratorics, skin of a young guinea pig is immersed in water
i . Scmhigii .il ttingnif is : Antibodies ap iear in for an hour, infection takes place through the
^ [
serum towards- the end of the first week of the Abrasions.
disease und increase till lllL- fourth week declining k[Hdemiolr }£ s : Leptospirosis is considered to
thereafter . Agglutinins may, however, he be the most widespread of zoonoses, being regularly
danemstrabk years utter the infection . Two types present in all continents except Antarctica .
of serological tests arc available , the broadly Pathogenic Leptospiras survive for long periods in
reactive screening tests and the serotype specific the convoluted tubules of the kidneys in natural
tests . hosts,, multiply and are shed in the urine. Animal
The broadly reactive or genus specific tests identify carriers often excrete upto 100 million leptospires
Leptospira! infection without indicating the exact per ml of urine. If the infected urine contaminates
infecting scrovar . The antigens for these tes.rs arc the water or mud that is neutral or slightly alkaline ,
prepared from the nonpathogenic L . biiSen Panic the leptospires survive for weeks . When people
1 strain. The rests employed .include sensitised come into contact with such water, the leprospires
erythrocyte lysis (SET) , complement fixation , enter the body through abraded skin of mucosa
agglutination and indirect immunofluorescence. .
ind initiate LO fee non. Certain occupational groups
ELISA Has been used to detect IgM and IgG such as agricultural workers in rice or cane fields,
antibodies separately, in order to indicate die stage miners ,aod sewer cleaners are more often exposed
of infection, A simple and rapid difi- stick assay to infection , and so leptospirosis is more common
has b«n developed tnr the assay of leptospira- JO them. Ijeptuspires mav be shed in the milk of
specific IgM antibody in human sera. lac fating animals.However, they die rapidly in milk,
The type specific tests identify the infecting and human infection through milk is TIDI known .
scrovar hy demonstrating specific antibodies . They are not shed in saliva, and so animal bites are
Macroscopic and microscopic agglutination teals not infectious. Arthropods arc not known to transmit
arc used for this purpose . In the former , the infection.
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* Spirochete 393
Several animals act as carriers. Rats arc vcrcrowdingh insanication , increasing rat
particularly important Ifi they are ubiquitous and population and the habit of walking barefooted .
carry the most pathogenic serotype ictcro - Prophylaxis As leptospirosis results from
haermirrhagiiie . Field mice carry gripporyphosa . contact of skin or mucosa with contaminated water ,
pigs pOtnOrta and dugs Canicula sem-t^fjes. However, general measures of prevention such as rodent
the HITH{ serotype may He carried Hv different control, disinfection of water and the wearing of
mammals and one mammal may tarty different protective clothing contribute to its prevention.
serotypes. While leptoapires are generally Vaccination Has been attempted with some success
nonpathogcnic in the reservoir animal , leptospirosis in dogs, cattle and pigs. Immunity following
is of veterinary Importance as infection of cattle vaccination or infection is serotype specific .
and pigs cause considerable economic loss . Infection Vaccination has also been cried in persons at high
among animals is also transmitted by urinary risk such a -i agricultural workers.
cuniammsiiurt of water and fodder. Human beings Therapy: l ^ ptospites are scnAltifUe ft ] penicillin and
arc an aberrant nr 'end ' host. There is no evidence tetracyclines, bur the treatment to be effective should
that human patients Infect others. be started earlv m die course of the disease. PtuLclUjn
From being predominantly a rural disease of '
is given IV, 1-2 million units 6 hourly for 7 days m
agricultural worker leptospirosis has , in recent serious cases . A mild Jarisch-HenKbeimar reaction
^
times dtD become an urban problem in the may occur in some, Doxycycliric 200 mg orally nn.ee
developing countries. This is perhaps doe to a week ! H effective in prophylaxis.
-
Table 42.2 Important leploapi al infections
Serotype ClimcaJ picture Animal Distribution
reservoir
lctcrohacrnorrhagLat Weirs disease
Can kola GanicoJa fever
.
Fever jaundice, titmorrhafle*
Influenza like, aseptic- ,
R*i
Dog
Worldwide
Worldwide
mejlingitU
Grippetyphosa Swamp or marsh Fever, prostration * ascpric- Field mice Europe., Africa*
fever meriinsitis S.E. Asia, USA
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Hidden page
CO Mycoplasma
Mycoplasma arc it group of bacteria that arc Mycoplasmaralcs , which contains the following
devoid of cell walls and so ire highly pleomorphic! families and genera;
with no fixed shape or size. They lack even cell .
1 Family Afycopltsm ,which belong
ttou to
wall precursors like muramie acid or parasitic mycoplasma!^ requiring cholesterol or
diiTninopirrctic acid- The cells arc bounded by a other sterols as an essential growth factor,lliis
soft trilaminar unit membrane containing sterols . contains cwo general
Because of their plasticity; they car pass through a. CcmiH hcoptasma which utilise glucose or
bacterial filters and have often been rristateri tor arginine but do rot split urea, and
viruses . The first member of the group was the b. Genus Lfaeaphisrnrf which hydrolyse urea.
organism causing bovine pleuropneumonia,isolated 2.. Family ."ifho/cpffl.^mjrfjrcFie, mostly saprophytic
by Notard and Roux (1R98). A simitar organism
was found to cause contagious agalactia in sheep.
..
mycoplasrnis which do not require sterols as
growth facror
When manv similar isolates were obtained from
Jr
3. Family Spiropiaamttaeete, containing tin- Genus
LULU rials human beings, plants and environmental
] , SpiropLsilia, which parasitise arthropods and
sources, they came Co tie caller! yrJermipm'nmnnfHi' plants. They are stem! dependenl. These are
like organisms'(PPLO). This unsatisfactory name helical in shape.
has heen feplat ed by the term Mjncnphcsma 4 . Family djUAV liUdtltMcie, containing the
from the fungus - likc form of the branching ^
genus Ari.kWfiisma, which am strict anaerobes,
filaments, pliama , denoting their plasticity of found in the rumen of cattle and sheep .
shape). Mycoplasmas may be saprophytic, parasitic or
MycopUsmis have been placed in tin- class pathogenic . More than 100 species of mycoplasma
Mollicutes (literally meaning soft s k i n o r d e r ? re known rn cause disease in a variety of
Copyrighted materia
196 H Teat & ook &l WKfflbwNjOf *
MYCOPLASMA
. \ .*-/ ,
0
,r .
;
;*
. V/ ''
. cJ >r-
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« /r ' ;.
iftf. ...
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p
i
. .V i
i
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/y
.
—
fhg . 45,1 Morphology of mycoplasma otlreme plcomofphism
mammalian, inject and plant hosts . Ahour sixteen Mycoplasma? may he cultivated in fluid or solid
species, belonging lo dlftt families are found in media. 1 hey are generally facultative anaerobes,
human beings (Table 43.1), growth being hotter aerobically. They grow within
Morphology: Mvco plasmas arc the smallest a temperature range of 22 -41 ‘C . tbe parasitic
"
m aerial
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39S * Tfijclboek a ( Microbiology
and agglutinates guinea pig erythrocytes, t lolonics Some strains of mycoplasma frequently isolated
on agar adsorb erythrotylM. The hemadsorption is from the urogenital tract of human beings and
enzymatic and occurs optimally it 37 °C , The cell animals form vtr> tiny colonies, generally 15 50
r
-
neceph > rs are destroyed hv neurarn Lnidase . It inhibit! ptdi in EEZCL They were called T Strain or 7 form
ciliary motility in hamster trachea organ cultures. mycoplasma? (T b * r tinyl/Fhcy are peculiar in their
M. pneumoniae is unrelated to other human ability to hydrolyse urea , which is an essential
mycoplasmas and may be identified hv growth growth factor in addition to cholesterol , 1 Etiman T
inhibition by specific antisera. As isolation is strain mvcnpla -smas have been reclassified as
difficult and delayedh PCR assay which is rapid l ' rexphy-mx urafytievw-
and specific is being used where tcasihlr.' , Genital infections arc caused bv M . hominis
. .
j
Serological diagnosis may be made by specific and U urealjrtkum Tliey an; transmitted by sexual
tests using mycoplasmal antigens or hv nonspecific contact, and may cause urethritis, proctitis,
methods. Among the former, immunofluDrescence, balanoposthiti? and Reiter's syndrome in men, and
hem agglutination inhibition and metabolic acute saljihingiris, pelvic inflammatory disease,
inhibition ure the rrm-st sons iridic tests . Complement cervicitis and vaginitis in women. They have also been
rotation and indirect hemagglutination testa are less assoviited with infertility abortion postpartum fever,
sensitive: .
^
churutfumkniilis and low biithiveiglil ot infants.
The nonspeeifiit: serological tests are Mycoplvi & ma mid HIV infection:
MG and cold agglutination tests. The Mycoplasma ? tend to cause more severe and
former is done by milting 5Cfi .it dilutions of the prolonged infections in the HIV infected and other
pa lien r 'a unheated scrum and a heat killed iiumunodeficicnt subjects .
suspension uf Streptococcus MGt and observing Mycoplasma as cell culture
agglutination after overnight incubation . Lt 37 fL . contaminant*: Continuous cell cultures
A title of 1:20 fir over is considered suggestive . maintained in many laboratories have been found
The cold agglutination test Is based on the to be contaminated with different species of
Copyrighted materia
t Mycoplasma * 399
iDfDOpliinna.'Ilx contamination nay originate from omaiubaois and termed them L forms, after 1.inter
the worker or front animal seTa or trypsin Used m Instilmer London, where the observation was made.
cell culture. Contamination generally docs not It was subsequently shown that many bacteria,
luoduce evtopathic effects but may interfere with either spontaneously or induced by certain
the growth of viruses in such full cultures uiid may substances Like penicillin, lost parr or all of their
ill (id produce misleading results in serological, tests. cell wall and develop into 1 . forms. Such L forms
MynjplaWtV growing in cell cultures have often mav he ‘unstable', when they revert to their normal
hcen mistaken for viruses - Rradieatinn ot morphology; or stable ^ when they Continue in the
myCOplasinas from infected Ceils is difficult , cell wall deficient male permanently Cell wall
-
irc atmenl ate the drugs of choice tor the deficient forms ( L forms, protoplasts, flphtiOplatfs)
treatment of mycoplasmal infections. Some may not initiate disease hut may be important in
ureaplasmas are resistant to tetracycline , bacterial persistence during antibiotic therapy and
doxycyclinc , the newer macrolides and quinoloncs. subsequent recurrence of the infection. It Hus been
Mycoplasma * and [ . forms of bacteria : suggested that mycoplasmal may represent stable
hlcincherfitrr (1935 ) found pleuropneumonia - L forms of bacteria hut genetic , antigenic and
like forms in a culture of Srnepfo6acj Jj' yff '
biochemical evidence are against the possihiliry.
Punhtr i t o i u h i L k
. .
Lin J 5 1985 Human, rnycoplafmaJ infections Serologic observations. Rev /nicer f .Ji 7 - 21 fy .
. ^^ . .
Symposium 1 3 The chaffing rule er mycoplasrnaE in nespiratury disease and AIDS CVifl /ritecf
'
17;f 5 uppl , l ) ,
.
Taylni- Kj iinsoTi [1 and J Bradbury 199S. Mywplanna distast * In Tbpfcyantf WiJwin'x .VIrcarpfcnj/qgy and Mkrvbiid
^
Jnfnrirjm, V' cdn. LondofcAri jld
Copyrighted materia
Actinomycetes
Actinomycetcs arc traditionally considered to he It causes human actinomycosis. Art inti mycosis in
transitional forms between bacteria and fungi- [ .ike cattle is produced by A. hovrs-
fungi they tunm a mycelial network of branching Actinomycosis The disease is a chronic
filaments but, tike bacteria , they arc thin, possess granulomatous infection occurring in human beings
cell waits containing marunlc it id, have and animals . It is characterised by the development
prokaryotic nuclei and ire susceptible ro of indurated swellings, mainly in the connective
antibacterial antibiotic ;!- They are therefore true tissue, ^suppuration and the discharge of 'sulphur
bacteria bearing a superficial resemblance to hingi . granules’.The lesion often points towards the skin ,
ActinomytCtics arc related to mycobacttti ^ and hading It) multiple sinuses.
curynebacteria, They are Gram positive , nonmotile, Actinomycosis in humtin beings is an
nonspoting, nomapsubted filaments rh;it break up endogenous infection. The actinomyces species are
into bacillary and coccoid elements. Most are free - normally present in the mouth , intestine and vagina
jKLiEicukrly in the soil. as commensal*. Trauma, foreign bodies or poor oral
Acfmomytctc* indttdc many genera of medical hygiene may favour tissue invasion . A- jsraefir is
'
interest SLLHTI as the anaerobic dCfinonytcJ, tbe most common causative agent - However, other
ArMchaia, 9i£dobtcrerrumT Rothia and aerobic actiiiomyecies such as A. iiaeslundii , A, vtscastis,
.\’cjcarJja , Aetinonudvra , Dermatophilut, and A UiJojtftjfytJcum, A. jntyerr, A. getcnctiunci and
StTcpjomyces , The major pathogenic genus AifuMaclcnumpropionicum may sometimes he
Actinomyces is anaerobic or mjcroaerophilic and responsible. Actinomycosis is usually a co-operative
nnnacid last, while JVOL- jnlia Speede* art; aerolite dtsea -se , the actimmivces being accompanied by
and may be acid fast. Some species of StrtjrnojDytcs utlicr associated bacteria which may enhance the
may L inise diheane rarely, hut their importanee is an pathogenic effect. These Lnelude fljricltab;iL7
"
' errujTP
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< Atlincmyctiies * 401
the; abdominal wall. SoiQCCEnicS die uifeLtion spreads methods. Ge! diffusion and immunofluorescence
to the liver vi ;i the portal win, {4) Pelvic. Many can differentiate A isrselii from other actiiionycetc
cases of pelvic actinnnuTcosis hnvc been Kported species and from other filamentous- anaenrhes that
in association with rbc use of intrauterine devices. may produce granules in tissues.
Actinomyces have been incriminated in F ^pidemiology: The disease occurs throughout
inflammatory diseases of the gum; ( in svifis and the world but its incidence i n the advanced con ntrics
^^
periodontitis ) and with sublingual plaques lending has been declining probably as a result of the
to root surface caries.. Actinomvcusis may also widespread use of antibiotics. Actinomycosis i*
present as mycetoma . more common in rural areas and in agricultural
Laboratory iNa nosis; The diagnosis is made workers. Young male persons (10^30 veajlJ old ) are
^
by demonstrating nctinomycefcs in the lesion hy most commonly affected. The reason for this
microscopy and by isolation in culture - The predisposition is not known. About 60 ]>cr cent of
specimen CO be Collected is pUS. In pulmonary the cases afe Cervicofacial and some 20 per cent
disease , s- putum is collected. Sulphur grannies mav abdominal. I let vie latino mere* is seen mainly in
m r
OkJ
Sulphur granules or pus containing
acrinomvcetcs arc washed and inoculated into
f
^
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Miscellaneous Bacteria
also present as abscesses, conjunctivitis, pharyngitis, nonsporing, noncapsulated Gram positiye rod, witli
urethritis, pneumonia , infectious mononudeosis-
like syndrome, endoc.Ltdnis or septicemia.
a tendency towards formation ot’ long filament .. It
LS microae.ophilie on primary isolation but on
-
MOM tinman infections arc caused by scrovar lubcultuiv, grows as an aerobe or facultative
l /2a nr l / 2h and 4b. Experimental inoculation m anaerobe. It grows on ordinary media . Black
Copyrighted materia
404 < Textbook of Microbiology *
colonics are developed in tellurite ircdiL Itfenrents saprophytic in water and soil . Human infections
glucose and lactose, producing acid without gas ; have been recorded mainly in the tropics and consist
sucrose and mannitol arc not fermented . Different nt skin lesions with pvemia and multiple abscesses .
antigenic types have been recognised .
E rhi&iopEthiJi: is a natural p &rasiLc oi many FUWGBACTERIUM MENINGOSEPTICUM
animals. Jr causes swine erysipelas and human FJJVUBACTERIUM mcningosepdcum is a Gram
erysipeloid , E luman infection usually occurs on the negative non mo rile tod , producing a yellowish
hand or fingers of persons handling animals fish pigment. It is oxidase positive, proteolytic and
or animal products. The lesions are painful, weakly termentalive. It is a ubiquitous saprophyte
edematous and erythematous, usually involving the i tottunistic infections. It has
capable ot causing o[|
local lytnphnodes and jisines. Occasional eases of been tcsponsible tor outbreaks of meoingitis io
endocarditis have been reported . The bacrUos it newborn infants. Infection in adults leads to a mild
aenaitive t < > penicillin , erythromycin and broad febrile ill lies?,
spectrum antibiotics.
DGNOVANIA GRANUL0MAT1S
ALCALIGENE 5 FAECALIS l CalymnritobactErnim granuhmatix)
The name JJacferjum faecalis tlkaligenes was Donovan 0 05 described the presence of
^ )
originally applied to an ill defined group of Oram characteristic intracellular bodies in smears from
negative bacilli indited from human feces , which ulcerated lesions of a disease now known as
did not ferment sugars bur produced an alkaline Douovanous. He considered the bodies to be
reaction Ln litmus milk. The term Alc\iKgcncs parasites . DonovanoELH is a venereal disease , first
described by McLeod in Indk in 1882 and seen
facL &il
* now refers to Gram negative , short ,
Ttonsporing bacilli , which Lire strict aerobes and do mainly in the tropics. The incubation period ranges
not ferment sugars. They arc motile by means of fn>m 1 It ) 12 weeks. It begins as a pamless papule
peritrichouj flagella . They ate usually oxidase on the genitalia , which leads to a slowly
positive. Nitrate reduction is variable. progressive, autoinocLiJable ulcers . The disease runs
Ait, /pectEuia a saprophyte found in water and a chronic course, Donovan 's intracellular bodies
soil contaminated with decaying organic matter . have since been identified as bacteria and named
They are also commensals Ln human and animal Dunovarna granuJeimurh - '
intestines. They have been isolated from a variety Diagnosis can he made hy demonstration of
of clinical specimens such as urine , pus and blood. Donovan bodies in. Wright-Giemsa stained
Thty have been considered responsible for a impression smears From the lesions. They appear
typhoid - like fever, urinary infections, infantile as rounded CQCObfKilli* 1-2 pm , within cystic space.;
1
gastroenteritis and suppuration in various pares ot in large mono nuclear cells . They show bipolar
the body. condensation of chromatin, giving a closed safety
pin appearance in stained smears . Capsules are
CHROMOBACTERIUM VIGLACEUM usually seen as dense acidophilic areas around the
Q lOOblUtrnun vrn /aneum is Gram negative , bacilli . They arc nonmotilc and Gram negative.
^ ] ,L
nonsporing bacillus, motile hy means of polar and They can be grown on egg volk medium and on a
lateral flagella. They are facultative anaerobes modified Lcvinthal agar. It is morphologically and
growing on ordinary media and producing violet antigcnksUy related to Idcbsielke.
pigment soluble In ethanol and insoluble in water Pathogenic tv is Limited to human beings .
)
and chloroform . They are oxidase negative and In trade rimiil inoculation of whole cultures or of an
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406 * Textbook ol M rotiiokigy »
Relapses arc common n untreated CIHI. The rod ) contains slender spirally curved Gram negative
disease can .1 KI occur A* outbreaks, m the absence Hods, 0.2 -0.5 pm thick and 0.5^5 uiii long. They
of rat hitC - t his ci»u) itill]ih first observed in are typii ally comma shape ;! hut mAY occur as lb Of
Haverhill, USA , Is called Haverhill fever or mnltispiral chains. Old cultures arc coccoid and
emhema arthricicuni rpfJemjamt , li is believed pleomorphic. They are nonsporing and motile with
to be caused also by consumption. of caw milk or A single unsheathed polar flagellum at one or both
water contaminated hv rat ;;.m
pules . Growth occurs under m icroacroptl i lie
Laboratory diagnosis iR by boiatu *r of the bacillus cunc . i Mns, 5% oxygen concentration being optimal.
'
-
I r i i i blood or oilier tjodi fluids , Smears of flic 01111
fluiL L may KIIOW pleomorphic Gram [legulive rods.
Many pathogenic species are thermophilic, growing
well at 42 ° C . Campylobacters do not attack
Agglu 1 : m :1 :m, CF and fluorescent antibody tests have carbohydrates but arc strongly oxidase positive.
been used for serological diagnosis. Campylobacter* first gained prominence in the
Spirillum minus is a short, actively motile 1970s as a common cause of human diarrheal
bacterium , 1 — —
. 5 * 0.2 0.5 )jm in ' .r.e , with two or
three regular spirals and 1-7 amphitrichous flagella.
-
di ease, affbi ting children and adults.They can, on
occasion, also cause systemic infections. They are
It is Gram negative but 1* better visualised by important veterinary pathogens. Campylobacters of
Ciiemsa or Fontana stains or by dark field medical importance are the following:
microscopy. It was first observed in a nit hy Carter
ill India. Japanese workers identified it as h r: .
-
Cau ing diarrheal disease: C- jejuni , C- cofj, t ,
suppurates at the unset of fever, with regional lymph - Species as it causes attacks of ni &Trhea worldwide.
ailenopathy. The HLibsccjncnt course is sil uilar to the The infection i* voonnti . :, the source being food of
streptobacillary type, Mortality rates of up to 10 per animal oriu: nh especially raw milk . It is part of tire
dent I lave bun reported , mainly due to endocarditis.
normal intestinal flora of domestic animals and
Laboratory diagnosis is bv the mirtOKopic b:’ ds, and is shed L11 thr. i feces- It can, be - wlated
examination of the blood and exudates from the frequently from stirface waters .
lesion , by intraperitoncal inoculation into guinea
Infection occurs by ingestion. The jejunum and
pigs and mice and by dcmonstrai inn of the spirilla ileum are the primary sites of colonisation, but it may
in their blond ami per . tones! fluid . Biological false
pnMiivc reactions for syphilis serology occurs in a
-pathogen
. -. i Lown the colon and
|ire L i hi It is an i
rectum.
and may Involve mesenteric lymph node
pwasive
*
proportion of RRF patients, more in tile spirillary and cause bacteremia. The incubation period is 1~7
form . days - The illness starts with fever, abdominal pain
Botli ty|jes ol RBF respond to penicillin and and warcty diarrhea. Stool contains leucocytes and
tetracyd . ne. Oral penicillin or doxycyelinc After blood. The disease is usually self-limited, though
rat bite is effective in prophylaxis , Campylobacter shedding may continue for weeks after
recovery Fluid and electrolyte replacement is all tliat
CAMPYLOBACTER is generally required. When needed, erythromycin is
The genus IdJ7ipvj'ob!
, L tcr ( Crcek, meaning curved the best antibiotic.
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* Miscellaneous Bacteria 407
LtbulMy Jiagnosi -s depends on isolation of humane, as ivcll as domestic animals anil birds . In
the campylobactcr from feces. Direct microscopic this situation , clinical disease is infrequent and
examination - phase contrast or dark licit ] usually confined to children, while older age groups
microscopy to detect the darting or tumbling arc immune due to subclinical in factions.
motility of the spiral rods, or demonstration of the The related genus Arcobacteria species (A
-
small curved rods in grained smears may be useful
for presumptive rapid diagnosis. Feces or rectal ^
buti/erj', A . Crvittuphila also cause diarrheal
d ]>ease. They are capable of aerobic growth .
swabs art pi ated on sc I ective medi a. In fuse of del av
in culturing, a Iran sport medium has to lie CAMPYLOBACTER F a r i a
employed' Campylobacters survive for 1-2 weeks This organism was isolated in 191 H by Theobald
at A °C in Cary El Lair transport medium but Smit h from i nfeeiious al H inion 11 L cattle and named
- -
glycerol s:i1 inc is not .arkfactnry. The pitting media Vibrio fetus , lr is a very important veterinary
commonly used arc Sorrow s , Butzlefs or
'
Campy pathogen . Human infection by C. telus may lead
HAP selective media . C , jejuni, as well as C roll to bacteremia, sepsis and meningitis.
and C. l.dj- j , are thermophilic .and Jo not grow at
25 rG . Inoculated plates are incubated at 42 °C in HELICOBACTER
an atmosphere of S% oxygen , 10% carbon dioxide Spiral, campylobiCttr Hike- bacteria were observed
and 8.5% nitrogen. Thermophilic tumpylnbactcrs in close apposition ro the gastric mucosa in several
ca n grow well ar 37 °C also hot i ncuhation at high er cases of gastritis and peptic ulcer, by Warren and
temperatures suppresses normal focal flora ro some Marshall in Australia ill 1983.They were original I v
extent . named C&tnpyl&bectet pylftri. As they differed in
Colonies appear usually by 4? hours. They are many respects from campylobaercrs, they have been
nonhemolytic, grey or colourless, moist, ami Jlat or redesignated as Hclicobactei pylori. It now appears
convex. Sugge ft Lve colonies are screened by Gram that helicobacters have caused human infection from
staining, motility and oxidase rests , Confirmation ancient times. By enzyme immunoassay ,
is. by further biochemical rests , including positive helirohacter antigens have hern detected in the
catalase and nitrate reduction tests.
C . coli causes an infection clinically
-
intesti n rs of pre Columbia n mu mmirs in (be 1 A.
Toddy, heUrnbacters colonise the stomachs of halt
indistinguishable from that due to C.JCfUTli ( ’. caii the human population of the world !
is commonly found in healthy pigs . It is I Icliicobacten inhibit die stomachs of different
differentiated from C. jejuni by the hippuratc animals, each with its own helieobacner species. / /.
hydrolysis test which is positive only in the case of pylori is adapted tti the luiman gastric mucosa. The
C, jejuni, only animal it infects is the monkey A larger spiral
C . lari also causes a similar diarrhea! disease, lr bacterium of uncertain taxonomy,'if. heiintMilf can
i n o be distinguished from C - fejuni and C. Coli by occasionally infect hmimns arui some animals like
its resistance to nalidixic acid. cats and dogs also . 11. cifiardi and II . fcnnelliae
C . jejuni and C. coli can be serotyped for are associated with proctitis in the HIV mfevted.
epidemiological puriMiHes.
C. jejuni is the most common bacterial cause of HBLICO&ACTBk I - OR ]
diarrheal disease in many developed countries - H , pylori is a Gram negative spiral rod, motile by a
more common than salmon elite or shigdlac . In unipolar cuft of lophottiehous flagella. h grows on
[ he developing countries, C. jejuni is endemic , chocolate agar or Campylobacter media under
asymptomatic infection being widely prevalent in microaerophulii.- conditions, with 5 -20% CO.. , and
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400 H Textbook d M crobiology »
pH 6-7. At 37 ;' C , no Ionics like 2 -7 Jays Iu immune responses , but they do not seem to be
develop. Coccoi . l forms appear in old cultures. It protective .
produce* oxidase, catalase , phosphatase and E E \ 11. pylori shows considerable genetic diversity,
A distinctive feature is the production of abundant as evident in molecular typing . The complete
unease , and this property lias been Used as a rapid genome of the bacterium has been mapped .
diagnostic test in gastric biopsy samples. It does Virulence has been associated with certain alleles
not metabolise carbohydrates
.
or reduce nitrate. in genes, such as cag {cytotoxin associated gene )
11. priori is global , with a prevalence of 30-60 and vac ( vacuolating cytotoxin gene ).
per cent— more in the developing than in the Diagnosric tests aie of two kinds, invasive and
developed countries. The sole source of //. pylori non invasive . Invasive tests Involve cndoscop : c
is the human gastric mucus. The exact mechanism biopsy of gastric mucosa, for examination by
of transmission is not clear, but it is likely to be microscopy, culture and urease tests . Microcopy' of
oral -oral or tcc^- l— tiral. Rwerty, overcrowding and biopsy sections by silver staining or of Grant
poor hygiene favour transmission, Wjtl : inprovements stamed smear- is a useful method. Culture is more
in lifestyle, tlx prevalence of cluldhood infections has sensitive, but requites expertise and takes 3-7 days.
declined in the developed countries. A Li [ of the biopsy material put in a urease indicator
After an incubation period of a few days, H . trediuni shows positive result in minutes .
pylori causes, in some persons, a mild acute gastritis Non invasive tests include serology ( ELISA ) and
which mav last for about two weeks. The infection
jf
the ' urease breath test’. In the latter, the subject
may he transient in some, but in most , it per- 1 sis drinks a urea solution containing labelled carbon,
for years or decades. Such colonisation is usually which can be detected in the breath . It is sensldve
asymptomatic, though chronic superficial gastritis and reliable, but needs isotope assay facilities.
may be demonstrable histologically. The bacteria SI . pylori is sensitive to several antibiotics and
are present only in the overlying mucus and do not to bismuth salts. The standard treatmenl is a
hvade the mucosa , Gastric antrum is the commonest combination of bismuth subsalicylate, tetracycline
ate of colonisation , though any part of the stomach (or amoxycillin ) and metronidazole for two weeks.
may he involved. The infection i.s strictly confined An alternative schedule employs a proton pump
to the gastric mucosa , in the stomach , as well as in inhibitor like omeprazole and clarithromycin .
areas of gastric metaplasia and heterotopia in the Treatment is indicated only for H pylori related
duodenum. The exact pathogenic mechanisms are gastric or duodenal ulceration and not for
not clearly understood . Bacterid protease , toxins asymptomatic colonisaLLon . Drug resistance and
nr ammonia released bv urease activity nr t
recurrences are frequent .
autoimmune responses to gastric antigens may all
contribute . LEGIONELLA PNEUMOPHILA
Peptic ulcer disease occurs in a proporrion of The name Legionnaires * disease was given to an
the infected . Chronic atrophic gastritis may be seen apparently new illness which broke out among
in the later stages. The infection is recognised as a members of the American Legion who attended a
risk factor for gas- trie malignancid such an convention in Philadelphia in 1976 . The disease
adenocarcinoma and ’mucosa associated lymphoid was characterised by fever , cough and chest pain .
tissue' ( MAI. E ) lymphomas. Such MA[Tomas Leading on to pneumonia and often ending fatally.
appear to he antigen driven and are found to regress The causa rive agent has been called Legionella
after elimination of H . pylori by Treatment , pneumophila . Subsequent investigations have
Infection induces IgM , IgG , IgA and cellular revealed that the disease is neither new nor localised .
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« Miscellaneous Bacteria 409
Infection w ith L, pncLr/nopMa is now known to Lcgioncllae arc widely d i - tributcd in natural
cause protean manifestations . rwn disci net clinical water sources, sueh as stagnant waters, mud and
patterns bare- beenidentified and dc - ignafcd as hot springs , where tbc nutrition a L and growth
Legionnaires disease and Pontiac fever, together
'
requirements for these fastidious bacteria arc
known as /ejfjcviei/usjy. provided by some types of algae . Legionellae survive
Legionnaires ( listasc: may he either epidemic and multiply inside free - living amefee and other
or sporadic . The incubation period i -. 2-10 days . protozoa . They also mulliply in some arlifici . il
'1'hc d - scasc presents with fever, nonproductive aquatic environments, which serve as amplifiers.
cough and dyspnea, rapidly progressing, if untreated , Human iiife- clinn is typically hy inhalation of
to pneumonia . Diarrhea and encephalopathy are aerosols produced by cooling towcrsh air
common . Last fatality maybe 15-20 per cent , the condiI innets and shower heads which act as
cause of death being progressive respiratory' failure disseminatora . Acrosolised lcgioncllae can survive
and shock . All age groups arc susceptible, though for long and can be carried over long distances . No
more cases haw: occurred in the elderly animal reservoir exists, and infection is limited to
Ptyntiae fever is a milder, nor fatal influenza'
’
human benign. No carrierstate is established. Mari'
likeNllness u ith fever, chills, myalgia and headache . to -man transmission docs not occur.
Outbreaks with high attack rates may occur. The outcome of inhalation of lcgioncllae
-
The discovery ot J . pneumophila led to the
isolation of many related hacteri ;:i, which have beer
depends- on the size of the infecting dose , virulence
of thestnun and resistance of the host. Known risk
placed in the genus LcgioncUa , under the family factors arc smoking, alcohol , advanced age,
T jcgLorctlaceae. Some 40 species of leg i nnellae have intercurrent illness, hospitalisation and immuno-
been recognised, many of them with multiple deficiency. Men are more often affected than
serogroups. The original isolate in tills genus is women , In the developed countries , legionedoi is
designated / „ pneumophila senogrnup 1 (SGl ) , accounts for 1 -3 per cert of community acquired ,
whieh accounts for nearly all severe infections . and 1CKJ 0 per cent ofhosptaJ acquired pneumonias .
Examples of other species char cause human Its prevalence tn the developing countries is not
infection less often are L . mit-thiclei, L . bozirmAnii . adequatelv known.
L . dusftti&ii and L. ur.u.ir.'Ji . Following entry into the alveoli through
j
^ , noncapsulated hacilii , 2-
I ^eginnellac are thin aerosols, legiondlae multiply in -side the monocytes
5 pm * 0.3-0.1 pmh coccobacillaTy in clinical and macrophages , Dissemination occurs by
material and assuming longer forms in culture . endobronchial , hematogenous , lymphatic and
Most ate motile with polar or subpolar fligeda , contiguous spread. Because of then intracellular
They arc Gram negative but stain poorly, location , humoral anybodies arc ineffective .
particularly in smears from clinical spec mens , Cellular immunity is responsible for recovery,
They srai - i better by silver impregnation , but arc Laboratory diagnosis is by the demonstration
best visua! ]sed by d 11 eCt fluorescent mil. i body ( DFA ) of legionellac i n clinical spec ] me ns, such as sputu m ,
staining witli monoclonal or polyclonal sera . bronchial aspirate , artd lung biopsy, by direct
They hare fastidious rcqr. jirements and grow on fluorescent amibodv test and culture , by the
complex media such as buffered charcoal , yeast idemificstion of leginne-lla antigen* in urine by Latev
extract ( liCYE) agar, with L- cystcinc and antibiotic agglutination or h’ LJSA , and hy the detection of
supplements , with 5% CO , at pH 6,9 , 35 =C and serum antibody by ELJSA or indirect
90% humidity, Growth is slow and colonies take immunnflunresccnt assay.
3— 6 days to appear. For treatment , the n e w e r microti des,
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.
* Misce laneous Bacteria 411
oxidase positive., nonmotilc* Gram negative rock , genus MonEetki They are part of the normal oral
with a tendency to occur as coccobacilkiy and flora , K- fctngflg has been associated with mdlrramJjtis
diplococcaJ forms, was formerly grouped under the
, and Sdktuoa of bones, joints and tendons
h u r d l e r I t e a d i m*
Bcrgone- Bcrran E and KJ Towns* 19%, Arirwhrimcfw *pp. AE nosocomial pathogen*, Clin Mictvbiol Rtf 9=148 .
Blawr MJ. 1998. liclicobMcnrr priori and gastric discasee, BMJ , 316, 1507.
fi laser MJ. t 990. Epidemiology acid pachophyaiolog) of Camp. pylori infections. Rev Infcci Dis 12:( Suppl.1 ) .,99 ,
1
Rickettsiae are smatl, < Iram negative bacilli adapted seen to have i three Layered cell wall, a trilaminar
to obligate intracellular parasitism , and transmitted plasma membrane and an outer slime layer.
by arthropod sectors, They are primary parasites Cultivation: Rickettsiae ate unable to grow in
of arthropods such .LH ]iceN ileus , ucks and mites, In cell- tree media. Growth generally occurs in the
which they are found ill the aluuentarv Canal . In cytoplasm of infected cell? bur in the case of the
vertebrates, including human ?, they infect the spotted fever richerts- iae , growth may take place in
vascular endothelium and reTiculuendnchelia] cells . the nucleus as well. Rickettsiae grow best in cells
The family Rickettnaceac is named after Howard that are not metabolising actively, The optimum
Tayh > r Ricketts who discovered the spotted fever temperature for growth is 32-35 ZQ .
rickettsia ( 190 b ) and died oi typhus fever contracted They are readily cultivated in the yolk sac of
during his studies. developing chick embryos , as first shown by COM.
They also grow on mouse fibroblast, llel .a ,. HEp -
—
The family current I v comprises [ It tee
genera Rickettsia. Ofienm. and Ehrlichia which
appear to have descended from a common ancestor.
Former members of the family, CoxieMa burnetii ,
2 , Detroit 6 and other continuous cell line? but
tissue cultures are not satisfactory for primary
isolation. Laboratory animals such as guinea pigs
_
which causes Q fever and ffodjiiimjen cjurnrjo;; and mice- ate useful for the isolation ot rickettsiae
causing trench fever have been excluded because from patients. They may also be propagated in
the former is not primarily arthropod -home and arthropods.
the latter not an obligate intracelhilar parasite, being Resistance; Rickettsiae are readily inactivated
capable ofgrowing ip cell tree media, besides being by physical and chemical agents. They arc rapidly
different in genetic properties. destroyed ar 56 aC and at room temperature when
separated from host components, unless preserved
GENUS RICKETTSIA in skimmed milk nr a suspending medium
TTlC gCTl- US Rickettsia consists of the causative containing -sucrose, potassium phosphate and
—
agents of two groups of diseases typhus fevers and
spotted fevers.
glutamate (SPG medium),
Rickettsiae arc susceptible to tetracycline,
Morphology: In smears from infected tissues, chloramphenicol and ciprofloxacin. Penicillin and
rickettsiae appear as pleomorphic coccobacilli. 0, 3-
0.6 pm n Q. S 2|im in sire. They are mm mo tile
sulphon amides are ineffective but pata
aminobcnxoK acid ha ? an inhibitory action on
-
and noncapsulated TTicv arc Gram negative, though ticket [siae. Sulphonamides may actually enhance
rhev do not take the stain well, ’ ihev stain bluish
i
the growth of rirkettsiae and worsen the condition
purple with Gicnisa and Castaneda stains and deep if adm imstcnrd to patients.
red with MachiavcUo and Gimencz stains, Antigenic structure: Rickettsiae have species
Under the electron microscope, rickettsiae are and group specific antigens. The iirimuiiodnniinflrii
Copyrighted materia
i Rickaitsiacoae 413
surface protein antigens (SPA ) of R. piawaztkii common in Russia and Eastern Europe. Napoleon 's
and Rr ( yphi have borh species specific and cross
reActive epitopes, Spotted fever ricketlsiae have
dominant outer membrane proteins (OMP) A and
B, the former being a species specific antigen tiering
retreat from Moscow was forced hy typhus fever
as an adhesin for hnsr cells , and the latter showing remarked, in reference to the outbreaks of lousc-
Limited cross reaction wjih SPA of typhus homc typhus and relapsing fever rampant during
nckcttsiac . The third surface antigen is an alkali the Russian revolution, that "either socialism will
stable polysaccharide found in some rickettsiac and defeat ihc louse or rhe louse will defeat (socialism'!
in some strains of Proteus bacilli , This sharing of Tn recent times, the main foci have been Eastern
antigens klnui rickettsiae and proteus is the basis Europe, Africa, South America and Ajia, In India ,
-
for the Weii Felijc reaction used for the diagnosis
of rickettsial infections bv the demonstration of
the endemic spot is Kashmir.
The causative agent of epidemic typhus is R,
agglutinins fn Proteus strains OX 19, OX 2 and pfpwTLzeJai named after von PtOWfik, who died
OX K. of typhus fever while investigating rhe disease.
Fslthojg« tie»ls: Packet [ hi at are transinittcL] In HLIMANH are the only natural vertebrate hnsts.
humans by arthropod vectors through their bite Several animals - guinea pigs, mice, cotton rats
or feces. On entry into ihc human body , the and gerbils - may be infected experimentally.
fickeltsije mull ink locally and enter the blood. Natural infection in flying squirrels Lias been
Thev hcLome localised chiefly in the vascular reported from south - eastern USA . They may
endothelial cells, which enlarge, degenerate and possibly act as reservoir hosts, infection being
cause thrombus formation, with partial nr complete spread by the squincl louse and flea.
occlusion of the vascular lumen. The overall The human body louse fWi’culus hum anus
pathological features of the rickettsial diseases are coiparjs is the vector. The head louse may also
similar and can be explained hy the damage to the transmit the infection but not the pubic louse. The
vascular endothelium . lice become infected by feeding on rickettsiacrnic
The long survival of rickettriae in\arious organs patients , The rideflsiae multiply in the gut of the
and Lymphatic tissues of infected men and animals
is a distinctive feature in pathogenesis and is of
—
lice and appear in the feces in 3 5 days. Lice
succumb to the infection within 2-4 weeks,
importance in rhe epidemiology of some ticltenial remaining infective till they die. They can traumit
diseases. the infection after about J week of being infected.
The lethal nature of the infection in the louse
TYPHUS FKVKU CIUJIJI* suggests that the association between ft- jwcwMcJbj
This group of diseases consists of epidemic typhuit anti its vector IR relatively recent Odd not well
rrcrudescent typhus [BrilI - Zinsser disease ) and established. Lire may lie transferred from pCDOD to
endemic typhus. person. Being sensitive tn temperature changes in the
Epidemic typhus: ( Louubome typhus . host, they leave the febrile patient or the cooling carcass
Classical typhus, Gaol fever) hits been one of the and parasitise other persons. Lice defecate while
great scourges of mankind, occurring in devastating tootling. Infection is transmitted when rhe contaminateJ
epidemics during rimes ofwar and famine, so vividly louse feces is nibbed through the minute abrasions
described by Hans Zinsser in his bonk, 'Rats , Liu caused byscratching - Occasionally, infection may also
and History'. The disease has bun reported from be transmitted by aerosols of dried louse feces through
nil parts of the world hut Lins been particularly inhalation or through the conjunctiva.
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416 s Textbook of Microbiology
Uiekutlsinl pox: The mildest rickettsial disease equatorial rain forests. The term ebiggor-bome
ot ' humans is a self- limited, nonfaral, vesicular typhiai has therefore been suggested as a more apr
exanthem first observed in New York ( 1946). The designation. Four factors arc essential for the
name is derived from rhe resemblance of the disease establishment of a mierefocus, of infection, viz.,
.
to chickctipox It is also called vesicular or coexistence nod intimate relationship among O.
varied!ifbmi rickctrsiosis. The causative agent is tsuttugtmushif doggers, rats and secondary or
R . ikon ( from akarr, meaning mite). The reservoir Cransitioral forms of vegetation (known as the
of infection is the- domestic mouse, Mus mustmlus sojFtottr -
re rod) .
and the vector is the mite , Lipaays»oides ( formerly The incubation period is -
1 3 iveeks . Patients
AJfodcrmirlpfajus) sajTgurneLr.!;, m which tTffBO'sariul typically develop a characteristic eschar at the site
transmission Occurs. R . aA.ir: has ulso been isolated of the mite bite, with regional lymphadenopathy
from wild rodents in Korea. The disease hus also .
and a macufopapular rash The disease sets in with
been reported from Eastern Europe and Korea. fever headache and conjunctival injection.
,
Copyrighted materia
i Rickflttsiaceae » 417
Encephalitis snd pneumonia may be seen in severe The third is "human granulocytic ehrlichiosis’
eases. The disease is not a serious problem in civilian caused by an organism either identical with or
practice but assumes IC .LC importance LII military closely related Co the eqUiOC pathugen , £. epur
^
medicine, especially during jungle warfare, ?s was (probably £. phngwYtophila ) - lr is transmitted by
-
recognised in the In do Burmese theatre in the
Second World War .
Lcodes ticks. Deer, cattle and sheep are the suspected
reservoir. Leucopenia and thrombocytopenia me
Considerable differences oust among different seen in ] i .U. icuts . < ocmra stained blood dims muv
strains of O, autsugamushi in mucigenic properties show morula form ot the ehrlichia.
and virulence, a factor that OntplicMn Htodiagnosis LJowcvcline is recommended for Ireiirmcnr of
and iinmunopropbylajciH . Three major antigenic ehrlichioses.
Kata ,
—
types have been recognised Karp, G illi a in and La hunt Dry diagnosis ; Rickettsial diseases
may be diagnosed in the Laboratory either by
isolation of the rickettsiac or by serology. As
GENUS EHRLICHIA riekerfSUK are bigblv infectious and haw caused
Ebcliihiac arc small , Gram negative , obligately seven!ct- riousond fatal infect ions among laboratory
inrracclluar bacteria which ham im iffinitv towards
Jr
workers, their Isolation should be attempted with
blood cells . In the cytoplasm of infected phagocytic utmost care and only LII Laboratories equipped with
cells , they grow within phagosomes as mulberry^ appropriate safety provisions.
tlke clusters called aiomla ( morula , meaning Rickettsiae may be isolated in male guinea pigs
mulberry). They lire tick - borni:. Similar organisms or mice from patients in the earlv phane of tLac
under the names of dnaplasroa, C'oWn? and disease. Blood dot ground in skimmed milk or any
NeorickemiiU had long been known to veterinary suitable suspending medium is inoculated
scientists an etoLugio.1 agurtv of ticldjome infections iittrapcritooealb The ANIMALH have to he observed
ot cattle and sheep. Three human infection ^ caused tor 3 -4 weeks and their temperature recorded daily .
by this group id organisms have been identified . Their response to rickettsial infection varies. In
The first of these human diseases, reported from Rocky Mountain spotted fever, guinea pigs develop
Japan in 1954, was a case resembling glandular fever, scrotal necrosis and may even die . With R .
fever who showed serological response against the iyph ) . R , conori and R jJatn. they develop fever
agent of canine ehrlichiosis. The etiological agent and tunica, reaction . R , prowazekii produces only
lias been named Ehnfichia tenuttsu (from ' jennefinf , fever u lrhouranv *
testicular inflammation. Smew
dxJlpfllKK word fetf glandular fever). It is endemic from the peritoneum, tuuia and spleen ot infected
in Japan and parts of South East Asia. It causes animals may be stained by Giemsa or Gimeoez
lymphoid hyperplasia and atypical lymphocytosis. methods ro demonstrate the rickettsiac-
No arthropod vector has been identified. Human Though laboratory strains of rickettsiac grow
infection is suspected to be caused by ingestion of profusely in the yolk sac of chick embryos, this
fish carrying infected flukes. method and tissue culture are not suitable for
The Second type of infection is 'human primary isolation. Egg and animal inoculation
mtmocvtic
J
ehrlichiosis’ caused by F . c/wffCCThtJS.
r
methods have been replaced by the faster and more
It is transmitted bv Amblvomma ticks. Deer and
J
sensitive cell cultures. Rickettsiae grow well in 3
rodents are believed n be reservoir hosts. Human to 5 days on Vcnocell MRG 5 cel! cover slip
: disease LH associated with leucopema , thrombt)'- cultures and can be identified by immuno -
cytopema and elevated liver enzymes. Multisystem fluorescence using group and strain specific
involvement and fatality may occur . monoclonal anti hod La.
Copyrighted material
418 * Textbook ot Microbiology
Seruhigical di :i gnus i £ may be bv the hcDfroplnlf by Proteus and at times in typhoid fever and liver
Wei I- Felix reaction or by specific tests using diseases. I fence it is desirable to demonstrate a rise
riduttiial antipens. Tk Wcil-Fclbc reaction is an
1
in titre of antibodies Fur the diagnosis of rickettsial
agglutination lest in which sera are rested For infection.
-
agglutinins to the O antigen* of certain n " LtiiotiLe
Proteus strains OX 19 f OX 2 and OX K. The test
The most Frequently used serological method
using rickettsial antigens is the complement fixation
was developed from ( he chance observation of Weil test. This may be done using the group specific
And Felix (1916) that a Proteus strain isolated from soluble antigen , or the type specific washed
the urine ( lf ; L patient of epidemic typhus was rickettsial antigen. The former lest is- in routine
agglutinated by the patient 's serum as well as by use but rhe latter is neces& ary for differentiation
the sera of other typhus patients. Tlte basis of die between epidemic and endemic typhus . Other
test is the sharing of an alkali stable carbohydrate serological tests me We agglutination of rickettsial
antigen by soinc rickettsime and by certain strains suspensions, passive hemagglutination of red cells
of Proteus, P. vulgaris OX 19, and OX 2 and P. sensitised by ESS ( erythrocyte sensitising
tnifabilis OiX K , The test is usually done an a tube
agglutination, though rapid slide agglutination
substance) , toxin neutralisation , immuno
fluorescence . Hid radioisorope precipitation.
-
methods have been employed for screening. Immunoprophylaxis: Rickettsial diseases may
bera From epidemic and endemic typhus be prevented by general measures such as control
agglutinate OX 19 and sometimes OX 1 also. The of vectors and animal reservoirs. Immunisation is
test is negative or only weakly positive in llrill- useful in special situations. Killed ami live vaccines
Zin ^scr disease. In Hckbome spotted fever both OX have been prepared against epidemic typhus . "Hie
19 and OX 2 arc agglutinated . OX K agglutinins earliest of these was phenoliscd intestinal contents
are found only in scrub typhus. The test is negative of hce infected pet rectum with R . ptvwfs kii
^
46.2 ) . ^
in rickettsial pox , trench lever , and. f lever ( Table
-
Table 46.2 WHII FBIIX reaction In rieksttaial diseases
DiSCMSC Aprfurimtiofl pattern with
OX 19 OX 2 OXK
Epidemic typhus + * -
—
Brill Zinsser disease Usually negative or
^ ^trve
r =
Endemic typhus
Tkkbornc sporred fever
4
-
HI +
+
-
# ±
+ +
-
-
Scrub typhus - - +++
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* Ricketlsiacoae 419
MLivnttin spotted fever. However, there is nu Lungs or intestinal tract . Person- to- perion
satisfactory vaccine available against any of the transmission is a rarliy, Ticks do not seem to He
rickettsial diseases, important in human infection.
Cur. burnetii is widely prevalent m birds and
GENUS COXIELU: Q FEVER animils in India , as shown by serological surveys,
Derrick ( 1935 ) investgating an outbreak nt hut human disease has been identified only rarely.
typhoid-like leVtr in abattoir workers in Brisbane, The human disease is ail acute systemic infection
Australia, transmitted rile infection to guinea pigs characterised by an interstitial pneumonia The
by inoculation of blood from patients. As the clinical picture is very variable and asymptomatic
etiological agent of the disease wan unknown, it infections very common . In chromic Q^ feve, tilt
was referred to as 'Query ' or fever. As Burnet coxiella spreads through almost all organs and may
identified the causative agent an a rickcttsia , it was cause hepatitis , meningoencephalitis or
named R, burnetii. At about the same time, the endocarditis . SpctlltaiXOtll recovery is usual . The
same agent was isolated independently by Cos in efiodella may remain latent in the tissues of patients
the USA from ticks, and was called R , diaporica, tbr 2-3 years .
the name referring to its ability to pass through Co* , burnerjj Li an obligate iutracellular
]Hires of filters impermeable to other rickettsiae .
The Australian and American strains were
pathogen, i > rim.arLIv infecting the monocytc
macrophage cells. It occurs as rods OJA).4 pm x
-
subsequently shown to be identical. As the Q fqver_ 0.4~l ,0 pm or as rphdej ( ]..SA >. 4 pm in cliameleT .
'
agent differed from other tickettsiae in many It is filterable . Generally regarded as Gram
features {smaller size, greater (WttUKC It ) beat and negative, A . it is better stained with Gimme? and
drying, major transmit on route being inhalation other rickertsiac strains
erf ingestionf (dependent uf arthropod vectors ), it In dried feces or wool it survives for a year or
has been separated from rirkettsiac into a special more at 4 "C and in meat .it least for one month . It
genus and rennmed Coxiclh burnetii. It has been is not completely inactivated at AO "C or by l 'ft
phenol in one hour . In milk it may survive
^
assigned to the group Vofo/Meferhtalongwith other
genera like Legionella and RrancisdU pasteurisation by the holding method, but ltic tlash
_
Q fcvcr is distributed worldwide, as a zoonosis method is effective. It grows well in the Volk SAC of
solidly established in domestic livestock. Wild chick embryos and in various cell cultures.
animals such as the bandicoot may be the primary Cm, burnetii shows phase variation. Fresh
reservoir. It is transmitted among them , and to cattle, isolate - arc in Phase I . It becomes Phase II on
sheep and poultry by ixndid ticks . Transnvarial repeated passage in yolk sac, but reverts to Phase i
transmission occurs in ticks . Cordelia are abundant bv passaging in guinea pigs . Phase 1 cells are
in rick feces and survive in dried feces for long autoagglutirtahlc and are phagocyiOScd in the
periods . They arc shed in the milk of infected absence of antibody. Phase [ activity is due to a
animals . They are particularly abundant in their periodate-sensitive trichloracetic acid- soluble
products of conception and con turn in ate the surface carbohydrate. Phase 1 is a more powerful
environment parturition ..
lit immunogen than Phase II and elicit* good antibody
Human infection TTULV occur occupationallv response to both 1 and II antigens. Phase 11 antigen
through handling wool or hides, meat nr other is more suitable for complement fixation tests . Q _
animal ]>roducT £ contaminated with the organism . fever sera do not cross - re aft with rickettsial or
Drinking infected milk can transmit the infection, proteus bacillus antigens.
Coxiella may enter through abraded skin, uUIOOSa , Lab oratory diagnosis is by serology, by
Copyrighted material
420 * T^ jrtbo & k of Micr 60iolM] y *
prolonged treatmentwith enmhi nations of During the E'irst World War, osier u million cases
tetracycline , cotrimoxazolc nr ritampicin may be
'
animals. Eilmtificution and classification of members tbeir lives. Vertical transmission does not occur in
ot bartoncllac , rickettsiae , chlamydiae and related lice.The causative agent was identified as a tidoetisU
'
bacteria often depend cm sophisticated molecular and nacneJ R . quinfana Ilnur tjuintun.- i , meaning
methods tike lb? SNA analysis. Is
fifth , referring To ' five -day fever’, a synonym for
trench fever ) . As it wag found ro differ from
BARTON H I. LA B : i 1.1 JFORMIM rickettsiae in a number of respects, including its
In 1870 , when the railway line from T . ima roOrovy ability ro grow in cell - free culture media such as
in Ptru was hting built, an outbreak of fever IdUcd blood igar, it was separated into a new genus
(huusaiid ^ oi workmen. The disease was called Rochalimaca. ( nfter da Rocha Lima , an cirlv
Omyir /era; which was seen in the mountainous Investigator ot rickettsial discuses ), In a subsequent
parts ot Peru , Columbia and Ecuador in South taxonomicaE s h i f t , i t h a s heen icclailLflcd ns
America. Some of the survivors developed nodular Bartonella and named B . tfuiawA,
ulcerating & kin lesions, called verruga peruansL The rhe disease Jicqucndy leads to ; L chronic or
common etiology ol ihese two conditions was latent infection. Recrudescence mavr occur as in
established tragically in 1385 by the Peruvian Hrill -yinsscr disease ansi relapses have been
medical student Daniel Carrion . He inoculated reported as king as 20 years after tlie primurv disease.
himself with material from verruga and developed
, The chronic intention and late relapses help to
Oroya lever from which he died , Oroya fever is maintain the bartonclla in the absence of animal
therefore also known as Carrions disease. reservoirs.
Oroya fever presents as lever and progressive french fever was thought ro have vanished With
anemia due to bacteria] invasion ot crvthmcvtcs. m J
the world wars. But isolation of li . qtimraria from
Mortality is high in untreated cases . A late sequel Tunisia and Mexico recently suggests chat the
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i R : d k en si ace a e » 421
clitica ?iHc may be more widely distributed than is agent. It can be demonstrated in lymph node biopsy
realised . Trench fever cases have been identified in smears and sections by Warthin-Srany staining.
some homeless persons living :Ln unsanitary" J3. henselae has been also linked with two other
conditions in the USA, conditions, seen more commonly ifi HIV infected
and other immunodeficienr persons. These are
*
name "cat smteh disease * but its etiology remained and spleen.
elusive. R- hCMCkc has been isolated from tin? blood Angiomatosis may also be due to B, quintans in
of patients , in blood media after prolonged some eases,- Another organism Atijpta fili* had aJso
incubation and is now considered as its etiological been proposed as a cause of cat scratch disease .
-
B
ScMttMG, lW8 A hifBOfy of taftoodlpm [CliriQn disease). AmJ Trap Med 17;503.
r
^
Walker DH andlJS Dnimiar I 996 Envcrgence of Ehrlichiosis at a human health problem. Lmcnjii r Tn / rtT Dtf Nol.
r
^ ^
Wnhlmd TTE, WTO, A himrleal mwiM: vf riekirnsial diseases with a discussion of the involved prnhJcTTus. ,/ inf Dm
127:585.
ZbUKT H . 1935, tfatfc HLM arici J iisiofw London: George, Routledpr -and Sons Ltd..
*
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Chlamydiae
Chlamydiae arc obligate intracellular bacterial C. rmc-homarfs strains form compact indsuns
piiJHMCfH <> t limuajiy , LLiUmuls and birds* With tfupkiti wLtk t !ie glycogen matrix , are sensitive to
for squamous epithelial cells and maerpphages of sulphonamidcs, and BA natural parasites of humans,
the respirator)' and gascroinEegtinai tracts. Due to usuallv causing locdlHd infections nf the eyes and
their fikerability and failure tn grow in cell-free genitals . C . pejfTad strains form diffuse vacuolated
media ^ they were considered to be viruses. Based i delusions wi thoot th e glvc ^ igcn matrix, a re ncsi stSITl t
on the human disease thev were then known to
ir
tosulphonamidcs and arc natural parasites of birds
cause, they were called psittaeo ^ is - and animals, capable of causing pneumonia and
lymphogranulom a - trachoma ( PLT} viruses, or generalised infections in humans . C. pneumoniae
noncommittally as TIT agents ' . However, they is ,in exclusive human pathogen with no animal or
differ from viruses in many respects. They possess avian host. It is a common cause of acute respiratory
both DNA and RNAt have cell avails anti
ribosomes , replicate by binary fission without an
disease worldwide .
Morphology and growth cycle:
‘eclipse phase ' , and are Susceptible to the usual Chlamydiae occur in two forms , the cJcmc/lfary
antibiotics and chemotherapeutic agents . They arc tody and the reticulate body (formerly also called
.
therefore accepted is bacteria. Unlike other bacteria, [ be 'initial body ) The elementary bnrly is the
'
thev d ( ] not have peptidugLycan cell walls, Thev extracellular, infective form . It is spherical particle,
lack enzymes of rhe electron transport chain and 200-300 nm in diameter, with a rigid trilaminar
so require ATP and outrtenr resources from host cell wall similar to the celt walls of C.Sram negative
cells . They have therefore been called energy bacteria, and an electron dense nucleoid . The
ptnsitts, reticulate body is the intracellular growing and
hi recognition of the pioneering studies of Sir replicative form, 500-1000 nm in size. Its cell wall
Samuel liodson on psittacosis, the name Bcdsunix is fragile and pliable , Leading to pleomorpbism.
was proposed for this group. However, they are Infection is initiated by the attachment of the
now officially classified ns- bacteria belonging ro elementary body to the surface of a susceptible
the genus Chlamydia , in the family Chlamydiaceac , epithelial cell , followed bv its codocytosu ( Fig.
under the order Uhhimydi tlcs .. 47 . Ij. Inside [ he host cell , the elementary body Lies
The genus Chlxmydia contains touf species : wirhm the endosontr, IJC Lng separated from the host
— £ aacbtmiMtis , C. pstemci, t\ pneumoniae, which
can affect humans; and [ he fourth species , C.
cell cytoplasm hy the crtdosomsl membrane
throughout its active growth cyde. By about eight
pecarum created recently to include some Strains hours, the elementary body within the endosome
effecting ruminant?. Species differentiation is based undergoes sphcrnplast - hkc transformation to the
on growth characters, nucleic acid profile, antigens , large retieulare body, which begins to divide bv
plasmids arid nature of the inclusion hndy. —
binary fission by 12 hours . By 20 24 hours, the
Copyrighted material
H Chlamydias 423
@
© © © c9
2 3 U 5
I
1
c o5> co
a
dy
<W
7 G
. -
L Elenwntir)' body t EBi: 2 E $ aJtwhssx to* Lril rcceplnr; J. Eii crtim cell fey cwkscyiMis. By ft ImirsL 4. EB reoffuiKd
'
. .
inut MIIIIIAI.^ ( RBJ: 5. Hast cell growtih in?%j by 12 twine R.R wnkqspMnj finim fis . By 24 tanmv nndunion body wish RR .
j!xl (tevdnpinif Eft ; 7 . By hnun. uicliiAiofi ftuly tonmjiinj;. imfcoHUb EB. Nuclrus pus&tard to periphery: ft, Py 4H hnun .
.
dejim iiiJ lysis v4 L'CII releuMng bft
with a scar ( Fig- 47.1 ). susceptible to ethanol , ether mid low concentrations
Copyrighted material
424 * Textbook oi Microbiology *
- |--
" J HTULLH 111 ri J.1111 [ v n jSMitLitrd ivilh I he cliscjM!
Copyrighted material
Hidden page
426 * Textbook ol Microbiology
or [ype-ipioc nuicrtf -IF. The CF rest is used fmgtre or fondles . FtieS may Transmit the infection
mainly in invasive chlamydial Infections
psittacosis and I ,CIV. A fourfold rise in titres is
— mechanically. If may also be carried by dust, in
which case infection may he facilitated by minor
diugrtostic- AH low titrv, grnup- specific .intibody abrasions caused by dust particles. The incubation
-
ITLJV be present m tliL sera of many persons due to
exposure to other chlamydias, a single CF antibody
period is Variable and influenced by the dose of
infection. Onset is insidious.
test is not diagnostic of psittacosis nr I ,GV unless Trichoma has been classified into several StagCS -
the rirn: is high - 1 :64 or greater. CP rest is of Thc earliest is trachoma dubium, where the disease
tittle value LII TR 1 C infections. i ] L which iniero-IF is jutf a suspicion. Protrachoma is the stage of
is more useful. Micro- IF tan test IgG and IgM Conjunctival lesion before follicles become visible
nntibodv sepa rifely. Titres of 1 or gtiea ter a re u &n ,il The inclusion bodies arc nor usually demonstrable
in infecred persons , Enzyme immunoassays also art in these early stages . Established trachoma
xvailubfe.The initial antibody response is IgM , progresses through stages 1 — IV. Infectivity is
which is replaced by IgG after about a month . maximum in the early cases . Stage IV is
Recurrent infection with the same serotype induces non infectious.
only IgG response . As llow tirre antibodies are Laboratory diagnosis : The characteristic
frequently seenin healthy individuals,, the diagnose it IOLIUMDIIV ( HfJbcn&cdter Avuacdt or HPbodiet )
criteriiL lor serology are Seroconversion, fourfold maybe demonstrated in conjunctival scrapings, after
rise in IgG t it re or presence of JgM antibody. High Staining hy
Gierma, Castaneda or Machiavcllo
title antibodies are usually Seen only ill infant methods. Because they possess a glycogen matrix
pneumonia , ntpuipris and LGV, they may be stained with iodine which enhances
Demi nutrition of hvpersensi tiviry bv shin ten ting the sensitivity' of smear diagnosis.
Fnci 's test ) w .ts widely used formerly for diagnosis
^
of LG V but has been given up because false positive
The chlamydia may he grown in the yolk sac of
6- ft days old eggs. Tile material is treated with
results arc very frequent. streptomycin or polvmyain B before inoculation .
The eggs are incubated af .15 ; C in 3 humid
CHLAMYDIA TRACHOMATIS atmosphere. Blind passages may be necessary for
C , aachotnatis is a leading cause of ocular and isolation. This method is seldom used now as it is
genital infections worldwide . rime consuming, cumbersome and relatively
Trnthomn: Trachoma is a chronic keratocon - insensitive,
junctivitis characterised by follicular hypertrophy , I issue culture using Stationary phase cells
papillary hyperplasia , panrus Formation. and in rhe ( ncmreplieating cells) is the method of choice for
[arc stages, cicatrisation. 'Hie name trachoma is isolation, McCoy cells rendered nonreplicating by
derived from [ be Grech rraAJzus ( rough ) referring irradiation nr anrirnctaboiitcs arc used. IIcLa or
to the roughness of the conjunctiva in the- disease. HL «11* treated with DEAE dextran may also be
Though Halhcrstaedter and ProwiL7.rk in 190" used. The inoculum has to be driven into the cells
transmitted the infection to orangutans and bv centrifugation upco 15 ,000 g to get a good
demonstrated in conjunctival smears the growth.
characteristic inclusion body that bw their names, TrciHmcnt ; Local application and oral
cultivation of the chlamydia became possible only administration of erythromycin and tetracycline or
half a century later, when Fang and colleagues other suitable antibiotics should be continued for
(1957) grew it in the yolk sac of eggs. several weeks. A sirglc-dosc azitromycin treatment
Infection is Iran 5 m it ted from eye - to-cyc by has been used with good results.
Copyrighted material
Hidden page
42S * Te- n tbook ot Microbiology
LYMPIItHiKANl t.OM\ VBNEREUM with litres of 1:64 or more in CF rest and 1:512 or
' [' more in micro- IF. Serological diagnosis LS
hi ' sexually transmitted disease, dunmtrisfd by
vUppurariw in uinul adcriritls , has been known in therefore feasible
^
the tropic* fhr a long time tnielt- r various names: A a iittrftdeful resL originally described by Frei
in 1925 was commonly used formerly Tbe crude
lymphogranuloma inguinale, porademns, climatic
tjftropical bubo . [ r is caused by the LCV serOvar; chlamydial antigen originally obtained From the
bubo pus, and later from mouse brain or yolk sac
of Chfoin, trachomatis , Ll , L2 and LJ most
commonly L2. LG V morns arc more invasive than
— cultures ( Lvgranum ). was inoculated irtradermally
in the forearm, with a control on the other arm.
P
-
patients develop h igh title - cJ L irvulati ng an tiln ulies ,
'
to a fatal pneumonia. Though pneurncmig is the
Copyrighted material
1 Chlamydlw * 429
possessed the group -specific antigen in common coronary artery atheromatous plaques , and the
with C. psjttaej and C, trachonwti' but could he
distmjjuishcd from both of them by species- specific
- experimental induction id atherftffia in rabbits
infected with the chlamydia strengthen the
,
KnuMpinen M and. P Sajklui IW5. ftwumanid AM fo Chlamydia pneumoniae. Cfa Infixt Dtf 21 Suppl . 3, S-244, "
Peding RW in4 RC Bnitihani 19%. ChLimydiae *s pathogens Emerging Infect Di* 2:4,
.
Unicellular Ttntronrganisnis may he classified in Viruses occupy the twilight zone that separates
-
descending order of complexity as i uJfcaiyorejj, such ihe 'living" I TO 11 the ‘nonliving’. The demonstration
]
as protuzua mid fungi „ and ptoLtryoicy , such as bv Stanley [19J5) that viruses could be crystallised
bacteria , mycoplasmas, rickeltRiae and chidmydiac - like chemicals , and the extraction by Gcirer and
Viruses do not fail strictly into the category of Schramm ( 1956 ) of 'infectious nudeie acid' from a
unicellular microorganisms as thev tlo not possess virus that could infect host cells and yield complete
a cellular organisation . Even the simplest of V i r u s progeny made it appear that viruses were only
microorganisms are cells enclosed within a cell wall , '
living chemicals'. Recent advances In molecLLljir
containing both types of nucleic acid ( DNlA and biology seem to make rhe distinction between 'life: 1
KNA ) , synthesising rheir own macromoJecnlar aiui 'nonlife' little more than a semantic exercise.
constituents and multiplying by binary fission . As the smallest Thing units', viruses offer the hest
Viruses, on the other hand , do not have a cellular models for understanding the chemistry of life'.
organisation and contain Only one type of duel tie The medical importance of viruses lies in their
acid, cither DNA or- RN
a -
'A lmt never
.. . - both
- - . Thev
'
/ ability to cause a very large number of human diseases.
are obligate intracellular parasites. They lack the Viral diseases range from minor ailments such as the
enzymes necessary fnr protein and nucleic acid common cold no territying diseases such as rabies or
synthesis and are dependent for replication on the AIDS. They may be sporadic like mumps, endemic
synthetic machinery of host cells. They multiply like infectious hepatitis , epidemic Ute dengue fever or
by a complex process und no L by bii wry fission . TJ Ley pandemic Like influenza. They may be localised to
are unaffected by antibacterial antihiotiw . The majioi circumscribed areas ( as some arbovirus diseases ) or
differences between viruses and microorganisms arc worldwide (as I Ierpcs simpdjex ] . Tlte control of bacte-
shown in Tabic 48.1 . In split' of these basic rial infection with anHbiotics has enhanced the role
differences. Viruses are generally tor side red of viral infections in human d isease, Viruses can cause
micrrjtjiganisms ir medical microbiology. cancer in animals and birds, as well as in humans.
--—
Bacteria + 4 * + 4-
Mycoplasma* 4 4 * + * 4-
Rickettsiae 4 4 * + 4 4
Chlamvdiac 4 - 4- * 4 4
Viruses - - - - - - 4
Copyrighted material
< General PrmcH« Q1 Viruses » 43 L
mav be examined under the electron microscope surface of the envelope . These structures are called
either unstaim.ed or stained. Lb this [rtethud , both pepltlfiter* ( from jrcphin , mean 11 ILT enveli jpe J. A virus
the shape and size of virions can be studied. may have more than one type of pcplomcr . The
Structure and shape: The virion consists influenza virus carries two kinds of peplomers -
essentially of a nucleic acid surrounded by a protein the hemagglutinin which is a triangular spike and
coat , the capsid . The capsid with the enclosed the neuraminidase which is a mushroom shaped
nucleic acid is known as the ilucl^vcap?. id . I be structure. Envelopes confer chemical , antigenic and
fund ion of the capsid is to protect the nucleic acid biological properties on viruses . Enveloped viruses
from inactivation by nucleases andother deleterious are susceptible to the action of lipid solvents like
>pyrighted material
H dr w
432 Tfljdhook o ( Microbiology
i
*
Lilr.NtKAl. OK VIRUSES
PEPLOMER -
B ENVELOPED
ICOSAHEDRON
ENVELOPE ~
—H - CAPSOMERE — -
-- NUCLEIC AC ID — -
i CORE
CAPSID - l - -
I
A, NAKED ICOSAHEDRON
-PEPLOMER
HELIX
ENVELOPE vz
- - CAPSOMERE *
i
: L - NUCLEICACID,
CORE
• y ~ - CAPSID
C . NAKED RIGID HELIX
.
A - naked icnviliednil virus . c-i^nsi^lin|£ nl nn inner cure <il nufEdc ;iftil L'rx kiNcd h_v a LUJISILI. Ii: _ li i in;ui_- uf i-djtauibiCft.
'
[) - envelope I i 31cMI virus in which Lhf Lu.huI.IT capsid. is pliable UIMJ is ciK'kiKcd wilhm an fin cl JH:
Fig, 46. t . Design And slfiiClure- nl virions
-
RLL L 1
an: . -
neuiritivarion of virus infecUvity depends on shaped, the tobacco rtVOssuC vir r 0 shaped.
antibndics c CJ 1 he sarface a tinge ns , UiologiciE Bacterialviiusethave a n r Kuphology il; .
properties sutb a* attachment t ( ] host cellsurfiace 45.2).
< > r licEiiagglLiimatum depend cm the envelope, Some Ghtmiuril proper ) h s: Viruses contain only
viruses pn ?- R £;sH additional ’.[ ructura] feanucs. Fur one type of nucleic acid, either single or double
cxitriple, fibrils protrude from the vertices of stranded DNA or KVA . In this respect, viruses are
adetifivirui particles. unique, for nowhere else in nature i - genetic
The overall sha ]>c of the vims particle varies in intorui.itKin solely carried bv RNA Viral nucleic
different groups of viruses. Most animal vim secure .uid - mrtv bn extracted bv I rvatment with detergent
-
roughly spherical. Some are irregular and or phenol and. in the case l "i it ISCH l
pleomorphic . The rabies virus is bullet shaped, example picornavirus, pj|Micnviru . rhe extracted
Copyrighted material
* General Properties cri Vtases 4,13
*
i
i i l
uJHaici ihi
*
i
1 4
j
11
1G
$
9 7
S
Fi y. 44.2 Casparalive sizes end shapes cl different groups c! viruses, 1* Poxviruses 2. Rhabdovlrus
. .
3 Herpesvirus 4, fletnovLrus 5 Tog-ovims 6. Adenovirus 7 Parvovirus &, Plcomevirus 9 Bacteriophage .
10. Coronavkus 11 Orthomyxovirus 12- Paramyxovirus ,
Copyrighted material
a
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ri
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43« s Textbook ! Microbiology *
n
10 20 40 00 160 »0 6 JO 1260
•
4}
“’
^ ’V.
? s.
P
* •} #
*
4 HA units
A
End Pain 1 HA . unit
Fig .43.3 Viral hantgglulinillon, Virus continuing' fluid is dMsd In doubling dilutions and 0.5% jusperrslon
of chick red CEEI? added. Wrws virus Is presenl theta Is dltluse widespread even pallet or the to Mom at 1 he
wells In (he plastic plate. Where no virus is present the calls settle down tn a button lake aggregate wllh
sharp edges.
me rely the viral nuclei c acid and capsid protein retroviruses which arc synthesised partly in the
hut also of enzymes necessary in the various stages nucleus. Viral protein is synthesised only in the
ofviral synthesis, assembly and iclease. In addition, cytoplasm.
certain Regulator proteins are also synthesised
1
Biosynthesis consists essentially of rhe following
which serve to shut down the normal cellular steps:
metabolism and direct the stL|uetiii.aI production 1.Transcription ol messenger RKA (JTLRNA) from
of viral components. The site of viral synthesis The viral nucleic acid.
depends un the tv] *? of virus . In general* roost DNA 2Translation of the mRNA into 'earlv proteins'*
viruses synthesis: their nucleic Itid in die host Cell These fearly or sonstnieturaJ proteins' are
nucleus . Tbt Exceptions are the poxviruses, which enzymes \vhich initiate and maintain synthesis
synthesise all their components in the host cell of virus components. They may also induce
cytoplasm. Most RNA viruses synthesise all their shutdown of host protein and nucleic acid
components in rhe cytoplasm. Exceptions arc synthesis.
orthomyxnviruses and some paramyxnviruses and .1. Replication of viral nucleic acid-
Table 40.Z ChflracteiiSliCs Ol hemaggluiinalion by viruses
Virus Erythrocyte ipccics and other conditio
*
Influenza virus Fowl* human , guinea pig, others; Elution at 37 " £.’
Parainfluenza, mumps, F4DV Fowl , human* guinea pij', others; Elution at 37 "C;
Hemolysin prevent
Meulci Monkey, 37 *C
Togavini*— severd of Arbwimr GiXsii, pigeon , urtr Jay oltl chick: pH and
rc rrLfMiTiLr in L- u-rnri-e l
Rubella
^
|> i £conp traeday old chick; 4nC
Enrcrovirus * same CoXMckir and I CIIO
'
I Ium .rn; 4 "C and .37 1
Copyrighted n i atonal
* Gerter &l Properties Viruses * AM
genorne, viruses use dliferent strategies for the polymerase) enzyme. Double stranded DNA is then
transcription of mRNA . Viruses hive been synthesised from the RNA ;DNA hybrid. The
categorised into six classes by Baltimore ( 1970) double stranded DNA form of the virus ( pro virus)
bused on rhier replication mechanisms , is integrated into the host cell chromosome . This
dual 1: In rhe c ? sc ot fully double stranded integration may lead to transformation of the cell
DNA viruses { such as adenn - , herpes - , and development of neoplasia .
papovaviruses), the DNA enters the host cell Maturation: Assembly of daughter virions
nucleus and uses the host cell enzymes tor follows flic synthesis of viral nucleic acid and
transcription .'Hie extracted DNA from these viruses proteins. Virion assembly may take place in the
is infectious. Wit ft hepadnivi ruses which have- a host cell nucleus or cytoplasm. Herpes and
partially double stranded DNA, the duplex is adenoviruses nrc assembled in the nucleus* while
completed by a viral DNA polymerase, inside the piedrna Hid poxviruses are assembled in the
-
host Lvropla -m . The m ,iCure DNA then moves into cytoplasm- At this stage , the noncnvclopcd viruses
the nucleus, to be transcribed by host transcriptases. arc present .intraccllularly as fiiUy developed virions
Extracted hepadnavirus DNA is not infectious , but iu the case of enveloped viruses, only the
Pbxvi ruses which replicate in rhe cytoplasm form nucleocapsid is complete . Envelopes are derived
mRNA usmg polymerases contained in rhe ifirion from the host cell membrane during die process of
itself lAoirirus DNA is not infectious. budding. The host cell [nembrane which becomes
Class 2: With single stranded DNA viruses (for I lie envelope is modified hy Incorporation ol virus-
example parvovirus), the DNA molecule moves into *pecific antigens - I lerpcs viruses assembled in the
the host cell nucleus and isconverted into the duplex '
nucleus acquire their envelope from the nuclear
form. Transcription is achieved by host enzymes membrane us diev are released into the cytoplasm
ChurI : In renvi ruses, the double stranded RNA enclosed in a vesicle. Myxcmrum bud front the
is transcribed to mRNA by viral polymerases . cell surface and their envelope is formed by rhe
Clas* 4: Depending on the method of mRNA modified cytoplasmic membrane of the host cell .
transcription , single stranded RNA viruses arc The incorporation of viral antigen (hemagglutinin)
classified into two categories. In the positive strand
, on the cell membrane endows the coll with rhe
{phj.s sfntnJ, positive sertSe) RNA viruses, the viral property of hemadsorption.
RNA iirselt act as the mRNA . Viral RNA is Release: In the case ot bacterial viruses, the
infectious bv itself and is translated directly into release ot progeny virions taken place hy the lysis
viral proteins in the host cell cytoplasm ( fi>r example of the infected lracEerium . However, in the case of
picorna-, togaviruses). animal viruses, release usually occurs without cell
Glass 5: Tlte nqptfve strand (znjntu sense) RNA .
lysis Myxoviruses are released by a process of
viruses ( for example rhahdo - , nrrhmnvxo- , budding from the cell membrane over a period of
parsmyxovirijac) the RNA is ‘antisense ' , with rime . The host cell is unaffected and may even
polarity opposite to mRNA,They possess their own divide, the daughter cells contiruling to release
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440 * Texlboc* oF Microdoiogy *
system generally consist ] ng of bicarbonate in type, usually derived from wncer cells, that arc
equilibrium with atmosphere containing about capable o t com i n uous set ill Cult i vati o n
S% carbon L!in KLIJC: . This is supplemented with .
indefinitely Standard cell lines derived from
UptO S CflEf or fetal L ;ilf serum . Antibiotics are human canceiy, such as lleLu, Hep-2 and Kfi
^ '
added to prevent bacterial contaminants and cell lines have been used in laboratories
phenol red as indicator . Such media wiil enable throughout the world lor many years. These cell
most cell types to multiply with a division rime lines may he maintained by serial suhcultivatinn
,
o t 2 4 -48 hours. The cell suspc nrion is dispensed or stored in the cold (- 70 *C) for use when
'
in bottles , tubes or Pfctri dishes. The cells adhere necessary. Some cell lines arc How permitted to
to the glass surface and on incubation , divide to he used tor vaccine manufacture* for dtamplt
form a confluent monolayer sheet of cells Vcrocell tor rabies vaccine .
covering due surface within about a week . Detection of virus growth in dell
Cell culture tubes ITIAV be incubated in a sloped cultures: Virus growth ill cell cultures Can he
horizontal position , either as ‘stationary culture' detected by the following methods :
or may he rolled in special 'roller drums' to 1 . CvTopjffm effect : Many viruses cause
provide belter Aeration. Some fastidious viruses morphological changes in cultured cells in
grow only in such roller cultures - which they grow. These changes car be readily
Uased on their origin, chromosomal characters observed by microscopic examination of the
and the numher of generations through which they cultures. These changes are known W cytopathic
can be maintained, cell cultures are classified into effects' ( CPE ) and the viruses causing CPE are
three types (Table 48,3) : called ' cytopathogenic viruses The CPE 3
.
1 . Primary cdl m /fufes. Time are normal cells produced by different groups of viruses are
freshb taken from the body and cultured. They
1
characteristic: and help in the presum price
arc capable of only limited growth in culture identification of virus isolates. For example *
and cannot be maintained in serial culture. enteroviruses produce rapid CPE with ere nation
Comrnon examples of primary cell culture -s are of celts and degeneration of the e ntire cel I sheet;
monkey kidney , human embryonic kidney, measles vims produces syncytium formation;
human amnion and chick embryo cell cultures. herpes vims causes discrete focal degeneration;
Primary cell cultures are usclul for the isolation adenovirus produces large granular dumps
of viruses and their cultivation for vaccine resembling bunches of grapes; and SV produces
^
production. prominent ectoplasmic vacuolation .
2. Diploid cell SiraJ /lS ' These are cells of a single 2 . Mt t \ boli . inhibition: In normal «11 cultures *
:
type that retain the original diploid chromosome the medium turns acid due to cellular
number and karyotype during serial metabolism. When viruses grow in cell culture
subcultivation Ear a limited number of times. cell metabolism is inhibited ami there is no acid ^
After about fifty serial passugefe, they undergo production . This can lie made out by the colour
' senescence ' . Diploid strains developed from ot the indicator ( phenol red ) incorporated in
"
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* General Properties of Viruses 441
multiplying in the culture* the crvthrueytcb will enumeration are electron microscopy and
adwjrh onto the surface of cclk. Thus ! H known hemagglutination. Ry simple negative staining,, the
as hemadsorption '.
' virus particles in a suspension can he counted directly
-
4 . in tcrk - ft'rfin The growth of a non cytoparhogenje
virus in cell culture can he tested by the
under the electron microscope . The virus suspension
can he mixed with a known concentration of latex
subsequent challenge with a known particles The ratio between the virus and latex
UYtopathngEnic virus. The growth of the first particles under the electron microscope gives on
will inhibit infection by the second virus by indication of the virus count . With
interference. herragglutiuating viruses * a convenient met find oi
5 . 7:. m.sifji'jrurron : Tumour forming (oncogenic) quantitation is the determination of
viruses induce cell 'transformation ' and loss of hemagglutination times. Hemagglutination is not
contact inhibition, so that growth appear? in a a very sensitive indicator of the presence of small
piled-up fashion producing ' micrommoLirs'. amounts of virus particles. Thus approx i mutely 1 0 -
*
6. Irnmunofluorvsctncr: Cells from virus infected influcntst virions are required to produce
cultures cm be stained by fluorescent conjugated macroscopic agglutination of a convenient quantity
antiserum and examined under the UV ot chicken erythrocytes (0.1 ml of 0.5 per cent
microscope for the presence of virus antigen . suspension ). However, because of its simplicity ,
This gives positive results earlier than other hemagglutination is- n very convenient method of
methods and, therefore, finds wide application virus assav,
a
in diagnostic virology.
AHSAV til 1 NKI : C;TEVIT \
Vi HA i ASSAY Two types of inicctivitv assays can be carried out -
The vims content of a specimen car be assayed in quantitative and quanta! assays. Quantitative assays
two ways: either with reference to the total vims measure the actual number of infectious particles
particles or with reference to the infectious virions in the inoculum * while quanta! assays only indicate
only - Two methods employed for total particle the presence or absence ol infectious viruses. Using
Copyrighted material
AAl 4
-
T salbook of Microbiology *
CHO« lOAELANTO:G
MEMBRANE
t ' fl SAC
LAVIT Y .
YO . K SAC & w
-
YA
ALBUMCM
.
Fiij. 4£ 4 Cross s«tlpn o1 J ten bay clu egg
serial JikuMn of virus suspensions and with the US a ntodiiicalioiL of the bacteriophage plaque assay.
.Liu of statist ic^J methods , reasonably accurate A viral suspension is added to a monolayer of
estimates nf injectivity can he obtained in quanta ] cultured cells in a. bottle or Petri dish, and after
SOWfl
J
. allowing time for absorption , the medium is
Quailtal ra of infect Lvity can he Lurried out removed and replaced with a solid agar gel , t< > ensure
in aniinnk, eggs or tissue culture. Examples of ' liar I TIL q >read of progeny virions is confined to
endpoints used for infect huy titration are the death the immediate vicinity of infected cells. In this
ot the animal, product ion of hema glLLtiiiin in system, each infectious viral particle gives rise to a
^
jliantoic fluid or the appearance of CfE in cell localised focus of infected cells that can lxe seen
.
cultures The titre is usually expressed us the 50 '
with rhe nuked eye. bm l : foci arc known AS
per cent infectious dos- c’ ( ITT,, ) per ml , which " plaques’ and each plaque indicates an infectious
-
indicates the highest dilution or rhr inoculum that virus ( big . 48.5}. home viruses which ure
_
Mould produce an effect in Tijper cent of animals,
ggs or Cell Cultures inoculated . IIT LH calculated
ov the application of statistical methods, such as
transmitted directly from cell to cell ( for example
herpesvirus) may form plaques even without an
agar overlay. Oncogenic viruses produce cell
that of Reed and Mucnch.
The quaniil .itive inactivity assay of viruses is
transformation which can he seen as micro
tumours, Hcncc they can be enumerated by the
-
similar to rhe estimation of bacteria! viable counts transformation assay.
bv colony counting. Two methods arc available - Viruses thatJdrm pocks on CAM ( lorexample
plaque assay in monolayer cell culture and pock vaccinia) can hr assayed by counting the number
assay on chick embryo CAM. Plaque assay vm of pork tbrmcd on CAM by appropriate inocula
*
introduced in animal virology by I >U lbCOCO ( 1952) of virus. This is known as pock assay.
Copyrighted material
* Geno ; al Properties d Virus** * 443
Tilt; mutants may he of various Types. Some demonstrated experimentally in vitro and in vivo.
mutations i >1 clinical ,in ; i laboratory Interest are This may be one of E he ways by which the pandemic
those affecting virulence, host range , antigenicity Strains of the inflmen ^:i vims originate in nfilure.
and pock or [llaquc morphology. A class of mutants When a cell is 'infected ' with ait active virus
that are of great importance in laboratory studies is and a different but related inactivated virus, progeny
the conditional lethal mutant. These are mutants possessing one or more genetic traits nt the
which are able to grow under certain conditions inactivated virus may be produced . This
(called permissive conditions), but are lethal, that phenomenon is tailed OWJWtMtitn or marker
is it cannot grow under certain oilier specified rescue. N’ew antigenic variants a\ the influe ru» virus
-
condition ; ( called non permissive or rcstric five
conditions).There are different types of conditional
causing epidemics, often do not grow well in eggs
as compared to established laboratory strains.
lethal mutants but the types most widely employed When such an epidemic strain tlbr example strain
7
in genetic studies are the ' temperature sensitive ( ts) AJ iv grown in eggs along with a standard strain
Copyrighted material
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446 Mitirp rlcv] >
* faxihoOK at ^ ^
( jj. VSSEIH: V tH > v \ \ |> viruses began lit be classified into groups- based on
N IMKPJ< . T
"
( AN HR OK Vim SK $ their physicochemical and Structural features.
Till about little.- was known oJ the basic
] 950 Nomenclature and classification arc n o w rhe of haul
properties of viruses . They were named responsibility of (he International Committee on
haphazardly, based on the diseases they caused or Taxonomy nf Viruses.
o.n rhe place of their inlvian, They were grouped Vituses .ire classified into two main divisidtii
according to assumed 'tropisms' nr affinity to depending on the type of nucleic acid they possess:
different systems or organs of the body.Thu human rihovi ruses are llirssc CQlit lining RNA and
* deoxyriboviruses arc those containing DNA .
viruses were classified as dcrmotnipic., that is those
producing skin lesions (upillpor, chiekcnpoitj Further classilicarui-n is basal nil other properties sncli
AS rhe srrandedness of nucleic acid , symmetry of
measles), neurnrropic, thal is thufie affecting the
ruu-Jeneapsid, presence of envelope, size and shujic Lif
nervous system ( poliomyelitis ,, rabies ) ,
ptieucintropic, that is those affecting the rapuntbty virion .i : d number ot rapsdtnca. Short tlc -< riptiors-
1 (influenia, cortution cold ) and vifeero tropic,
trn. of the major groups of viruses arc given helow,
that is those affect jpg visceral Organs (yellow lever ,
D N A VIRUSES
hepatitis)- Hawden { 1941) made the pioneering
suggestion that viral nomenclature and codification Moxviridne JnmiT : ' These arc Urge , brick
should be based on the properties of viruses and shaped or nvr>id viruses (300 * 240 * HW p ). with
not upon host responses-. From the early 1950 s, complex structure , hiving a lipid containing an
roar, Line nr two lateral belies- and a enrr
outer
carrying i single linear molecule of double stranded
UNA . Multiplication and maturation take place in
the cytoplasm. The family is divided inm several
genera,
Herpcsviriilac fnmily: These arc medium
saved viruses L-Lintaining linear double stranded
DNA . The iweahcdraJ nuclcocapud (100 nm) has
UJ 2 eapsomers and is Surrounded hy a lipid
eoiuaiiliilg envelope . Multiplication lakes place in
111 L auckun and [liaturatinn by building through
1
VI maerial
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Virus-Host Interactions: Viral Infections
Virus- host in tersedons may be considered at antigens may confer new properties on the cells.
clif -Ferenr levels - the cell, the individual arid the For example, viral hemagglutinin appears on the
community. surface of cells infected with Influenza virus and
At tile cellular Level , vital infection ItdyonK i causes adsorption of erythrocytes to the cell surface
bmad xpertnim at effects, ranging from m apparent ( hemadsorption ) , Virus coded antigens also appear
cellular damage to rapid cell destruction , Some on the surface of cells transformed by oncogenic
viruses, like poliovirus cause cell death ( cytoeidal viruses .
effect ) oi even lysis (cyrolysis ). Others may cause Certain viruses such as measles , mumps ,
cellular proliferation {as molluscum com agiosum ) adenoviruses, cytomegalovirus and varicella virus
or malignant transformation {..is oncogenic viruses). cause damage to the chromosomes of host cells .
In some instances the virus and ho*t cell enter into Chromatid gaps and breaks in cliromosotnc 17
3 peaceful coexistence , both replicating occur frequently in cultured cells infected with
independently without inv cellular injury , a adenovirus types 12 and ill.
condition known as ‘steady state infection '. In tissue The most characteristic histological feature in
culture viral infection may lead to readily observable virus infected cells is the appearance of uhittaan
.
cellular changes ( cyiopathic effects) These may not bodies. Inclusion bodies arc structures with distinct
paraltel the changes produced in the infected size , shape, location and staining properties that
animal , as in the latter situation infection is can he demonstrated in virus infected Cells under
influenced by the various defence mechanisms of the light microscope. They may be situated in the
the body. cytoplasm (as with poxviruses) , nucleus
Cellular injury may be due to a number of causes. ( herpesviruses ) or both ( measles virus ), lliey arc
Early or nonstiuccura] viral proteins often cause a shut - generally acidophilic and can be seen as pink
down of host protein and DNA synthesis. Large structures when stained with Giemsa or cosin
amounts of viral ui . iciomalcculcv that accumulate ill methylene blue stains. Some viruses (for example
the infected cell may distort the cellular architecture adenovirus) form basophilic inclusions .
and exert a toxic effect. The permeability of plasma Demonstration of inclusion bodies helps in the
membranes may be altered , releasing lysosomal diagnosis, of some viral infections. The presence of
enzymes and leading to autolysis. intracytoplasmic eosinophilic inclusions ( Negri
Many viruses produce alterations in the bodies) in the brain cells of animals juttific ^ the
cytoplasmic membrane of infected cells. Some (such presumptive diagnosis of rabies. Vaccinia infected
as respiratory syncytial virus) cause fusion of cells show rather smaller multiple inclusions known
adjacent cell membranes, leading to pofykaryocytosis 55 Guarnicri bodies. Large inclusions ( Bollinger
or syncytium formation. Virus coded antigens may bodies ) are seen in fbwtpOOL Inclusion bodies in
appear on the surface of infected cella. These molluscum contagiosum ( molluscum bodies ) are
Copyrighted material
450 * Tsidbook of MfemtMi3Jogy
very Large (2D~30|iin) and can he readily -seen under secondary diseases, ending fatally after many
,
Copyrighted material
i Virus-Host Inieract .ons : V- ral Infections 451
blood. Rabies virus travels along the nerves to the SltJMrirANtlH OF THK l t d '
ll A TI O N
—
Lind produces lesions In remote target sites, the
incubation period is longer 10- 20 days , as in
chickcnpox or polioolyelitii. There are, however,
exceptions to this rule . In arbovirus diseases, as in
SPREAD OP VIRUS IN LHIK BODY yellow fever or dengue , the incubation period may
,
The manner in which the infecting virus sjueads be shorter \. S 6 days), probably because the virus
in introduced directly into the bloodstream by the
from the poi nt of e ntry, multiplies in sites of election
mid causes lesions in target tissues was fiist studied insect vector*. The incubation period in type R
by Fenner ( 1 48 ) using mousepox: as the hepatitis may be 2 ~ f > month* and in slow viral
^
experimental model ( Fig. 49.1) . The mousepox infections, many years. Papillomas and molluscvim
-
vims enters the - kin, where it multiplier initially
and proceeds along the lymphatics to the local
coniagiosum have Long incubation periods, probably
because the viruses myltipty slowly:
nudes. After usultiplication in the lymph nodes, the HOST Itiispoissns T O VIRUS
vims, enters the bloodstream ( primary vireima) and INEECTIOMK
is transported to the spleen and liver which act as
the ‘central foci ' for viral multiplication After The outcome of a virus infection is influenced by
attentive multiplication LO the centra! foci, there rhe virulence of the infecting strain and the
occurs a massive spillover of rhe virus into the resistance offered by the host. Mechanisms of host
bloodstream (secondary viremsa) . This heralds the resistance may be immunological or tMQfpeciflc.
onser of elfe -Lcil symptoms ( the prodromal phase The latter includes various genetic and
in eruptive feverc). The virus reaches the target physiological factors such as interferon production,
organ ( skin in eruptive fevers ) through the body Temperature , nutrition and hormones.
bloodstream. Multiplication in the target sites Immunity in virus infections: Virion in
*
produces the distinctive lesions . With minor general are good antigens and induce both humoral
modifications , this model holds good tor most and cellular immune responses. The multiplication
systemic virus diseases . The reasons for the of a vin '- in die body during infection induces int
1
difference in the foci of multiplication and tmget only a t \ .unlit atively greater iinn JILL FMpGrje nut
organs in the case of different viruses are also liberates and makes available to the immune
obscure. system the whole range of virus antigens, including
Copyrighted materia
4S2 * Textbook of Microbiology
Sbtfn inyKiQfi
Or ^ =
mdiLp lci irirjn
lymph d« -
«
14 mijijpficfltian
8 »
Primary
vifaerma
2
s
/ p Q&s
3 i Mollification
Mulirp in
CentfSi foo"
< and sptean
§ 4-
2
Secondary
5" '
viraemia
>* Antibody *n
> *um
§js
S vv lryj erf
^
& l foot - primary lesion
s<
"a
Early rash +
9^ L pBpulH
3 10
U»te rash-
11- .
JCBr itKXl
F: g 49.1 Pathogenesis of mousepox e model lor acute exanthemata of humans jailer Fanrter )
neutralising. Antilsodik's to surface antigens vary in Some viral infections cause a suppression of the
then ncu. tr3ili -. iiig ability. For instance, two types of
'
mmune response. Measles infection induces a
surface antibodies appear following influenza temporary depression of delayed hypersensitivity to
—
infection antihcm &ggluri n i n and antineur
ami ^ idase- 'l l- c termer can neutralise inactivity tmt
- tuberculin. Infection of adult mice with lymphocytic
^
the latter cannot. Antircuraminidasc antibody can,
choriomeningitis or leukemia viruses inhibits
anfibodv resjvrnHe to other antigens. HIV strikes at
however, i nh i h: t the release of progcnal virions from the centre of the immune system by infecting the
infected cells. Some antibodies can paradoxically CD4+ helper T cell.
enhance viral infecrivity, Humoral antibodies may In general, viral infections are followed by solid
Homelim.es actually contribute to pathogeH.esis . immunity to reinfect ion, which may often be
Antibodies mav cause complement dependent injury 1 ifelong. Apparent exceptions I i ke the common cold
to cells or induce an immune complex type of r issue and influenza arc not due to lack o! immunity hut
injury. The enlianced severity of respiratory syncytial to reinfection being caused by antigenically different
viral infection in early infancy is believed to be due vi ruse 5, L ive vi ms vact i ties also induce more durable
to the presence of passively acquired maternal protection than bacterial vaccines.
antibodies. In older children who have no antibody,
the virus causes a milder disease . The pathogenesis N o n tinlimnological K e s p u n s c s
of some viral hemorrhagic fevers is immune Jr Ph$gOCytO$!5: Polymorphonuclear leucocytes do
thrombocytopenia , Most extrahepatic lesions in not play any significant role in the defence
type R hepatitis are due to damage caused by against viral infect inns. To fact , more viral
immune complexes. diseases are characterised by a
Cell mediated immunity is ofcrilivaJ importance polymorphonuclear leucopema. On the ocher
in viral infcctions-The earliest indication of cell hand, macrophages phagpeytoHC viruses and are
mediated immuniiy in viral infection! was the important in clearing viruses from the
demonstration of delayed hypersensitivity following bloodstream.
vaccination in immune individuals. Similar skin 2. Body temperafurr Fever may act as a natural
reactivity is also seen in mumps. The normal defence mechanism against viral infections as
resistance to vi ms infections shown by most viruses are inhibited hv temperatures above
again miglobulincmics is ascribed to their cell 39 C . An exception is herpes simplex which is
mediated immunity; though it may also be due to usually reactivated by fever to produce lfever
interferon or Other non immune mechanisms. blisters '. Herpes febtilia is a frequent
Individuals with deficient cellular immunity show accompaniment of fevers caused by pneumococci ,
a heightened susceptibility to infection hv the streptococci , influenza virus and tnalana
-
herpesh pox and me - ivies v: rusr The administrtU ion
of antilymphocytc serum inducts fatal infection in
parasires, hut for some unknown reason, is very
rare in other fevers (typhoid, tuberculosis).
mice Injected with a sublethaldose oftbeectromelii 3* fformwffi Corticosteroid administration
virus. Cell mediated immunity is considered to play enhances most viral infections. Coxsackie virus
a m.ijor role in recovery from viral infections in B1 does not normally cause disease in adult mice
which viremia is not important and in which hut will induce .i fatal infection in. mice treated
mfccted cells have viral specific antigens cm their with cortisone. Normally mild infections such
surface . In some virus infections cell mediated as varicella and vaccir. ia may be lethal in
immunity may contribute to tiHSue damage, as n patients on cortisone. Injudicious use of steroids
lymphocytic choriomeningitis ill mice. i n the treatment of herpe tic keratocon i unctivitis
epithelial cells following stimulation by viruses or been reported in che use of interferon as an
polynucleotides. It is i glycoprotein. anticancer agent * particularly in lymphomas but
Gamma interferon (IFN-y), formerly known as there have been reports of toxic effects in cancer
immune interferon * Is produced by T lymphocytes* patients given high doses of interferon.
on Stimulation by ant inerts or mitogens. It a Although interferon was first recognised as an
glycoprotein , Tt is more concerned with antiviTal gcnt ; iris now known lobe a more general
i inmunomoduktoiy and intiprolilcntivc function! ^
regulatory peptide belonging to the class of
than with antiviral defence. It alsodiffers from alpha cyn>£inr& The main biological effects of interferons
and beta interferons in having a separate cell are the following:
receptor. 1, An:mral effects : Induction of resistance to
Interferons are inactivated by proteolytic infcctinps-
enzymes but not by nucleases or ]i [';iscs. Thev resist 2 Anrimicmbid effects: Resistance to intracellular
,
heating at 56— 60 —
for 30 60 minutes and are
stable over a w ide range of pH ( 2-10) , except
infections , for example toxoplasma, chl . Lnnvdi . L ,
malaria ,
.
gamma IFN, which is labile at pH 2 They have a 3, Cellular effects: Inhibition of cell growth and
molecular weight of about 17,000* are nondialyaable proliferation: and ofDNA And pro!ein synthes is;
and nunsetlimentable ( IQOJOQO g). They are poorly increased expression of MHC antigens on cell
antigenic, so no routine serological tests arc surfaces,
available for their detection and estimation. 4, Immunoregulatoiy effects: Enhanced cytotoxic
Interferon assay is based on its biological activity activity of NK, K and T cells; activation of
such as the ability to inhibit plaque formation by a macrophage cytocidal activity modulation of
sensitive v irus. The potency of 1 FN is expressed as
^
antibody formation; activation of suppressor T
International Units (1U ) per ml. cells; suppression of UTH.
Many properties of interferon make it an ideal
candidate for use in the prophylaxis and treatment LABORATORI DIAGNOSIS OF VIRAL
of viral infections; it is nontouic * nonancigenic, ll> t s a -: AS KM
diffuses freely in the body and has a wide spectrum Technical difficulties in virus isolation and
of antiviral activity, The rmijor drawback initially identification* the length of time required for these
was its species specificity - interferon produced by procedures and the lack of specific therapy lor viral
nonhuman cells was not clinically useful. This was infections have contributed to the sparse use of
overcome to some extent by producing interferon diagnostic virology till recently. The situation has
from buffy coat leucocytes from blood banks * with changed in recent years, With the development of
NDV or Sendai vims as the inducer. Now, human rapid techniques for the diagnosis of many vims
interferon is available in unlimited quantities infections and the availability of Specific drugs
following its commercial production by cloning in against at least a few viruses, diagnostic virology is
bacteria and yeast . Even so, its initial promise as an fast becoming 1 routine procedure.
antiviral agent has not been fulfilled . Local The demonstration of viral infection in selected
application of high doses has shown some benefit groups of persons ( screening ) is an important
against upper respiratory infections * herpetic procedure Ln the prevention of some diseases (such
kcratitis and gen ital warts. L. i mited success has also as screening for HBV and HIV in blood donors).
been reported against generalised herpes infection Etiological diagnosis of viral in lections is useful in
in immunocompromised hosts * and against hepatitis many ways . It is of vital important in some cases * as
Ji and C infections. Some encouraging results have in rubella in pregnant women . It helps institution
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m + Textbook o Microbiology
*
ut early specific therapy as in herpetic encephalitis the capability of the virus laboratory. The
and. lesions of the eve. It serves to define the etiology appropriate specimens should be collected Grom
of vague syndromes such as upper respiratory patients* preserved and transported to the laboratory
infection or aseptic meningitis. It is essential for in the proper manner along with pertinent clinical
the detection and prediction of epidemics and the and epidemiological information (Table 49.1 ),
identification of antigenic variation in viruses. It is In the laboratory the following methods are
invaluable in the prompt control of outbreaks . It commonly employed; microscopic demonstration
may lead to the discovery of new viral infections. of the virus or its inclusion body, demonstration of
Successful diagnosis at viral infections depend
, the virus antigen, isolation and identification of the
,
as much on the awareness of the physician as on virus, or detection of the specific antibody.
Central nervous system Feces, blood Brain biopsy ( IF -Sc EM); Paired sera
(for arbovirus isolation ) CSF ( EM &L IF)
CSF, (brain biopsy,
throat swab, rectal swab )
circumstances
IF = Jrnmun0fluAKKflkCC; EM Electron Microscopy, ID Immunodiffosion; LM = Light Microscopy
Tor diagnosis of rabies only
( Adapted from W1 IO)
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f -
Vir U B t+osl Inleracliofis : Vital Inlad uns 457
Microscopy; The demonstration of virus the mere recovery of a virus from a patient th »cs
elementary bodies by examination of stained smears not justify the assumption that it is the causative
is now seldom employed. The detection of virus by agent nf the patients illness . Many viruses ( For
electron micTORropv is being Lined increasingly- In example adenoviruses, enteroviruses) are frequently
some diseases , it used to be the only diagnostic found in normal individuals . "lire results of isolation
method ( for example viral diarrhea ), Demonstration should always be interpreted in che light of the
of the inclusion body is a routine diagnostic method clinical data. Demonstration of an immunological
tor rabies in dogs. The microscopic diagnosis ol response to the virus isolate in tlie patient during
rabies has hecn rendered very sensitive by the course of the disease reinforces the significance
fluorescent antibody techniques . 'Hie use of direct of die isolation .
and indirect fluorescent antibody techniques for the Scroingienl diogiUMids: The demonstration Lit
examination uf material from lesions, as well as for
the early demonstration of viral antigen in tissue
-
a ri - c in titre of antibodies to a virus during the
course of A disease is strong evidence that it is the
cultures inoculated with Specimens has enlarged etiological agent. For this , it is essential to examine
the scope and greedy increased the speed of virus paired *erfl, the 'acute' simple collected early in
diagnosis. the course of the disease and the 'convalescenr '
Demonstration of virus antigen: In ases ample collected 10-14 days later Examination of
where virus antigen is abundant in the lesions, its a single sample of serum for antibodies may not be
demonstratiori by serological methods such as meaningtul except when IgM specific tests are
precipitation in gel or immunofluorescence offers done. The serological techniques employed would
a rapid method of Lliagnosis , Highly sensitive depend on the virus bui those in general Use are
serological [ M T S such a s con nterimmuno - neutralisation, complement fixation, El . ISA and
eEcctrophoresis , radioimmunoassay and enzyme hem agglutination inhibition tests.
linked immunosorbent assay have found wide Mnlccultir diagnosis; The availability of
application in diagnostic virology for the detection molecular methods has transformed the diagnosis
of viral intigeiu in clinical samples. Molecular of viral diseases, enlarging the scope, sensitivity and
methods such as probes and polvinerase chain specificity of such tests .
reaction provide rapid , sensitive and specific
information about the presence of viruses in clinical IMMUNOPROPHYLAXIS OP VIHAI
sample s. Thev are fait liecoin i ng routine diagnostic D I S K \ * E -. S
methods in the affluent countries, hut in the poor Prolonged and effective immunity is a characteristic
nations, their availability is limited . of most viral infections. Viral vaccines also confer
Isolation of virus For virus isolation it is solid protection and are, iq general, more effective
imperative that fbe specimen be collected properly than bacterial vaccines. Viral vaccines mav be live
Jr
and transported with Least delay to tire laboratory. at killed (Table 49.2 ) . Live Vaccines are more
As niost viruses ire heat labile , refrige ration is effective than killed vaccines . Tlie smallpox vaccine
essential during transport . The methods used for lias been used at the sole tool for the global
isolation depend on the virus sought. In general , eradication of the disease. The early live vaccines
they consist of inoculation into animals, egg* or were developed empirically from natural viruses (as
tissue culture, After the specimen is processed to Jcnncr 's cowpc* vaccine) or by attenuation by serial
remove bacterial contaminants. The isolates are passage (as yellow fe ^ er vaccines). The basis of the
identified hv neutralisation or Other Suitable latter technique was ail unconscious selection of
serological procedures. It hit to he emphasised that avimlent mutants . With the development of more
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45« + Textbook Of Microbiology *
Copyrighted materia
Hidden page
m H Itaxtboofc a\ Wic 'st dogy >
own thymidine kinase fl ISV, V- Z ) are for more Foscarnct Triaadium phosphnnnfnrmate )
(
susceptible than tKobe which do not (CMV- EBV ), specifically inhibits DNA polymerase of the herpes
The related drug Ginci ^iimr LS more active against simplex virus and has some effect against hepauus
CMV. If and HIV also.
The widely publicised drug Azidorhymidinc Suramin developed as an anti parasitic drug in
( Zidovudine, AZT ) used against LLIV infection is 1916 was found to inhibit reverse transcriptase
a rhymidint analogue which blocks the svnlhe -- is activity and so was one of the first drugs ut-cd against
of proviral DNA by inhibiting viral reverse AIDS- Because of toxicity and inadequate efficacy
transcriptase. AZT is used widely m HTV infection , us use was discontinued.
h . IE is toxic and costlv
.s . Interferons: The discovery of interferons, with
A series ot Jideosynudetjsides DiiJ .drt £>.Sirie,
1
activity against a wide range of viruses raised hopes
StJWJtJjnf , Lamivudine ) have been of their application as anriviral drags. However,
svnthesised and found tn possess anti - Hl \ activity most trials have been unsuccessful. Eenefici .il effect
by blocking reverse transcriptase . The second group has been obtained in persistent infections such as
of drugs used m Hl \ infection is protease inhibitors hepatitis B and C, laryngeal papilloma and against
(SarjttjjMVjr; /JjfojiJt irh ftidjViiiVJ-r) , CMV infecth ins in transplant recipients. High doses
/Eifctfwmj { VitAZoh ) is a synthetic nucleoside of interferon lead to toxic effects .
related to guanosme. It shows activity ags.inst many In spite of intensive efforts, progress in the field
DNA and RNA viru-ses. Administered as an of antiviral chemotherapy has not been satisfactory
aerosol, it has been effective in the treatment of Various (actors contribute to this. Many compounds
respiratory syncytial viral infection and also in show antiviral activity in tissue Culture but most of
mllurns. Intravenous ribavirin has been reported to them prove to be ineffective or toxic in animal tests.
be effect! ve agai ost Lass* fever and other hemorrhagic The available drags have a narrow range of activity.
fevers. They are seldom able to eradicate the virus from
i liiur A man rad: nr i Aifamantananune the host so recurrence is common. Viruses develop
hydrochloride, Symmetrel ) blocks host cell resistance to the drugs and break through infection
penetration bv influenza A urus but nor Bor C. A takes place even duemg t r e a t m e n t . The AIDS
derivative rimantaibne is less tDxic and equally pandemic has been a catalyst in development of
effective, A second line of drugs against influenza antiretroviral drugs . It is hoped that better
employs neuraminidase inhibititmr understanding of the molecular and cellular biology
Enviroxinc and related chemicals have shown -
of viruses and of virus host interactions may lead
to the development of more effective antiviral agents.
activity agtimsr rhinoviruses.
Further Reading
Collier L andj Oxford 3000. Hvman Virology, London: Oxford University Prrse .
Fields DM et al . 19%, pyroJqgy 3 J tdn. Philadelphia: Lippmoolr- Raven .
Harper UK . 199 J . AfofeorJar Vlm/q T. Oxford: Bios .
^
Zuckerman AJ ft all 3000 . Ciinjcif Vjra/op; llh ed . Cbicheiterjohn Wiley.
Copyrighted materia
L J Bacteriophages
*
Bacteriophages (commonly abbreviated as phages) are Phages occur widely in nature in close
viruses tliat infect bacteria. Twurt ( l 9 l5) described a association with hacterij. They be readily
can
degenerative change in staphylococcal colonics isolated from feces* sewage and other natural sources
isolated from calf lymph, which could be transmitted
, ofmbted bacterial growth. Early hopes that phages
serially by application of culture filtrates from the could be used in the treatment of bacterial infections
original growth. d’Herelfe (1917) observed that have not been fulfilled but these viruses have
filtrates of feces cultures front dysentery patients Contributed much to microbiology. As phages could
induced transmissible lysis of a broth culture of a be grown easily on bacterial cultures, they provided
dysentery bacillus. He suggested that the lytic agent
was a virus and gave it the name bacteriophage.
the only convenient model for the study of virus
host interactions at the cellular and molecular levels
-
\Z _ B
c
D
'
6
Flg.SQ . 1 Morphology oP bacteriophage . A. hevagonat head , B. DMA cote, C. demarcation between head and tailr
. F. tail litres . G- prangs ; Right, process of injection ol phage DNA into host cell
D. tail, E . baa ? plate
Copyrighted material
462 * Textbook d Microbiology
before The dcvrloprOCnt uf cell culture IftlutUJUtS bacterial genome , replicating synchronously with
made similar studies with animal vim ^- es ] n.sn ^ iL )lc . it, causing no harm to the host cell ( Fig. 50.3),
Phagts piay an important mV in the transmission ! lie eyeduf Replication of a virulent phage can
of genetic information between bacteria by the be considered in the hdlowing stages adsorption,
process of transduction . The presence of phage penetration , synthesis of phage cornpon*nt 5 n
genome integrated! with bacterial chromosomes assembly, maturation arid release of progeny phage
confers on bacteria. certain properties by a process particles .
known as phage convttsii JJI. Phages have been used Plixgt' particles Come into Contact with bacterial
ns cloning vectors in generic manipulations.. 'I'hc cells by random collision . A phage attaches to the
presence of high corcci' iirnitioiLH of phage particles , surface of a susceptible bacterium by its tail.
upiu 10s per ml in some natural waters suggests Adsorption is a specific process and depends or
that they may have ; L role in the control of bacterial the presence of Complementary chemical groups
populations in such environments. The specificity on the receptor sites of the bacterial surface and or
ot the host range ot phages is the basis of phage the terminal base plate of the phage. Under optimal
typing methods by which bacteria can be identified condition ?.. adsorption is- a very rapid process, being
and typed. complete within minutes (.iertaip oofcctoro, such
,
Morphology ; Certain hactLTHiphages thaL irtlkt as cations , are necessary for adsorption . The
£, coli , called the T even phages (T2, T 4, T6) h
have been studied in great detail and traditionally
serve as the prototypes in describing the properties
of bacteriophages.
T even phages have a complex and characteristic
morphology. They are tadpole shaped , with a
hexagonal head and u cylindrical mil . The liead
consists. of a rightly packed core of nucleic tcid
(double stranded DNA ) surrounded by ii protein
coat nr capsid . The SLAV of the head Varies hi
different phages from 2H n. m to 100 nm . The tail JS
composed of a hollow core, a contractile sheath
surrounding the core and a terminal base plate 1
which has Attached to it prongs , rail fibres or both
1
n - 1 Sl
.
( Fig. 50.1).
Though im > si bacteriophages have the
morphology and structure described aljuve, phages
that are spherical or tilamcntous and possess
single stranded DNA or RNA have also been
identified .
1 . iff eycks Phages exhibit two dif 'fercm types
of lifecycle. Irt tile virulent Or fyiic cycler ,
intracellular multiplication ot the phage culmin -Ate -s
in the Ivsis of rhe host bacterium and the release of
m .
Fig . 50 2 Bacteriophages [ t 200,040) Multiple intention
progeny virions , In the temperate or fysOgvnk tytJe PI a bacterial ceil ( lysis from Mlhouti ( Source;
the phage DNA becomes integrated with the HlCED )
* Bacteriophages 463
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phage DMA . D replication ol phage DNA, E. production of phage components, F. assenbly of phage ,
G . release of progeny phage, H. integration of phage DMA with host chromosome, I. binary fission of lysogenic
bacterium , J . daughter bacteria Carrying prophage, K . eiCl$lQn ol prophage, L. same stage ± 6 C. ( A Ip C
injection; D ID G lytic ; H to J lysogenic cycle ; K tt> L induction. )
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synthesised separately in the bacterial eel ] . The the host chromosome and replicates synchronously
UNA is condensed into a compact polyhedron and with it. This phenomenon is called JyKjjejry md a
’ packiged ' into the head and , finally, the tail hacte ri urn that carries a prophage with i n i rs ge iiome
structures are added - Thi* assembly of the phage is called a fy*qgEajv bacterium. Lysogenisarion does
components into the mature infective phage particle not upset the bacterial metabolism.
is known as maturation. The prophage crullers certain new properties
Release of the mature progeny phages typically Lin the lysogenic bacterium . This is known as
occurs hy lysis of the bacterial cell . During rhe h yugetuc conversion or pJutgr conversion. Tiny is
replication of the phage, the bacterial cell wall is due to the synthesis of new proteins dial are coded
weakened and if assumes a spherical shape . Phage tor hi the prophage UNA. An example is Toxin
'
filliyma act on the weakened cell wall causing it production by The diphtheria bacillus . which is
,
to burst or lyse resulting ill the release of mature determined by The presence in it of the prophage
daughter phages. herd. The elimination of the prophage abolishes
The interval between the entry of the phage the toxigenicity of the bacillus.
nucleic acid into the bacterial cell and the During the multiplication of lysogenic bacteria,
appearance of the first infectious intracellular phage the prophage may become 'excised ' Emm occasional
particle is known as the tcllpst phase. It represents cell s.The exci sed prophage init iates lytic repliCation
the time required tor the synthesis of the phage and the daughter phage particles arc released ,
component* and their assembly into mature phage which in tec r other bacterial cells and render them
particles. The interval between the infection of a lysogenic. This Is KNOWIL as spontaneous induction
bacterial cell and the first release of infectious phage of prophage . While this is a rare event , all lysogenic
,
particles is known as the inenr/xnaci. I tin mediately bacteria in a population can be induced to shift to
following the latent period., die number ol phage the Ivtie cvcle hy exposure to certain physical and
particles released increases tor ;L tew minutes till chemical agents . Such i mind rig agents include UV
the maximum number of daughter phages is rays, hydrogen peroxide and nitrogen mustard .
attained. This period, during which the number of A lysogenic bacterium is resist , mt to reinfection
infectious phages released rises , is known as the by the same or related phages. This is known as
rf >e - period. The average yield of progeny phages fupericfecrirxD immunity.
per infected bacterial cell is known as the buTJf Bacteriophages may act as carriers ol genes from
size. This is estimated bv experiments in which one bacterium to another . This is known as
infection is established with ore phage per fransdircrion . Two types of transduction are
bacterium and the release of infected phage particles recognised - In restricted transduction, only bacteria I
is estimated serially over a pencil of time. The genes contiguous to the prophage are transmuted .
result* of such an experiment plotted on a graph is For example, rr JUSLIUCIION hy the pmpha c htaibdu
known as the OIK -step growth curve ( Fig , 50, 4) , .
in E calr K 12 transfers only the gal gene ^ '
Lyngenio cycle Unlike virulcnT phages which ( determining fermentation nt galactose ) , which is
'
produce lysis of the host cell, temperate phage* the bacterial gene contiguous to the prophage . On
enter into a symbiotic relationship with their host the other hand, any bacterid gene inaylfe transferred
cells without destroying them . Following entry into in generalise! trmfductioruTniwducrion has been
the host cell, the temperate phage nucleic acid demonstrated in many genera of bacteria and
becomes integrated with die bacterial chromosome. constitutes one of the most important mocha nis ms
The integrated phage nucleic acid is known as the of genetic exchange among bacteria in nature -
prophet . The prophage behaves like a segment of Plasmid mediated drug resistance in staphylococci
Copyrighted material
466 * Textbook o4 Microbiology *
in an example of a medically important property staphylococci. Adapted phages, aedve only against
that is transmitted bv transduction .
•iff
fresh isolates possessing the Vi antigen, are used
Phage particles cxhibil general ^ Tibili’ V of type For phage typing of typhoid bacilli . Staphylococcal
and a low rate of hcrirablc vari.irinp. phage typing is a pattern method using a set of
,
Copyrighted materia
* 6 ac1eriophages 467
defective pbsgy genomes, able to code only h>r parti; bacteria are then streaked at light ingles to the
of phage particles. original inoculum . After incubation, the pattern oE'
The synthesis t > f baddunlins is determined by inhibition of the indicator strains represents the
rhe presence in bacteria of colicin-ngcnic factors bacteriocin type of the lest bacterium (Fig. 50.5).
Further Kending
Anderson IT. 1981. Reflection! on phage genetics. Ann RsvGenaia 15 :^ 05
"
Dav M. 199S. Baaenodns and bacteriophages, In TipTey & H ifemr 's .Hdi'cnjfctA y 4rtd Mitwhitl ( nfL-nion .-. , VruL. 2.
I.nndnn: Arnold-
' ^
MjdirwE CECctal («1E ). 1983, .JXfletenopA c T 4 , Washington D- C; American Soriery tor Mkrobtoiogy.
1
svrrahtet ia
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4 Poxviruses * 469
.
Flg.51.1 Structure of vaccinia virus The nucleic acrid is
ar contained within a dumb -beN shaped core ( C ).
membrane which surrounds a biconcave 'nucleoid ' FHiring Inio the concavities d Mia core are iwo lateral
containing the DNA core On either side of the
* .
bodies (LB) The virion is enclosed within a protein
nucleoid is a lens shaped structure called the lateral shell which has an irregular surface .
body (Fig, 51.1 ). The virion measures about 300 * have been identified . These include the L$ antigen
200 * 1G0 nm and so can be seen under the light (a complex of two antigens, the heit labile L and
microscope . Variola virus was first demonstrated the heat stable S antigens ), agglutinogen , and
microscopically hv Ruisr in 1BE 7. Paschrn in I 90fi hcm .igglmiinir , which is responsible for rhe
developed a staining technique for the virus particles agglutination of erythrocytes of those fowls which
and demonstrated the elementary bodies ( Paschcn ate also agglutinated uunsjiet ifu- jllv by tissue lipids.
bodies) in smears bom smallpox lesions . Cultivation and host range: The variola
Physical and chemical properties: and vaccinia vimses can he differentiated hv rheir
Poxviruses arc stable and if protected from sunlight growth characteristics and host range .
may remain viable tor months at room temperature . Chick embryo: Both viruses grow or the
In the cold or when freeze dried , they survive for CAM of 11-13 day old chick embryo producing
years. They are susceptible to ultraviolet light and pocks in 48-72 hours. Variola pocks arc small, shiny,
other irradiations. They are resistant To 5(M> glycerol white , convex, non - ueCTOtic , nun - hemorrhagic
and 1 % phenol but arc readily inactivated by lesions . Vaccinia pocks arc larger , irregular, flat,
formalin and oxidising disinfectants . The virion greyish , necrotic lesions, some of which are
consists essentially of DNA, protein and lipid . hemorrhagic ( Fig. 51.2). The viruses may also be
Though enveloped, the vims is not inactivated by differentiated by their 'ceiling temperatures' , the
ether. The virion contains a multiplicity' of enzymes. highest temperature above which pucks are nut
The entire multi plication of the vims takes place produced.1"he ceiling terrperatures art 41.(1 UC fqr
in the cytoplasm of the infected cell. vaccinia, 38 "C for variola major and 37 -5 "0 for
Antigenic structure: All poxviruses share a variola minor.
common nuclcopmtein ( NP) antigen . By Tissue cutaz/r; Variola and vaccinia viruses can he
immunodiffusion some twenty different antigens grown in tissue cultures nf monkey kidney, HeLa
Copyrighted material
m * Toxibocfc of Micros: D - cgy *
-•
** j
7
F
. 'I
Lv , _ c- J
I
and chick cmbiyo colls . Cfytopsthic effects are generations are unlikely to witness the disease but
produced by vaccinia in 24-4B hours and. more its disappearance has been too recent for it ro be
slowly by variola . Eosinophilic inclusion bodies - inquired altogether . A brief account of smalljiox is
-
Guarmeri bodies can be demonstrated in stained therefore being presented. ( For more details about
smallpox* the 3d edition of [ bis textbook may he
preparations. The inclusion bodies consist of
aggregations of virus panicles in a matrix. Vaccinia consulted -)
but not variola virus produces plaques in thick Smallpox was an exclusively human infection,
embryo tissue cultures- with no animal reservoir. There were no carriers
Animiis: The vaccinia virus can infect a wide range as the vims was eliminated completely from the
ui animain experimentally. Monkeys , calves , sheep patient on recovery. 'Hie source of infection was a
and rabbits can be infected by scarification leading patient in the early phase yf the disease* though
Co vesicular lesions. The variola virus produces Infect ivity extended from the appearance of buccal
similar lesions only in monkevs. Scarification of mucosa! lesions (enanThem s) to the disappearance
rabbit cornea with variola virus leads to keratitis ot all the skin lesion (exanthems). Infection usually
and sections of the cornea will show typical occurred only in close contacts. Virus entered the
Guarnicri hodics , Intranet instillation of the body by inhalation. After ini rial multiplication in
variola virus in the monkey produces a self-limited the local lymphoid tissues, the vims reached the
attack of smallpox with generalised skin lesluns. reticuloendothelial ceils , where further
mulriplication took place * leading to a severe
SM A I T PCIX virertiLj with seeding of the mucosa and skin
Smallpox has been eradicated.The last natural case heralding the clinical disease/ I'he incubation period
of variola major detected was Saiban Bibi * a was around 12 dav$,
’ *
Bangladeshi woman found with smallpox un ibe l he single crop of centrifugal exsnthenns passed
Karimgani railway platform in Assam on 24 jVlav, through macular, papular * vesicular and pustular
1975 - The Last Case < tf variola minor occurred in stages, before scabbing and healing by scar
Mcrea , Somalia, in October 1977 . The coming -
formation in 2 4 weeks . The exanthems varied io
FIC r
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Herpesviruses
The herpesvirus family cuntMIW ( iVet x hundred ifefilierpesi'irtJies, which replicate slowly ( more
spcc i e s of enveloped DNA viruses that affect than 24 hours), have a narrow host range, grow
humans nnd animals. ' [’ hey are L- h & ractcrised by best in fibroblasts- with a tendency In produce
their ability to establish latent infecfifmj* enabling enlargement of infected cells (cyromegaly ) and cause
rhe virus to persist indefinitely with in infected hosts latent infection of s,divan' gland and other organs.
and to undergo periodic reactivation. In culture ^ the cytopathic effect is slow and the
The herpesvirus cups id is icosahedral, composed virus remains cell associated, for example
of 162 capsomers, and enclosing the core containing cytomegalovirus .
the linear double straoded l ) N" A genome. The ( 1 jnimLificfpe.svTruse.s, which have a narrow
nucLeocipsid i* surrounded by the lipid envelope host range, replicarc in lymphoblastoid cells, are
derived from che modified hose cell nuclear specific for either li orT lymphocytes and frequently
membrane through wlti ^ li the naked virions bud cause latent infection in lymphoid tissue , for
during replication . The envelope carries surface example le- pstcm-TJarr virus.
spikes, about S nm long, Between the envelope and tight different type? of hepesvinuta are known
the capsid is an amorphous structure called the whose primary hosts are humas. They have been
tegument , containing several proteins . The officially designated ‘Human hcrpcsvini* types 1-
enveloped virion measures about 200 nm and the $ but their common names continue to lie in
naked virion about 100 urn in diameter. general use, except for types 6, 7 and S (Table S 2.1 ).
Herpesviruses replicate in the host cell nuclcus. The herpesvirus family has no common group
They form tfowdry type A intranuclear ( Lipschurc ) antigen and the different herpesvirus species do not
inclusion bodies . Like other enveloped viruses, show any significant antigenic cross reaction, except
herpesviruses arc susceptible to fat solvents like between Herpes simplex types 1 and 2 .
alcohol, ether, chloroform and bile salts . 1'hey arc
heat labile and have to be scored ar -70 "C. HERPES SIMPLEX
The family Herpesviridae is divided into three The her ]ieH simplex vims ( HSV ) occur* naturally
sub fa nil lies based on biological „ physical and only in human , but it can produce experimental
genetic properties : infection in many laboratory animals. There are two
Alphahcipcrvimscs , with a relatively short rypes of the herpes. sim|ilrx vims . HSV type 1
replicative cycle ( 12 -1 S hours) , x variable host ( Homan herpes vims type 1 or Hi IV type 1 ) is
range and a tendency to cause latent infection in usually isolated from lesions in and around the
sensory ganglia . In culture the \ are rapidly niotith and is transmitted bv direct C ^ nt ? Ct or
cytopathic and infectious viruses may be released droplet spread from cases or carriers. HbV type 2
ir ^ m cells, for example herpes simplex virus , [ ] I E I V type 2 } is responsible for the majority of
varied la - zosler virus . g e n i t a l herpes in feci ion * and in c o m m n r l v
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* PoUYiruSiU* ¥ 475
Human herpesvirus type 1 Herpes simplex virus rvpe 1 alpha qtioljflX neuft> rl
*
Human herpesvirus type 2 Herpes simplex virus type 2 alpha . cytolytic neuions
Human herpesvirus type 3 Varicella zoster virus alpha . tyrolytic neurons
Human herpesvirus type 6 Human B cell lymphotropjc ben hinphopKiliimtive lymphoid iissues
virus*
Human herpesvirus type 7 R K yinis* beta lymphopFoliferatlve lymphoid tissues .
larger pocks resembling variola , infection with type 2 strains . Transmission occurs
d ) Types 1 siraiiw replicate well in chick embryo by close contact and may be venereal in genual
fibroblast celts , while type 1 strains do so poorly . herpes. The virus enters through defects in the skin
4 ] The infectivity of tvpe 2 is more- temperature or mueous membranes and multiplies locally, with
sensitive than that ot type 1 . -
cell 'To cctl spread . The vims. enterv cutaneous nerve
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* H&rpe
^viruMS 477
ateu with wide spread ulceral ion . A clinically hepatitis . Erythema multi forme rrav be seen in
indi & tinguishabk picture is also produircd by association with HSV infection. Disseminated HSV
vaccinia virus infection, both designated Kaposi s infection UI .LV occur In patients with
rMrkctlifoim eruption. immunodeficiency, malnutrition or Ibums-
jMucoy .t }: The buccal mucosa is tlis site most GaiitAl: In rhe 1970s genital herpes became the
commonly ifFcctfd, GingivQttomatitis a nd most rjpidlv increasing venereal disease, particularly
phiryngiris. are [ fie most frequent conditions in in the USA. In men, the lesions occur mainly on
primary infection and recurrent herpes labialis in the penis, or in the urethra causing urethritis . In
recurrent infection. The vesicles nuy ulcerate and women , the cervix., vagina, vulva and perineum are
become secondarily infected. affected. When only the cervix is involved, the
t T/ltfut/pmc: HSV infection is the- most common infection may be asymptomatic. The primary
^
cause of corneal blindness in some developed
countries. Acute kcratoconjunctivitu may occur by
infection is usually more icriouc, accompanied by
systemic fen to res like fever and malaise , It is
itself or by extension from Facial herpes - Follicular followed by several recurrent episodes whieh arc
conjunctivitis with vesicle formation o i l the Lids is milder. The vesiculo- ulcerative lesions may be very
another manitestafi on . The cornea may he invotved , punfnl. Rectal and perineal lesions occur in
with typical branching dendritic ulcers . homosexuals . Both types of ) ESV tmy cause genital
[ )ehr i de me nt, topical antiviral dr\Lg$ an d i nterlc n trt lesions , though HSV 2 is responsible more
help in healing. Steroids are contraindicated as they frequently and causes many more recurrences .
lead to deep stromal involvement and healing may There have bee n several reports of ai L JMSociit M > n
be delayed, with scarring and corneal blindness. between I CSV 2 ,uid carcinoma of the cervix uteri
Chorioretinitis and acute necrorising rectnicis are but a causal reLiionshlp has not been established.
uncommon but serious manifestation. CH CIHUL' Ttmiphcetm] infection with HSV 1
Nervous system; HSV encephalitis though rjie , is ^
Or 2 can trad to congenital mallurmalions , but this
the most common sporadic acute viral encephalitis is rare. Infection may occur during birtli , particularly
in most parts of the world. HSV encephalitis has it the mother has genital lesions due to HSV 2. In
an acute onset, with fever and focal neurological such cases, cesarian section may prevent infection.
symptoms. Brain biopsy was employed in diagnosis Postnatal infection is more commonly due to
ftir instituting early specific therapy. This is now HSV i . Neonatal herpes may be confined to the
replaced hy demonstration of HSV DNA in CSF eyes , mouth or skin , but is more commontv a
by PCR , which is a very sensitive test in rhe acute disseminated disease having multi - organ
stage . HSV meningitis is a self - limiting disease , involvement, with oi without encephalitis. The
usually resolving in about a week, without sequelae . mortality rate is very high ,ind survivors may have
The CSF shows lymphocytic pleocytosis and may neurological impairment.
yield the vims in culture. Laboratory diagnosis : The dlagnos is of herpes virus
HSV can cause sacral autonomic dvsfuncrion B
infection may be made by microscopy, antigen or
and also rarely transverse myelitis or the Guillian- DNA detecrion , virus isolation or serology.
liarre syndrome . HSV has been implicated in the AJrrfij.wnpr: The Tzanck smear is a rapid, fairly
etiologv of Rel I 'H palsy. sensitive and inexpensive diagnostic method. Smears
Vi&Cttntl: HSV esophagitis may cause dysphagia, are prepared from the lesions, preferably Irum the
substcrnal pain and weight loss. It may involve the base ( if vesicle ^ and stained with 1% aqueous
respiratory tract causing tracheobronchitis and solution of toluidinc blue '0 ' for 15 seconds .
pneumonitis. HSV is an uncommon cause of Muldnudeared giant cells with faevred nuclei and
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478 i T&xtbaak ol Microbiology »
homogeneously nec1
. ground glass' chromatin
"
may help iin less serious conditions . Valacidovir
{ Tzanck cells ) constitute a positive smear . anrl famciclovir arc more rftcctivc oral agents.
Intranuclear type A jadwibn Undies may be seen When resistance to these drugs develop, drugs like
in Giemsa stained smears. The vims particle may toscamet which arc independent of viral thymidine
also be demonstrated under the electron kinase action may be useful,
microscope . It is nor possible to different Late
between herpes simplex and varicella zoster by HERPESVIRUS 5IMIAE: B VIRUS
micron copy. The herpesvirus antigen may be This vims was isolated by Sabin and Wright ( 19.14)
demonstrated in smears or sect iotas from lesions be from rite brain of a laboratory worker who
the fluorescent antibiwJy technique. The fluOttStCiit developed fatal ascending myelitis after being bitten
antibody test on hrain biopsv specimens provides by an apparently healthy monkey. It came to be
reliable £ nd speedy diagnosis in encephalitis. PCR known as the ' R ' virus from the initials of this
based DNA detection bis replaced brain biopsy. patient . Many nimlircases have been reported since
.srjlatmji: Inoculation in mice and on chick then. Herpesvirus si m i ae i nfects old world monkeys
'
j
embryo LAM is insensitive and has been replaced in the same manner that herpes simplex infects
by tissue culture for virus isolation , Primary' human humans, the infection usually being asymptomatic .
embryonic kidney human amnion and many other The typical lesions produced are vesicles on the
cells are susceptible, but human diploid fibroblasts buccal mucosa which ulcerate shedding the virus
are preferred. Vesicle fluid, spinal fluid , saliva and and inferring contacts, 'Though most human cases
swabs may he used. Typical cytopalhic changes may have followed monkey hires, the infection in some
appear as early as in 24-48 hours but cultures should was acquired through che handling of monkey
be observed for two weeks before being declared tissues .
negative. Drug susceptibility too cm be tested in Herpesvirus simiae is similar to herpes simplex
cell cultures . virus in its properties . The [ Wo are antigenkallv
Differentiation between HSV types 1 and 2 mar related but the herpes simplex virus antibody does
be made by a variety' of serological techniques, by not protect against herpesvirus simiae infection . A
nucleic acid hybridisation or by restrict ion fcrfmoLised vaccine has been tried experimentally
endonuclease cleavage and electrophoretic analysis in laboratory workers at risk.
of viral DMA or viral pnotiens. The disease in humans is usually fatal, 'l'he rare
Strategy: Serological methods are useful in the patients who survive have serious neurological
diagnosis of primary infections. Antibodies develop Sequelae . The official name for R virus i -.
within .t tew days of infection and rise in title of Ccrcopfthcaac herpesvirus 1.
ami bodies may be demonstrated by ELISA ,
neutralisation or complement fixation tests . In VARICELLA ZOSTER
recurrent ot reinfection herpes, there mav he little As early as 1 RB9, Von frokay had suggested that
change in the antibody litre . varicella { chickenpox ) anil herpes 7.nstcr a r e
< jhanatherapy; Idoxyuridine uved topically in dif feren t man i festatirms of the same vi rus infection .
eye and skin infections was one of the first clinically Virotogieal and epidemiological observations have
r m"
successful antiviral agents. The introduction of proved this concept. The virus is. therefore culled
acyclovir and vidarabine enabled the effective varicella aosCer virus ( VZV ). Chidcenpox follows
management ot deep and svstemic infections. Early primary infection io a nonimmune individual, while
treatment with intravenous acyclovir lias improved herpes zoster is a reactivation of the latent vims
the outcome of encephalitis . Oral and topical use when ( be immunity has fallen to lnelfective levels.
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•« Hwpwtruwa * 479
Thus ^ thicIccupM is Bcaft3gh/ hut not luster. Contact mainly the trunk and sparing the distal parts of the
wirh either zoster or chickcnpox may lead only to limbs , and is very superficial without involving the
chtclccnpox hut not zoster deeper layers of the skm , resembling a dew drop
,
VZV is similar to the herpes simplex vitus in lying cm. the skin . The rash appear* in crops during
its morphology. It does not grow in experimental the first three nr four days of the disease, so that
animals or chick embryos. The virus was first lesions of varying agp can be noticed or the same
isolated by Weller in human embryonic tissue patient. It matures very quickly, beginning to crust
culture . Ir can be BTOWH in cultures uf human within 4E hours.
fibroblasts, human amnion or HeLa cells. The When varicella occurs in the adult, systemic
cytopathic e r k .. i > are similar TO, but less marked symptoms may be severe , the rash very profuse and
than those produced by the herpes simplex virus. the entire disease much more intense than in
In cultures the virus remains cell associated and children. The rash may become hemorrhagic and
does not appear free in the medium . By using highly occasionally bullous lesions appear. Fitted scars on
specilic .mlisera , iL is possible td distinguish between the skm may reninm after recovery . Varicella
herpes tirus types 1 , 2 and varicella zoster viruses. pneumonia is more common in adults, and often
Only one antigenic type of VZV is known. fatal in the elderly. Other complications like
Varicellfl (oliicluflpnx}! Chicken pox is one myocarditis , nephritis , acute cerebellar ataxia ,
of the mildest and most common of childhood meningitis and encephalilis may ensue . Secondary
infections. The disease may, however, occur at any bacterial infections, usually due to staphylococci
age - Adult chickenpos, which is more serious, is or streptococci , may occur. Rey syndrome may
rather common in some tropical areas. follow varicella ir some cases wiih a history of
The source of infection is a ch ickenpax or herpes administrai ion of salicylates, But in most cases *,
, jr
zoster patient . Inactivity is maximum during the ckickenpox is an uneventful disease and recovery
ir trial stages of the disease when the vims is present the rule . One attack confers lasting immunity.
abundantly in the upper respiratory tract. The buccal Chkkenpox in pregnancy can be dangerous for
lesi i ms wl rich appear in the early stage of the di *ease both motherand bahy. The disease tends to be mure
and the vesicular fluxl are rich in vims content. severe in pregnant women, with enhanced risk of
Infecti s ity wanes as the disease progresses and the
'
complications like pneumom . L. The baby may
scabs are virtually nonin fee ri < i.us- There arc no develop two types of complications, depending on
animal reservoirs of vail -dla.
L the pei iiod of gestation when foe woman develops
The portal of entry of the virus d rbc respiratory chicken pox . If maternal varicella occurs during
tract or conjunctiva . After an incubation ped ' d of the first half of pregnancy, the fetal infection may
about two weeks ( 7— 23 days ) the lesions begin to usually be asymptomatic . Some infants may develop
appear. The patient is Considered to be in lev Lx ms the fetal varicella syndrome, manifesting as
during the two days before and five days after the cicatrising skin lesions , hypoplasi . L of Limbs ,
onset of the lesions . In children , there in little chorioretinitis and CNS defects. Some babies may
prodromal illness and the disease is tir - r noticed not exhih i t any defects, but may carry latent VZV
when skin lesions appear. Buccal lesions may not infection. When maternal varicella occurs neat
be TII it iced . The rash appears usually on the trunk. delivery, babies may develop congenital ( neonatal )
The evolution of the rash is so rapid that thcvariuois varicella, wi tli in two weeks of b irth. If the mother's
stages - macule, papule, vesicle , pustule and scab - rash began a week or more before delivery, she
cannot be readily followed in individual lesions. would have developed . iiudnuLie - which would have
The rash is centripetal in distribution, affecting been passed , along with the virus , to the fetus
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490 * Tf *1(JOOk o' Microbiology »
tram placen rally. Such A biby, though infected, passive protection ir immunocompromised children
usually '
clinical dincase. It the mother
escapes. exposed to infection , hut its availability is limited.
develops chicken pax lens the - i week (or wlrinn 2
, It Is not useful in treatment.
days) ot delivery. the b*by would have received
"
nee , the babies ate to be given VZV anti sc mm and of pneumonia and disseminated disease.
chemotherapy immediately after birth.
Laboratory diagnosis: Uiagnosis is usually HBHT &S ZOSTI - K (SHtNCLfiS, Zn ^ A )
clinical . Multi nucleated giant cells and type A Tlie name is derived from Horpcufi, meaning to
,
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462 * TemtJMk of Microbiology *
Primary
. infections in older clii.3d.rcn and adults of infection. Once infected , the person carries the
arc usually asymptomatic. However, a heteropEnle, virus for life.
antibody negative, infect iotis moncmucleos] ; may -
be seen . I'll is is more common frlkvwi ng transfer ion
Prev«mbn and treatment; Prevention is
indicated only in high risk cases such as organ
of CMV infected blood ( post- transfusion transplants, immunodcficicnt persons and in
moronudeo M ). -
In the immunocompromised host, CMV can
premature infants . Screening of blood and organ
donors and administration of CMV
cause severe and even fatal infections, 'this occurs immunoglobulins have been employed in
in transplant recipients , cancer patients on prevention. Acyclovir is useful in prophylaxis hut
chemotherapy, and moft. particularly in the E [ IV not in treatment . Ganciclovir and foscarner have
infected. CMV is an important pathogen in AIDS- been fiiund effective and arc used for the treatment
In AIDS patients, the already weakened immune nf CMV disease in AIDS patients.
response is further damaged by the nonspecific No vaccine is available . Experimentally, live
CM1- inhibiting effect of CMV. One of the attenuated vaccines (Towne 125 and AD 169
glycoproteins on the surface of CMV acts as a strums ) and a purified CMV polypeptide vaccine
receptor for the Fc portion of immunoglobuli n have been found to be immunogenic but not
molecules. This leads to masking of the virus by effective in protecting immiinodefLcient subjects
attachment of irrelevant immunoglobulin molecules, from CMV LnfccfioTi -
preventing access to specific ami - CMV antibody.
Laboratory diagnosis; Diagnosis may be -
EPSTEIN BARR VIRUS
established by recovery of the virus from the urine, In 1956, Burkitt described an unusual lymphoma
saliva or other body fluids by inoculating human among chiidrcn in certain parts of Africa and
fibroblast cultures . A simpler bur less reliable suggested on epidemiological grounds that the
technique is the demonstration of cytomegalic cells tumour may be caused hy a mosquito borne virus.
in the centrifuged deposits from urine or saliva. This led m several attempts at isolating viruses from
Demons [ ration of an ; ibodv is useful in [ he such tumours. A number of different viruses,
diagnosis of primary infection but not in apparently ’ passenger viiuscs", were isolated from
reactivation. Serological tcchniqties in use include cultured lymphoma cells. One virus observed in
CF> I HA, IF and EDSA, Antibody detection may the cultured lymphoma cells by Epstein, Bair and
be necessary far screening blood or organ donors. Achong in 1964 was a new type of herpesvirus,
Epidemiology! CMV spreads slowly and named the EB vims, specifically affecting cells of
probably requires close contact for transmission. It the B lymphocyte lineage. Only human and some
may spread through salivary or other secretions or subhuman primate B cells have receptors [CD 21
by sexual contact. A special method of transmission molecules) for the virus, EBV infected B cells arc
- -
i j by blood transfusi m or organ transplants. The
virus has been detected in saliva, urine, cervical
transformed so that they become capable of
continuous growth in vitro.
secretions, semen, blood and milk . Congenitally Epidemiology: The Epstein-Barr ( EB) virus
i nfectcd i nfants have viruria for uplo 4-5 years.They is ubiquitous in all human populations. As with
.
aie highly infecti rus in early infancy. About one other herpesviruses, infection with the EB virus
per cent of neonates in the USA are infected with leads to latency periodic reactivation and lifelong
CMV. In the developing countries, the rate may be persistence. The EE vims antibodies are present in
much higher . Upto SO per cent of adults show about 95 per cent of adults. In the oveicmwdrd
CMV antibodies, indicating the high prevalence developbig world, the EB vims infection occur? in
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* Herpesviruses 4fl3
droplets and aerosols ace not efficient in clones may replicate unchecked , resulting in
Transmitting infection . Intimate oral contact, as in lymphomas. Hypercndemic malaria prevalent in
hissing, appears to be the predominant mode of Africa is believed to be responsible tor the immune
transmission . This accounts for infectious impairment in children with Burkin 's Lymphoma.
mononucleosis being called the ‘Idling disease'. The frequency of lymphomas sett ] in many types
Infection may also follow blood or marrow , of immunodeficiencies, most typically in AIDS,
transfusion but these are rare event*. may have d similar pathogenesis. Nearly half the
f' H virus infection may lead to the following lymphomas seen in immuDodsficicnt subjects
clinical conditions; contain EB virus DNA sequences.
1. Infectious mononucleosis. Gercdc and environmental factors arc said to
2. EBV associated malignancies; be .important In tile nasopharyngeal carcinoma seen
a . Burkin's lymphoma. in men i > t Chinese origin . EB virus DNA is
b . 1 ympbcimas in immurnN.tefLviert persons Huch regularly found in the tumour cells. These patients
as AIDS patients and transplant recipients. have high levels of EB virus antibodies. Genetic
c . Nasopharyngeal carcinoma in persons of Influence is best illustrated in the X - linked
Chinese origin . lymphoprolifrrative (XLP or Duncan ) syndrome
Pnthogcnusis; " Hie virus enters the pharyngeal associated with extreme susceptibility to EB virus
epithelial cells through CR 2 (or CD 21) receptors, infection ,
which art the same as for the C’ dd component of
complement . It multi [dies locally, invades the I !N h KCTIOUH MONON ( C l.flGS LS
( GLANDULAR FIIVEH )
bloodstream and infects B lymphocytes i. n which
two types of changes are produced. In most cases, This is in acute self-limited illness usually seen In
the vims becomes Latent inside the Ivmphneytes, non immune young adults following primary
which become transformed or fnrjntfrtaJist.ii ' so that infection with the EB virus . Tin: incubation period
they become Capable of indefinite growth hi vitro. is 4-8 week*. The disease is characterised hv fever,
J
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< Hapatvliuvn 4 BS
Human Herpej Virus rih fn.- ^ HHV 7 also appears to be widely distributed and
A herpesvirus, first iosolitud in l 9flb from the transmitted through saliva. Jr shares with HIV the
peripheral blood of patients with lympho - same CD4 receptor on T cells and could therefore
prol rerarive disease, was called the human B
^
lytnphotfopic vims , It has been renamed J JHV-6,
- contribute to a frirther depletion ol CD4 1 Cells in
HIV infected persons. It has been said to cause
some cases of exanthetn subitum.
--
Thi i iubiiiuiros and spreads apparently chn ^.j rh saliva
in early infancy. Two vari .mts recognised, A and B, ^ In 1994, L>N A sequences presumed to represent
Variant B is the cause ofmild but common childhood a new herpesvirus were identified from tissues of
illnrsK ' nsaittheiti subitum’ ( rtrtcolsi infantum of > Lxt I L - Kaposi 's sarcoma from AIDS patients. This has
disease i . In older age groups, it has l >een associated been named HHV fi. This has subsequently been
with infectious mononucleosis syndrome , focal identified also in Kaposi's sarcoma in persons not
encephalic ^ and , in the i - nmu node fie lent, with infected with HIV. Jt has been therefore referred
pneumonia and disseminated disease. to some limes as Kaposi V sarcoma -assuoa fed
HHV 7 was isolated In 1990 from peripheral herpesvirus fKSHV ), but an etiological
CD 4 cells of a healthy person - Like HHV 6, relationship is yet to be proved .
Rowley AH e ( al - 1990 , Rapid dctectiim H *«pa simplex -. - i r -±ia UNA . Jjneer .135.
Strauss M - . er al . 1993. Epsnein-Barr virus infections . Ann Jnr Med 118:45.
WtlleeTH . 19 B 3. Varicella and Herpes- Zoster: changing concepts. New Eng } JlJed. J0913& 2 , 1434.
Adenoviruses are a group of medium sized , Adenoviruses appear to have a spare capacity
rLonenYeioped > double stranded DNA viruses chat to cam DMA insert up to 7 kb and arc being
1
share a common complement fixing antigen. They investigated as potential vectors in gene tberapv.
infect humans, animals and birds, showing strict Morphology: Adenoviruses arc 70-75 nm in
bast specific in'. size . They have ,t characteristic morphology. The
In 1L >3 , Rowe and associates grew surgically capsid is composed of 152 eapsomcn arranged as
^
removed human ad end id tissue in plasma eldt an icosahedron with 20 lri &rtgui])r facets and 12
cultures and nociced that the epithelial outgrowths vertices . Of the 252 capsomers , 24Q have six
underwent spontaneous degeneration resembling neighbours and arc called hexnns, while the 12
viral cytopatbic change. Thin wan neutralised bv capsomers at the vertices have Five neighbours and
human sera . A viral agent was shown to be arc called pentuns. Each pen top unit consists of a
responsible for this degeneration. Tins was the petunn base anchored in the cap - id and a projection
1
prototype of the group of viruses subsequently or fibre consisting of a rod like portion with a knob
designated adenoviruses because they were attached at the distal end. Thus , rln - virion has the
originally isolated from the adenoids. I liUcman in appearance of’ a space vehicle .
195 *1 isolated a related virus from the throat
washings of military recruits with acute respiratory
illness .
Over SO serotypes? of adenoviruses have been
isolated from human sources . Most uf the recent
serotypes; were recovered from AIDS patients.
Adenovirus infections are common worldwide
mostly in children . Many infections arc
asymptomatic [’ he virus may persist in the host
,
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* Adenaviru &frS m
Tabta 511 Classilicetlen of human adenoviruses
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4 SE H Ten [ book 01 W crobiology *
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* Adi IOVKU fct!fi- * 469
nasopharyngeal and ocular infections are useful. independently as they lack enough DNA , They can
I ' rrtphylaKie: Specific prevention required multiply only in cells simultaneously infected with
only for flic control of outbreaks i :i closed
communities, ^ in military recruits. Killed and live
adenoviruses and are called adcno associated
viruses {AAV ) or adcnosatellite virases. They have
-
vaccines have been used in them for prevention of been clarified as the genus depenefonn ^ {referring
ARD, with some success. No vaccine is available to their dependence on adenoviruses) under the
for general use. family Parvoviridae. They can be detected by
electron microscopy and complement fixation or
ADENO-ASSOCIATED VIRUSES ( AAV / immunofluorescence wirh specific anri -icra. Types
Electron microscopy of adenovirus preparations 1, 2 and 3 are of human origin and cause natural
have revealed small icnsahedral viral particles, 20~- infection while type 4 is of simian origin . Thrir
25 nm in diameter They are unable to replicate pathogenic role is uncertain.
Further Reading.
J3 . SC 1.1990 Adencmruiiex. In C_ .
De Jong J L aL 19K 3 _ FiuiieliftLis jtLertnrifu
x JVIjndpl ft jl. / Practice of In fc-CTJCHH IA IEWKE V sdn . New Ynrfc Wiley.
'
J UIIL . . jurat
The familv Picomavindac comprises a large dumber be sc ] adapted. Progress was also inhibited by the
of very small ( pko, mealing small ) RNA viruses, dogma then held that polioviruses were strictly
They art- nonenveloped viruses, 27-30 nm in size, nemolxopK, multiplying only in the neural tissues.
resistant to ether and other lipid solvents, Two The demonstration by Endcn , Wtllcr and Robbins
groups of picamsviruies are of medical importance , ( 1949 ) chat polioviruses could grow in cultures of
the cntcrcvaufet that parasitise the enteric craft non-rte ur J ] cells from human embryos, producing
and the rfujwvinisffs that infect rhe nanal mucosa. cytoparhk effects, was a major breakthrough . The
Two other pieornavirus genera of veterinary Nobel Prize was Awarded ro them in recognition
importance are *phr/tovi'rusej causing the foot and of rhe seminai importance of this discovery in the
mouth disease of cattle, and cardrcrniuse* of mice, development of virology as a whole.
including the encephalomyocarditis virus . A new type of virus was isolated by Dalldorf
and Sickles (194S) from the feces of children with
ENTEROVIRUSES paralytic poliomyelitis , from whom type 1
Enteroviruses of medical importance include: poliovirus was also isolated . The virus caused
,
Pbfitmmi types 1 _ 3n Coxsackievirus A types 1 -24, piaraivsis on inoculjtion into suckling mice. This-
Coxsackievirus 13 types 1 -6 , Eehovim; typos 1-34 was called the coxsackic virus, as the patients came
and Enterovirus rypej 6 H - 71 . from the village nfCoxhackie in New York. Many
Paralytic disease of children ( m /autii/c partJysjs) similar viruses have since been isolated from the
has been recognised from very early nines. I lowever, frees and throats of patients with different diseases
it wan only towards the end of the nineteenth as well as tram healthy individuals. They have been
century that poliomyelitis { polios = grey; myelitis designated asconaddc viruses, classified into groups
= i nflunmaftion oft he spinal cord } w as eh aractensed A and U based on the pathological changes
as a separate clinical entity capable of causing produced in suckling mice.
LfljectiuilS ill which paralytic cas- cs are tar The in traduction of tissue culture techni <|ues
outnumbered by silent in apparent infections, in diagnostic virology led to the isolation of several
Landstciner arid Popper (1909 ) reported cytopathogeuie viruses from the feces of sick as
experimental transmission of the disease to monkeys well as healthy persons. Thcv were called orphan
hy inoculation of spinal cord and fecal extract viruses as they could not be associated with any
fdtrates from fatal cases of poliomyelitis. The particular clinical disease. They came to he known
exjterimental study of the disease was restricted as by rhe descriptive Term ' enteric cytnpatbogenic
monkeys were the only laboratory animats human orphan ( ECHO) viruses ' . Several orphan
susceptible to the virus . Armstrong ( 1939 ) viruses have also been isolated from annual feces.
succeeded in adapting a poliovirus ( type 2 Lansing The classification of enteroviruses as
strain ) ro cotton rats and mice hut few strums could coxsackieviruses and eehoviruses was not
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4 n . - -
* TaxjtbQOfc OF MI J : IJ LMIJ lij U y »
The vims grows readily in tissue cultures of seroconversion alone. It is only in 5-10 per cent
pi i mate 0 ri i.ii , Pri unary monkey kidney cultures are chat any sort of clinical illness results. The
^
'
used fur diagnostic cultures and vaccine production. incubation period is about 10 days on the average
The infected cells round up and become refractilc but may range from four days to four weeks. The
and pyknotic. Eosinophilic intranuclear inclusion earliest manifestations are associated with the phase
bodies may be demonstrated i n sfs i ued prepare ions. of primary viremia and consist of fever, headache,
Well- formed plaques develop in infected —
sore throat and malaise lasting 1 5 days. Tkis is
called the minor iifness and in many cases may he
monolayers with agar overlay.
Pathotfencftis: The virus is transmitted by the the only manifestation (abortive poliomyrlitis ). If
fecal-oral route through ingeS' Lun. Inhalation or
entiy through conjunctiva of droplets ofrcspiratoiy
secretions may also be possible modes of cntiy in
close contacts of patients in the early stage of the
—
the infection progresses, the minor dlncss is followed
5 4 days later by the major illness. The fever comes
or again (hiphasic fever ), dong with headache, stilV
neck and other features of meting iris. This marks
disease , The virus multiplies initially m the the stage of vital invasion of the central nervous
epithelial cells of the alimentary' canal and the system. Sometimes the disease does nut progress
lymphatic tissues, from the tonsils to the Feyeris beyond th i s stage of wcptic meningitis ( nonpwaJyr.'e
patches. It then spreads to the regional lymph nodes poliomyelitis ). In those proceeding to paralytic
and enters the blood stream ( minor or primary poliomyeliiis, flaccid paralysis develops. Paralysis
vjVejKjd ) . After further multiplication i 11 the is local in distribution ini dally hut spreads over the
reticuloendothelial system , the virus enters the next 3-4 days. Depending on the distribution of
bloodstream agii i n ( nrajeror secojjdajr virem W paralysis, cases are classified as spinal, bulbar or
is carried to the spinal cord and brant. Direct neural bulbospinal. Mortality ranges from 5-10 per cent
transmission to the central nerwus system may also and is mainly due to respiratory failure. Recovery
occur under special circumstances , as in of the paralysed muscles takes place in the next +
poliomyelitis following tonsiLiectomy, 8 weeks and is usually complete after sir months,
In the central nervous system , the virus leaving behind varyingdegrees of residual paralysis.
multiplies selectively in the neurons and destroys Laboratory diagnosis: Virus isolation in
them. The earliest change is the degeneration of tissue culture is the best method for specific
diagnosis. Many specimens can be used, Including
Nissl bodies {chromatnlvsin ) - Nuclear changes
follow. When degeneration becomes irreversible ,
the necrotic cell lyses or is phigocjtnued by
.
blood CSF, throat swab and feces. Virus can be
isolated from blood during the phase of primary
leucocytes or macrophages. Lesions are mostly in vircpiL, 3-5 days after i nfccricm, before neutral ini ng
the anterior horns of the spinal cord, causing flaccid antibodies appear. But this is of little practical
paralysis, but the posterior horns and mtcrmedi .Ltu importance. Unlike other enteruviruses, poliovirus
columns mav also be involved to some extent. can seldom be isolated from the CSF hue can bn
Pathological changes are usually more extensive obtained from the spinal cord and brain ,
than the distribution of paralysk In some cases, postmortem. The virus can be isolated from the
encephalitis occurs primarily involving the throat in the earlv stage of the disease. Virus
brainstem but extending upto the motor and isol.ition from feces is usually possible from over
premotor areas of the cerebral cortex. 00 per cent of patients in the first week, 50 per
,
Clinical featuret: Following exposure to cent - t il ] 3 weeks and 25 per cent till six weeks. As
,
. -
poliovirus 90 95 per cent of susceptible ndivi duals fecal excretion maybe intermittent, best results are
develop only inapparejif iji /ecfion, which causes obtained by testing fecal samples collected on two
L l
jpyriaruea natena
• Iricomavlmwe 493
separate days , as early in the illness as possible. Breast milk containing IgA antibody protects infants
Fruluiigd fecal excretion may be seen in the from infection.Poliomyelitis tends to be more severe
i mmurmdefic i en t, but permanent carriers do out and virus shedding mure prolonged i :i those with
occur. impaired humoral immune response. The virus also
After appropriate processing to destroy bacteria induces cdl mediated immunity but its importance
[centrifugation, treatment with ether, addition of appears to be uncertain as persons with defective
antibiotics), specimens are inoculated into tissue cellular immunity arc seen to respond normally to
culture. Primary monkey kidney cells are usually puli,*V:rtiS infection.
employed , though any other human of simian cell ProphylflJcir Passive immunisation by cite
culture may be used. The virus growth is indicated administration of human gammaglobulin is of little
by typical cytoparhic effects in 2 3 days. - value .
Identification is made by neutralisation rests with Attempts at active immunisation with vaccines
pooled and specific antisera. It must be remembered date from 1910, Soon after the discovery of
-
that the mere T olation of poliovirus from feces docs
not constitute a diagnosis of poliomyelitis as
poliovirus . The early vaccines were crude
suspensions of the spinal card from infected
symptomless infections are so common Virus . monkeys, inactivated with formalin ( Brodie and
isolation must be interpreted dong with clinical Park) or riL inoleate ( Kolmer). They were not only
'
-
disadvantage in the advanced countries. Ptr' hii| s , it
"
corresponding to the sire may be more susceptible
to viral damage due to reactive hyperemia - 4) Severe
may even be beneficial and may help to extend the
\~ .iL L iiiLk coverage in coumiies where vrild vims is
' '
muscular exertion or rraumadui mg the preparaJytic
endemic. Ideally, however, i r i s desirable to stage increases the risk of paralysis .
vaccinate the whole community , n one time so chat
igT
Poliovirus type 1 is responsible for most
natural diRaeirunarion is prevented. The strategy of epidemics of paralytic poliomyelitis. Type 3 also
administering the vaccine to ail children in a region causes epidemics to a lesser extent . Type 2 usually
on the same day { pulse stnmwthition: ) has been causes inapparent infections in the western countries
found to be useful in the developing countries. but in 1 nd i a paraiys i K due to type 2 is quite common.
Eradication of poliomyclilia: By global Immunity is type-specific but there is an significant
immunisation with OPV it was considered possible amount of cross protection between types 1 and 2,
to eradicate the disease . The World Health between types 2 and 3 and little or none between
Organisation Assembly in 19fiS had proposed tvpes 1 and 3.
global cradicat ion of poli omyclir is by the year 2000-
Poor progress in immunisation in many countries COXSACKIEVIRUS
has been a set back to this objective. The prototype strain was isolated by Dalldorf
Epidemiology: Poliomyelitis is an exclusively and Sickles ( 1943 ] from the village of Coxsackie
human disease and the only source of virus is in New York. Several related viruses have been
humans, the patient of much mote cominoiilv the isolated si nee then from different parts of the world ,
symptomless carrier. Patients shed rhe virus in feces The characteristic feature of this group is its ability
for varying periods, about SO per cent for three weeks to infect suckling but not adult mice , Based on the
and a small prnporriun far 3-4 months . No pathological changes produced in suckling mice,
permanent earners occur, 1 lowcvcr, the virus may coxsackieviruses are classified into two groups, A
persist in the environment (sewage) for upto six and B.
months. Virus shed in throat secretions during the Properties of the virus: Coxsackieviruses
early part of the disease UI .LV also be a source of are typical enteroviruses. Following inoculation in
infection for the contacts of patients. suckling mice , group A viruses produce a
Infection is, in general, asymptomatic. The ratio generalised myositis and flaccid paralysis leading
of subclmical to clinical infections has been stated to death within a week. Group B viruses produce a
as 100 or 1000 to 1 . The outcome of infection is patchy focal myositis, spastic paralysis, necrosis of
influenced by the virulence of the infecting strain, the brown fat and, often , pancreatitis, hepatitis,
the dose of infection and the age of the individual, myocarditis and encephalitis- By neutralisation tests,
adults being more susceptible than children. The Group A viruses are classified into 24 types and
following factors may influence the incidence of group B into six types. All types in group B share a
paralysis: 1) Pregnancy carries an increased risk of common complement-fixing antigen - Coxsackic A
paralysis, perhaps due to the associated hormonal 23 is the same as echo 9 and Cox&ackie A24 the
changes. 2 } Tonsillectomy during the incubation same as ECHO 34. Some coxsackieviruses ( A 7,
period may predispose to bulbar poliomyelitis. 20, 21, 24 and B 1, 3, 5 6) agglurinalc human or
T
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< Pjcomsviruses 497
mutes. Adult mice are nm susceptible. Suckling called because it was first described on the
hamsters can be iiif^ued experimentally. Danish island of Bornholm ) is a febrile disease
A|J coxsackic E viruses grow well in monkey with stitch-like pain in the chest and abdomen ,
kidney tissue cul hurt , while ingroup A, only types caused hy group B viruses. The disease may
7 and 9 straw well. Group A 21 virus grows in occur sporadically or a; epidemics.
HeLa cells. 6. Myocarditis and pericarditis in the newborn ,
Clinical features: Coxsackieviruses produce associated with high fatality may be caused hy
a variety of clinical syndromes in humans ranging group B viruses . The disease may sometimes
from trivial to fatal ] niece mos. The (bllowing types occur in older children and adults also.
have been recognised: 7. Juvenile diabetes has been claimed to be
1. Herpangi us (VB > icular pharyn i r is ) i 5 a common associated with coxsackie B4 infection but a
^
clinical manifestation of coxsackie group A causal role for this virus has not been
infection in children . It is a severe febrile establishcd -
pharyngitis, with he .1 Hue hr , vomiting and pa: n .E Orchitis due to coxsackievirus has also been
ill the abdomen . The characteristic Lesions are reported -
small vesicles , on the fauces and posterior 9 . Transplacental and neonatal transmission has
pharyngeal wall , that break down to form ulcere , been demonstrated with coxsackie B viruses
2- Ascpt iv men ingitis may be caused by most group resulting in a serious disseminated disease that
A and all group B viruses. A maculopapular ash [ ] 1 , LV include hepat i; is, meningoencephalit is and
may be present. The ih:- ease may sometimes adrenocort i call involvement.
occur as epidemics . Type A 7 had caused 10. Type B viruses have been associated with the
outbreaks of paralytic disease in Russia , Scotland condition called posfriraJ farigue syndrome, hut
and elsewhere, the virus for a time having been neither the condition nor the association has
erroneously referred to as Poliovirus type 4, been clearly defined.
3 . HandjFoot “ and - Mouth- Disease { HFMD) was Laboratory tlin noMB : Virus eolation from
identified in I960 as an exanthematous fever ^
the Lesions or from feces may be made by
affecting miiinly young children , characterised inoculation in to suckling mice. Idenl ification is by
by clusters of papulnues i c LLL .LT le > ions on the ski n studying the hiRtnpathnlogy in infected mice and
and oral mucosa. It occurs as sporadic cases and by neutralisation tests . Due to the existence of
as outbreaks . Coxsackie A-16, 9; B 1- 3 were several antigenic types , serodiagnosis is not
Common causative agents inn Lilly. It was a practicable.
henii n illness resolving in 1 - 2 weeks . The Jt- pidtrtjicHrtgvt Tike other enternviruses,
^
situation changed drastically in the 1970 rs with coxsackieviruses inhabit the alimentary canal
-
enterovirus 71 becoming a causative agent , primarily .ind are spread hy the fecal-roral route .
Coxsackie B virus cpiikmics tend to occur every
causing extensive epidemics with serious
compl icsti ons like ascptiic meningitis* 2-5 years. Young infants arc most commonly
encephalitis, flaccid paralysis , pulmonary affected . Vaccination is not practicable as there are
hemorrage, with many fatalities, particulirilv several serotypes and immunity is type specific.
in East Asla frumTaiwan to Siilgaput6 - HFMD
I opyri
S
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498 < Texlbook of Microbiology »
health)' individuals. ' ITICSC viruses were not pathogenic Laboratory diagnosis: Feces , throat swabs
for bboab anirnakTheywere neutralised spooled or C5 E maybe inoculated into monkey kidney tissue
^
human gamma globulin. AH they could rot he cultures and virus growth detected by Cytopftlhii:
associated with any particular clinical disease then, changes- The large number of serotypes makes
they were ailed orphans. They have been given the
, identification bv neutralisation tests laborious. IVis
r
descriptive designation 'enteric cytopathogcniL human maybe simplified by hemagglutination and the use
orphan viruses' and arc generally known by the sigh of serum pools for neutralisation. Serological
echovi roses . Similar 'orphan:' Anises have also been
1
diagnosis is not practicable except in case of
isolated from manv animats.
a
epidemics where the causative serotype has been
Properties of the virus: Echoviruses identified.
resemble other enteroviruses in their properties. By Epidemiology; Like other enteroviruses,
neutralisation tests, they have been classified into echoviruses inhabit the -alimentary tract primarily
34 serotypes. Types 10 and 28 have been removed and are spread hv the fecah-oral route. Epidemics
from the group , the former becoming a rcovirus may occur, especially in summer. Vaccination has
and the latter a rh in <>virus. not been attempted .
.
Some echoviruses ( types 3 , 6, 7 , 11, 12 13, 19.
.
20 ! 21, 24 , 29 30 and 33) agglutinate human
erythrocytes . Hemagglutination is followed by
MEW ENTEROVIRUS TYPES
.
Ot the new enterovirus types, 68-71 type 6fi was
elution , rendering the cells inagglutitiablc by echo isolated from pharyngeal secretions of children with
or ooanckijeviruses but not by myxoviruses . pneumonia and bronchi ns.Type 69 is not associated
( ifXfrwth nnd 1IL > SI ranf [c All echoviruses with any human disease . Type 70 causes acute
well in human and simian kidney cultures
grow
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lO Orthomyxovirus
in
The name Myxovinu was used origin illy for a respiratory tract which occurs in sporadic, epidemic
group of enveloped RNA viruses chincttriscd by and pandemic forms. The name ' influenza' is said
their ability' to adsorb onto mucoprotcin receptors to have been given by Italians during the epidemic
or ] erythrocytes, causing hemagglutination. The of 1743 , which they ascribed to the malevolent
name referred To the affinity of the viruses Co mucins
r
influence of the heavenly hotlies or of inclement
( from mt ra , meaning mucus). Included in chis weather . The modern histeiry of the disease may be
^
group were influenza, mumps, Newcastle disease considered rodate from the pandemic of 1 RH 9-1 H 90,
and parainfluenza viruses. The subsequent during which Pfeiffer isolated Hiemophilua
recognition of important differences between influenzae and claimed that it was the causative
influenza viruses and the other viruses in this group agent.The most Revere pandemic occurred in 191 K -
led to their being reclassified , into two separate 1919 , when it was shown that PfeilVerfs bacillus
-
families orthomyxoviridae eon* sting of the]
Property Orthomyxovirus n
JT MI d
. .r
UiJTJUJm U B
-
SlTEi? Etf Yfrkfl 80— 120 nm 100- 300 nm
Shape Spherical; filaments in fresh isolates Pleomorphic
Genamc Segmented; « ight pittC* of RNA Single linear
molecule of RNA
Diameter of mideocapsid Lf mm 18 nm
Site of synthesis of ribonudeoprotein . Nucleus Cytoplasm
Genetic reassortment Common Abienr
DNA -depcndent RNA synthesis- Required for multiplitftiiW Nor required
Effect of Actanomjdn D Inhibits multiplication Docs not mhibi. t
Aniigtnic stubility Variable Stable
Hemolysin Absent Present
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502 * Textbook or Microbiology
discovery by Hint, and by McClelland and Hare not causehuman infection. But they may play an
,
(1941) chit influenza viruses agglutinate few ] important role in the emergence of pandemic
erythrocytes.The property of hemagglut i nut in m was influenza.
found to be a common feature of many other ’ 'iruscs,
Francis and Magill (1940 ) independently INFLUENZA VIRUSES
isolated a serotype of Influenza virus which was Morphology: The influenza virus is typically
antigenicalfy unrelated to the strains known till then.
Thin was designated influenza virus type B , to
-
spherical , with a diameter of 80 120 nm but
plcomorphism is common. Filamentous forms, upto
distinguish it from the original serotype, which was several micrometres in length and readily visible
named type A . Taylor (1949) isolated the third under the dark ground microscope, are frequent in
serotype of influenza vi ms, type C . The classi ficat: on freshly isolated strains.
ot influenza viruses into [he three serotypes, A , El The virus cote consists of ritxmucleoprotein in
and C , is based on the antigenic nature of the helical symmetry. The negative sense single
'internal or nbonucieoprotein and the matrix ( M )
1
stranded RNA genuTne is segmented and exists ax
protrin antigens - LIJTIL; pieces. Also present is a viral RNA-dcpcndcnt
Influenza occurs also in animals and birds in RNA polymerase which is essential for transcription
nature . Indeed, the avian influenza virus was of the viral RNA in infected host cells. The
demonstrated as early as in 1901, when Centanni nudeocapsid is surrounded by an envelope, which
and Avonuzzi showed that fowl plague was a viral has an inner membrane protein layer and an outer
disease . However, as fowl plague ( avian influenza) lipid layer. The membrane protein is also known
is a septicemia, so different clinically from human as the matrix or ' M protein ' composed of 2
influenza, the association between the two remained components, Ml .end M2. The protein part of the
unknown till 1955, when Schaefer demonstrated envelope is virus coded but the lipid layer is derived
-
that the fewl plague viius was an igenicaHy related
to type A influenza virus. Shope (1931) isolated
from the modified host cell membrane, during the
process of replication by budding. Projecting from
the swine influenza virus- Not only did the swine the envelope are two types of spikes ( peplomers) :
disease re- Kemble human influenza clinically hut heifl & jizglutiijin spikes which are triangular in cross
there was also epidemiological associ , i:inn between section and the mushroom shaped ficLrramwifda.se
the two. It was widely held that the virus spread to peplomers which are less numerous ( Fig. 55.1).
swine from man at the Ume of the 1918 pandemic. Resistance: The virus is inactivated by heating
Influenza viruses have also been isolated from at 50 °C for 30 minutes. It remains viable at 0-4
horses, whales and seals. for about a week. It can be preserved for years at
Birds, particularly aquatic birds, appear to be -70 °C or by freeze drying. The virus survives slow
the primary reservoir of influenza viruses and drying and may remain viable on to mites such as
natural infection has been identified in several avian blankets for about two weeks. Ether, formaldehyde,
species . In birds it is usually an asymptomatic phenol, salts of heavy metals and many other
intestinal infection. The cloaca of healthy wild birds chemical disinfectants destroy injectivity, Iodine is
is the best source for isolation of av:.un influenza particularly effective,
viruses. Ah isolates from oonhuman hosts belong HcmaggluTinafiug, enzymic and complement *
to type A - Influenza vims types B and t - appear to fixing activities of the virus are more stable than
be exclusively human viruses and natural infection inactivity,
with them has not been identified in animals or Huinagjluti upturn: Hemagglutination is an
birds. Ordinarily, nonhuman influenza viruses do important chatactefisric of influenza viruses. When
I rianiec ceria
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OrthHnyxavkus t 507
especially m type B infection in children, which C strains agglutinate only fowl whs at with amirem
miv cn h present as an acute abdominal emergency
'
. to types A, K indC . Subtype identification is made
The uncomplicated disease resolves withiri about by hemagglutination inhibition test. Some of the
seven LI .LV - . recent type A strains can be isolated by direct
The most important complication i
which is mainly due to
- pneumonia,
bacterial super infection or ,
allantoic inoculation of the clinical specimen: into
9-11 day old eggs. However, type B and C viruses
rarely , caused bv the virus itself. Cardiac will be missed if only allantoic inoculation is used.
complications ,. such as congestive fa Hurt: or Inoculation into monkey kidney or other suitable
myocarditis and neurological involvement , such as continuous cell cultures, such an baboon kidney, is
encephalitis, may occur rarely the preferred method where the lability is available.
Influenza, particularly infection with type B, has Inoculated cell cultures art - incubated without scrum ,
been associated with Rcyc's syndrome. It especially and i u the presence oF trypsin, which increases
affects young children and is characterised by acute sensitivitvar of isolation . Incubation at 33 "C in roller
degenerate change ^- in the brain, liver and kidneys. drums is recommended. Virus growth tan be
Type B infections may sometimes cause identified by hemadsorption with human O gimp,
gastrointestinal symptoms (gastric flu ). iowl or guineapig erythrocytes., Rapid rtsults can
Laboratory diagnosis: of be obtained by demonstraring virus antigen in
the nrw antigen : Rapid -
Lliagno is on influenza may infected cell cultures bv immunofluorescence.
-
IT
be made by demonstration of rhe virus antigen on 3. Serology? Complement Fixation and hem
the surface of the nasopharyngeal cells by agglutination inhibition rests are employed for the
immunofluorescence. Detection of the viral RNA semlogical diagnosis of influenza - It is essential to
by reverse transcriptase polymerase chain reaction examine paired sera in parallel, to demonstrate rise
is extremely sensitive , but is of limited availability. in Here of antibodies.
-
1imAttior) of the nrus: Vims l otarion is obtained
readily fmm pa Herts during the fir^ t two or three
Complement fixation tests with the RNPantigen
of Influenza virus types A, B and C are very usefeJ
days of the illness but less often in later stages. as the antibodies are formed during infection only,
Throat garglmgs are collected using broth saline and not following immunisation with inactivated
or other suitable buffered salt solution . If the vaccines. Complement fixation can also be done
specimen is not processed immediately! it should
be stored at 4 ^ Ch or if the delay is long, at -70 cC .
using V antigens for the demonstration of strain
specific antibodies. Because of its complexity, CF
-
The specimen should be ( reared with antibiotics teats are now used only rarely.
to destroy bacteria. Isolation may be made in eggs Hcmagglutinatlon inhibition is a convenient and
or in monkey kidney cell culture. sensitive test for the serological diagnosis of
The material is inoculated into the ammatic influenza. However, it has some disadvantages. As
cav i ty of 11-13 day old eggs, ir i ng at least six eggs the antihemaglutmin antibodies arc subtype -
per specimen. After incubation at 35 °C For three specific, it is necessary' to use as antigen the strain
days, the eggs are chilled atid the amniotic and currently causing infection. The may nr drawback is
allantoic Quids harvested separately The Quids aie the frequent presence in the sera of nonspecific
tested lor hemagglutination using guiiteapig arid inhibitors of hemagglutination. The sera , suitably
-
fend ells in parallel, at room temperature and at
4 ^C . Some strains of the influenza vims tvpc A
treated for the removal of nonspecific inhibitors-,
are diluted serially in hemagglutination plates and
agglutinate only guineapi cells O i l initial isolation. the influenza -vims suspension containing 4 HA units
^
The type B virus agglutinates both cells, while type added to each cup. Fowl red cells are then added.
separate , and also to keep pip away from them. ( H ) antigen , beroepidemiological (seroarcheo-
The practice nt keeping several species of birds logical ) studies indicate that the severe pandemic
along with chicken* ill live bird markets is of 1889 was caused by a virus with rhe antigenic
potentially dangerous . structure H 2NE ant! that this was followed in 1900
A unique feature uf influenza epidemiology wai by the subtype I 13 IV 8 which led to a moderate
that once art antigenic variant emerged, it displaced pandemic. In 1918 came the most severe of al!
completely the pre -existing strain , Thus when Al pandemics, caused by the ' Swine type" H l N l
( HlNl ) strains atwc in 19*16-47, they became the ( formerly Hsw Nl ) virus. Mild epidemics occurred
only viruses causing human disease , and the around iy33 and 1946 associated with minor
previous Af > (HONI) strains disappeared completely. variations in the H antigen ( from HEW to HO in
The Al strains were displaced by Asian N I 2N 2) 1933, HO lo Hi in 19+6 ). The next severe
strains in 1957 and they, in turn , by the A2 Hong pandemic came in 1957 with the H 2N2 (Asian )
Kong ( } ! 3 lM' 2 ) strains in 196 E . However, thin rule subtvpc. This was followed in 196 E by the H 3N2
has not been observed in recent -vears. Even after
X
( Hong Kong) virus leading to a moderate pandemic .
the re'emergence and wide dissemination of the The year 1977 saw the reappearance of the H l N l
HlNl strain in 1977 , the A2 Hong Kong H 3N2 virus. Thus the sequence of variation in the H
Strains continue to he prevalent. The reason for this
coexistence is rot known ,
— - — —
antigen baa httn Hl * H 3 * Hi » H 2 > HJ
111 from 1689 to the present time (Table 55.3) ,
There is considerable evidence to suggest that From 1977, both H 3N2 and H l N l viruses have
there occurs an orderly reeve ling of the virus beer circulating together . Table 5 5 - 3 lists the
subtypes at Irate with regard to their hemagglutinin sequence of appearance of these various subtypes.
Table 55.3 Calender of appearance ol influenza A virus subtypes ( from 1 SS9 )
; Data wlcre 1933 based on Serci neciogf i
^
Dare Antigenic subtype Remajr&f
- ^
( WS strain lWS Epidemics of ’AO* strains
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Paramyxoviruses
The family PiramjntovLridue comains important the host cells, which is the essential early step for
pathojrc ns of infants ajid children, responsible tor infection. It also bring about cell- lo-cell fusion,
* major part of acute respiratory infections *
causing Large giant cells or syncytia, which are
( respiratory syncytial virus and parainfluenza characteristic of paramyxovirus infections. The F
viruses) and also for twin of the most contagious protein also mediate the liemnlytic activity of
*
diseases of childhood ( measles and mumps ) . paramyxoviruses.
Though much less cochniont infections may also The family Paramyxnviridae in divided into four
occur in adults . genet a— Kubulsvirua f 3 a rainfluenza rirus,
Paramyxoviruses resemble orthomyxoviruses in Norbiliivirtu and Ptieumannu (Table 56.1),
morphology hut are larger and more pleomorphic .
They are roughly spherical in shape and range in. RUBULAV1RUS
size from 100 to 300 urn, sometimes with long M l MPS VIRUS (G K M S R l Bt I. A V I H I s >
filaments and giant forms of upTa H00 Aiti The . Mumps is an acute inleCtious disease commonI v
helical nudcaclpjid is much wider than in iffecring children and characterised by
orthomyxovi ruses, with a dismetc rnflSnm (except nonsuppurative enlargement of the parotid glands.
in fVitLrnwnj- LT!; where it is 13 mrtJ. The geuOnte is As epidemic parotitis, it had been described by
a molecule of linear single stranded RNA. Unlike Hippocrates in the fetch century BC. The viral
the orthomyxoviruses, in which the segmented etiology of mumps was demonstrated by Johnson
nature cif the genome facilitates genomic mid Goodpasture ( 19J4) bv its experimental
rcassortments and antigenic variation so typical of transmission ro monkeys. Habd in 1945 cultivated
Influenza viruses, the paramyxoviruses with their the virus in embryonated eggs. In 1955 , Hcnle and
unsegmented genome do not undergo genetic Deinhardt grew LI in [issue culture .
revoruhniaCioTL 'i or antigenic variations . Hence all Properties: The mumps virus is a typical
paramyxoviruses are antigenitilJv stable. paramyxovirus possessing hoth HN and F proteins.
The nncleoclpiid is surrounded by A lipid It agglutinates the erythrocytes of foiwl, guiueapig,
envelope which has rhe matrix (Ml protein at its hum ans and many other specin. Hemagglutination
base and two types of tmumembrane glycoprotein is followed bv hemolysis and elution at 37 C. The
'
spikes at the surface. The longer spike is the virus can be grown in chick emhrvps - in the
hemagglutinin (H), which may also possess amniotic cavity for primary isolation and the
neuraminidase (N) activity and is hence known as allantoic cavity after adaptation. Eggsare inoculated
H or jHN protein. It is responsible for adsorption at b-S days and incuhated at 35 °C for five days
of the virus to the host cell surface . The second before harvesting.
spike is the I' ( fusion) protein, responsible for fusion Cell cultures are better suited for isolation -
"
of the viral envelope with the plasma membrane of primary monkey kidney being the preferred cell.
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< Pgramyxgvirysgs 513
Neuraminidase + 4 - -
InrrjetlliLdar inclusions in c c NX C
cytoplasm {CV nucleus (N)
The cytopathic effect is slow and consists of EpididymO -orchitis is a complication seen in
syncytium formation and the presence of acidophilic about a thirel of postpubertd male patients. The
cytoplasmic inclusions. Growth is hqst identified festis becomes swollen and acutely painful, with
by hem adsorption . accompanying fever and chills . Orchitis is usually
The mumps virus is labile , being rapidly unilateral but when ir is bilateral and followed by
inactivated at room temperature Of by cynosure to testicular atrophy, sterility or low sperm counts may
formaldehyde , ether or ultraviolet tight . It can be result .
—
preserved it 70 "C or by hjfphili nation..
Tire mumps virus is aniigeniically stable and only
The central nervous system is involved til about
60 per cent of cases, as indicated by pleocytosis in
one serotype exists.Two complement fixing antigens , the CSFbut only about 10 percent show symptoms
can be recognised , is in influenza viruses -the of meningitis- Mump bus been reported to cause
soluble (5) antigen and the ' viral' ( V ) antigen. about 10-15 per cent of cases of "aseptic mcningi ti s',
Climeal features: Infection is acquired by Mumps meningitis and meningoencephalitis usually
inhalation , and probably also through the conjunctiva. re salve without sequelae but deafness may
The virus replicates in the ujjper respiratory tract arid sometimes result . Mumps meningitis may
cervical Lymph nodes and is disseminated through occasionally occur in the absence of paioiitis, when
the bloodstream to vinous organs. "I he incubation , diagnosis rests solely on laboratory evidence . The
period is long, about 12-25 days. virus can be grown readily from the CSF in the
Parotid swelling is usually the first sign of early phase of meningitis.
illness chough it may sometimes be preceded by Other less common complications are nrthriris,
prodromal malaise , Parotid swelling is unilateral oophoritis, nephritis , pancreatitis, thyroiditis and
to start with Luc may become bilateral . It is myocarditis.
accompanied by fever, local pain and tenderness bur Kpidemiology; Mumps LS endemic worldwide
the skin over the glam! is not warm orerytliematiOUS. but has become Less common in the advanced
Parotitis is nonsuppurative and usually resolves nations due to im muni Ration. It often occurs as
within a week. However, involvement of the epidemics in children 5-15 years of age, and also
extraparnrid sites may be mote serious. Suck in young people living in groups such as in army
involvement may sometimes occur even in the camps. Household spread is common . Humans are
absence of parotitis. the only natural hosts. The source of infection is a
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516 * .
TGKttaok ol M crabiolcigy *
hemagglutination inhibition . -
150 300 rnn . The viral envelope has two
Serological diagnosis in hampered by wide —
glycoproteins the C protein by which the unus
antigenic cross reactions. Haired sera can be tested attaches to cell surfaces, and the fusion ( ]’) ptitein
by neutralisation, ELISA, HI or CF for rise in litre which brings about fusion between viral and host
of antibodies, cell membranes. The F protein is also rcspoisiblc
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i Paramyxoviruses » 517
—
occurring in the fir** few months of life. Most
patients recover it ] 1 2 weeks but those with
immunodeficiency or cardiac defects mav have
protracted iUncs* and high death rattS-
cell and syncytial formation but cytopathic effects
take about 10 days to appear. Earlier detection of
viral growth in cells is possible bv immuno -
fluorescence tests. Rapid diagnosis nf RSV infection
RSV is an important cause of otitis media in can be made by the immunofluorescence tost on
JP
young children . A relation between RSV and the smear* of nasopharyngeal swabs ,
sudden death syndrome in infants has been Serological diagnosis is by demonstration of
proposed bur not proven . rising antibody litres in paired scrum samples bv
In adults RSV infection may present as a febrile ELJSA, CF, neutralisation or immunofluorescence
common cold. It can cause pneumonia in the elderly, tests.
Epidemiology: RSV is global in distribution , Prophylaxis No effective vaccine is available.
lr causes annual epidemics in the temperate regions Attempts at immunisation by formalinised vaccines
during winter and in tile tropica during rainy had to be given up as the vaccinees developed more
seasons. Outbreaks are common in children s wards, serious illness than the controls on subsequent
nurseries and daycare centres. Infection is most exposure to the infection.
common in children six weeks to six months of Treatment: Management of RSV infection is
age, with the peak at 2-3 months. The newborn!; primarily by supportive Care . Administration of
are believed To be protected hv high levels of ribovirin Hv continuous aerosol has been found
maternal antibody. beneficial in hospitalised patients , decreasing the
The virus is transmitted by close contact and duration of illness and of virus shedding.
through contaminated fingers and fomites . Coarse
droplets of respiratory secretions discharged during GENUS MORBILUVIflUS
coughing and speeding arc more efficient in Mli \ SI KS C Its -
III . CI . A )
spreading the virus than fine aerosols. The Measles is an ancient disease but for a long time
incubation period is 4-6 day*, Virus shedding may no dear distinction was made between measles and
persist for 1-3 weeks , though children with other exanthematous diseases, including smallpox.
defective cell mediated immunity may continue to It was only in 162V that measles time to be
sited the virus for months. considered a separate entity. Thomas Sydenham in
Reinfection with the virus is not uncommon but 1690 gave the first dear and accurate description
the disease so produced is milder than in primary of measles in the English language.
infection. The role of the antibody in protection In lf )4 b an outbreak of measles occurred in the
against the infection is not clear. Secretory IgA is remote Faroe Islands,, affecting 75 per cent of the
considered more important than circulating IgG islanders. The classic study of this epidemic by Peter
in protection . Cell mediated immunity appears more Panum, a Danish muJicid student, laid tile basis of
important than humoral antibodies in recovery from our scientific knowledge about measles.
infection . RSV does not induce high levels uf The viral etiology ( it measles was established
interferon production. bv Goldberger and Anderson in IV 11 by
Laboratory dLadnoaiu: RSV can he isolated transmitting the disease to monkey* through the
from nasopharyngeal swabs or nasal washings, inoculation of filtrates of blood and nasopharyngeal
Samples should be inoculated in cell cultures secretion* from patients- The virus was isolated In
( HeLa or HEP-2 ) immediately after collection, monkey and human kidney cells by lindcrs and
freezing of clinical samples may destroy the virus. I cblcs in 1954,
In cultured cells, RSV causes characteristic giant ^
Men ales virus: The virus has the general
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518 * ‘raxlbOOh OF Microfcwtogy *
morphology ff pifUflJMtwiniStt ( Fug - 5ft.2 ) It it u few develop Complications whit- h may he due to
'
roughly spherics] but often pleomorphic particle,. the virus ( croup, bronchitis ) or to secondary
—
120 250 run in diameter. The rightly coiled helical
nuricocapsid is surrounded hy the lipoprotein
-
bacteria] infection (pneumonia* otitr media ). Ranch ,
the virus may cause a fatal gi .Liu cel ] pneumonia ,
'
The measles virus is antigcnically uniform - It Measles induces labour in some pregnant
shares antigens with the viruses of can inc distemper
and bovine rinderpest.
Clinical features: It lakes about y-1 l days
from the time of exposure to infection for the first
signs of clinical disease to appear. These consist of 2
prodromal malaise , fever, conjunctival injection ,
cough and nasal discharge . After 3-4 days of
prodromal illness, the rash appears. A day or two '
before the rash begins, Koplik 's spots develop or
the buccal mucosa and occasionally on the
conjunctiva and intestinal mucosa, The prodromal
illness subsides within a day or two of the
appearance of the rash. The red maculnpapular rash
of measles typically appears on the forehead first
and spreads downwards , to disappear in the same
sequence 3-6 days later, leaving behind A brownish Fig. 56.1 Measles res 1. Membrane ( Mi protein 2 Nuc -
discolouration and finely granular desquamation . leotapsid 3 . Lipoprotein membrane. 4. Spikes-
Most patients recover uneventfully but quite a hemaggluiinin .
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Arboviruses
-
ArixwimsM (ttthnpod botDC viruses] axe viruses
<rf vertebrates biologically transmitted by
disease caused ( Yellow fever ), the place of isolation
of the virus ( Kyasanur Forest Disease ) or the local
hematopKagnn-us insect vectnrs. They multiply in name for the disease (Chikungunya ) . They arc
bloodsucking insects and are transmitted by bite to classified according to their physical and chemical
v£ i"i:ebr& T £ hosts. Insect viruses and viruses cif features into CMNNOMINAL families. Arhoviruses have
vertebrates that are sometimes mechanically a
been placed in Toga- , Flivi-, Bunya-, Reo- and
transmitted by insects do not come into this
IT
Rhabdovirus families (Table 57-1), Within each
category. Inclusion In this group is based on family they are classified into genera, and antigenic
ecological and C|i i Jen .loLoi-iL - aJ considerations and groups, based cm serological relationships. Some
hence it contains members that are dissimilar in viruses arc ungrouped -
other properties. With better understanding of the Arhoviruscs have a very wide host range
physical and chemical properties of individual including many species of animals and birds. The
viruses, they are reassigned to more defined ability to multiply in arthropods is their special
tixodomical groups . Though tax on Clinically characteristic. The most important arbovirus vectors
unacceptable, the name Arbovirus' is a useful art mosquitoes, followed by ticks. Phclhofomus,
biological concept. COR -older and G m/cidae are less common vcctors.
'
A similar ecological group is that of the In the laboratory, mice are commonly used for
rodentborne ( robo ) viruses which are maintained growing arboviruses, intracerebral inoculation in
in nature by direct transmission between rodents, stickling mice being the most sensitive method for
and sometimes infecting other species, including their isolation. They can be grown in the yolk sac
humane, by direct contact without the agency of or chorioallantoic membrane of chick embryo, in
arthropod vectors- Robovi ruses, I ike arboviruses, tissue cultures of primary cells like chick embryo
belong to different taxonomical families, some of fibroblasts or continuous cell lines like vero or
them in common with arboviruses. 1 IcLa, and in cultures of appropriate insect tissues.
Arboviruses art worldwide in distribution but Most arboviruses agglminate the red cells of
are far more numerous in the tropical than in the -
goose or day old chicks . Hemagglutination is
temperate zones. Over 500 viruses have beer listed influenced by pH and temperature , the optimal
in the International Catalogue of Arboviruses requirements varying with different viruses.
published in 1965. MOST of them cause silent Spontaneous elution does not occur.
infections in rodents and ether wild mammals but Hemagglutination is inhibited specifically by
about 100 of them can infect humans. In India, antibody, and nonspecific ally by lipoprotein
over 40 arboviruses have been detected, of which inhibitors In serum, brain and other tissues.
more i ban 10 are known to produce human disease. In general, arboviruses are labile , being readily
Arboviruses have been named according to the inactivated at room temperature and by bile salts,
.
/"H
LJ opy righted
B w
jr
material
$ 22 * Textbook or Microbiology »
hemorrhagic few
Bunyaviridae Bunyimrus California eruxphaliris Qiopoucht, Turlock
!
ether iind other lipid solvents , Infecrivity may be arthralgia; encephalitis; hemorrhagic fever; and the
-
retained nr 70 °C or hv lyophilisatLOii . characteristic systemic disease , yellow fever (Table
.57,7 ) . All infections occur with varying degrees of
Antigenic Structurei Three antigens are
important in serological studies - hemagglutinin , severity', subclinical infections being very common.
complement fixing and neutralising antigens all
integral parts of the virus pm tide . Considerable
- Arboviruses also cause a number of veterinary
diseases such as Eaton, Western and Venezuelan
antigenic cross reactions occur among arboviruses. equine encephalitis in horses in America, Rift
The plaque reduction neutralisation test { PRNT} Valley lever in sheep and cattle in Africa, Blue
shows the greatest specificity. tongue in asses in India, Africa and America,
Pathogenesis: The virus enters the body Ganj am d isease of sheep in India and African horse
tbrough the bile of the insect vector . After sickness in horses and mules in Africa and Asia.
multiplication in the reticuloendothelial system , 1 aboratory diagnosis: Diagnosis may be
viremia of varying duration ensues and, in some established hy virus isolation or scmlngy. As all
cases, the virus Is transported to the target organs , arbovirus infections arc viremic, blood collected
such aa tlie central nervous system in during the acute phase of the disease may yield the
encephalitides, the liver in yellow fever and the virus. Isolation may also be made from the CSF in
capillary endothelium in hemorrhagic fevers. some encephalitic cases but the best specimen for
Arboviruses cause the following elinieal virus isolation is the brain . Specimens are inoculated
s y n d r o m e f e v e r with or without r a s h a n d intracerebrally i n t o suckling mice. The animals
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4 Arbcv iu
^CS * 525
lesser extent in complement fixation tests . The when the disease Erst appeared there in 1963
neutralisation test is more specific. They produce but have been extremely rare afterwards. The
epidemics of encephalitis in America and dengue ’ fever is typically biphasic with a period of
like fever in the tropics.
Enoephaililis viruses; Three members of this
—
remission after 1 6 days of fever. The vector is
Aidei legypli . Nt> animal reservoir has been
group, Eastern, Western and Venezuelan equine identified . Antibody to the virus has been
encephalitis viruses, cause encephalitis in horses demonstrated in horsey cattle andother domestic
and humans , Eastern equine encephalitis ( EEE ) am main but its significance is not known. No
occurs along eastern Canada , USA and the vaccine is available.
Caribbean , causing sporadic cases and small 2. 1 fYUTtg-nyong virus . Th is vims was first isolated
^
epidemics. Western equine encephalitis ( WEE ) is in Uganda , litis is confined to Africa, is closely
more widely distributed in America and causes large related to the ehikungunva virus antigenically
epidemics. Venezuelan equine encephalitis [VEE) , and causes a similar disease. This is transmitted
prevalent in Central and South America, usually by the Anopheles species. The Mayaro virus
causes an influenza - like illness, with encephalitis causes a similar disease in the AAfcsr Indies and
in a small proportion of cases. Several species of South America.
Culcx and Anopheles mosquitoes are the vectors 3. Semi1.1, : Fonstvirut : This vims was. first isolated
and wdd bLrds the reservo i ns. Formal ] nised vacci n en in 1942 in Uganda from Aedes mosquitoes has
have been developed for EEE and WEE and a not been associated with clinical illness in
Live attenuated vaccine for VEE, humans though neutralising antibodies to the
virus have been demonstrated in Africans, The
Viruses Causing Fchi Je Illness Sindbis virus , originally isolated from Culex
J. Chrkungunpa wirm.. This virus was first isolated mosquitoes in the Slndbi -i district of Egypt in
from human patients and Aedes aegypii 1952 , has subsequently been recovered from
mosquitoes from Tanzania in 1952- The name other puts of Africa, India, Philippines and
'
chikungunya' is derived from the native word Australia. In Africa , it is known to be associated
for the disease in which the patient lies H doubted with febrile illness in human beings. In India,
up* due tD severe joint pains. Epidemics of antibodies have been detected in human sera
dukungunya have occurred in many African but no association has been established with
countries . In 195 B, the vims caused a large human disease . The closely related Ross River
epidemic of hemorrhagic fever in Thailand . The virus has been associated with epidemic
virus first appeared in India in 1963, when along polyarthritis in Australia.
with dengue, it caused very extensive epidemics
in Calcutta , Madras and other areas. FUVW1 RUSES
Chikungunya outbreaks occurred at irregular Ike family Flaviviridae eontamh only ore genus,
intervals along the east coast of India and in .
FiavivituS They are somewhat smaller than
Maharashtra till 1973. Since then the virus has alphaviru«5 , being 40 nm in diameter, The name
been quiescent. /IsV7 virus refers to the type species, the Yellow fever
The disease presents as a sudden onset of fever, virus ( FJavus, L = yellow ).
crippling joint pains, lymphadenopathy and There are over 60 arthropod -borne flaviviruses,
conjunctivitis, A maculopapukr rash is common Reprecentarbre members af th i g r o u p are
and some show hemorrhagic roam lest mums . dhtriliutcd in all parts of the world* covering all
Hemorrhagic lesions were common in Calcutta the zoogeograph ic regio ns. They may be considered
Lu u 1 1
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« AftHwusH p 537
In India, I .Lpan-e -sc encephalitis was first cattle do not develop circniia, they do not -contriluitc
recognised in 1955 when the virus was isolated to spread of the virus. The high cattle- pig ratio in
from mosquitoes of the tufev Hshnuj complex from India hasbeen suggested as a factor limiting human
Vellore during an outbreak of encephalitis in Tamil infection.
Nadu , The vims continued to be active in Tamil Prevent ive measures include mosquito control
Nadu and Andhra in subsequent years also, causing and locsti i ig pi ISeries away from human dwellings.
illness mainly in children, indicating the endemic A formalin inactivated mouse hr ,Liu vaccine using
nature of the virus. Most of the cases occurred the Nakayama strain has been employed successfully
between October and Nowmbcn for human immunisation in Japan and. lit a small
Jipanestr encephahLi ^ remained confined to the scale, in India also. Two doses at two weeks" interval
southeastern parts of Indu Till 1973, when it caused followed by a booster 6-12 months later constitute
A large outbreak of encephalitis in West Bengal , a full course . Immunity produced by the vaccine is
The epidemic affected adults also, with mortality shortlived. A live attenuated vaccine has been
rates approaching 50 per cent , suggesting that the developed in China from JE strain SA 14- 14- 2,
virus was freshly introduced into the area. Cases passed through weanling mice The vaccine is
occurred mainly between |une and October. Fro FT produced in primary' baby hamster kidney cells.
1976, there have been periiwiical outbreaks of the Administered in two doses, one year apart, the
d ease in various parts of India— Dihrugarh
^ vaccine has been reportedly effective in preventing
( Assam ) in the cast, Gorakhpur ( Uttar Pradesh) clinical disease.
. .
und E E LCV H 11 ,L in the north .mdGiia and Maharashtra Vaccination nf pigs has been proposed in view
in the west. In the south , outbreaks have occurred of their importance as amplifier hosts. During
in Kolar in Karnataka1 various areas in Andhra major epidemics , slaughter of pigs have been
Pradesh, I’irunelveli and South Arcnt in Tamil employed as a measure of conti inmentr A million
Nadu, in Pondicherry and lately in Kerala . In pigs were reportedly slaughtered in Malaysia in
addition, sporadic cases have been reported from 1999 to stop an epidemic of encephalitis.
different parts nl the country, excepting the Yellow fes er: Yellow fever was recognised as a
northwestern states . Japanese encephalitis has clinical entity as early as in the seventeenth century
become a mu jot public health problem of national and was familiar to pirates as the 'Yellow Jack'. Jr
importance in India ( Fig, 57.1) , is a native of Africa and was transported thence
The natural cycle of the virus has been worked along the trade routes to Europe and America . The
out in detail in Japan. Herons act as reservoir hosts most serious epidemics occurred in the Western
and pigs as amplifier hosts. Human infection is a I Hemisphere' Central .America and the Caribbean,
tangential 'dead- end’ event and occurs when the and even as far north as New York. Since the early
I n 11! i*. J iI if. i L .LL J I LI igk den xi 1 y. Tl i L natural
11:J enl EL 1
L ( ' ' ' ' largely
twenik rh century, the rii & euiE IL.M: Keen
cycle in India also may be similar. Natural in lection confined to certain areas of Africa and South and
has been demonstrated in Ardcid birds (berons and Central America.
egrets), 0 ' wdl as hird- ro- bird transmission through Carlos Finlay in Cuba in 1331 suggested that
QiJct tnVaen/orbync/ius, Other birds such as ducks, yellow fever was spread by Acdes aegypti
pigeon) and sparrows may also be involved . mosquitoes. In 1900, the US Army Yellow Fever
Vertebrate hosts may Include cattle and buffaloes, Commission, under Walter Reed, confirmed this
besides pigs - The rmiior vector Culcx observation and demonstrated that Aedcs
tritucif '. fyi hynchus has a predilection for cattle and
'
mosquitoes were infected by feeding on human
bites them in preference to humans or pigs, but as pati ents during the early vi remi L phase of the disease
>pyrighted material
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530 4 Tpittbaok o1 Microbiology
no doubt, has checked the entry of the virus ln.to in Thailand, have since occurred in many countrics
India through legitimate passengers. It is likely that in Southeast Asia and the Western Pacific . They
stray virus introduced may have been kept uut, due arc mote common in previously healthy children
to the prevalence in the local Aedes aegypti of in the indigenous populations of endemic areas.
] Jetigue virus, and of anybodies to a wide range of They may be a hypersensitivity or enchancement
arboviruses in the local population. Another reason response to sequential dengue virus infection in
could have been that in Africa, yellow fever was persons sensitised by prior exposure to other
ifi.iinlv in the west , and in India , Aedes mosqiutoea serotypes of the virus.
were along the east coast , so that even stray Dengue virus is transmitted from person to
importations of vims by sea may not have found person by Aedes ae ti mosquitoes. The extrinsic
suitable vectors. TIIK is no longer valid as yellow ^
incubation period is fi-10 days. No vertebrate hosts
fever has in recent years have caused epidemics in other than humans have beer identified.
East Africa , and Aides mosquitoes have spread all Dengue was initially confined to the east coast
along the west coast of India. If ever yellow fever of India and has Caused epidtitt ica, some t i me i along
gets established in India* the consequences could with the chikungunya virus, as in 1963 when
be catastrophic. extensive outbreaks affected Calcutta and Madras.
Dengue: Dengue virus in vilely distributed Subsequently it has spread westwards and in the
throughout the tropics and subtropics. (The name 1990s Surat and Delhi had major epidemics with
\ lengue’ derived front the Swahili KJ deiygap£ pQt deaths due to DHF and DSS. All four types of
meaning a sudden seizure by a demon. The term dengue virus are present in this country.
' break - bone fever’ was coined during the Occasionally, more than one type of the virus has
lJhiLadelphi.i epidemic in 1750). Dengue fever is been isolated from the same patient.
clinically similar to the illness caused by Demonstration of circulating lgM antibody
chikungunya and O' nyong nyong viruses. Four
* provides early diagnosis, as it appears within two
types of dengue virus exist: DEN 1 first isolated to five days of the onset of illness and persists for
from Hawn in 1944, DEN 2 from Nrw Guinea i n one to three months. IgM ELISA test offers
1944 and DEN 3 and 4 from the Philippines in reliable diagnosis. A scrip imiriunoehrornaiographie
195 b. Immurin is type specific so that it is passible
' test for IgM is available for rapid diagnosis.
fora person to have four separate episodes of dengue Control of dengue is limited to vector control
fever. Dengue has been increasing worldwide over as no vaccine is currently available.
the last few decades and today ranks as the most
important wetorborne disease, with about 2.5 TICIBORNB GROUP
billion people in 200 countries at risk . These viruses produce two clinical syndromes,
Dengue presents clinically after an incubation encephalitis and hemorrhagic fevers.
period of 3-14 days, as lever of sudden onset with Tiokbome encephalitis viruses: A number
headache, retrobulbar pain, conjunctival injection, nf viruses belongi ng to the Ru»i; m Spring Summer
-
p.Lin in the back and limbs ( break bone fever ),
lymphadenopathy and maculopapular rash . The
Encephalitis ( RSSE ) complex cause encephalitis
along a wide area ot the northern lindm.viH from
fever is typically biphasic (saddle . back) and lasts Scotland to Siberia.The names given to the disease
for 5-7 days. Dengue may also occur in more serious vary from one area to another depending on the
forms, wirh hemorrhagic manifestations {dengue variations In the prominent clinical features. Thus,
Hemorrhagic fever) or with shock (dengue shock in Scotland, it is called 'louping ill' as the disease
syndrome] . These complications, first recognised occurs primarily in sheep in which it causes a
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532 * Textbook of Microbiology *
V
—
taj E
- JT* --
%4T
_ ijr __
4
v1- i
Fkg. 5?. 2 DI $|ribu1iQn of Kyasanur Foresl Disease (Courtesy : Nafmnal Inslilule of Virology, Pune ;-
fienus I hUboviniB: Tlte major members of
BUN YAVIRUSES
This familv containing over 300 species is the
’
this genus are the sandfly fever and Rift Valley fever
viruses.
Invest group of arboviruses. The virus is about 1.00
f Webufomufi fever or sandfly fever, also known
rtm in diameter and has A complex Structure, with ^ -
as Pappataci fever and three day fever, is a self-
a triple segmented genome of single stranded RNA.
Mosr bunyaviruses ue masquitobomc. Some are limited, nonfatal fever nansmined hv the bite of
transmitted by sandflies ( forexample, Phlebommus the sandfly Pftiefcotetfnu paparasii, It occurs along
^
fever) or ticks (Crimean Congo hemorrhagic fever). the MetijterrjiTUfan Coast and Central Asia,
Some arc established pathogeny causing natural extending as far east as Pakistan and North Wsm
diseases, and even epidemics and epizootics while India. Cases have also been reported from South
many hive been isolated only from insect vectors and Ccn tral America.Twenty ant i genic types of the
and have not been associated with any human or virus exist , of which only five cause human
animal disease . Bunjnvimgcs are HO named from - -
disease — Naples, Sicilian, Punta Turu f Chagics,
the type species Bunvamwtra virus isolated from Candlru.HIT»c vjrus has been isolated from sandflies
mosquitoes in Uganda in 1946. and patients in India. No vertebrate host other than
The family Bunyaviridae contains four genera humans has been identified. Vbcife is evidence for
of medical importance— Bunyavirus, Phlebovjrus, vertical transmission of the virus in sandflies.
Nairovirus and Hantavirus. A number of viruses Rr /r Valley fever is a inosqpirobot tie virus
are yet ungruuped. causing enzootic hepatitis in sheep Laid other.
Gtitlus Bunyavirus: The genus contains over domestic animals in Africa, ft is named after Rir t '
150 specie , of which only a few cause human Valley, Kenya,where it was first recognised., Human
*
infect ions. The clinical disease caused is infection causes a disease resembling influenza. In
encephalitis, aseptic meningitis and fever, The 1977-80, Rift Valley fever caused extensive
California encephalitis gjooip of viruses are endemic epidemics, with many deaths in Egypt and an
in the USA. Large epidemics of fever with aseptic outbreak ofliicniorThagie fever with many deaths
meningitis have been caused by Ovopouehr virus in 1997-9? in Kenya. In 2000, it spread outside
( member of the Simbu group) in Broad- The midge Africa for the first time, causing epidemics in Vemen
CuJrcojde paracusia is the major vector for the and Saudi Arabia with hundreds of dcsths-
*
Oropouchc vims . frMus Naimvinu: The uenus is named after
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H Arboviruses * 533
endemic in Eiitcm Europe, Central Asia and many Europe, and Prospect Hill virus isolated from voles
parts of Africa. Cattle , sheep , goats and other in the USA, which has not been associated with
domesticated animals act as natural reservoirs. It is human illness.
transmitted by Hyalornma ticks. During the acute Hantavirus species arc natural pathogens of
phase of the disease, the blood of the patients is
highly in feet i uus and direct Transm issitm may occur
—
rodents field mice [ Apodemus agntr.' < ) being the
js
ff
when a large number of US soldiers serving in physical findings. In severe cases, tachypnea ,
Korea got the infection mu it has been prevalent in tachycardia, hypotension and hypoxia lead to death.
Scandinavia , Russia and China for centuries.
The disease occurs in two forms the milder
epidemic nephritis ( EN ) common in Scandinavia
and the more serious epidemic hemorrhagic fever
— The disease is caused by a newly identified
hantavirus, the Sm Nombre { meaning nameless )
virus, which is associated with the deer mouse and
other rodents of the sigmodontine subfamily. No
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H RtabdOVifUHE > 517
cytopafhic effects are not apparent and the yield of Pasteur.'Vhc boy survived, Thfr dramatic event was
vnus is low. The fined virus strains adapted for a milestone in the development of medicine.
growth in human diploid cell, chick embiyo and Pathogenesis Human infection is usually
veto cell cultures are used for the production of caused by the bite of rabid dogs or other animals.
vaccines, The virus present in the saliva of the animal is
deposited in the wound . If untreated, about half of
RABIES such case; may develop rabies. Rarely, infection can
-
Rabir ' has been recognised from very ancient rimes
as a disease transmitted to humans and animals by
-
also occur following non bite exposures such as
licks or aerosols or transplantation of cornea or
the bite of 'mad dogs' . The name 'rabies’ comes other virus Infected tissues. Humans appear to
from the I . itin word rahfdus, meaning mu. I , derived possess a high degree of natural resistance to rabies.
from the Sanskrit root mbhsSf , for frenzy. Reference The extent of inapparent or abortive infect i ' in with
to rabies occurs in the Mesopotamian laws of rabies virus in humans is not known but the finding,
Fshnunna ( Circa 2200 BC ) . The disease was in a survey, of rabies antibodies in * ix per cent of
tradiiinnally associated with the appearance of the veterinarians without any history of anrirabic
Dog Star Sirius in the dog days af summer when vaccination suggests that it does occur.
dogs were considered to be prone to spells of The virus appears to multiply in the muscles,
'
-
madness . The di -sease in human being is called connective tissue or nerves at the s- iCc nt deposition
hydrophobia because the patient exhibits fear of
water, being incapable of drinking though subject
-
for 48 72 hours. It penetrates the nerve endings
and travels m the axoplasm towards the spinal cord
-
to intolerable thirst . Rabies in animals i not called
hydrophobia because they do not have this peculiar
and brain. The movement of the virus in the Ubtls
ii p; LH!- ivc, at a speed of about 3 mm per hour . The
/"H .
LJ opy righted
Bw jr
material
538 T xiboei of MitrcSio
i
^ *
milk and urine. Vircmia is not clinically significant and in those who have received postexposure
though ir has been donon ^tnled under experimental vaccination. Patients who survive the stage of acute
conditions. neurological involvement lapse into coma , which
In humans chi - incubation period is usually from m a y last for hours o r days. Death i s due t o
1-1 months, though it may be as short as 7 days or respiratory arrest or other complications.
as long us dime years. The incubation period is Some persons exposed to real of imaginary risk
usually short in persons bitten on the face or head, of rabies develop a psychological disorder which
and long in those briten on the legs, This may be
milted to the distance the virus has to travel to
-
ha ? been called tyssaphobia or h ydrti photlop hobia.
Patients present with anxiety, irritability and
reach the br ;Lm . The incubation period is generally exaggerated hydrophobia , They are afebrile.
shorter in children than in adults. Serial ion and reassurance are generalk all that are
The course of the disease in humans car be called for.
—
classified into four stages prodrome , acute In dogs, the incubation period is usually 3 6
weeks but it may range from 10 days to a year. The
-
encephsill IC phase, coma and deftch. The omei is
marked by prodromal symptoms such as fever, initial signs arc an alert, troubled air and a change
headache , malaise, fatigue and anorexia. An eftrly in disposition w i t h restlessness , snapping at
symptom is often a neurluc type of pain or imaginary objects, licking of gnawing at the siie of
paresthesia and fascicularion at the site of virus the bite. After 2-3 days of tliri prodromal stage,
entry. Apprehension, anxiety, agitation , irritability, the disease develops into cither the furious or dumb
nervousness, insomnia or depression character:?- r type of tallies. In filrioux rabies, which is much
The acute neurological phase usually begins convulsions and death follow, The second type,
with hyperactivity, which is characteristically dumb rabies, is the paralytic form In which the
intermittent, with bouts of bizarre behaviour, animal lies huddled, unable to feed. The dog may
agitation or seizures appearing between apparently not hilx but attempts to feed it are dangerous. The
normal periods . Such hyperactivity may be dumb form Is as infectious as the furious type. About
spontaneous or pteripasted by external stimuli . The 60 per cent of rabid dogs shed the virus in saliva.
pathognomonic feature is difficulty in dririkiug, Rabid dogs usually die in 3-5 days.
together with intense thirst. Patients may be able There have been reports of persons developing
to swallow dry solids but not liquids. Attempts to rabies after being bitten by apparendy healthy dogs.
drink bring urt such painful spasms of the pharynx However, in countries like India where stray dogs
and larynx producing choking or gagging that are so common, it is not always easy to exclude the
patients- develop a dread of even the slight or sound possibility of such patients having been bitten earlier
of water ( hydrophobia ). Generalised convulsions by other animals. The presence and prevalence of
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4 RhandouiruFieF: BAl
. .
I Ltnir LIIJJI -LIII India manufacture BPL vaccine . strain) grown on human diploid «Els ( Wl dfl
3. inf int brain vaccines; Tire cncephalitogenic or MR.C 5 ) and inactivated with beta
factor in brain tissue is a basic prorein associated proplolactone or tri- n -butyl phosphater It is
with myc - in , It is scanty nr absent in the highly antigenic and free front s-erious Side
'
nonmyelinated neural tissue of newborn an i m a Is effects. Tts only disadvantage is its high cost .
So vaccines were developed using infant mouse, Other equally effective and more economical
rat or rabbit brain . Occasional eases of vaccines have been developed. Ihcsc include;
neurological reactions Lave occulted following primary cell culture vaccines grown on chick
infant brain vaccines also- Infant brain vaccine embryo, hamster ki dney and dog kidney cells and
is impractical in India due to the very large conr .' jjunus cell culture vaccines grown on Verio
quantities required. cell line derived from the kidneys of vervet
Neural vaccines are Lins at is factory for many monkey or African green monkey ( CcjTopjYjftecws
reasons. "J’hcy are poor immunogens as they contain artfrinpr).
mostly nuclcocapsid antigen, with only small In India the following cell culture vaccines are
quantifies- of glycoproLi; i 11 tj,. which is the sole available: Human diploid cell { HDC } vaccine
protective antigen. They may contain infectious chick embryo cell ( PCEC } vaccine and
agents which may not he inactivated during vaccine Purified verv cell ( PVC ) vaccine. All three of
preparation and storage. They are encephalitogenic. them are equally safe and effective.
Neural vaccines have been abandoned in the 3. Subuoii raceme:The glycoprotein subunit on
developed countries. The onlv reason for their the v ] rus surface, whith i the protect i ve ann:gcn,
continued production and use in a few developing has been cloned and recombinant vaccines
countries is that they are cheap. produced. They arc still in the experimental
stage.
-
NON NEURAL VACCINES
VACCINATION SCHEDUI L -. S
1. Egg racemes: (a ) Duct egg vaccine prepared
from a feted virus adapted for growth m duck Antlrabic vaccine should be administered when
eggs and inact i rated wi [ h beta propiolactone was person has been bitten, scratched or licked by an
used, hut was discontinued because I its poor animal which is rabid or cannot be apprehended.
immunogen kiry, A purified, more potent duck When the biting animal can be observed, it should
egg vaccine was developed, but was supplanted
bv.
JT
tissue culture vaccines which became
available then. ( b) Live attenuated chick embryo
not he destroyed but should be kept for ten days.
The observation period often days is recommended
hecausc the vims may be present in tile Sldivfl 3 4
_
vaccines; TWO types of vaccines were developed days before onset of symptoms and the animal
-
with [ lie Flury strain rhe Low Egg Passage —
usually dies within 5 fi days of developing the
—
( LEP) vaccine at 40 SO egg passage level for
immunisation oi dogp and the High Egg Passage
disease. If the animal remains healthy after this
]K rind , there is no li * k of rabies and vaccination, if
{ HEP) vaccine at ISO passage level for cattle already starred , may be discontinued - This, of course,
and cats these are nor in use now.
, does nor take into account the rare possibility of
2 - Tissue culture ncc. us: The first cell culture the carrier state in dogs.
vaccine was the human diploid «11 ( HDC ) In cases where the vaccine is started with the
vaccine developed by Koprowslcy, Wlktor and hiring an i mil kept under observation , art alcernat i ve
Plotkirt . It IH a purified and concentrated recommendation is to stop treatment after five days.
preparation affixed rabies virus {Pitman-M' lore The animal is observed for a further five davs*
vaccine being starred again if die animal becomes f All - nicka] and wolf hires
,
ill ar dies during [be period. g. Any Class 11 patient who has not received
NtLIlMl VfljMiilttK The dosage [ ]f the vlccirte treatment within 14 days of exposure.
depends on the degree of rink to which the patient The ren >mmcndcd schedule of vaccination for
has been exposed. Accordingly; pat ients are classified the different classes is as follows:
as follows: Semple vaccine BPL vaccine
Class I: Patients in whom the risk i -. estimated to
Class I 2 ml x 7 days 2 mi x 7 days
be slight. These include: L iass II
'
5 ml x 14 days 3 mi x 10 days
a. Licks, including direct contact with saliva on Class III ID ml x 14 days 5 mi x 10 days
definitely remembered fresh cuts or abrasions The above schedule for the BPL vaccine is
on all parts of the body except the head, face, recommended by the Pasteur Institute, Coonoor.
neck or fingers. The Central Research Institute , Kasauli ,
b. Licks on intact mucous membrane or recommends a slightly different dosage for its
conjunctiva. vaccine, ( The manufacturer instructionsshould be
c. hides or scratches which havciai -ed the epidermis followed in even' case ). ^
but have not drawn blood , on all parts of the body The immunity following vaccination with
except the he .L:L 1.LCC . mck or fillers. neural viaincs is expected to last far six months
d. Consumption of unboiled milk or handling raw only and any exposure later should receive fresh
flesh of rabid animals. treatment . The vaccine is ad mi nistcEcd
Class II : Persons who arc e& Timated to he at subcutaneously on the anterior abdominal wall.
moderate risk. These include: Anti Tab i c Vaccine may cause certain adverse
a. Licks on definitely remembered fresh cuts or reactions. These range from minor local reactions
abrasions on the fingers-. to serious ncuroparalitic cam plications. The latter
b. All bites or scratches on the fingers which are may he of the neurihe type, the doreilumbar type,
not lacerated, not more chan half a centimetre the Landry type of ascending paralysis or
long and have nut penetrated the true skin. encephalomyelitis. The etiology of neurological
c . Hites or scratches on all parts of the body except complication is believed to be immune response to
the head, face, neck or fingers which havedrawn the injected brain Tissue resulting in organ specific
blood but excluding bites which have five teeth iinmunologicaJ damage .LH in experimental allergic
marks or more, or in which extensive laceration encephalomyelitis. These complications usually
has occurred . occur within 1-4 weeks of commencement of
Class III : Persons in whom the risk is estimated vaccination. The incidence of complications varies
to be great . These include: with different vaccines, one in 2000 to one in 12,000.
a. Licks on definitely remembered fresh cuts or When such complications are noticed during the
abrasions on head, face or neck. course of vaccination , further vaccination should
b. All hues or scratches on the head, face ur neck. he withheld and the patient started on
c. All bites or scratches on the fingers which are corticosteroids. If further vaccina tion is considered
lacerated, mure than half centimetre long ur ,
imperative, non -neural vaccine should be used.
hive penetrated the true skin. Severe exertion and the use of alcohol during
d. All bites penetrating the true skin and drawing vaccination have been said to increase the risk of
blood , when there are five teeth marks or more. neuralogical neactiinns.
C, All li i tes on any part of the body L .LUH i ng extensive
'
( lull culture vaccina: All three cell culture
laceration. vaccines available in India ( HI JC , PCEC and PVC )
opyrighted materia
< Hhabttoviruws * 543
'
+
I
t $ Ik *
F|gP 53 - 2 Negri btwltes : Oiiuio inclusiors in Hie Cytoplasm ! neurons In Tahiti dog train
have the same dosage schedule, which is the same Pittivr immunisation : Antirablc scrum is
for both adults and childrer . manufactured by hyper immunisation of horses .
Pre -exposure prophylaxis requires rhrw doses Cnidc equine antirabies scrum is not to he used as
of the vaccine injected on day 0, 7 , 21 or Q, 28 and it is liable to induce anaphylactic reactions, Purified
5fi. A booster dose is recommended after One year equine rabies immune globulin ( LRlG ) is much
and then one every five years, safer, though not completely free from risk. Human
Fostexposure prophylaxis requires five or six rabies immune globulin (11RIG ) is fret from the
doses, on days 0, 3, 7, 14, 30 and optionally 90. danger of ttneidtatkm but should be ensured free
Tins course is expected to give protect ion for at Inom ElIV and hepatitis viruses. HRIG is much
least five years-, during which ]TL'rioJ any further costlier than tRlG,
exposure may need only one or two brmsrrr doses Passive immunisation is an important adjunct
(on days 0, 3) depending on the degree of risk . fn vaccination and should lie invariably employed
After fi %' e years, it is advisable to give a fitll five whenever the exposure is considered of high risk .
injection course if exposed to infection , The recommended dose of H RIG is 20 lU/kg body
The vaccine is to be given IM or SC in the weight, half the volume infiltrated at the site of
deltoid region , or in children on the anterolateral the wound and the other half injected in the gluteal
aspect of the thigh. Gluteal injections arc to be region. Passive iinmuni -sarton should be given before
avoided as they are luund to be EeaS jrtiiminugCniC. or simultaneously with the first injection qf the
It has been shown that a dose af 0 , 1 ml vaccine, but not after it. In persons receiving the
administered intradermailv is as effective as a 0, 5- serum and vaccine , a booster dose of cell culture
1.0 m! dose SC ot IM and that i uwnurii &adon may vaccine on day 90 may he given.
thus he made more economical . However, this is Recommendations for postexposure prophylaxis,
not rccommrnded a routine practice , as as endorsed by the WHO in 1988,, are shown In
imradermul tnjtxtion is technically difficult , and it LVaccj' ne faifores' ( persons developing rabies
will be ineffective if this dnse is given even after a full course of immunisation ) are not
subcutaneously by mistake. uncommon with neural vaccines , while they are
pyriq
544 H Textbook of Microbiology
--
Tab lc 50-1 Recommendalion for postexposure prophylaxis ( WHO]
extremely rare when Immediate Inral treatment has not beeo recorded, though the virus is present in
betel followed by rabies immunoglobulin and a full the saliva of patients. Therefore, there is nn danger
course of i cell culture vaccine. In view of the safetv in examining or nursing hydrophobia patients
of the cell culture vaccine, U would be advisable to provided suitable precautions arc taken. An unusual
recommend the vaccine even when there is the mode of tranum iss- ion of rabies has occurred in some
slightest risk of exposure to rabies. recipients of corneal grafts- The donors had died
Vaccine lor animals: Anlirabic* Lmmunisarkm of unsuspected rabies and the infection was
in animus is to be done as pre-exposure prophylaxis. transmitted through the cornea.
PostcxpoHure treat me ot is nut generally of much Rabies virus is present in terrestrial animals in
use, Neural vaccines arc not satisfactory as they are all parts [if the world except Australasia and
not adequately immunogenic, need nuiiciplf doses Antarrica, and some islands like Britain. Two
and have to he repealed every six tn ( jn. Clie . -
epidemiological types of rabies exist , [/rharg
transmitted hv domestic animats like dogs and cats;
Concentrated cell culture vaccines containing
inactivated virus are nuw available , which give and syfvaitru , involving animals in the wild, such as
gnod protection after a single IM injection . jackals, wolves, foxes, mongooses, skunks and bats.
Injections are given at 12 weeks of age and repeated -
Most cases of human rabies follow dog bitc fi but in
—
at 1 3 yeux intervals. Rabies vaccines mav he given endemic areas almost any animal can transmit
rabies . In India , anti Tabic treatment in to he
separately or as combined vaccine for immunisation
against other common veterinary infections also. considered following rhe bite of any animal except
'DwalitienC Until recently, rabies was considered .-
rats. Where urban i r i mestic rabies has been
to be invariably fatal and no serious attempt ar controlled , as in the USA, the majority of infections
treatment was made , apart from sedation. It has are due TO bices bv wild itiimali .
now been demonstrated that Complete recovery Carl The primary source of lire rabies virus in nature
occur from established rabies , with iotensive seems to he in the mustel 'Js and vtverrids, rhe
supportive care and management of complications. ermine in rhe northern coniferous forests, the skunk,
No specific an 11 rabies ugeiU is available. mink and weasel in North America, the mottled
Epidemiology : Human rallies is A [lead end. pole car in the USSR, rhe elver and pole cat in
Direct person - to- person transmission of rabies has Africa and the mongoose in Asia. Rabies vims has
Copyrighted materia
i Rhabaaviryses * 545
been isolated repeatedly from the bruin jind salivary shed the rabies vims as sympenniless carriers over
glands of apparently healthy wild rodents.The virus a period of several months.
survives in this reservoir population by achieving Rabies alio occurs in insectivorous and
i start: of latency with occasional activation nucb
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546 i Ttfidbaoh Oil M . ::rnbiotogy
no native mustelhd nr vivemd population has no clinical rabies after being bitten by a bar. It is
rabies . Eradication of rabies from countries Like classified as Lyssavirus serotype 4.
India wirh abundant wildlife may not be practicable. 5 . 6. Rabies- bke viruses isolated from European
bats have been classified into two groups:
RABIES RELATED VIRUSES European bat lyss &vi rus types 1 and 2 . They can
The genus Lyssavints consist* of the rabies virus infect humans, as was found in the UK in 2002,
and other serologically related viniHS. Lyssavirusts when a wildlife worker fell ill with 'rabies' and
have been classified into seven serotypes. diLid. Tins was the first "rabies death in the UK
1
2. The Lagos bat virus, classified a* Lyrsivirus 7. Australia was considered free of rabies and
serotype 2 . was isolated in 1956 from the pooled related viruses till 1996, when a lyssavirus was
brEiins of frugivnnous bats from Lagos Island, isolated from a frtigivorous bat . Since then a
Nigeria, It causes a rabies-like illness following number of similar isolates halve been obtained
intracerebral inoculation. Negri bodies are found from ; Litferent types of bats in Australia. Fatal
in infected monkey brain but not in mice or dogs. infection* have occurred in persons having
3-. The Mokula vims , first isolated in 1968 from contact with bats. The virus antibody is widely
shrews captured near Ibadan , Nigeria, has later prevalent among Austral i:tn bats which appear
been found in many wild and domestic animals to be carriers. The virus, named A u f ndfim bat
^
m Africa, It was also recovered from two lyssavieus is closely related to, but distinct from
children with central nervous system disease, the rabies vims. Antirahic vaccine and scrum
one of whom died. A case of laboratory infection appear to protect against experimental infection.
with the v: nis occurred in a person possessing The relevance of rabies related viruses in human
high tittes of antibody to the rabies virus. 1c is disease is not dear, though some of them have
classified as Lyssavlms serotype 3. caused illness and death in humans. They are
A . The Duvenhage virus was reported in J 971 from considered to represent a biological bridge between
the brain of a man who died in South Africa of the rabies virus and other rhabdoviniscs.
Further Reading
GM . 1991 . The Nimfil I h .-.icn of
]i , ii.Lr
FiEhijcsii . CRC Pre -^.
BD wd LE RubLnaun I 99.V Rubies, Vpw fi- nglj Afwf 329:1632.
Rupprecht CE ei al. 1994. Lyaavinuea . CufWrlf Tfiprcs in Micrvbioi Immunol 167 .
Smith JS ct al . 1990. Unexplained rabies in three irtimLgrjnTt in The United States. New EngJ Med 324 :1990.
Smith JS. 1996. iVew aspects of rabies. Clin Miembio! Re* 9:166 .
WHO. 1992 . Expen CflunturnM u»t Rxhiex , 3rb Kep«) rT . Technical Serin No . 924 , Geneva.
Copyrighted material
Hepatitis Viruses
.
A hepatitis A second type of viral hepatitis , TYPE A HEPATITIS
transmitted mainly hy inoculation was originally Type A hepatitis ( infections hepatitis} is a subacute
olnserved in persons receiving serum inoculation disease of global distribution, affecting mainly
or blood transfusion. This had been given various children and young adults.
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550 i Textbook of MicrDtupIpgy »
third ( ]t the wi?rld|' population is csti mated to he Hepatitis It virus ( HBV ): HRV is a 42 nm
have been infected by hepatitis R virus ( HBV ). DMA virus with an outer envelope and an inner
About a quarter of them hecome HRV carrieni. A conL , 27 n:n ul di&neterj enclosing the viral, gtibtmW
quarter of these develop serious liver disease , and a DNA polymerise ( Mg 59, 2), Because of its
including chronic hepatitis, cirrhosis and primary unique features. HRV is assigned to a separate
hepatic career. As there in an effective vaccine family .' iepadnsviridae (hepatotropic 1 > N A viruses),
against HBV, hepatocellular carcinoma becomes which consists of two genera, O r r h o£ VJJTAS
the only human cancer which in vac cine - CI in tain mg HRV as welt as the wnodfhuck and
preventable . The WHO estimates that HBV ground squirrel h e p a t i t i s viruses , and
infection causes more than a million dearths a year AvjhepadridWmS cunt .Lining the Pekin duck and
worldwide . grey heron hepatitis viruses. HRV is HepaflntviniS
Clinical features: The incubation period is type 1 ,
-
long, about 1 6 months. The clinical picture of The discovety of HBV was serendipitous. In
hepatitis R if - similar Co that of type Ar hut it tends
to be more severe and protracted. The onset i ?
-
1965, Rlumhcig, tudvi n g Inj ma n se rum Lipoprotein
allotypes, observed in the serum of an Australian
insidious and fever is not prominent. Ejttrahepatie aboiLgmeh a new antigen which gave a dearly
complications I Lice arthralgia, urticaria and rarely defined Line ol precipitation with sera from two
polyarteritis or glomerulonephritis may occur . hemophiliacs who had received mulii [ile blood
These are ascribed to circulating Immune complexes transfusion*. . his was- flamed the Australia jtn ycn.
containing the viral surface antigen. By 1969 the 'Australia antigen was found to be'
^
About 90-95 per cent of adults with acute associated with serum hepatitis. IL was mbsequendy
—
hepatitis R infection recover within 1 2 months of
onset and eliminate the virus from the body within
shnwn to be the surface component of 1 J B V .
Therefore the name Australia antigeo was changed
about six months , remaining immune thereafter . To hepatitis B surface airmen [HBsAg ).
Mortality Is about 0.5 to 2 per cent , but may be
-
more in post transfusion eases. About l pet cent of
patterns particularly those having simultaneous
EftwSrtQpn i HHsAq ;
della virus infection develop fatal Ruminant Nuclwcap
iHBfcAgl
^d C- .r ^
i
y
hepatitis.
A proportion of cases fl- UJ per cent ) remain
chronically infected - I'hcy may be asymptomatic
carriers or may progress to recurrent or chronic
liver disease or cirrhosis. A few of them may develop
hepatocellular carcinoma after many decades.
The pathogenesis of hepatitis appears to be
immune mediated. Hepattxylcs cany viral antigen!;
and are subject to antibody -dependent NK. cell and DNs* P&yfW
Plus SlrfiifcJ
-1 DMA
cytotoxic T cell attack. In the absence of adequate Mims SlmndJ
immune response, HBV infection may not cause
hepatitis, but may lead rn carrier state. Therefore
infant ? and irmnunodeficicnt persons arc mote
likely to become asymptomatic carriers following
infection. Fie. 59.2 Hepatitis S virus struclurs
i r i
* Hepatitis VirUi&S
* 551
nonparticulate nuclenvapsid protein. HBcAg and The genome has a compact structure with four
552 * Texibook o Microbiology *
'
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* HG|M1I1IS Viruses f 555
L»f the high oust of imported vaccine. Now Thai the its antibody. sutti - HBc appears in serum a week Or
meant is manufactured in India , and is available two after the appearance of J JBsAg. It is therefore
at lower cost, it should be possible tn include this the earliest antibody marker to be seen in blood ,
in the national immunisation schedule. long before anti - fEBe or anti - HBs , As anti-HBc
1 , Elbetrulor \ din nosi ^: Specific diagnosis of remains: lifelong, it serves as a useful indicator of
^
]ic[iatiri. s J J rests on the serological demonstration prior infection with HBV, even after all the other
of the viral markers- It is therefore necessary to viral markers become undetectable. Initially, anti'
,
understand the sequence of their appearance in HBc is predominantly IgM, but after about 6
blood ( Fig. 59.6), months, it is mainly IgG . Selective tests for IgM
HEsAg is the hret marker to appear in hlood oi IgG anti - H Re therefore enable distinction
after i nlwtinn, being detectable even before delation between recent nr remote infection respectively,
of transaminases and onset of clinical illness. It HBeAg appears in blood concurrently with
remains in circulation throughout the icteric or HBsAg, or soon afterwards. Circulating HBeAg
symptomatic course of the disease. In the typical is an indicator of active inirahepatic viral replication,
case, it disappears within about 2 months of the and the presence in blood of UNA polymerase ,
start of clinical disease, but may sometimes last for HBV DNA and virions, reflecting high infectivity.
6 months and even beyond . When it is no longer The disappearance of EIBcAg coincides with the
detectable , its antibody. anti-HBs appears and fall of transminase levels in blood . It is followed by
remain* for very long period*. Anti- HBs is the
protective antibody
the appearance of anti HBc. -
For the diagnosis of flBV infection detection
HR cAg isnor demonstrable in circulation of HRsAg in blood is all that ordinarily necessary.
because it is enclosed within the HRsAg coat , but The simultaneous presence of IgM anti - HBt
JAUNDICF
LIVER ENJYMEE 1
Anrti - HRt
nBeAq
AnliHBA
.
*
f
\ \\ /
.* * n wm M . .
wA pc **.
^ -
a %
^ it
1 '
0 j
12 6 2Q 2* 26 32 36 50 ICG
* I I h
Week! iflei ftntechnr
Copyrighted material
556 4 Texib&ofc of Microbiology
indicates recent infection and the presence of IgG Active immunisation h more effective. The first
anti-HBc remote infection, Occasionally when the vaccine introduced in 19fl2, was prepared from
level of H RsAg LH too lew to be detectable, diagnosis pooled plasma of healthy human carriers with high
has to be made by testing for IgM anti-HBc. level antigenemiia. The 22 nm HBsAg particles
HBeAg provides information about relative separated by ultracentrifugation were treated with
in fee rivitv. Its presence denotes high infertility and proteinase, urea and formaldehyde and used as the
its absence, along with the presence of anti-HBe, vaccine. This was immunogenic , but became
indicates low inftetivity. As it i* invariably present unacceptable because ire source was human
during acute hepatitis, its testing is indicated only plasma, limned in availability and not totally free
in chronic infection and carriers. from possible risk of unknown pathogens. It
-
The presence of anti HBs without any other continues to be used in tome countries because it is
serological virus marker indicate? immunity cheaper.
following vaccination. Table 59.1 shows the The currently preferred vaccine is genetically
interpretation of various serological patterns in engineered by cloning the S gene of HBV in baker 's
hepatitis B. yeast. It consists oi nonglycosyiated I IBs Ag particles
Like HBeAg, HBV DNA is also an indicator alone. It is given with alum adjuvant, IM into the
of vital replication and inactivity. Molecular deltoid or, in infants into the anterolateral aspect of
methods such is ON A : DNA hybridisation and the thigh. Gluteal injection is not recommended as
PCR, at present used for HBV DNA testing ate it may result Ln poor immune response. Three doses
highly sensitive and quantitative. HBV DNA level giver at 0T 1 and 6 months constitute the full course.
in serum reflects the degree of viral replication in Seroconversion occur* in about 90 pc cent of the
the liver and so helps to assess the progress of vaccinees. A special vaccine containing all antigenic
patients with chronic hepatitis under antiviral components of HBsAg (Pre-Si , Prt - S2 and S) has
chemotherapy. been developed, which gives greater seroconversion.
Prophylaxis! General prophylaxis consists in Screen version can be checked by testing for anti'
avoiding risky practices like promiscuous see . HRs which is usually detectable for about 5 years.
injectable drug abuse and direct or indirect contact Clinical protection is believed to last much longer.
with blood, semen or Other body fluids of patients Booster doses are needed only for those at high
and carders. Health education, use of the disposable risk
syringes and needles, screening of blood, semen and For nonimmune persons exposed to HBV,
organ donors, have all helped to an extent, bur these combined immunisation is recommended. For
alone cannot eliminate the risk altogether, babies bom to carrier mother?, a single injection of
particularly in the devdopuyg .
countries The only 0.5 ml of HBlG given IM immediately after birth,
certain method appears to be universal is followed by the full course ufvacdncat a. different
immunisation. anatomical site, the first dose being given within
Both passive and active methods of 12 hours of birth . When HBlG is not available,
immunisation are available. Hyperimmune hepatitis the vaccine given alone has been reported to provide
B immune globulin (HBlG) prepared Bom human protection.
-
volunteers with high title anti HBs, administered
IM in a dose of 300-500 i.u. soon after exposure to
Treatment; No specific antiviral treatment is
available for acute HBV infection. Interferon alpha,
infection constitutes passive immunisation. It may alone or in combination with other antiviral agents
not prevent infection, but protects against illness such as lamivudine and famcyclovir, has been
and the carrier state. beneficial in some cases of chronic hepatitis.Them
i
[J LJ
J riantea feria
* Hepatilis Viruses 557
is no effective treatment tat the carrier state, though high risk. Sexual transmission is probably less
^ pnntuneauK mnlution takes place in some of important. Vertical transmission from mother to
thCtflr baby may take plact-
Thc infection occurs throughout the world, with
TYPE C HEPATTHS carrier rates varying from 1 2Q per cent. HCV
Attempts to identify the group of 'non -A nou - infection is prevalent in India too, with an estimated
s' viruses by experimental infection in chimpanzees 12.5 million cases- A quarter of all chronic hepatitis
led to the discovery of hepatitis C vims ( HCVJ. It eases in India is believed to be due to HCV
is now the commonest cause of past - transfusion infection*
hepatitis in the developed countries. Hcpnlilis C virus { HCV ): The virus has
Clinical features: The incubation period is not been grown in culture , bur has been cloned in
long* 15-160 days , with a mean of 50 days. The —
.EscfiericJuz coii. HCV is a 50 60 run virus with a
acute illness is usually mild or anicteric. Overt linear single stranded K.NA genome , enclosed
jaundice is seen in about 5 per cent of patients only * within a core and surrounded by an envelope,
The important part in type C hepatitis is the chronic carrying glycoprotein spikes . HCV resembles
Illness. About 50 to SO per cent of patients progress fhvivinucs in structure and organisation, and has
to chronic hepatitis, with some developing cirrhosis been classified as a new genus W( jM(iviras in the
and hepatocellular carcinoma , family Flaviviriiae.
lipidtinioluiiy : HCV infection is seen only in Thc virus shows considerable genetic and
humans. The source of infection is the Idige number antigenic diversity. At least six different genotypes
of carriers, estimated to be about 200 million and many subtypes have been identified, indicating
worldwide- In general the epidemiology resembles high mutability Some genotypes are seen
*
-
Table 59.1 Inlerpn&talfon of common, serological patterns in HBV mfeelion
Virus/ Antib&dy Mlfkttt
Interprets lion
HBsAg HBcAg snd - HBc unti - HHs
+ IgM m Amhc HBV infection; highly infectious
+ l G Lare/chionk HBV Infection or carrier state;
*
* highly infectious
p- IgG * w- Latc/chronic HBV infection or cumer state;
Low infcctivity
+7 - IgM + /- Seen rarely in early acute 1 IBV Infection ;
infectious
IgG */ - Remote HBV aafedkicis infetrivity nil or
very Low
= *
+ “ J
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558 4 Textbook of Microbiology *
antigens used art variant structural and non- Its mode of transm i ss ion K the same as for HBV.
structural proteins cloned in E. CGJJ . There have Two types of infection arc recognised, coinfection
boon three successive ger it ration > of such antigens, and .‘ irf 'tTjrift' L rHur . In coinfection, delta and HBV
'
,
introduced Do improve iicrts ] ti^ iity and specific!ry of are transmitted together at the same time . In
Krotagb-a] diagnosis , Even the third generation supennfcctian, delta infection occurs in a person
ELISA currently i :i use, employing NS- 5 region already harbouring HBV, Coinfection clinically
protein and sy ntheri c pept i dea becomes pc * 11 ive only presents as acute heparm;- IS . ranging horn mild ro
months after the infection and shows mmspecific fulminant disease . Super imfecLKsn usually leads to
reactions. Confirmation. % iinmunoblot assay is more serious and chronic illness, with deterioration
therefore recommended . In HCV infection of the underlying HBV infection. No association
antibodies appeal irregularly and late, limidng r I itir has been noted between HDV and hepatocellular
diagnostic utility, carcinoma .
Ideoiiticatniii of HCV RNA in blood provides Delta amiigen is primarily expressed in liver odl
more sen* i ri ve and speci tic results wi TO i n a few days nuclei , where it can be demonstrated by
of -exposure to HCV. Molecular methods like PCR immunofluorescence . It is only occasionally present
and branched DNA assay arc employed for the in scrum. Anrfeddta antibodies appear in senim
purpose, and can be idem ified by ELISA . The IgM antibody
Prophylaxir Only general prophylaxis , such appears 2 3 weelu after infection and is soon
as blood screening , is possible , No specific active replaced by the IgG antibody Ln acute delta infection.
or passive immunising agent is available . However, in chronic infection , the IgM antibody
Trefllraenti Ptolongcd treatment tvirh interferon persists for years . Delta RNA sequences have been
idphu, eilher alone or ML combination with antiviral cloned and DNA probes have been developed for
agents like ribavirin has been reported to be useful the ra|ud identification of deha particles in
in some eases, circulation. The woodchuck has been found to be
a suitable experi mental model for the study of HDV
HPED ( DELTA) HEP ATTTB infection.
In 1977, Rfewtto and colleagues in Italy identified HDV is distributed worldwide but is more
a new viral antigen in the liver cell nuclei of patients common m certain endemic areas . In the
infected with hepatitis B virus. This lus been shown Mediterranean countries, where it is endemic,
co be due ro the hepaiotropic virus Delta or LiifecLion is spread commonly by nonpcicutancous
Hepatitis D Virus ( HDV ). Delta is a defective mutes, cspccialy by close personal contact . In the
RNA virus dependent on the helper function of noncndemic areas, such as Northern Europe and
HBV for its replication and expression. Therefore, North A me [ feu , infection is more often through
it has ild independent existence and can survive and blood and blood products and is commonly seen in
the host,
-
replicate only a.; ( png as HBV infection pCTMSft itl drug addicts and hemophiliacs. In [ reduction of
HDV into nonendemic areas where HBV infection
,
HDV r i Spherical , .lb 11 ITL particle with an i 5 common may lead to outbreaks of severe hepatitis
outer coat composed of the hepatitis B surface with high mortality.
antigen surrounding the circular single stranded No specific prophylaxis exists, but immunisation
RNA genome . Though it resembles some plant with the HBV vaccine is effective as HDV cannot
viruses, such as viroids or satellite viruses, it has infect persons immune to HBV. Screening of blood
been proposed to be classified in a new genus donors for HBsAg automatically limits blood home
Deltaiirus, because of its special features. HDV infection.
^
J
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TaWe 53.2 Viral hspatilis ifpm : comparalive features
s
A B C D E
Virus- HAV, 2 7 n m RNA,
Pleornavirus
HBV, 4 7t a t i DNA
{Hepadnavirus)
-
HCV, 30 Mfim
RLNA, FkvdviriiJis
.
HUV i $ mm
Defective RNA.
HEVn 32- 34nm RNA
Herpesvirus
{ Hepatcmruii) -
( hepamini-s ) Delta.vii. ru5
Age Affected Children Any age Adults Any age Young adult*
*
15-160 30 - 180
Textbok
IntiabiCitin 15 -45 3EMS0
Period(dap )
1S
*0
Ontti Acute Insidious Inekti&u* Insidious Acute of
Illness
Carrier scare
Mild
Nil
Occasionally severe
Common
Moderate
Present
OccaskariHlIy severe
Nil
except in preg
Microblgy
*
& Specific praphylj-xis Ig md Vaccine ; Ig and vacc i ne Nit HBV vsrixine Nil
p
leut i
* Bopflftta Viruses 561
Alter HJr 1997- Anrte nnn A- E hfipflirttiai in the USA and Elbe rale ofkepaddr; G ifeiis iofeelkn, NfiW EngfJMed 336:741 -
British Midkal Bulletin , 1990, Hepatitis 2:46
Brown JL. 1995, Hepabria C. J fnfict 3KW5,
-
Collier L (ed ). 1996. Heparirie A and E Tbpfch &pls'VEJfsoiriiAfianbwJqgv and Mm a] /n/pctj»ons 9*«hi , London;Arnold
'
^ .
Dhenstag JL and Kj IsseUbachEr 1998. In Acute Viol Hepatitk. Hinisons jFWncfpJks aflbwal AfoJrdne 14* cdn. New
Yoflc Mc^Gcnf HiU.
Hbllinger FB andTj Liny 2002, Hepatitk B vims, In Field’s Virology, Vbl 2. 4* ed . Philiiddlphia: Williama and WffldnL
1989, Type D ( Delta ) Hepatitis, J Amer Med A*wc ML1321.
Krawoyraiski 1993. Hepatitis E. Hepa udogfr 17:932
EC
--
[ .cn :: r $ M and DL Thomas 1997. VfiGdm to prewnc viral hngbm NewEnglJ Mtd 336cl 96.
MtiPtrd ME and JR Gels 2003. Suppressing Hepatitis R . NgwBngJ Med 348; ML
.
Sarin 5K and AK Singil (« d*). 1996. Hepatitisi & in /ndis. New Delhi - CBS Publishers ,
Van DuiHM P m al 1.997. ImgnfiQn of heparin* B vaednarion into narimaJ imniunLy arinn nmruillIKt. iJAI / 314il 033-
. .
rabbit papilloma are potentiallymalignant , Polyoma which is a more moist, soft, pedunculated wart
and the vacuolating virus SV 4f) produce malignant found on the external genitalia is usually due to
tumours when inoculated into newborn mice or types (J and 11. This may be transmitted venereally
hamsters, These viruses havebeen widely employed and may occasionally turn malignant. There is a
in the study of viral oncogenesis. close association between specific HPV types and
Myoma virus often causes latent infection in genital malignancies in both sexes. HPV types 6
laboratory mice. However, when inoculated into and 11 are associated with intraepithelial neoplasia ,
newborn mice , it produces a wide variety of while HPV 16 and 18 sue ctiologically related to
malignant rumours. Hence the name polyoma. more severe invasive malignancies such as uterine
The simian vacuolating vims CSV 40) was isolated Cervical cancer. CnfaCtnr? appear to be important
from uriinoculated rhesus and cynomolgus monkey in the induction of HPV associated malignancies.
kii.lncy tissue cultures. The virus did not produce any Human papovaviruses have been isolated from
cytnpaihic effects in the original cultures but when a number of patients with impaired immunity. The
| C vims was isolated in 1971 from the brain of a
fluid from such cultures was inoculated into kidney ,
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* Miscellaneous Viruses 565
- I- T
i' V.
-A r*
>
- ft »
,TH .
Sr V '
.
- .-
r Jr 6
k .J
i ' 1
^ -
-
loo nm
.-
* * * 00 om
*t
J
/- - i SiKrw * » JQp
«8-
r
-s
* .- ar
- «
i -
.
:^ -
>? UP
l^r _ 35! '
j
1
^J\ . ®
.
..
-
p
»^ jS
* JHI
,j
.
* SI
" ¥aH; Iq
’W
'
. JJ
.-
r: #J§
J
-#.
1 r - wa v:
w -r
* mV *
)
It 4
jr '4
Wh
ur ?
4
^
/if
& i
.
F q &D 1 Electron Tiicrograph & pi viruses associated with diarrhea ft 20O QOQI 1. Rgtavirga. . 2- Adengvirua ;
.
3 SmalE round structured virus [Mcrvialk -like calcivirusesi : 4. Corgnevirus
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* Miscellanea us Viruses * m
coronavinis' in memory of Dr Carlo Urban! who a common complement-fixing antipen but can be
identified rhe new epidumu , initiated cAfiy ^trps dilTefmtiated by neutralisation ( ES( t . Reoviruses
_
'
for its Control and died of i [ within a month! aggiuti natc hum an tryth rnryte v Hcmaggluti nation
REOVmiDAE is type specific and is neutralised by the specific
antibody. Rrovimses- have not been proved to muse
The [anuiy Reorirldac derives its name from the any human disease.
jirmoiypi; virus wltieh was known as the Respirator
1
stranded RNA in 10-12 pieces , a feature unique multiply in insects and ndrcbmte* thns Qualifying
among animal viruses , They are nonenveloptd a ]td as arboviruses. They are responsible for veterinary
restslant to lipid solve nis . Tlit fan i ily contains it ] ret diseases such African horse tick] Less and blue
as
—
genera Rcovim^ , OrMvmn and Retinnu. tongue. The only known orbivitus infection of
human beings is Colorado tick fever.
flEO VIRUS ROTAVIRUS
The genus Rcovirus contains three mammalian These double willed viruses prune nt a eharaeteriStic
serotypes ( Reovirus types;\ y 2 and 1 } which possets appearance under the electron microscope ,
t O
* f i
®S&
|
o si'Sk o
Hi
6 5
2
3 4
Fig . 40.3 : SAftS - a $ H c i M Ipti cororisvitus. [ diagrammatic reptescniatiofl) 1 Envelope : 2. RbA -. Kud?oeap?ld
nueleoproleifi . A , spike glyenpruleln ; $. HoftiagglutinIn- acetyi ealafafit glycoptoEstn; &. aroaN envelope
glycopr & toin ; 7. membrane glycoprotein.
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< M '- scsIlaneaus Viruses * 573
Rotavirus vaccina have been developed, bat are adenoviruses can he only grown with LliftiL-ultyin tissue
not in UK row. culture ' Iliey have been designated types 40 ami 41 .
,
OTHER VIRUSES CAUSING DIARRHEA Adenovirus associated diairltwiL lias been seen more
often in summer months.
Reside rotaviruses, the following viruses arc known Astro virus: These star shaped, 2 % nm isometric
or ^iLspccled to cause diarrHcal disease : particles have been associated with sortie epidemics
Norwalk vime A 27 rm virus was shown to of diarrhea in children . Similar viruses have also
be responsible for an epidemic of gastroenteritis been identified in lamb and ealf diarrhea .
affecting school children and teachers in Norwalk, Corondviruc These are well established causes
Ohio* in 1972. The virus induced diarrhea in of acute diarrhea in calves , piglets and dogs. They
human volunteers. Serological surveys have have been observed in human feces also but their
shown that infection with Norwalk virus is relation to diarrhea is unceitiLtl.
widespread in many countries . Extensive epidemics HenJrji a. nd Xipnh viruses: Two new
of Norwalk virus diarrhea associated with zoonotic viruses, closely related hut distinct are
consumption ofrawoyseen have been reported from Hendm U994) and Nipah (1999) viruses named
Australia and America. It appears to be an important after the place they were first isolated in , in
cause- dt LJI .LRRLIL~ JI iit adults aod children. Australia and Malaysia respectively. They have
The virus can he demonstrated in feces by cJeCtTO© been classified under the family Fammyxovi ridae.
rtiiooseopy, Antibody to the virus can be detected by They arc prevalent in Australia and in Malaysia,
immune electron micmsjctipy and radioimmunoassay Indonesia. Philippines arid the Pacific Islands .
It Jus not yet boat propagated in cell cultures. Little Their natural hosts arc fruit bats in which they
i s known about the properties of tlie virus. 11. lilts been cause silent infection. Symptomatic infection occurs
included in the family Cdicftinditc; consisting of in horses , pigs and many other domesticated wild
small round RNA viruses, 22-30 nitl in size, many animals. Human infection is usually saibctinieal , but
ofwhtL^ h have been reported from diauheal feces. may he a fiulikc fever which may lead to fatal
The name caUcivirus LF derived from the presence encephalitis, sometimes ILS outbreaks . Hendra virus
of 32 cup shaped depressions on the vims surface outbreaks have been minor, but nipah virus bw
(fnom CJJIT, meaning cup). caused large outbreaks with case fltalitY rates of
Adenovirus Several outbreaks of L arrhea in up to ?0 per cent in persons in contact WLth pigs .
children haw both associated with the presence
^ Large scale slaughter of pigs had been undertaker
of latgc number* of adenoviruses in feces. These in order to stop outbreaks.
Further Heading
LSerlin 11 iftd U Dciselbufgef 1996 . Rotavirus paih^flenHis . Lancer 21 R : £99.
Ciitcf MJ and \VD Cubitt 1995 . Norwalk and related Cun- In&er iiii 8:401
.
Fields BN er al . 1996. Virology edn , PhUaddphia;Lippinccirt - Rivcn.
Johnson KMet al. 1939. Clinical virology of LlW fmr.J Infect Dis 155 :45*.
Kapikian AZ - 1994 , Vlr^l infa-titm* ipfthe gatizoijirHnii tract New York; Mircel - Drkkrr
Lee HW and Li Van der < Irom 19 H 9. Htiruvrhagi .: fever with nenal syndrome. /Ye r Aled Vjni/ 16:62.
Holmes KV. 20U 3. SAftS . New En$ J Med 348: 1948. ^ ,
Pnisancr SR . 199R Priona of humans and animals. In Tofitcy tnd W41h? fl * MuQWfrbygyurf MicraMaf Jnfec CIOFFT, 9 r edn .
London: .Arnold .
Richmond JK and DJ Baglole 2003 , Lu;i Fm(. Hr MW / ttttUTl.
Wenzel RA and MB Edmond 2003. Mansions SAKS. New ] MeJ .14S: 1 SM 7 ..
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I
Table 61.2 Lia1 cl onCDgenic viruses possess oncogenic potential {Table 01.2). The
RNA VIRUSES viruses associated with cancers in human beings
I . Rc trovi ruses 1. Avian leukosis viruses are shown in Table 61.3. Both KNA and DNA
1. Murine leukosis viruses viruses art oncogenic. While all oncogenic RNA
3 . Murine mammary viruses (formerly called oncornaviruses) belong to
tumour virus
a single family ( retrovirus), oncogenic viruses occur
4. Leukosis -sarcoma vims
of various animals among ail major groups of UNA viruses, except
5 . Human T cell leukemia parvovirus. Retroviruses arc responsible for naturally
viruses occurring leukemia and sarcoma in several species
DNA VIRUSES of animals . Among DNA viruses , some
I . PipOvavirus 1 . Papillomaviruses ol herpesviruses and hepadnaviruses cause malignant
human beings , rabbits
tumours in their natural hosts.
and other animals
2. PoSomavirus
A Simian vims AO ONCOGENIC DNA VIRUSES
4. BK and JV viruses V A P u V A V i m. S E S
IT . P& Kvirui 1 . Molluscum coniaginisurti Papilloma viruses cause benign tumours in their
2. Yaha virus
3. Shope fibroma
natural hosts but some of them (e.g., condyloma
Ill . Adenovirus Many human and acuminatum in humans, rabbit papilloma ) may turn
nonlwman types malignant. The association between human
TV. Herpesvirus 1. Mareki disease virus papilloma vims ( HPV ) infection and cancer of
2. Lwcke’s frog tumourvirus cervix uteri, particularly HI > V types 1.6 and IS ha*
3. Herpes virus pau, papio, been established. The continuous cell line He La ,
attic* and saitnui
Epstein-Barr virus
4. derived many decades ago from a cervical
5 Herpes simplex virus
. carcinoma and used widely in various laboratories,
typci 1 und 2 has been found to contain HPV- lfi DNA. In
6. Cytomegalovirus general , infectious vims particles cannot be
V. Heparitii B and C demonstrated in tumours induced by DNA viruses
limn but papilloma in the wild cotton tail rabbit is an
(Table 61.1) . Transformation from normal to exception . Rabbit papilloma virus , or DNA
malignant cell is a multisKp process anil may he extracted from it , can produce papilloma in rabbit*
partial or complete. For example, some viral agents following subcutaneous injection.
C ; m ' immortalise ' infected cells, SO that they become Tbe polyoma virus causes natural latent
capable of continuous multiplication in culture, infection in laboratory and domestic mice .
without possessing other features of malignancy. However, when injected into infant mice or other
Transformation is recognised primarily by a change rodents, it induces a wide variety of histologically
in morphology of cultured cells. Transformed cells diverse tumours . The virus can be cultivated in
are altered in shape and lose the property of 'contact mouse embryo fibroblasts or baby hamster kidney
cells, in which it induce* transformation . The
1
inhibition HO that , instead of growing as monolayer,
they grow piled up, one over another, forming polyoma virus produces a hemagglutinin .
'miciotumours'. Foci of transformation can easily The papovavirufes BK and JC, which cause
be made out: and are used in the assay of oncogenic widespread asymptomatic human infection, can
viruses, such as cite Rous sarcoma vims. induce tumours in imjmunodelkient subjects.
About a quarter of the 600 or so animal viruses Simian virus 40 ( SV 40) was discovered in
Copyrighted material
576 ^ Textbook oi WicrDbiology
cultured cells from several species , including human ncurdymphomatosis of chickens . Mo infectious
cells. Millions of doses of polio VKOSt prepared virus particle can be isolated from the lesions or
in monkey kidney cultures that may have haibourrtl seen under the election microscope . However, sick
SV 40 virus had been used before the virus was hirds shed large quantities of virus from their
discovered. These individuals have been followed feather follicles. The virus is a typical herpesvirus.
up for over 25 years and no SV 4Q - related tumours MarekV disease can be induced in young chicken
have been reported . There was considerable hy the injection of the virus. The virus grows well
apprehension when die oncogenic effect of SV 40 in chick embryo fthroblasts producing cytopathic
was discovered. However there is no evidence that
change* but no evidence transformation . Manet's
^
the injection of vaccine containing SV 40 has disease car lnL prevented by a live a virulent vaccine .
induced cancer in humans. Tins ii tile fiftst inn, tirtce of LI L 3 iJ.hgo .mt disease
POJCVIM 5 being controlled by a vim] vaccine.
h . I . ucke' s tumour of fm su A herpesvirus
Three 1 embers of tile pOJtvitUS group induce
[]
Nonpritmres are unsusceptible . suggested LLH n primate model for the study of
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i QntpgeriLC Viruses 57 7
virus but cell lines established fmm them contain stranded RNA molecules . The icosahedml
5-20 per cent of cells that produce the virus. The nucl cocaps id core encloses the helical
virus multiplies only in human lymphoid cell lines. rlhomiLleuprotein and is MiHDundcd by an envelope
Serological surveys show chat Infection with the composed of glycoprotein and lipid.
virus is worldwide. Infection is usually The characteristic feature of retroviruses is the
asymptomatic . In young adults without pne- existi ng presence within the virion of the unusual enzyme
antibodies, EB virus infection induces infectious RNA dependent DNA polymerase or reverse
mononucleosis. Lymphoma is believed to occur transcriptase (hence the name retro , meaning
when the infection takes place in children whose reverse). Unlike the classical transcription of generic
immune systems are compromised, as tor instance, information from DNA to RNA, the reverse
by chronic malaria, EB virus associated lymphomas transcriptase cn-cyme prepares a DNA copy of the
have been reported in transplant recipients. EEJV retroviral RNA genome— initially an RNAtDNA
has also been linked to nasopharyngeal carcinoma hybrid and then its double stranded DNA form.
in the Chinese male population in southeast Asia The double stranded DNA form of retroviral
and East Africa, genome, called the provt /us, is integrated into the
e. Herpes simplex nnd canter cervix: DNA oftbf nltected host Odll, It is front the prnvims
An association has been proposed between herpes that all retrovirus proteins are translated . Infection
simplex type 2 infection and cancer of the uterine with oncogenic retroviruses does not lead io
cervi though not proved. It has also been suggested cyrolysLS or death of infected cells but die peprinu
^
that herpes simple--* type I infection may be remains integrated with the host cell DNA for the
associated with cancer of the lip . Herpesvirus rest of the life of die cell .
type S has been linked to Kaposi’s sarcoma . While all oncogenic RNA viruses belong to the
f. Cytomegalovirus infection has been family Retntviridae , all retrovirus- es are not
associated with carcinoma of the prostate and oncogenic, The family /teflrQvjriJaf is classified into
Kaposi Sarcoma. three subfamilies.
1. OnCovirinac comprising all oncogenic RNA
HEPATITES B VIRUS viruses ( formerly called oncornavirus).
HBV has hcen chimed to be directly or indirectly 2 . Spuimnirinxe containing the nononc-ngenic
' foamy viruses' (
involved in the etiology of hepatocellular spuma = foam ) causing
carcinoma . Studies in many countries have asymptomatic infection in several animal species,
demonstrated an excess prevalence ot markers of and presenting as contaminants of primary cell
HBV infection in patients with primary cultures in which ihevinduce Ibaittv degeneration.
hepatocellular carcinoma as compared with 3 . I enmirinne including the viruses causing ‘slow
^
matched controls or with the general population. i rtfoctions' ( fantut = slow) i n animals ( see chapter
Hepatitis C virus infection lias also been reported 60) , as well as rht human and related animal
to lead to hepatocellular careinoma. immunodeficiency viruses ( see chapter 62) .
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57S * TOKIDOOK of Microbiology *
Retroviruses arc widely distributed * being found quantifies of interleukin - 2 < I'hc closely
in nearly silt vertebrates, including am mils , birds related HTLV-1I is also u with T cell
and reptiles. R .Lsed on tbc host range and types i >f mjljgruiLCy. HTLVr infection it tu be spread
disease caused , oncogenic retroviruses can be through blood transfusion and methods of
considered under the following groups : transfer of leucocytes .
1 . I 'ltL \ \ i in i euko^ ia ( iotTiplux: A group
"
HitSl Specificity: RetToviruM usually infect
of indgenivaUy related viruses which induce avian only one ho t species, the specified ^ being
^
leukosis ( lymphomatosis, myeloblastosis and co]iditioned mainly by the i u st r )c e of viral receptors
erythroblastosis viruses ) or sarcoma in fowls ( Rom ( i n the host celt surtacc. ! icndi on their ability
sarcoma virus* R 5V ), to grow in cells from different . . . retroviruses
2 . M u r i r i L I euka[HG Viruses: This group have been classified into (1) ecotiopi ( multi fliving
consists of several strains of murine leukemia and in cells of native host species nl ' in pic
sarcoma viruses* named after the investigators who ( multiplying in cells of native i Iron species);
first described them (e .g. Gross* friend * Moloney* and (3) xenotropic ( m ldptymg mi? to ceils of
Rauschef ) . foreign species but not of native host species).
O M b i m r i v j m I i n i r i n v : r i ^ ' i! I I I I L T ; This
r Virus ' i wmission; Two : pi of retrovirus
virus occurs in certain strains of mice having a high transmission occur. Exogenous ernoviruM are
natural incidence of breast cancer. It used to be spread horizontally. Most cutcugeni retroviruses
known as the Hmilk t actor' or LRinner virus'. It .ire exogenous. Endogenous rerrnvi are
multiplies in the mammary gland and is transmitted transmitted vertically from parent to offspring* by
from mother to offspring through breast milt Mice the pro vims integrated with the germ Line cell
can be infected by oral * subcutaneous or genome . The endogenous reiiovirus provirus
iritrjjieritonf .Ll mutes . MamtUiy cancer occurs otilv behaves tike a cellular gene ind is subject to
in susceptible strains of mice, after ; L Latent ]ieri( jd
of 6-12 months.
-
4 . Lcukugi & Kstnit)ina viruses ol other
mirunlsl A number ot viruses have been isolated '
from leukosis and sarcomas in various specie of
5.
-
animals cat , hamster , rat, guinea pig and monkey.
Human T cell leukemia
( lymphotropiii ) vinisrt : ( HTL\ ) :
Retroviruses named humanT cell leukemia viruses
were isolated in 1980 from cell cultures from adult
patients with cutaneous T cell lymphoma ( mycosis
fungmdcO and leukemia (Senary syndrome ) in the
USA. Similar viruses have hcen isolated f r o m
patients with a d u l t T cell leukemia in japan and
the Caribbean . HTLV lype ] it pnesent worldwide
hut the disease is limited to endernit areas. Besides
Fig. 6i .t Mouse rnarmriity cancer , stained wih
adult T cell leukemia , HTLV- 1 Ins also been uranyi acetate and lead nitrate . melure
associated with tropical spastic paraparesis * a bud dine E type virus ( arrow ] intracellular
demyelinating disease- The vims preferentially
infectsT4 (CD 4 ) cells . Infected T cells express large
Immalura A type virus (1 rlpl
iCcurk sj -: Carter RssE- .srtli
* 1 .
(* ISO OQO )
Mumbai,]
' !
* Oncogenic Viruses 579
A LTR LTR
itWV
B LTR LTR
C LTR LTR
snR
line
. .
Fig 61 Z Provirus genomic structure ol different s of reTrcvirusfS
-
A. BasiK irtmvinis Jjemimc e -J'. Avilli li .ilstuLia viruses, sLiiw transforming viruses
'
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580 * TembooH oF Microbiology
transforming viruses are unable to replicate myc to DNA-binding proteins, all concerned with
normally because they carry on their pnaitir an regulation of normal cell growth .
-
additional gene* the viral oncogene { V onc gene)
which replaces some of the genes essential for viral
A useful method for the study of oncogenes is
transfection. Certain mouse fibroblast ccD lines, such
-
replication. Such Y onc viruses can replicate only
if co- infected with a standard helper retrovirus.
as NIK 3T3 , can take up foreign DN A, incorporate
them into their genome and express transfection.
The Rous sarcoma virus which carries the By this technique, DNA extracted from human
oncogen ic sre ( pronounced 'Sark1) is the best known rumour cells has been shown to transform 3T3
among acute nans tor riling viruses bur it is different cells, and such transforming genes have been shown
in being replication competent, that is it can . to be identical with cellular oncogene*.
replicate normally because it posseses the full
complement of gag, poL, and me genomes. Most ANTE- ONCOGENES
acute transforming viruses axe replication defective, A class of genes has been identified in normal
retinoblastoma ( Rb) gene, the loss of which is
ONCOGENES associated with the development of retinoblastoma
-
Viral oncogenes ( V onc) , commonly known as
‘cancer gen^ arc genes which encode proteins
1
in children. The p53 gene appears to be a tumour
suppressor gene with a wide range of effects.
triggering transformation of normal cells into cancer Specific chromosomal deletions, recognised in
cells. Oncogenes are not csscn ri al for the repli cation association with certain types of human cancers may
of the vims and mutants lacking them occur, which reflect che loss of tumour suppressor genes.
replicate normally without being oncogen ic.
Genes closely resembling viral oncogenes are MECHANISMS OF VIRAL ONCOGENESIS
found in normal as well as cancer cells. Oncogenes While it is known that oncogenic viruses are able
isolated from cancer cells arc called cellular to transform etUs in Culture and Induce tumours in
oncogenes (C-onc). Similar genes found in normal animals, under natural or experimental conditions,
cells are called prom-oncogenes. They are not of
, the exact mechanisms of viral oncogenesis are not
vital ORIGIIL. On the contrary vi rai oncogenes appear well understood. Malignancy is a stable heritahfe
to be of host cell origin. Cellular oncogenes contain change and , as such, should be the result of a
intnons characteristic of eukaryotic genes, whereas modification of the host cell genome .
viral oncogenes do not. Apparently viral oncogenes In the case of oncogenic DNA viruses, the viral
originated at some distant past from proto- DNA ( or a portion of it ) is integrated with the
oncogcnes by recombination between retroviral and host cell genome.The viral DNA being incomplete
cellular genes. Or 'defective , no infectious virus is produced.
1
Proto oncogencs are widespread in vertebrates However, under its influence, the host cell undergoes
—
and metazoa from human beings to fruitflies.
They arc well conserved in their genomes ,
malignant transformation . A virus transformed
cancer cell is in many ways analogous to a
suggesting that they serve some resentin ] functions bacterium lysogeniEed by a defective phage. In both
in normal cells. They have been found to code for cases , the cell is not destroyed and no vims is
proteins involved in regulating cell growth and produced. Acquisition af new characteristics by the
differentiation. The presumed functions of many transformed cell resembles lysogenic conversion in
oncogenes have been identified. For example, the bacteria.
oncogene sne related to tryioeine-specific protein
kinases, sis to a platelet derived growth factor, and
—
In general, retroviruses induce tumours by two
mechanisms cither by introducing into the
/” *L
u. name* ri -
J V I
i Oncogenic Viruses S 81
Table 6 l.i Sflme oncogenes' and ( halt chrwioMrtL* l location in humans
V,rirHJOinCOg-CDC Origin Nmturml Human gene Chfomnsomal location
tumour in humtn beings
V- re chielspn C arc 20
V-^ra*
^
cellular genome a new transforming gene gene product may lead abnormal growth .
to
( oncogene ) or by inducing or altering the expression Recombination between retroviral and cellular
-
of a pre existing cellular gene , Several molecular genes , promoter insertion, chromosomal
mechanisms have beer suggested for the conversion
of benign proto-oncogenes to cancer genes. The
-
t ran location , gene amplification and mutation are
some of the genetic processes relevant in this
genes may get overevpressed and the overproduced connection.
Further Rtiidiiijj
Bnhdjj JM. 10 R 9 . The mulet-idlr gerittu:£ ufOUBK Siicnif 235: 305.
Friend , SH etal. 1900. Oncogenes and tumour suppressor genes. New Engl J Med 318:618 .
Nejl MS and JA Wyke 1990 . Viral onooRenicity In JbpJ r and Wiltons Microbiology and Microbial Infections , 9 , k edn .
Vc' l . l . LondoruAmcdrL ^
Varmus Hr 1980 - ftctrovinises . Sdener 7-
WeinbeiR RA 1998, tlndinf ; the antioUCpRtiK , Siieur 4 I!?9: i-i.
njrr
-
Vfanbrip RA . 1989 . Oncogenes , intioncogenes and the molecular basis of carcinogenesis , CIIKIT fie-v 493713,
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4 Human ImmunotteficienCy Virus: AIDS 583
o
i
-
o
>
FlJ, 62.1 Slrutture Ol HIV (dbflrammiUc repre $flrvtatlcn| l. Etivdopfl glycoprtfeln spike ( gp 120),
E- Tfansmemljrance pedicle glycoprotein (gp4i|, $ . Outer Icosahedral shell of nuclecicapsid (p16), 4, Cora
shaped cone ol nucleocapsld \$ 24\- , 5. Inner core. 6. Viral proteins associated with RNA . 7. Viral iRHA,
.
0. Reversa transcriptase, 9 Envelope lipid bilayar .
in 2003 , Three mill inn patients died of AIDS in which is integrated into the host cell thro mosunue .
2003. The provirus can remain latent for long periods ,
though it influences host cell functions. At times,
HUMAN IMMUNODEFICIENCY VIRUS (HIV) in response to viral promoters, the provims initiates
HTV, the etiological agent of AIDS, belongs to the viral replication by directingsynthesis of viral RNA
Icntivinjfl subgroup of the family R-ntruvifidae. The and other components
lentivirus subgroup includes the causative agents During viral replication, when the naked virus
of the slow virus diseases visna/ maedi in sheep and buds out through the host cell surface membrane ,
infectious anaemia in goats and horses. Besides it acquires a lipoprotein envelope, which consists
HIV, the related animal immunodeficiency viruses of lipid derived from the host celt membrane and
also are assigned Co this group (Table 62.1 ). glycoproteins which are vims coded. The major
virus coded envelope proteins ire the projecting
SJTftUtTUKB knohkke spikes on The surface and the anchoring
HIV is a spherical enveloped virus, about 90-120 [ ransmcmbranc pedicles, llic spikes , constitute the
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c Hyman Immunodeficiency Virus: A.IDS £85
prevalent ail over the world belong to HIV type 1. Group M only. Even subtype differences may cause
HIV strains, first isolated from West Africa in 19B6, discrepancies so it may be
advantageous to use
which react with HIV type1 antis cnun very weakly antigens containing the prevalent subtypes in
or not at ill have bun termed HIV type 2. The different countries.
envelope antigens of the two types one different, The subtypes seem to vary in frequency of
though then COrt polypeptides show some emss cransmissibility by different mutes. The suhtypes
-
reactivity. HIV 2 has only 40 per cent genetic
identity with HIV-1. It is more closely related to
common in Asia and Africa (C and E ) are more
readily transmitted by heterosexual contact than the
simian immunodeficiency virus than to HIV-l. It American strains (subtype B ) which are
is much less virulent than HIV- I . It is largely preferentially spread through blood - by injection
confined to West Africa, though isolations have and homosexual contact ( homosexual transmission
been reported from some other areas, including is considered to be bloodbome as the virus is likely
western and southern India. tn enter directly into the blood through minor
-
HIV l strains have been classified into at least
ten subtypes based on sequence analysis of their
mucusal lean ).
Differences in growth characteristics are
gag and rnv genes. TTiese subtypes are designated sometimes observed between HIV isolates from
as A to J and constitute Group M ( for ‘nuiicr’), asymptomatic carriers and from AIDS patients. The
-
which cause the large majority of HIV 1 infections former grow slowly and infect only the peripheral
-
worldwide. A few HIV 1 strains isolated from West
Africa (Cameroon, Gabon ) do not fall within the
blood lymphocytes, while the latter grow faster and
yield high litres in established cell lines of
Group M and have been designated Group G (for lymphoid and monocytoid. origin. Strain varLutidns
-
'outlier'). Some later isolates of HIV 1 from
Cameroon, distinct from M and O groups have been
may account for differences in clinical course of
HIV infected persons.
called Group N (fill new), Resistance: HIV is thermolabile , being
HIV-1 subtypes show a geographical inactivated In 10 minutes at 60 :: C and in seconds
distribution, though this is often blurred by viral
trafficking. All known HIV virus groups and
-
at 100 °C, At room temperature (20 25 aC ) , in
dried blood it may survive lor upto seven days. A,
subtypes are present in Cameroon , West Africa, At autopsy, HIV has been isolated from various
which may perhaps be the site of origin of the tissues upto 16 days after death.lt withstands
virus. Subtype A is the most prevalent, being found lyophilisation. The virus in lyophilised blood
worldwide, while B is the most common in the products can be inactivated by heating at 68 °C for
Americas and Europe. The common subtypes in 72 hours and in liquid plasma at 60 aC for 10 hours,
Afnca are A, C and Dh while in Asia the common
subtypes are E , C and B . Subtype E is ihe Table 6S,a Major atrtlfltfla al HIV
commonest in Thailand. In India and China, A Envelope antigens;
subtype C is the must prevalent. -
1. Spike antigen gpl 20
( Principal envelope antigen )
Antigenic difference? between HIV strains may
2. Tran ? membrane pedicle proieiri ” gp 41
be important in serudiagnosis. Infection by HIV 1 - B. Shell antigen
or 2 may not be identified unless the corresponding
type is represented in the test antigen. Even with C. Core antigens:
-
1. Nudrocapsid protein plB
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m * T£ xJbD3k Gf Micmtunlcgy
pcrsisfcrit generalised Lymphadenopathy, A[D $ - hndy weight. The common opportunistic infections
relatcd complex ( ARC), ultimately terminating in arc oral candidiasis , herpes zoster, hairy cell
fall blown AIDS , with it? characteristic infection;; Eeucoplakia , salmonellosis nr tuhercnlo-sis .
and malignancies. The median rime between Generalised lymphadenopathy and splenomegaly
primary HIV infection and development of AIDS are usually present . ARC patients arc usually
lias been stated as approximately 10 years . About severely ill and many of them progress to AIDS in
5-10 per cent of the infected appear to escape a few months.
cli nical AIDS for 15 yearn or more . Diey have heen
termed ' lung term survivors' or ' lung term
5, AIDS: This is the end stage disease
representing the irreversible breakdown of immune
-
nonprogrepsors '. The mechanisms for such defence mechanisms , leaving the patient prey to
prolonged survival are not dear, though many viral progressive opportunistic infections and
and host determ inant* may he responsible. malignancies.
This period of clinical latency however dues The clinical severity of AIDS varies with the
out mean microbiological latency an virus type of infection or malignancy present. In early
multiplication goes on throughout . ') IK host mounts AIDS, many patients are ill only during episodes
an immune response against the virus , both of infection , which may respond to treatment.
hu moral and cellular, wh ich can only limit the virus Between episodes they may be relatively well and
Load, but not clear it completely. A chronic persistent able [ L. I resume normal life. Patients with Kaposi 's
infection with varying degree* of viral multiplication
is the result. The CD4 + T cell count decreases Table $2.$ Immunological abnormalities in HIV
steadily; from over 10OQ per microlitre to about in lection
500 or less by the stage of acute infection. When [ . Featuresthat characterise AIDS
the count falls to 200 or less, clinical AIDS usually 1 Lymphopenia.
.
sets in . For this reason the case definition bv GDC 2. Selective T cell deficiency’
.
Reduction in number nf T4 [CD4 ) "
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590 * Tsutbook ol Microbiology
pathy leading to loss of higher functions, 1. Antigen detection: Following a single massive
progressing to dementia. infection , as by blood transfusion , the vims
7 . I Vdkltrie AIDS: About a third to half the antigens may he detectable in hlood after about
number of babies born ro infected mothers arc two weeks, The: major core antigen p24 is the
infected with HIV. Virus transmission may occur earliest virus marker to appear in blood and is
to the fetus in pregnancv as early as the first the one tested for, IgM antibodies appear in
trimester, but infection is more common perinatal ly-
Many of the infected children may not survive for
—
about 4 6 weeks , to he followed by IgO
antibodies [ Fig, 62.3; Tabic 62.6).
a vtar . Children may also acquire the infection from If the infecting Jose is small, as following a
blood transfusion or blood products. needle - stick injury, the process may he
There are manv. differences between adult and
X
considerably delayed. The appearance of p24
pediatric AIDS. Children develop humoral antigenemil and viremia, followed by IgM
immunodeficiency early , leading to recurrent antibody response coincides with the acute or
bacteria ] infection!. Failure to thrive , chronic seroconversion illness . Afterwards, free p 24
diarrhea , lymphadenopathy, tuhrrculosis and antigen disappears from circulation and remains
opportunistic bacterial infections are common absent during the long asymptomatic phase, to
manifestations in pediatric AIDS. Lymphocytic reappear only when severe clinical disease sets
in [end la] pneumonia in seen mostly in children,
while Kaposi sarcoma , toxoplasmosis and
-
in . However intihody bound p24 antigen
continues to be demonstrable, after dissociation..
Cryptococcosis are less common than in adults. The p24 antigen capture assay ( F.L1SA ) which
uses anri - p24 antibody as the solid phase can be
LAHOKATOKY D I A C^O S I ^ used for this. The test is positive in about 30 per
Laboratory procedures for the diagnosis of HIV cent of HIV infected persons. With prior
infection include tests lor unTTlUOOdcfiCrenty Et5 well
as specific tests for HIV.
-
dissipation of the antigen antibody complex,
the positive rate increases to about 50 per cent .
A . Immunnli itnl nests: The following In che first few weeks after infection and In the
^
parameters help to establish the immunodeficiency terminal phase, the test is un i fermly positive. The
in HIV infection. test is most useful in persons recently exposed
1 . Total leucocyte and l ymphocyte con nr io to risk of infection, in whom antibody test ia
demonstrate leucupenia and a lymphocyte count negative. It LS oirrentlv used for screening blood
usually below 2COQ/ mm \ donors in the USA, along with HIV ELISA.
2 . T cell subse [ assays . Absolute CD4* T cell count 2 . Virus hoL non:Once infected with HIV, a person
will he usually less than SCKVmm '- T 4:TS cell remains infected for life . The virus is present in
ratio is reversed . circulation and body fluids, within lymphocytes
5. Flatekr count will show thrombocytopenia, ur cell-free. Virus litres parallel p24 titm, being
4 . Raised lgC and IgA levels. high soon after infection, low and antibndy-
5. Diminished CMJ as indicated by skin rests . bound during the asymptomatic period, and
6 . Lymph node biopsy showing profound again high towards the end . An infected person
ahnormalities- may therefore be infectious throughout , the
B, Specific tests for HIV infection : 7
infectivity being highest in the early phase and
These include demonstration of HIV, its antigens again when the perwn becomes terminally ill.
or other components and antibodies and isolation The virus is present in many parts of the body
of the vims, and Can be isolated from the p e r i p h e r a l
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* Human immunodefiofmcy Virus AIDS m 595
have spread to Europe from America, us well is of mitochondrial DMA from human and
directly from the (briner African colonics of the chimpanzee viruses . It shows rhar the progenitor
European nations. of HJV-1 entered the human fropulatiou from
Conclusive evidence hus now been obtained chimpanzees of the subspecies Pan tragladfTti
from molecular studies, including genetic typing troglodyte living in equatorial west Africa
(Cameroon*, Gabon, equatorial Guinea) , where the
vimh it considered, on other evidence , to have
M
emerged in humans . HIV's are believed to have been
3S
present in monkeys for over lQ0 h 00Q years ,
NS
Transmission to chimpanzees is a far more recent
.J
Of3 M
* s,
^ killing an.d eating monkeys. Human infection could
have conic from chimpanzees who were hunted by
ftp 12 11 them and killed for meat. The cpz 51V may have
taken root in humans by becoming HIV through
IPS
ffil
“ = mutation or recombination.
W *1 r* * - i
PH
Pi
— flp 1 w
90120 =
pfiS
piv- 4^ - *
-
P 5A P Sf
TlLLCHt] It U VT - -
gp-41
HIV- 2
ip J i . p Jj l and p 6 f . Examples «
typical results
are sera ngs . 4 , 5 . £ for positive .. 7 & for . «C I " *P % I IH
negative and ft , 21 tor week positive rations, '
iWi wniTTwr
pyriqrEGt IQ
596 -
* Tc^ltioc k of Microbiology *
The three groups tit HIV-1 {M , O, N } miy women. However, the situation is very different in
represent independent transmissions from Africa and Asia where men and women are equally
chimpanzees to tinmans. The source of HIV 2 Has
already been established as SIV from the sooty
- affected - In some places, more women arc femnd
infected due to the high rate of infection in
mungaher rmsnkev Ctrcocebus a tvs. pnxSticuMs. Transmission m the developing countries
The virus has spread virtually all over the world, is almost always heterosexual and can take place in
though the prevalence rates in different countries both directions.
vary widely. Initially North America , Brazil , The besr method of checking sexual
Western Kurnpe, Australia, Centra] and West Africa transmission ( if infection is health education
had high prevalence, while Eastern Europe and regarding the danger of promiscuity and other high
Asia, were only sparsely affected . However this risk activities. Some changes in life style and sexual
soon changed . By vigorous measures and active attitudes have already taken place in the USA and
public participation, the developed countries have the incidence in homosexuals has come down.
succeeded in reducing spread of the infect ion. Hut Persons indulging in high risk sexual practices and
in many countries of Africa and Asia, the infection spouses of infected persons should be counselled
,
has spread unhindered reaching epidemic regarding safer methods. The use of condoms
sex '
proportions. Differences also exist, both in the offers considerable , though not complete ,
modes of infection and in clinical manifestations, protection. The risk of HlVtransriuSsion increases
between the affluent and developing countries.The with multiple planners.
epidemiology of AIDS has been studied mostlv in
—
the developed nations and only sketchily in the third
world - HIV is spread only hy three modes* sexual
contact with infected persons ( heterosexual or
homosexual ) ; by blood and blood products ; and
Table G 2.7 Common modes of transmission of
Type*
HIV and Iheir TEiahve risk
of expoturt
Approxiuiitc cfuitK of
infeawa per txpvtuiv
from infected mother to babies ( intrapartum ,
.
perinatal, postnatal} There is no evidence of HIV I ScaTyaJ inf -ercminse: -
O. l UW
anal^vagma] ora1
transmission hv other means including casual
contact or through insect* , The mode* ol
I] Blood and blood ^ * 90%
products. Factor VII
transmission ol HIV and their relative risks are CTC Blood transfixion .
5 Q-9Q%
HIV is primarily a sexually transmitted infection. don-jition: sem^- ri , comw.
Inline USA it was transmitted predominantly among bone marrow; kidney etc
male homosexual*. The danger of infection is more
IV Injections and o.s-i m
injuries; shared needles
for the passive partner because mucosal tears are by drug addict
very frequent during anal intercourse and virus laden Injections with unsrerile
lymphocytes in the semen can directly enter through syringes and needles
these . One reason for che high incidence of HIV .
Needle- s rjck and other
infection in male homosexuals may be the large iniunes in health staff r
Surgical wounds
numbers of sexual partners they are reported to V Mother to baby 3096
have. In the affluent Countries, homosexual and Trutplacenol
bisexual men ire infected far more often than the At birth
heterosexuals. For this reason, infection was found After birth
predominantly in men and only occasionally in Bteasr milk
>pyrighted material
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* Tesrtiook nl Microbiology
-
5 million HIV infected people, the second largest
such population alter South Africa,
of infeci ions and tumours; ( 2 j general management;
(3 ) immunorcstomivc measures; and (4) specific
PrapklriMs; The prevention of AIDS rests at anti -HTY agents.
present on general measures such as health IWnpt diagnosis and appropriate treatment of
education, identification of sources and tliminairon opportune he infections and tumours In the early
of high risk activities . No specific vaccine is stage or AIDS can be very useful and the patient
'
available . The bi b mutability, dr.nefte antigenic mav be able resume normal life in between
to
^
types and subtypes, long latency and persistence in
if
f l i r t h e r It t a J i n n
ftmder SC arnlTC Mtiilpii led}. 1993, JblfuK of AIDS Mrdkine . London: Williams and Wilkins.
^
Fauci AS . :: . l HC 1-ane 199 R , HIV Ubease , En Harrison* Principles of Internal Medicine, l 4, h edtL VoL 2 . New Yorlc
i
McGraw - Hill .
CenneH for DL
. -
iase CoriTrtJ nu: Pyrvcnri . ni , 1993. Revised clawifiniticHL system for HIV infection Morbidity Mortality
Widely KL-| M: I . Ll
Copyrighted materia
CO Normal Microbial Flora of the Human Body
CO
lInman beuigs [ike other animals, harbour a wide On the contrary, the opportunistic pathogens
JTT .LV o t microorganisms both on and in their bodies. among tliem cause disease when the body's defence
The normal m i cnobial flora are more or less consta n r mech an isms faiL Thei r abnormal multip]ication c an
for each species and arc broadly divided into cause diseases such as enteritis and endotoxin shock,
residents and transients. The former constitute a PenidJlinaJc producing organisms can aggravate
constant population which cannot be completely infection by interfering with therapy. Certain
removed permanently while the larrer vary from streptococci of the mouth cause dental caries.
time to time and are temporary. The residents In environments Laden with pathogens for
prevent permanent colonisation of the body br - example, hospitals, a shift in the normal flora of
other organisms. A knowledge of the normal flora the individuals there can cause an increase in
of rhe body is essential ro an understanding of the carriage of antibiotic resistant staphylococci. It has
interaction of human beings and their pathogen also been shown that such people can he
laden environment."The normal microbial flora play rccolonised with penicillin sensitive staphylococci
an important role in body economy. They can of strain 502 A which arc harmless and thus
(1 ) become pathogenic when host defences falter overcome the damage done , When large numbers
( 2 ) prevent or interfere with colonisation/!nvasion of people congregate from different parts of the
of the body by pathogens > (3} raise the overall country as in army camps , the new recruits
immune status of the host against pa I hope ns having experience increased colonisation rates of Nafferi*
related or shared antigen ? , and {4 ) cause confusion
, meningitidis and Group A Streptococcus and
in diagnosis due ro their ubiquitous presence in viruses ^ uch as rhinoviruses and adenoviruses
the body and tlieir resemblance to some of the sometimes resulting m epidemics.
pathogens. Members nf the nnrmal flora form part
and parcel of the host and include saprophytes „ NORMAL FLORA OF THE SM %
commensals , fa cultstive pathogens, and true The human skin is constantly and Lontinuouafy
pathogens. bombarded by organisms present in the
The microflora of rhe intestinal tract synthesise
wf
environment. It is also contaminated by one 's own
vitamin K and several B vitamins which supply on secretions and excretions, the extent depending on
OCCSHftQ the body's needs . 1 he antibiotic substances
'
the individual's personal hygiene . The Dora depend
produced by some , fur example,. coQicins , haw a on the area of the body, the clothing one wears,
harmful effect or pathogens . The endotoxins one 's occupation and environment. Transient
liberated by them may help the defence mechanism mktoflora tend to occur more frequently on the
of the body by triggering the alternative skirt.
complement pathway , as long as they arc not Cultures front the skin have frequently
produced in excessive amounts. demonstrated diphtheroids ( including
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600 < T^ rfrnok ol Microbiology K
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'i MnmaJ Microbial Flora d the Human Body » 601
enterococci , colon bacilli and staphylococci. In including lactobacilli. G .irJ. vaginalis , alpha
artificially fed {bottle fed ) children L, acidophilus hemolytic streptococci and Bacfero.'Jes species.
and colon bacjlli and in pan by enterococci, Gram Chkrn. nachumatis and UftaplaantA NREALYTJ cum
positive aernhic and anaerobic bacilli . With the may also be present. The female urethra is either
change of food to the adult pattern, the flora change. sterile or contains a few Gram positive cocci.
Diet has a marked influence on the relative The vulva of the newborn child is sterile but
composition of the intestinal and fecal flora. after 24 hours it acquires a varied flora of
In the normal adulth the microorganisms on the nonpathogenic organisms from the skin, vagina and
surface of the esophageal wall are those swallowed intestines. The nature of the flora in the vagina
with S .LIL 'LM and food. Because of the low pH of depends on the pH of its secretions and its enzyme
the stomach , it is virtually sterile except soon after content. In the first 24 hours it is invaded by
eating . In patients with carcinoma of the
Stomach OC achlorbvdri .i nr pylon -c obstruction ,
micrococci, enterococci and diphtheroids. In 2 3 -
days , the maternal culrin induces glycogen
there is proliferation of Gram positive cocci and dept iM Cion i n t i LC vaginil ep i theliurn . This facilitates
bacilli. the growth of a lactobacillus {Doderlien 's bacillus)
The number of bacteria increases progressively which produces acid from glycogen, and the flora
beyond the duodenum to the colon , being for a few weeks is similar to that of the adult. After
comparatively low in the small !uterine. In the adult the passively transferred estrin has been eliminated
duodenum there are HF-106 bacteria per gram, in in the urine, the glycogen disappears, along with
—
the jejunum and proximal ileum 1(F 10* batten A
per gmm , and in the lower Ileum and cecum
Doderlien's bacillus and the pH of the vagina
becomes alkaline. This brings about a change in
bacteria per gram of contents . In the the flora to micrococci, alpha and nonhemolytic
duodenum and upper ileum , lactobacilli and streptococci, coUfbrms and diphtheroids. At puberry
enterococci predominate but in the lower ileum the glycogen reappears and the pH changes to acid
and cecum the flora resemble the lucal flora. There due to the metabolic activity of Doderlien's bacilli,
are about 10" bacteria per gnam of contents in the .
E eoli and yeasts. This change probably helps in
colon and rectum, constituting 10-20 per cent of -
the prevention of colonisation by pos .iblc harmful
— —-
the fecal mass In the adult normal colon, the
resident bacterial flora are mostly {‘96 99 per cent )
anaerohes anerobic streptococci anaerobic ,
Durham* rubes contain E. CKJ /J - From the number enteroviruses and other cyiropathogenic viruses front
of positive tuba obtained, results are read nfF the warer bur rhev do nor form part of routine tesrtrg.
probability ts ’cs . Further confirmation of the As a general rule, it is assumed that the viruses in
presence of E , coli can be obtained by testing for water are destroyed by eh lor i ration , when the
indole production and citrate uriliutum. concentration of free residual chlorine is at least
L
) A /:‘mh . anr filtration method A measured
' 0.5 mg per liire, for a. minimum contact period of
volume ol water is (titered through a millipore filter . 50 minutes at pH below 4 and a turbidity ol
All the hacteria present anq retained on its surface, 1 nephalomctrle turbidity unit nr less-
It is placed on suitable media face upwards and
incubated at the appropriate temperature, and the PROTOVOA IX WATKK
cola diet that develop on the surface of the membrane Entamoeba histolytica , Ciiardia species and
arc counted. After 1 ft hours of incubation the ftoiantrebum coir can contaminate drinking water
presumptive coliform counts and E . coli counts can However, there is m > good indicator for pruioioal
be directly made.
3 . Detection of fecal sirepiocnoci -
- con Cam i rati on of water Coliform courts arc rot
reliable as indicators of protozoal contamination
Subcultures are made from all the positive bottles of chlorinated water as they are more resisrant to
mf
ir the presumptive coliform test into tubes chlorine than are coliforms-
containing 5 ml of glucose azide broth - The
presence of Srr. focc-Alit is indicated by the production BACTERI0L0GY OF MILK
of acid in the medium within IS hours at 45 C . "
T V I ' I' S u f. |1 \ C T E H I\ I X Mu K
Hie positive tubes should be plated onto These ear be diLss- ified as- below:
MaeConkey 's agar for ennfirmarinr - 1 . Acid forming bnclcria: The commonest
Millipore membrane technique can also he are lactic streptococci including Srr. hetia and 5rr
adopted for this purpose. faccaJji. Ijcrobjcilli are also found. Tb»e ferment
'
4 . Examination for Cl - perfmgent; This laetoHe in the milk , producing aeids- , mainly lactic
is tested hy incubating varying quantities of the
acids, which lead to the formation of a smooth
water in litmus milk medium { anaerobically ] a[
gelatinous curd.
57 " C for five days- and looking for storm v
2 . -Atknli forming bacteria: These consist
fermentation . of alkaligercs sjip, -some aerobic spore bearera and
5. Tests for pathogenic bacteria: Linder Achromobacter. These render the milk alkaline.
special circumstances, specific pathogens such as 3, Gai*forming bacteria: Coliform bacilli
typhoid bacilli or cholera vibrios may have to he are the com monesi . Others are Cl. perfringens and
looked for in water, This used to be done by adding Q fctifyricum* Acid and gas are produced , A smooth
the water samples to tenfold concentrated liquid gelatinous curd riddled with gas bubbles. is formed.
media , incubating and sub Culturing onto Cullfonn bacilli am responsible for the ropiness in
appropriate solid media . A simpler and more
sensitive method is ro filter the water sample
mult -
4 , Proteolytic bacteria: Sporc - bcaring
through manbnne filters and incubarc the filters aerobes such as B. subtilis and B . cereos, Proteus
cut appropriate solid media. vufgam, staphylococci and micrococci come under
this category.
VIRI SES WATER
IN n . Inert baclerin: bacteria which produce no
Methods are available for the isolation of visible change in milk arc called inert. These
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Bftc e 'iQlQ? / d ter . MilH fend Air 609
further Rcadiaf
Linton *\I I and HM Dick (cds ). 1990. Bacteria in the Environment. In Toplcy &. Wc/soni Pdodfim of Bacteriology
J
\riroiogy and fmmunrrv, Veil I General Bacteriology and Immunity. Section B. 211-291. London: Edward Arnold .
^
World Health Organization, Geneva: GupdWjnes fer Drrnijng Vtirer Quality.
'
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* Mecical Mycology r 615
I -
- *
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616
^ or Microbiology
* ToxlbOd
Table 55.2 Clinical types of dernfialophytoses and Trentnu ' iil : Topical antifungal agents arc usually
their common causative agents effective. 7? rubrum infections may be resistant to
jDlSCilSC Common causative agents treatment . Oral grbeofulvin is the drug of choice.
T. i*di$ X! mbnutu E, fimwm arc normal inhabitants of the skin and mucosa.
Ectotlmx ha.ir Mktosp&tmn speriei, Candidosis isanopportunisticendogenous infection ,
infecliurt T rufcnjjT] TlffKiuiyqpJtyw
F the rnm monest pred i sposi ng factor being diabetes .
Endorhrix halt T ^choenJemi , Tr hymirojii, Cutaneous candidosis may be intertriginous or
infection T, viuheemn parooych i al.The form cr i s a n c rythc rnatous, sealing
or moist Lesion with sharply demarcated borders ,
Growth is slow and colonics may .LpjiCiLr onlv in where papular Lesions arc most prominent.The sites
1-3 weeks. ilflpcred are those where the skirt is macerated by
Epidemiology: Dermatophytes!B occurs —
perspiration groin , perineum , axillae and
infra mammary folds. Paronychia and onyehia are
throughout the world but certain types of disease
seen in occupation* th.ir lead to frequent immersion
"
and some specie* d fungi show geographically
restricted distribution. Social and mltuml patterns of the hands in water.
also influence deroiatoph ^ toses. Tories petlix , so Common mucosal lesions arc va firjjris
common in the temperate climates where all went characterised by tun acidic discharge and found ^
shoes ]* fare m die tropes where most walk barefoot.
• frequently in pregnancy , and oraf fjfirusft found
Many factors, such as age, hormones and intcrcuncnt commonly in bottle fed infants and the aged and
diseases, affect rhe susceptibility to dermatopliytosi;; . debliiated. Creamv white patches appear nn the
Depending on theii natural habitat , tongue or buccal mucosa, that leave a red oozing
dermatophyte* may he dlassfied as anthropophilic, surface ou removal.
zoophilk JIUI gcophiliL species. Human beings arc Intestinal candidosis. is a frequent sequel to oral
the main only hirsts for n nth r lipophilic antibiotic the rap v and may present as diarrhea not
dermatophytes. T. rutrain, E. fbcouian and M. responding to treatment , bronchopulmonary
auJoufrtJj are examples . They Ciutc mild hut
chronic lesions. Zaophilic species are natural
eandidosis is seen a & a rare complication ot pre
existing pulmonary or lytttmic disease. Systemic
-
parasites of animals. Examples arc 7 ' rerrumsum infections, such as septicemia, endocarditis and
in cattle and A/. CAtiii in dogs a[]d cats. Human meningitis may occur as terminal complications in
infections with zoophilic dermatophytes cau ^ c severe generalised diseases such as leukemia and
severe inflammation but are more readily curable. in persons on prolonged immunosuppression
Geophilk species, which occur naturally in soil , Candida granuloma and chronic mucocutaneous
are relatively Less pathogenic lor human brings* candidiasis arc serious manifestations seen in
Examples are A/, gypseum and T. jjethii. ImriuDodeJicicTideE.
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* Medici Mycology * 617
>
phenomenon ) .
)$s
Agglutinins appear in the sera of patients but as
thev are frequent in normal persona also, they are
not helpful in diagnosis. Delayed hypersensitivity
to Candida is so universal that skin testing with
m Candida extracts is used as an indicator ni the
functional integrity of cell mediated immunity.
Management of cindliiotif it mainly by
removing the predisposing causes . All Candida
v strains art sensitive to Nystatin bait as it is poorly
* '
%
absorbed from the gut, it is not useful in systemic
diseases. Amphotericin R, S - flunrcKTtnsine and
clotrimazole may Le used for disseminated
candidonih .
4
>
\ i \ *
pyngh
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620 ^ Teubcok o \ M ' crobiology
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Fig. 65 - 5 Sporothrix {Sporolrichum/ schenftii: culture mount showing line branching hyphne and pear shaped
com dia borne in rosette I ire clusters at tips of lateral branches and singly along sides of hyphae
friable polype usually confined ro rhe no&e, mouth dtsidioboltti haptosporus , a saprophytic
or eye but rarely seen on the genitalia or ocher phycomycctc found in decaying vegetarian and in
mucous membranes. Though the disease wan first the intestines ol many reptiles .ind amphibians . If
identified in Argentina, rhe large majnricynf cases has been suggested that the infection may be
come from India and Sri Lanka. While the disease acquired by insect bites.
is generally confined to MUIIOUH membranes ,
< ] H Y I P T( X : < H I T I O S I S
.
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* Medical Mycology * 621
r r ' lAfcTSft
preparations - The fungus grows readily on
Sabouraud 's agar forming smooth, mucoid, cream
coloured colonies-. The ability to grow at 37 ®C
* *
r* C and hydrolyse urea differentiates C- mwfojmm.sfrom
* : jinnpathogenic cryptwocci . Pathogenicity can be
V
demonstrated by intracerebral or intraperitoneil
H
y
»
*
#
A inoculation inco mice , which develop a fatal
infection . Capsule ted budding yeast cells can be
h& n
/
* demonstrated in the brain of infected mice .
i
Two perfect stages of the fungus have been
discovered.They belong to the class Basidiomyeetes
wt TL «- '
and have been termed FHobstldidh DCofomans
and F. bnsilispora.
? - - 7 ^S
*
Four Knlogkil types of ttyptocoecal capsular
-
polysaccharide A, BH C and D - have been
Fig .65.1 D ncsponclium Mature Sporangium ideiititled. Demonstration of the capsular antigen
(com tally loeatcdi in the stroma of a lesion.
by precipitation can sometimes be valuable in
cancer clinically. BorKH ^ nd joint? may be involved. diagnosing some cases of vryptncnccaI meningitis,
Cutaneous cryptococcosis vines from small ulcers when the CSF is negative by smear and culture.
ro large granulomas , Ciyptocoeczl meningitis is the Amphotericin 13 , o -lluofOCytosine. clotrimazole and
most serious type of infection and can resemble miconazole are useful in the treatment of the disease.
tuberculous or other chronic types of meningitis. Cryptococcosis is worldwide in distribution . As
Its onset it iAtidimi and. the cOUfJc slow .ind it Was originally reported from Europe , it used In
progressive, [ r is ofren seen in AIDS . he known HR 'European blastomycosis'. Several
Diagnosis is established by demonstration of cases of cryptococcos i s have been idenrifled in 1Lidia ,
Cipsukttd, budding yeast cells in the lesions and this being the onlv deep mycosi -s common in this
r . **
rs
. .
Fig S 5 fi Cryptococci s nfiafai st CPUs surrounded by
a Ijtg o capsule-
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624 4 Textboah DI MicrotJiO ogy
Amphotericin B has been found useful in Systemic aspergillosis occurs as die following
therapy. clinical types:
1. Pulmonary aspergillosis
OPPORTUNISTIC SYSTEMIC MYCOSES
Some saprophytic filngi which arc ubiquitous in
.
a. Aspergillus asthma
b. Bronchopulmonary aspergillosis
the environment are important in medical mycology c . Colonising aspergillosis ( AspeiyiLlnma )
for two nelsons , Firstly, they are common laboratory
contain LTia. nts tin culture media— AHf >Crgilhli ,
2. Disseminated -aspergillosis
PcniKiUivm , Mucor and Rhizopus species grow on Aspergillus asllima occurs in atopic individuals
virtually anything , Secondly, they can produce following sensitisation to inhaled aspergdlus spores.
serious and even fatal infection in persons who are Er bronchopulmonary aspergillosis , the fungus
otherwise debilitated . Aspergillosis and grows within the lumen oi the bronchioles, which
mucormycosis are Lcnportanr opportunistic systemic may he occluded by fungus plugs. The fungus can
rnycuses - be demonstrated in sputum . The condition Is made
worse by the development of hypersensitivity to the
A S P K t t t j t L I . UK IK
fungus . Colonising aspergillo- is usually develops
Aspergill! and fleniclltia constitute the commonest in pre - existing pulmonary' cavities , such as in
MOULDJ teen on dump bread nr almost -Uiv other tuberculosis or cystic disease . The fongus grows
organic matter. Qf the 300 odd species of aspergilla, into large "bails' (aspergilluma}. Surgical removal
A . fomigafus is highly pathogenic for birds, and becomes necessary as the disease commonly causes
occasionally Causes invasive disease in human massive hemoptysis. In invasive aspergillosis, the
heings - A few other species may also cause fungus actively invade* the lung tissue. Disseminated
opportunistic human disease . The commonest aspcigillosis involving the brain, kidney and other
human disease caused by aspergillj is otomycosis . organs is a fatal complication sometimes seen in
vm
jf
(
i
antibiotics, steruids and cytotoxic drugs. without cvclohcximide . Mucor shows branched
ST
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626 4 Tesftbook ot Mierobiotagy *
ft 0 v:
* J
r
I f
.
r
fig. eS-14 Common contaminant fungi. culture mount) . PetfcMium showing the 'peniciitus or brajahh cgnsisung
'
-
of dui ns nt spares ertehding from I he ends nr shori br inchgs oi conicfinphares . Aspetgfflus showing
unbranched non ^Bplite conidicphores. terminal mg in a gtobose vesicle bearing phtallds from which arise
chains gl conldis . JWucor showing nonseplate mycelium without rhliolda irootlikc- structures) .
SflOrdtitplophorea, which miy be branched terminate m forge globose sporangia containing numerous spores.
,
RhUopus showing nennseptate mycelium with rhlzolds . UnbrAnthad sporangiophcirea arise opposite rhizoids.
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4 Medical Mycology r 627
Mycctism has been known from ancient times, com and peas. IT is highly toxic to animals and
several varieties of poisonous- mushrooms having birds, and probably to human beings as well It can
been identified as inedible, Mycctism may cause cause hepatomas in ducklings and mss* and its
gastrointestinal disease1 dermatitis or death. The possible carcinogenic effect in human beings has
-
hallucinogenic agents (d lywrgic acid, pwilocyhdn )
produced by the Psihcybe species and other fungi
caused great concern. There have been several
reports of aflatoxicosis from India, involving human
have Attracted much attention in recent years.
-
The best known my corns in is aflatoxin '
produced by Aspcrgiltus ffrvm It is frequently
beings and animals.
Ergotoxicosis (ergotism ) is due to the toxic
alLdoids produced by the fungus C/awceps ppuin,
present in mouldy foods,. particularly in groundnuts, while growing on the fruiting heads of rye .
F u r t h e r keailulft
Evans EG V et al (eda). 3989. Mndol Mfrologft Oxford: Oxford University PVess.
Kibbler CC ec ah 1996. fVincipIcj and Aactrcc osf Climod Chkhem Wiley.
-
Kwun Chung KJ and JE Bcnnen 199i . MadSssalMycohgyr, Philadelphiar Lea and- FGfei cr-
^
Riduidson MD and DW Warnock 1993, Fungi/ /nfretfon. Oxford- Blackwell .
Rippon JW . 1981?. Medical Mycology. Philadelphia-; WB Saunders.
ANTIBIOTIC SENSITIVITY TESTS drying the plate 0 ? "C for 30 mins), antibiotic
from rare exceptions like Strep. pyogenes, discs ( four or five per 10 cm plate) arc applied with
Afiart
pathogenic bacteria exhibit very great strain Sterile forceps. After overnight incubation , the
viriftidpi in susceptibility to antibiotics anti degree of sensitivity is determined by measuring
CbClDOtllcriptUtk agents. Thin is particularly
the zones of inhibition of growth around the discs.
marked in the case of Staph. aitmrs and Gram Growth will be inhibited around discs containing
negative bacilli. Therefore , it is essential TO antibiotics to which the bacterium is susceptible
determine die Susceptibility Ot isolates of pathogenic
"
but not around [hose TO winch it is migrant.
bacteria to antibiotics that arc likely to be used in The diameter of the zone of inhibition is
treatment. Antibiotic sensitivity tests are of two types, influenced by a variety of (actors, such as diftusibility
diffusion rests and dilution tests. of the drug, the disc concentration, the nature and
Diffusion test: Here the drug is allowed to composition of rhe medium, its thickness, presence
diffuse through a solid medium so that a gradient of inhibitory or sti m uUtory substances, pH and rime
is established, the concentration being highest near of incubation . It is , therefore , necessary TO
the site of application of the drug and decreasing standardise all the variables. It is also necessary to
with distance. The test bacterium is Seeded Ort [he check rhe potency of the discs periodically using as
medium and its sensitivity to the drug determined control a standard bacterium of known sensitivity,
from the inhibition of its growth. Several methods such as Staph, aureus Oxford strain (NCTC 6571).
have been used fur rhe application of the drug. It There are several recommendations regarding
may he added to ditches or holes cut in the medium the antibiotic concentrations to be used in discs.
or to hollow cylindeni ( Heady cups) placed on it. The Kirby-Bauer and the ICS methods art in
The method most commonly employed is to use common use , Table 66.1 shows the disc
filter paper discs, impregnated with antibiotics. concentrations and the critical zone sizes for
The dj.se diffusion method uses filter paper discs, antibiotics in common use.
b . O rnm in diameter , charged with appropriate A suitable method for routine use in diagnostic
concentrations of the drugs. The discs are stored laboratories is the technique originally described
dry in the cold , They may be prepared in the by Stokes . This incorporates built - in controls
laboratory or purchased commercially. A suitable against many variables and therefore provides
dilution of a broth culture or a broth suspension of dependable results, A standard sensitive strain of
the test bacterium is flooded DO the surface of a bacterium is inoculated in the middle tliird of the
solid medium (MutOfir- Hinton ugar or nutrient culture plate. The standard strains used are Staph ,
agar). The plate is tilted to ensure uniform spreading aureus ATCC 25973 . K . cnii ATCC 25922 or
and the excess broth pipetted off. Inoculation may Ps.acruginoaa ATCC 27853 , depending on the
also be performed by spreading with swabs . After bacterium to be tested . The test bacterium is
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630 * Medical Mycology
inoculated, After overnight incubatmn, the versions of sensitivity tests are available and are in
'minimum inhibitory concentration’ ( MIC) is read use in large laboratories.
by noting the Inwest concentration of the drug that Antibiotic aaauya in body fluid*: The & c
inhibits growth . The "minimum bactericidal arc required to verily whether adequate drug
concentxatinn (MBC) is the W»t concentration concentiarinns arc achieved in blood and other body
of the drug that lulls the bacterium. It can be fluids, and to guard against excessive blood levels
estimated by subculturing bom the broth tubes that of potentially toxic drugs. The assays are generally
show no growth on to suitable solid media. done by making semi dilutions of the specimen
The ‘igar dilution’ method is more convenient and inoculating standard suspensions of bacteria of
when several strains are to be tested at the same known MIC . Assays can also be done by the agar
time. Hem * serial dilutions of the drag ux prepared diffusion method - This depends on the direct
in agar and pouted into plates. The advantage is relationship between antibiotic concentration and
that many strains can lx inoculated on each plate the diameter of the zone of inhibition with a
containing an antibiotic dilution. Automated Standard sensitive Strain of bacterium.
Purther itfe . id i
L«uan V. 19 Bb Antibiotics in laboratory Medicine. Baltimore: Williams and Wilkins.
Murray P et al («h). 1995. Mwual of Clinical Microbiology. Wuhlngran: ASM EYC&L.
Willi um RJ and D1 Heymann 1998. Contajnmcnt of antibiotic resistance. Science 2?9il 153
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Is
co - Immunoprophylaxis
-- J .. DPT- 3 GPV 3P -
( I ) routine immunisation, which forms part ( it’ basic 9 monitu Mettlei
health carei or ( 2 ) immunisation of individuals or lib-24 inentht .r , P ,P I 3 IJT, OPV
selected groups exposed to risk of specific infections . -
l fii years
( wJiKKd entry)
P B P H I P m
ROUTINE IMML NISATION
10 ynn . TT
--
< k <> fi i
decline in the recorded incidence of VPDs* as well 4 A second dcra of TT to be given after
tstc inuiich tu thuac willi rccutfd dr
as of infant and child mortality, for example * it has hutory of prior DPT DT w TT
been reported that in 1992 alone* 1.7 million lives immunisation.
5. Fw pnzvenhun of tetanus in the necmaEe
of children under five year? were saved , sis compared primariEv, but dro in iJic mother
to the mortality figures tr 1984, the year before
UIP was started.
Copyrighted materia
i>32 ^ Texluook of Microbiology
Immunisation with three dows ofOPV has not in cost of the vaccine as a result of indigenous
been consistently effective in India and other manufacture, has made mass vacanafi on mote feasible.
developing countries, with hi li rates of MM It V -.i L L i i i f ; The composite tneaslct-
- ^
seroconversion faiLures. Thi is sought tn be met mumps- rubella vaccine is employed in the affluent
through the strategy of lmop uph rounds by giving countries but in the developing countries only the
OPV to all the children in an area on the same day, measles vaccine is given at nine months, the earliest
expecting natural spread of the vaccine virus among age when it is likely to he immunogenic in the
the children to reinforce immunisation . These presence of maternal antibodies in the baby.
rounds arc preferably held during October to April, Whenever possible, a dose of MM R vaccine may
as the polio season in Indio :.s from May to October, be benefic ial at 16-24 months or later, not only to
-
with a peak in July August, reinforce immunity against measles but also to
-
E Jdh’rvrit cocritrie eriijiluv different immunisation protect against mumps and rubella.
schedules depending on thrir priori tics. Varicella vatu: inert Chiclrenpcot is very roiild
disease in children, but in adult it can be serious
1 NDI \ mi \i . IMMUNISATION and even fataL In most parts of the world, chickenpoK
Vaccines offered under national programmes art is very rare in adults, but in some areas m the tropics
limited bv. economic considerations and so some it i ^ not uncommon . The age of incidence of
important vaccines may be omitted because they varicella is reported to be rising. Varicella vaccine
are costly These maybe supplemented by individual had been used for many years in
initiative, whenever possible. immunocompromised children. Recently, with the
Hepfttitia h vaccine: Many developing development ofa more stable and effective vaccine,
countries, including India , have hii t cmk minty for its scope has been extended for general use for
^
this virus. Perinatal cfansmissidn and acquisition of prevention of varicella and herpes zoster The live
the virus infection in the first five tars of life are attenuated vaccine is recommeded as a single
r
^
common in such areas, in contrast tY> low endemic subcutaneous dose in children 9 month? to 12 years
areas where infection is usually acquired in of age, and as 2 doses at an interval of at least 6 weeks,
adolescence or adulthood from satual or household in those older, Pregnancy is a contraindication.
contactst contaminated needles, blood or blood Typhoid vaccine, Typhoid fever continues to
products or occupational exposure. Besides the be a maj^ KT public health problem in the developing
morbidity and mortality due to acute and chron ic vi rus countries. Immunisation against typhoid is areal
infect ion „ chronic carriage which may be very need, particularly in view of die spread of drug
prolonged i -. itself a serious public health problem - It mi slant typhoid strains . The original typhoid
has also become an economic problem as carriers are vaccine is not width used because of its uncertain
jf
domed entry oremplcymcnt in many foreign countries. benefit and frequent adverse reactions. Two recent
Inclusion of the hepatitis B vaccine in routine typhoid vaccines, the live oral Gal-E mutant
childhood immunisation will therefore be benehdaL vacc ine and die i mectabfe pui lied Vi polysaccharide
]
The feet that a quarter to half the adult dose of the vaccine may be acceptable because they offer
vaccine is adequate for children bongs down the prolonged protection and are free from reactions .
cost . Till it becomes part of the national Thev are recommended for Immunisation of those
a
immunisation schedule, it would be desirable to five years old or above and so may be employed at
have the vaccine admi matured tn as rn .mv children
im
school entry
and adults as possible by individual immunisation Immunoprophylaxis of individual diseases has
or through voluntary agenesis. The recent reduction been described in the respective chapters.
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Tetanus an a result of ]i i > > ii i C al iflfei^lionf iS iWW Staph. bacteremia is seen commonly
fire hur should he kepr in mind and toxoid in patients with artificial heart valves. Bacteremia in
administered to nonimmune patients before elective those with valvular defects may lead to endocarditis.
surgery. Many oases of neonatal tetanus have
occurred due to the use of contaminated umbilical DIAGNOSIS AND CONTROL OF
cord ties. H O S M T A I . I \ F K t :T I O N
2 , I rinjiry trsiel inftctionif Even with Hospital infection may occur sporadically or as
adequate precautions, catheterisation in hospitals outbreaks . Etiological diagnosis is by the routine
leads to LirinuTT infections in about Two per cent; bar tend logical methods of smear , culture,
with Indwelling catheters, the tilt gtn'S Up to 50 identification and sensitivity testing . When an
per cent or more. E. coli , Profm-s, Ps . teusvginott outbreak occurs, the source should be idem tried
and other Gram negative bacilli are the causative and eliminated . This requires the sampling of
agents . Mixed infection is common. Infection can fioysible sources of infection SUCIL as hospital
he prevented by strict asepsis during catheterisation. personnel , inanimate objects, water, air nr food .
Indwelling catheters arc to be used only when Typipg isolate - phage , hacteriovin, antibingram
unavoidable , and then only with proper closed -
or biotyping from cases and sites may indicate a
causal connection . Obvious examples of sources of
drainage.
3, Respirator} infections'. Aspiration in hospital outbreaks are nfrsll carriage of
unconscious patients and pulmonary' ventilation nr staphylococci by surgeons or pseudomonas growing
instrumentation may lead to ousccoinial pneumonia, in hand lotions. Cirrieiy should be suitably mealed.
particularly in those with pre - existing Sterilisation techniques have to be tested. The
cardiopulmonary disease. Multidrug resistant iVapfi. cause of infection may be a defective autoclave or
auJTWS and Gram negative bacilli arc the common improper techniques such os boiling infusion sots
pathogens. Antibiotic treatment is unsatisfactory. inward sterilisers. A cardial analysis of the pattern
Postural drainage is useful in the prevention and of infection may often reveal the source bur
management of mch oses. sometimes it eludes the most diligent search .
4 , Buotcncmia and MpTioentia: These may IT must be emphasised that control of hospital
be consequences of infections at any sire but are infection should be not merely i spasmodic exercise
camirKmlv caused hv infected intravenous carmulae . to be employed when an outbreak occurs but rather
The longer the cannulae life kept in situ, the greater a permanent ongoing activity :n any large hospital,
the risk of infection . 'Gut -downs’ on the leg veins Evciy maior hospital should have 'infection control
in infants or children with diarrhea generally get
1
teams consisting of microbiologists, medical and
left in place for long periods, the site being bathed nursing scoff and hospital administrators. Besides
in diarrheal SUHJIH . Phlebitis sets in with Consequent investigating and controlling outbreaks, their
bacteremia . Many a child admitted with diarrhea functions include formulating appropriate
thus dies of septicemia . Gram negative bacilli arc guidelines tor admission , nursing and treatment of
the common pathogens. 'Cut-downs’ arc safer on infectious patients, aurveillance on sterilisariun and
the arms than on tegs. Intravenous rehydntiem in disinfectant practices , determining antibiotic
diarrhea should be restricted TO emergencies and policies and immunisation schedules, and educating
should be replaced bv oral fluids as early as possible . patients and hospital personnel on infection control.
Infection can ho prevented by proper skin toiler Such measures help in reducing the Incidence of
before 'cut -down ' and rhe i ] se of stainless *ted hospital infections, even if they do not eliminate
needles instead of plastic canmilic , them altogether.
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H Huso . tn infection ¥ 637
Unfortunately, In many hospitals, infection prescribed LEI the rules , which have come into- effect
control is attempted by resorting to more and, more from 1 January 200.T . The occupiers halt to obtain
of antibiotics. This is not only futile but may even
r m
due authorisation from the prescribed authority
be positively harmful by encouraging selective after selling up the retjuireil waste management
cdoniiiJticm by imJtincsLHiant pathogens. In the timd facilities.
analysis, prevention of hospital infection rests on a
proper undemanding of aseptic practices and Qi i v r m ANJI TVPHS OK
meticulous attention to hygienic principles. Sir UloMBDlCAL WASH:
William Osier's aphorism that 'soap, water and "
Hie amount of waste generated in hospitals under
cymmonsense are the best disinfectants applies even Indian conditions has been estimated a-s ] to 2 kg
"
today in the contest of hospital infection . per bed per day. This is composed of different rypes
f > ( infectious waste , not all of which is infections.
legally responsible lot maintaining the conditions volume, rttrive reusable material and ensure safe
^
Copyrighted material
Hidden page
INI >KX
A aerobes 21 anabooeptor 102 124 k
adoptive imuuiinigaHOfl 177 alpha methyl cbpa 122 antibiotic 39, - jjj.
J monie eria
4 TEXTBOOK OF MICROBIOLOGY >
autoimmune 371 viral ( v) 504, an antineuramintdase 453, 504 , 508
blocking: ( n agg]urinatingj Vi 291-91 - antisera. 32
9g. 99. 149 WiistriTkiiin 302 antisepsis 21
sLytcjEfcjjik: 160 antigen-antibody neaetioni 97- ltW anriaraphylolysin (anriaJpbalysin} 199
dependent cell mediated agglutination reaction antistreptolysin O titration test 103,
cytotoxicity ( ADCC) cells 126 antiglobiilin ( Coombs ) test 33 206
humoral 452, .453 passive agglutination test 1QQ an rite ran i,is immunoglobulin , human
!
j
/ name eria
4 INDEX ft 641
iihkie'i foot 614 reaction) H gene 53, 5ft, 60
Miipy 164, 3S1 . eett envelope 12 generic mechanisms of drug
itHMUxn 2, 62 «11 wall & 1L 12 niPtintt in bacteria SS
SLlypiraJ SBjjnfaaCIHtt ( ariuilyiTKMift/ defideni ftwms J99 gene iransfer S3
- genome 53, 54
—
unclassified/ non tuberculous cell wall deficient bacteria 13
mjrabaercTia) 351, 361 cytoplasm 15, H genotype 53, 54
-
,
Australia antigen 5 5*0 551
Australian ' X disease 526
1
cytoplasmic inclusions 12 14
cflapmaaL lAasma membrane 12, 13
- iusertion KqueflOH 6Q
innons 51
All E Dill] 169 episome* H mucagctu 54
aucoanribodics, cold HI Fimhri 15 mutation 51
autoclave 25 27 *26
^ ^
(uirncrtne effect 144
fbgdla 14 -1 ft mutational v? transferable type of
drug resiseanire 52.
autoimmune diseases
rnH
^ocapsufes ll
nucleus 14 molecular genetics 60-61
class ini cation of 171 spore liL blotting techniques 62
entnia for 169 types of bacterb DNA probes 62
mcduuiem of aurtoimmuiitsHbon 120 carbon dioxide, requirement genetic engineering 53* 5iL 6D
aurojmmunity 1 ft 9-7 S of 22 molecular epidemiology 63
autojnterfcrence 445 colonies 12 niicfeotides S X 6fi
autolysis 2 t 7 continuous culture of 1£
female 16
- PCR 62
uutertrophs 2D phenotype 53
aviuleiKTvirusc 44fa freeze drying ( IjxsphilUadofO 22 polypeptide SI , 53
avian leukemia virus 579 Gram positive n Gram protein synthesis 52
avian leukosis 52ft Kgldw 2 replica plating technique 55
growth and mi drip!ration of l£ replicons* independent 53
B growth curve 14 resistance fnuwfcr factor 52
idealised bacterial cell Ijf 12 transduction 55
B-cclI activating factor 1.44 UVtVBcytOfPlNlIkic indusions 11
R-etll grnwih brim 115, HI transmission of gen«k
invohinon forms 2£l material 55
R-eells 115, 1S2
.
R virus 47B 494, 4%
Babw- Ernst granules { m also volutin
male 16
microscopy £
moLunint 22
tnmpoHUe genetic
dements
granules ] 231 transposition 60
nucleus 14 tnuispovons 6Q
ha he no. 41fi nutrition 2Q
bacillary angiomatosis 421 momma S3
bacillary dysentery 3ft5' BB =
osmotic effect cm 23
cuygen requirement and
bacterial idenrifieaEion 44 4ft
bacterial lysis 461
-
bacillary pdiosi-s 421 metabolism of 21
bsdle CalnHEie ct Guerin (BCG ) 354 bacterid tMonomy 48-51
pleomorphtsm 12 classification M.
bacillus 14, 15 1454, pH KnalinCj 23
^
jn[lmcL5 241-46 ncifflcncluvre 5D
shapes 10 ape»cLe£ EtcHieejits 4fl
differentiating features MW of Z
between an thru and type OIITULECS >Q
temperature requirement of 21 bacterial vaginosis 410
Hnthracoid bacilli 246 bacterial genetics 51-64
medusa head appearance of 242 bacteriodn 57> 301. 466
chemical structure of double
eereus 246
unadfid BNA 52 -
typing 47 467
bacteriological index 370 » 325
lichmlformis 2ft codons SI
tapsubtUH 2M IwiLtcrialyHB 110, 113
phlcgmonis emphysematosae .249
FtcarorhcrmophilujiE 22* 2£
coldnogenwz (col) factor 52
conjugation 56
bacterinphnge 4 , 55a 450 67
assay 466
-
subdLis 64 24ft. 6fl5
^
deoCTribofinick-k: Mid ( DNA.)
double helix 5L 52
.
iiTi i' vf •: LUJVE 464
badnacin 300, 204, 205, 2lfl, 261, 26i recombinant dna til lifeeyek 442
bacteremia 70, 197 , 200„ 216.219, enzymes- 5A morpholiDgy 462
im, 237, 296., 297, 3.11 , Hi puma 53.56 typing 466
hacieria exons 55 bimteriuria 27 ~~ 77
anatomy of 22 txErachjomoMiiud genetic baricroides 267-69, 4008 ft^ll
morphology and pbvrio bogy 7-21 efemenu S3 bactcroidks liaplis group 26 ft . 269
bueroides. niel Miogefikus -ordis
captide 1L L2 , 14
capsule dwelling (qpjellutig
F facEur 56, 5Z
fllKtHtlHI HSLT 55 gfotip 26
^ ^
: i
yrrante aieria
«
642 * TEXTBOOK OF MICH06I0L0GY *
balantidium vnli t£lS HnunMbXt pmmgiop of 1SZ cams 145
Baliensp-Bcthesdh group 28ft. 291 inheritance LH.7 mclitcnsi* 348
Barnxndb badUifomis 42ft blood transtiisiofi 181 BBtnBB 346
banunella iM blue pus 12Q twis 346
bortondlosis ( Carrions disease) 120. blue tongue drug 522, 521 sun M&
baEsichobolu-s haptospoms 62Q body EDUK brucellins 150
basidiomYtctcs 611 ,. 621 boivin antigen 12^ 291 brucellosis 25Q
bandiojpuncs 6-11 Bollinger hodia 449 bufTalo pux 468
haney bacillus 366 Bolivian hemorrhagic few 569 buLbolderia
B cells ( lymphocytes) 90, 121, 122. bone marrow culture 2918 ccpacca 321
characteristics of 125 ^- -
booster dtwe 7 jJ L 2£d
IkideNjfifigffFil modium 339. 341, 342
mallei 321
pseudomaUei 121
growth factor (bgf ) 111 WIeKlIa 119 44 BtiflyuMHi vim* S32
marunrion 124 bronchkepcjea 344 bunyavMdLae 447r 522-24 512-14 p
n r
yrigm i
ri
cuanoeiDlHjmHC antigen 191
cardiobacterimn homini 410
-
dijwf hone typhus 416
INDEX y
chancre 379
!!
colostrum
chronic fatigue syndrome: 464 commcn^J" 64
chanuTHd 33? = chrunic granulofrHta (gumnrme?.) 3W
chandipura vim* 533 534
Chang CVHL EDC Ml - chrome granulomatous ctiMuue 15?
chrnc i m IIL u nosuppm si ve therapy
common cold ( sec also rfmoviiuses) 42Z
compknaencaDon 445
complementuirn*’ determining region*
Chediak-Higiwhi Fyudrume 153, 157 LSI 137
chemiluiTunncence immuiH ay IM
chcmo- organotrophs 397 ^
chronic obstructive pulmonary disease complement (C) aysrem h 110-16
(COPD) 402
chcmotacrtic factor 1.43 acrivarion 111
dtfVtt utiluuitiun EfcSB 46, 2 .1, 2*M)
chemotrophs 2i(
chicken chuJetfa 2
ritrobacter 274 28Q.
citron bodies 256
- alternative C pathway 112
biological effect? erf 113.
bacillus 2 cladosporivm 61B 619 buqtihem of Hi
cascade 112 ,. 113
?
.1 ,
J rionte ate nai
644 4 TE5CTBOOK OF MCTOBJiOLOGY t
deficiencies 11 5
general properties 110
M 3, 444
•
conjugation 57
conjunctivitis
methods 39 43
anaerobic dU
- detmit-6 cell line 441
deutoromycetos ( hsphomyceiM/fongi
acute follicular 485 continuous 4fl imperfccd ) 611
acute hemorrhagic 498 pour plate 39* Mi demin 232
angular 410 stab 32 dapsone 325
fnllirttlar 477 streak smote 32 dktwcbm 385
cenwirotive eruyenes M. mw plate dHJ Dkk tec 201
contact dermatitis (hypersensitivity ) culture media, types 33^ :15 didcmyadcnDSLne (DDA) 459
i59, i6.i , m cutareous hmopfau hyptam ii-irtY 166
contact inhibition 575 cyrocidal (cytolytic ) rears 103 ^ didemeytyridine ( DDC) 459
dkleiwinQsne ( DDI ) 459 -
contagious disease 66 cytokines 143* 455 dkne% m.ettwd 396
cnfitpgintki neumlymphomatcwi* 576 cytolytis 112. 449. 4,52, 511 Djp| « B syndnrMine 154
oragiouf p&i&niar dnulitB 4faSp 472 cytolytic metim 1M. dilute carbnl fkurhsin. stain 388
comagiLLm vivum ] cyun dawia JJH, 4S1-S2, 577, 635 dimne^phsc fungi 619, 621, 622
Cwmb? teFt 99, 172,
191, 349, 3«
mmm ^
clinical features 431
laboratory diagnosis 4B2
dkiiirochloTCiJbeinjzeQe 186
diphosphopfiidincneucIntidAse
coproantibodies 31 5 cvtupclTiic dfeis (CPE) 440. 487.. 506 (DPNiie) 2HZ
cold factor 171, 352 qrtopltttn 12, 13, 121 435 p diphtheria 606, 60S, 633
commeal agar 6-11 cytoplasmic knchitians 12 a 234
eulnoieoy
IOK 240
JjpMwk 240
—
coio navi ruses 448, 569 r 573
onrwehfl£teriu:m 231 4Q
-
cjToaine arabiimide 141 , 459
cytotocric reactions 16fl 165,
CMpdc-DteK medium 611
>
B
mm& 04, 140a J33, 237 39
J61.332
diphtherions 466
diphfemids. 240.334
-.
cqui 240 diplncwnn ( see pnesumooeccus ) 216-
monutissEimuin 240 D 21
parvum 2Mi dakar vaccine 529 1 dipbid cell strain 440
p-ssudodiphtheriticu.iia 2441 O antigen 136 dip dide culture mediEwk 277 .
Dvrianiec ri
* INDEX *
drug resistance ( R ) factor 57 cm rn mopoxvinruK 468 erysipelas 202, 208. 404
mutational £8 enteric fever ( typhoid fever ) 295- 03 ^ eiysipJothrii rhuskpithiae 404
trjTLNluLihli.: (epnsi i mal/i ntuitionsJ 5ii bacteffcophage typing 300 erytluuiu
dubos medium 152 8 di&gnotis of carriers 30Q anhritkiim epi <kmicuni
dysgammaglobubneinia 154 drug RUftinoe 1L12 ( Haverhill fever) 406
dysgonk 2S2 epidemiology 296 chronicum migrahs (annular skin
labonnEEKry diagnosis 297 lesion ) 389
prophylaxis liVl mu hi. forme 4ZZ
E
early antigtiis 484
^ ^
treatment302
nt robacto 281-84, 603, 635 eryrhri. ru l 345 , .347
-
iiEKhssum 33®, 3311, 3 62
carty proteins 452. aefogenes 231 erythroblastosis fetalis 165, 188, 190
Eaton agent 398 cloacae 281 ttyffort&L«fflQ ris virus 578
tbenhdk typhi J9fl
Eh nuclear antigen 434
_
entoobacreriatceae 771-SU
] USKI li . aCirnj 271
tfydiTDCfft ( E) antibody ( A ) complex
ill
ebok fever 569 enterococcu* 203. 234 aychrqgenic fdiclk, scaikiaml) mxbs 2Qi
echo viruses 491L 49h enterotcodni 1%. 273. 2K 6 E$therichid ccJi 3L 54-57, 59 67,h
eclipse phase 43BB 465 enteiwimses 434. 436, 442. 445, 447, flg, 224, 252, 271 , 272-BO , 286, 287,
Ectliyiiniij gnngtfenresum 320 490-500. 635 326, 346, 462, 466, bOiL 602, 635,
ecnjchm hair infection 615. clinical s)rndiamc 497 6.36.
Eczema herpcticum 476 envirnurinc 460 antigenic stETacnure 273
Edmcmstoiir-Zagrdb strain 520 enzootics 526, 531 bncKrhemicaJ reactions. 2Z1
ifidwititibdli 2!? 274* 2SQ enzyme immunoassay (eia ) 106 . clinical in&eiicMii 275
efferent Inhibition 128 enzyme Linked immurujtocbem assay cultural characteristics- 212
egg yaUk medium react ion 46 ,
(ELISA ) 106, 457 582, 590 .
enteroa j reganve ( EAEC ) 279
Egyptian ulcer 231 enzyme multiplied immunoassay ^
eniemhaiwiTha c ( EH EC ) 222
Ehrhch phenomenon 2J2J
Ehrlichia 412, 1LZ
technique ( EMIT) lfifi
enzyme neucialisation ccs;s 508
^
enterobvuivE ( ElEC) 278
HiKcTopathogHiijc (EPEC) 228
aenrteBu 417 .
et ^ inyphilia tropical 382 enterEHEraiggriir: ( E^ PEC) 278
EjchwaJd-5 ilmsct cffccr 178 L- qrfarii.Vj.ThiE L lici iiul jdiC tiij:;:tn|
'
r?i of enterotoxinmethodfs for
Eijikman toe 604 anaphylaxis , ( ECF - A ) 363 detection 223
EiknelLa ennodpens JLH) eosinophilk: hyaline indusksei hi id i LN 473 virulence factors 223
- —
cledrobn mu clodiifluson ( see
antigen anybody reactions )
epidemic
diarrhea of infant mice ( ED1M)
Escherichia 272
^
esophagitis, HSV 477
election rnkporseopv 431 551 568*
, , 572 KSS (emhtcscyte sensitising
57ft diseases Zli mbstance ) 418
tlek pel pm E pi Cation 2M hcmoflrhagic fever 525 esthiMKiK 42$1
ekrncnmy bodies 422, 4J1, 4612 kierrtiileiinju.nctivitis ( EKC ) 4B8 ether 432, 434, 502, 513, $18, 563
elution 434, 435, 503 nephritis- ( EN ) 533 ctiivknc diiminc tetracetic acid
EMJH medium 390 nephmsnnephritLi iil ( EDTA ) 212
EM USA 12±
mctphalid* 397, 475, 477, 478, 479,
parotitis ( fcsse JIMS mumps ) 512-13
pfeurndyinia 497
eubacterium 266 267
eugonk 352
.
.
492, 4%, 522 523, 524, 525, 536, polyarthritis 525 eukaryote L d3ll
539 typhus 413 exaltation fix
central eurepean 525 epidermuphytun 614 exanthem subitnm ( roseola infantum
fail 523 episodic angioneurotic edema 114 or aixih dbease ) 4R 5
hsv 477 episodic lymphopenia 1 exfoliative toxin 197
vaedme 527 cpisemes 34 exophiaJa wcmcckii 612
ciKephaJornycUtis 140, 143 epitope £li exmxin a Hi
encephalopilhy’ 343. 344. LLEi epizootics 328, 329„ 53lp 534, 545 exotuKins 67, 68
ndmk disease? ZQ
cr-idcri me ( or nib-uiuj] ) typhoid 297 574, 522
-
Epstein Bair vims 12 3, 474, 482-85, , expanded programiiK on
immunisation 631
endemic typhus 413 equine encephalitis virus 52.3, 525 , expanded rubeUa syndrome 564
cndofhrix hair infection 615 equine rabies immune globulin experimental aJkrgic
eikdcrinufri 67, 63 (imp si] encepba]omyelirii& 173
Entamoeba! histolytica 6Q5 6.15
Entamoeba maritirurtnlhim 481
P
erwinia 233
-
ergotism (efg oroxkosia ) 627 expkht cuilures 439
extrapuLmunary' infeerculoaia 1. 60
j name nai
646 4 TEXTBOOK OF MICROBIOLOGY *
F rularenrsis 12L 331, 332 Mh
P versus host .re-action 180
fab fragments 85 freeze diving 434 Gmm stained smear technique % 111
fattia sp 402 freeze etching & Graves disease (see rhyxotooskosis}
favut (Trichophyton fchonleinu) 6l0^ Fra * test 42 b, £2& gritSth typing 201
614, Mi] Freunds adjuvant 140.169, 173 gri&eoiuJ. 61b
k (rapiiflnt 85 Fuller’s method 2li5 Grim nJuite test 212
_
fclix lubes 229. fungi 143, 229, bl0-27 -
group specific nudeopiutcin antigens
k rii ) L'ijCri >.H 11 21 fiuigi impeffeeri (dcuCCTLiniyCetesi/ 577, 522
Fernandez 's reaction 121 hyphomjttMes) 611 growth inhibition ter 397, 398
fanmaternnl A HO incoiinpnbi% 182 fusiform badlh 235, fcQD Guamlen bodies. 449, 470
FfrrtW 56 fasubacterium 267, 268, ZZD GuillakHtfane syndrome ( idkspmfhiic
fibrinolysins 207 fliJH-wpiriKhcCnsis 388 polyneuottii) 173.477
fibroma 468 gummata (chronic granulomam) 370,
fibronectin (binding protein ! 127 G 321
FICVTC bouionneuse 414, 41 b gi£f\cf 11
Fikiba-iidiclla banllispora 621 grille rrtuCanl wiodrte 612 Id
Filobas.jdjelIa nco-formam fall gammaglobulin 153, 154 haernogogus apegizzimi 529
fitdes 334 gAA&dbfrii1
hacmaphysalis
fiLoviridjic 448, 569 gSnjam diseww £rflimp) 522 Icachi. 416
filtration 2H ganjam virus 522, 511 spmigera 531
fimbriae 272, 223 gatdnetelk vaginalis 229, 410-11, fefil
Fira-Hugh Curtis syndrome 427 gas gangrene 248. 249. 251. 252 =
ticks 530 31
hacmciphilu* 224, 11,3-38, 60fl
Dahlia (see bacteria cell will )
flavivirus 447,. 525-26
flai'obatleriuTH 603
anaaobie myoma 252
endogenous 254
prophylaxis and therapy 25b
-
acgypticus 337
aphruphiluK 118
ducreyi HZ
lEbCELiii iM.' piicum 4414 gauspak tytteEti 41, 269
^
fleabotne typhus 4111 GB vims 559
haemoljtLcuE 338
influenzae 311
flea index 328 generic engi neering (see blCttria] p M aherEiol yr ic us 117
fb? h eating butfwia 208 generic*) panunfluenzae
Fletchers medium 390 genital chkmydiasb 427 paraphrophilus 116» 339
flocculation M genotypic muring 445 HafFkine 's ,acdnc 130
fluctuation test 55 gentamicin 4Q3 , 405 ^
haiViia 281
flyaKtoem antibody test UK dffij gcotrkhum tiflQ HiJtKrscacdicr Pkcrtrt ^.Lk (HP) bodm
Ciepmeaual antibody test 104. german merles, ( we also rubella ) 563 425, 426, 126
fluorescent dye*. 351, 357 germicide 2i halophibc vdbmos 316
-
5 fluoxoujraciJ 390
5-fluorocytosiine 617 , 619, 621
ghan focus 355
giardia sp. 6il5
hand , foot and mouth disease 497
hantavirus 447a SH
fluey stmias 439, 5.16, 541 Cirms stain method 412. JlZ hanraan virus 533N
IbILioiJjria 589 girmaa stain 124, 178, 186, 3&EP 1W , haploijpe 112
t i.4iscLLii.Li ( harmod mdru m. ) 618
Fontana’s method 378 »
food poiaotiing 252, 27 H£
540 ^ . .
396, 424, 426, 449, 47 480 519
passive 172, 239, 329, 397, m timMUOt sentitHpR fklftr (HSF) 34T! h }JK* IgK sypchtime 158
*i£il 4 U
hemagglutinin 504
hi&riucytE (m mat rt phage)
(
hiffmcompatihLlity antigen* 1 7 H
hypenetisiiivLry 92, 10 L 159 6B, 261
atopic 164
-
filarnenttMi’. MB hirtupluma 623 classification of 159
^
hemolysins 6 195-96,, 251
hemolytis 110. 3 88, 250. 258
alpha, hera 258
tapsititum 623
duboisli 623
hisroplasmotis 623
delayed (or cell mediated ) 159
immerduaCE 1.59
immediate vs delayed 160
hemolytic disease of newborn 16-5 , afiican 623 types of mctirnnF and their
mm hisfnry nf hact nil ]logy 1-6
"
feature!!: 1.60
hrcnmnirhapir femrs, viral 563 HIV siiFertixm 89, IKE, 545 hvpnthyruidiKm ( myxedema) 172
hcinorrhicic Fever with renal HLA molecules 12L 13Q hypa*anthine ph »hinribnsvl (
--
Instiviruatt 594 primary 133
herpes simplex 476* 478.481 pathogenesis 586 secondify 133
herpcFvind ^e 474 85 resistance 5S5 immune RNA 132
herpesiimses. 474-35 tiles, of isolation 590
immune system 117-3T
herpesvirus simile ( B virus ) structure of 5H 3
immune tolerance 122, 123
(eirenpithicine herpes virus, 1) 478
herpes zoartr (shingles, MM ) 4flO
h*rpt* irater, nph[ hafinicu* 481
human herpesvirus type 6,
human leukocyte antigen ( HLA ) 130
complex Uffl
^8 485
immunisation
oumbanj&d 78
nie< ceria
MS 4 TEXTBOOK OF MICROBIOLOGY »
Iral 7g gusumieri bodies 449 morphology ID2
cntiiui iTitnr
^ of 78 HtncjtQplwHC 449 onhomjfluvmu and
BTTTifBiriifiMfrTwiglufttiin (IK ) 11 5 intranuclear 450 paramyxovirus 501
immunodeficiency diseases 152-58 inclusion conjunctivitis, monara! form pandemic 5012, 505
primary LtnmunodifiCLeftCLes 152 of 427 psAfOcernt 50S
dutlfic&tkn of 111 indole production test 45 , 273, 278, prophykm 511
disorders of uompkmem 153r ISfi 2811, 319 sporadic 508
cbsoniers of phagocyrais 3LS2 infection 64-70 treatment 511
secondary immunockfianiaH JLifi clarification of 64 types A B, C 502, 305, 507* fII
Lmmujsodiffusicm fprecipitation in stead).' state infection ( eeDuibu mterference 441, 443
»6036 injury) 449 anesrfimrs (IFN ) 143. I45r, 445, 454, 460
radial m i l j immunity 384 interleukins (IL ) 124* 127, 144 45 -
immuiindectTnn mpcfoBUQpy Mft mode* of transmission 66.69 intrauterine infections 89, 481
immunodectrophorcsis SI microbial parhogemcit factors 66 intravascular coagulation,
immunocnTyme tnt 1139 IABHOD 61 ^ disseminated 114, 127
inU}iui>0tKn.nic £*n« toft 104, 22®
* bacterial appendage? 63 iodine 31, 502
233* 245* 277* 382„ 39Db 392h 197, * baduml products 68-69 staining 424
398, 40 L 405 , 409, 417 , 416, 420, bacteriophages 68 iddophofca Jl
4B0, 484, 507* 5K 515, 5 T7* 519, communicability 6fi idoxuiidinc ( 5 - iodfl - 2 deoxvundIne )
*
r
pyrigniet i
ri
« INDEX t fi49
kerion 614 icremhemnrfhagrie 389 lymphocyte tnmsformafion tets 372
kingelk 410 intrarogans 389 fymphoqrosk producing factor 340
-
Kirby Bauer method 628 laboratory diagnosis 391 lymphocytic dhoriomeningirii ( LCM )
kflMinp d isease 4ft 3 serological diagnosis 392 virus 447, S/J1
kkbsKlla 15, 2BO-81, morphology 390 lymphogranuloma
—
KJcbs Leefl1irr bacillus 231
Koch's phenomenon 4
pathngEmdty 391
prophylaxis 333
inguinale 428
venereum ( LGV ) 424 228 k
classification 371
culcivjciun 3711 ^ iwusis- lem PunsEii ( LJ ) medium 26 ,
[. lysogenic haurteria 56, 462
epidemiology 372 11352, 367 lymgenic conamion 2hx 232 , 465
Lwkes tumour frogs 226 lysogeny 5 465
immunity 372
laborafcsTY diagwris 374
LucHami medium 193. L2R Imigemsadon 223 ^
Ludwig's angina 2QR lyuoi 32 329
morphology HU ^
lumpy skin disease 46 B Ivsosomes 21
prophylaxis 1Z5. lupus vulgaris 362 Jpostaphin 194
redsranee 371
Ijgrwmm 22B lysftcyme 12
beph 3xpi. nL 3R9 Lyme disease 389 lyssaphotwa ( hjdrophobkiphdbia) 528
antigenic properties 390 lynnphadeiicipathy associated virus lyraavirus 447, 545
odfirfal characteristic? 3*90 {LAV ) 582 lytic (ar virulent ) cycle 56^ 462
epidemiology 392 lymphoblasts L2i 'Ij'sis from Tvithout ' 564
Copyrighted materia
650 4 TEXTBOOK OF MlCHOBlOLfKiV
M 446, 447, 449, 451 458, 517-520, lethal dose (MID) 69, 238.239
,
BMKMJOCletff 326 h
in tbeusc ( histiocytes) 74 L 12fk methanamine silver stain 611 monkey fever 531
mad cow disease 567 nKthartobaetmA 266 monkeypox 468, 471
maduranijcosis (nuduia foot ) 402,. 618 mdtjfctt blue rtduLiicHni EE?W 46, 449,
606
monoclonal antibodies 122 135 .
madurdla 61S manocytt (see DHCRHIUHBJ)
maisdi ( progressive pneumonia ) 567 methyl red ( MR ) re*t 45.273. 3%, monokines 143
MAI complex 366 MTadyean's Traction 242 monoma-fphism 51
major harimompaiibLliry complex
{MHO
antigen
m
122.
^-
mcrrMfc hj]!! . : bacteria 23 6flfi
microbial flnr ? of human bodv,
nnrmd 599 602
^ Momrk CTTA medium 306
ManrAmridd hsdlius 410
moraxelb 230.600
genes 129 micrococci 194, 200* 600, 601, 605, iruwbiDirkrujt 447, 512, 517
molecule 129 micmconLdia 612 63 3, 61 4
V mctfgrmelia 272.274 , 262. 2S3
restriction 1 V, I „ 02 niernn Z morgani i ( formerly Prorms
msuor outer membrane protein miaophage 74* 122 maiganu )
(MOMP) J24 microscope
dark tldd (dirk ground ) 8, 314,
rnorpohnfogksd irrfew 370 32S .
fWHudi ( aiptcgillut, nuoar) 607, 610,
nsai-hi-wraj furfyr 612
mrfigfwwy 386 , 391 , 406, 301 624,
immwB response in. 178
knimianolGgy of 180
malignant edema, 254
direct examination 311
electron 8.
interference S
-
mousepox 451, 466
.
M proteins 9Q 205 206.209 402.
504, 512.513
.
optical (or light) 371, MS murker 494
malignant pustule 244
Malkin BEST 322 phase contrast 9^ 407 ^ mucormjTOSdB 624, 625
maTta fever 2» 345 polarisation 8 nSHiW spedcs 612, 624 625
^
mammary tumour virus (of mice) 578 micrtwpwuiiT 614 Mueller-Hinton medium 22.1, 225 ,
numiomc rear 361, 518 microfilmours SIS 223
Marburg disease 569 Mddkhuk'i medium 352 multiple myeliHiui 158
Mnki disease 576 migration mJiifcision factor ( mif ) 143 mumps 143, ]2L 436.448, 453, 458,
MnHFiw monkey mammary
Linde r 576
mitk 605 60?-
h ft jitter if ik ^gii^aiJ ^.'om inhaininrs 606
501 512
»
Mdnod1. l medium 37* 232 minimum infecting dose {MID ) 68 muv muscuJus 416
measles 3.143, 152, 158 , 436 , 440, inhibirorv ctHKeniruifHi ( MIC) mutation 443
630 mitogens 142
r
vrianiet i
ri
4 INDEX i 651
Mnumj grajiLilocoq udjts £M
^ N nrtrtrtJk virus 571
nmoorvnial infection 634-39
tny
^dbcJib gravis 172
mycelium *10, 614, 625.
Npgkr* * medium 3£
nuelcocapfiipd 431, 435, 437, 446. 512
Magler reaction 251, 252 , 256
mycctism 627 nairoviru* 447 nuU cdls 12i
myccToma 402 618 r Nfljrobt sheep disease virus 513, numenr agar 1Z
mycobacterium naKphaiyTipsal carcinoma 4S1. 574,
africanum {'alrkan type1) 351, 526 O
354, 36B native ( N ) antigen 491
asiaticum 365 , 368 Oaklcy-Fulcbjopc procedure 96
natural killer ( NK) cells 126 , ociiLnmycarsis 627
atypical 365, 366, 36ft, nccr-nrising LTIJLEU
avium 366 Ogiwa and wrocypw 3QZ
uteriju 212 old tuben.TJin ( OT) 361
hdnei 36ft hsmm im Omsk hemorrhagic fever 522, 531 ,
btjrvift 351, 1
. 66
iejuniris ( aiceriris flcoodmw, oncogenes
hufgli
piRhel ) 252 cellular nnenpene* (c-^ine!i 580
huiyfLcum rerinitii 477, 4ftl
chdonds 351. 367 proto oncogenes 5 Ril
negri bodies 449, S 36 , 539, 540, 543 sre cmL ogeji S 5 SO
clAssification of differentiatum 146
* '
^
viral oncogene ( v -onc} SBD
between M marinuiYi jnd M NeLH-McKnrr fhinica} redi-lion 415 an.ti -nncngei¥rs SSQ
uker-ans 166 Jirisser 's Ftrun l 3 j 211 chfamr^onrul lncUion in human
ffllbur 368
formicum 367
3i 222- SH
eatarrhalis {Branhamelta
m m m 601 beings SSI
mechanism of oncogenesis 581
gnrdonae 365
hjcm&philum 36S
eatarrhalis) 43.129. 2M
dAnatill dunuxerisek* ot 229
oncogenic viruses 574-S1 -
intraccllulare 366 ashicLfeEAd viith human cancer 5 / 6
N flm 221 dna vinisa 525
kansasli 165, 366, 369 N flavcKmE 221 ma viruses {formerly
Iqnc 370-76 N gonorrhoeae 225-29 oncomavimses) 577
,
11ELU r i
ria
§52 H TEXTBOOK OF MICROBIOLOGY
papilloma virus 446.563. 571.574
pfipDVtVtfUM 437, 444 P 445 , 446,
~
pett pnini medium ! ~T !
^ * i»«W 357
Htnoffh
-
plasmids 14 53,56^ 52
plasmdj'Sis 23
.
5 f 2 _ 575 ftp's punches ] IT 12& 2 % Vfl
• .
plssmoprysis 23
and cmc-er of the uterine PfsiffcnUa ill platelet activating factor (FAF) 161
cervix 562 PftiffeA barillas 333, 501 ptomorphism 17, 51
paracoccidioidcs brasilic-nEis 622 FfcLfFeA phenomenon lilt plesbmneias 3fllr 317
paracoccidioidomycosis 622 phuoMiMphjdaxiE 171 shigeUkiLdes 312
pUHokof 2 7 [ „ fiin phttobyphomycosit 615 Pkt medium 242
pninfluenu virm 515-16 pfcuKe pleuropneumonia- like organisms
m
^
pn rtk poliomyelitis 490, 422
pnpqffmridte 427. 447
ptqgmffrinv 468
466
ttOTveiuijQrt
DNA 462. ifil
463. 465
(PPLO) .
psin^osii- lyiTiphogriiEi.gbma
trachoma ( PUT) 422
piOtyphoid 290. 606 susceptibility test .107 pfieumoeoecus (air. pMumgniaae ) X
parmcriiua (m milkerPE nodes) typing 466 14, 216-21.224
paroxysmal cold hcvnogtobinruria 169, .
phagocytosis 126.205 225, 226, 223 pnciunocyFtii car ini: 582.389, 592
LZ1 dividers of 153 pocumolysln O 219
pariusymtfd WKtnn) hemogfebinurti phagplywsome IX 121 pneumovirus 447 , 512, 516
114 phagosome ( vacuole) 74^ 126 pock assay 448
pgHOTiliklfiC 447 phenetk system 42 poliomyelitis (uafifctirik paralysis) X
72* 76. 23B , 44Sr 458, m 491
"
pneumonic 327
^ 1M
-
VtmmutB tbulua (PfQ Beac»D ffl,
,
pyrign
i J J
i
ri
4 INDEX * 653
progressive multifocal pyoern typing 320 rhipkephaJus ^snguk^ua Ticks 4!l 6, 53.1
-
Leukoeivcephal* AEHY ( PML) 562,
566, 56E
j i%
- pyoderma 2u 4, .208
pyrimidine* 397
rhizopus. 624 625
ribosOnnix Li
-
prokaryotes 7^ 430. pyr wsc 204. 2115 ribtnnrus 446
properdin
pathway HI
rickemiu m, 412 21
dari 415
-
M?
Q
system
. Qfeirr J12. J 1 S. J 19-30.606 bufflitii 419
prophage 5 465 conori 415
^
prcrpiuriibaeterium 240.267. 269. fifiO
qurilung reaction 218. 335
quinsy 2lM parkeri
pruSodEmu: diieue 70 prowazelui 413
Prospect Hill viruses 522 ill
proFtaglandinrs 161
. ft
nckensii 415
Siberia 415
proteinase 207 rabbit fibrnnu 574 576p tsutsugamushi ( R oricntalis) 416
protens- 272, 2?i 275, 282, 231, 413 . rabbotpsK. 46E typfcu ( ft moused ) 417
418, 467, 60ft 601, 605, 625, 635 . rabies IQi 430, 432,, 439, 440.447,
535-46
weiWeLx reaction in rickettsial.
6JII diseases 41.8
'hauch.' farm 2fl2 rabies relaxed viruses %46 rickettsial pox 416
ohne haudf form 2fi3 lyasavirus 546 RLd»L Walker test 13
mirabilis 283, ilfl nluet virus 535-37 Rifr Valley fev« 522, 524, 521
mtirgiinii 233 radial immunodiffusion 96 . 508
radkiautive antigen - hiiiJjr&g test
. dngwwm ( tinea ) 612, 614
stnins Ritter's disease 131
ox H 40, 418 radicallergosorbent test {RAST ) 164 RNA viruEcs 447, 569
o^ 2 413, 431 radioimmunoassay ( RL.\] 105.1 Robertsons cooked meat medium > S,
WE k 413, 413 221.457 ., 572, 198.205. 24S, 350.256. 2.57,
Mul ix 418, tfiS Ramsay Muni syniirume 481 ruboviruscs 521, 533
protista 7± 4£ ^ Rants and Randall i method 205 ruchalimaea quintBita 412 420 421 - -
protoplasts 10* 12* 199 ratb-,re fever {RBF) 405 RsxJcy MounEadn sponed fever 414,, 416
pro trachoma 426 mgin antibody 164 , 379, 33L 382 Roa-Waaler test 100, 174
provklenck (formerly PfOBcus receptor djestroyiug cmtvme (RDE) Russ River virus 524, 525
inconstans) 274 , 283. 434.503 MSWUi 10 !107, 448, 5ZL
-
aJk alifaciens 283 rouomhdnckiit DESSA technology 61 *
nxis sarcoma virus ( RSV) 438, 578
rengerai 2B3 feenmbarmtnnn viral M3 routine test dose ( RTD} 466
stumtii 283 Reiters syndrome 229 398, 427 . card test (rapid jih&m t in ) Ml
cpf
^
provirus 577, 578, 531
pfHjutne phenomenon 94, 382
pscujJciinrhraji Ktd ]]i 246
relapsing fever 382, 3R6-88
.
mn'iros 436 443, 571
resaiuriri test 606
ruhelb 89, 148, 451, 455 54 563
-
66 632
rubeola {see measles )
^ ^ -
pwtjjdolywgeny 354 reservoir hoars 65 rubivnrus {rubella vinii) 447, 523, 563
pseudomonas 2X 275.319-23, 6CX). resistance transfer factor (RTF} 57 Runt diseau ( M lyrnphuUl otgsnt,
603 625.63S. 636
- respiratory syncytial virus 5 l 2, 516 central )
icrugirwH (ps. pyoevanea b. .- RVtridiiHi endonucltaw Russian spring summer encephalitis
fpxymm) 22, ST, 191* 319 21.
466, 600, 635
-
polyincirplruam CR>1 P) 179
reticula re body i :mrial. body) 422
(ESSE) WLplm 524, 53 . 531
RSSE vaccine 531
a
ntkukr ' eneas of de Vkal 153, 156
ceparia 321
mallei 321
makophila 321
^^
rctiwirus 433h 436, 575 577-Rfl
Bcnomk structure 579
. S
pscudomallfi 322 base speaficirv 578 Sabin 's vaccine 76, 458, 494
morphology 474 S HDuraud's glucose agar medium 611
peeudomycekum 610
-
pscudoviripon 445
virus transmission 578 ^
Sadis buffeted glycerol saline medium
psilncybc sp- 627
reverse transcriptoe 437 r 577
reversed passive kfet agglutination
> m
pattuau 422„ 42S Salk polio vaccine 491
( RPLA ) teats 196 salmonella 27k HI* 790-304
psyrhrophilic bacteria 22
purified protein derivative ( PPD) 361 Ktycii xyndnTme 507 antigenic structure 291
purpura 330
Rrjnokk-Br-aude phenomenon 617 andgenic variations 292
R (actor 304 bacteriiDphage typing 300
fbnmboLytopenic 165
puumala virus 522., 5.13 rtuhdorifusa 535 46 -
diinofiporidioaia 617. 63-0
biochemical reactions 291]
Ktuftm^WkiS schesiw 291
pyocm 466
.
duxwviruEes 4-34 447 499 . .
paxaryphii Ag B C 293-301
L ' ii
i J riahte feria
654 1 TEXTBOOK OF MICROBIOLOGY
[MEhogHlLCLty 225
ptuRC cnnverakn 3£H
sindbis virus 524, 525
Sjogren's syndrome 173, 174 chemical agents 24.3Q
-
sterilisation 17* 22* 27 36
jyrn pathetic EPphthalmia 17.1 prophylaxis 260 transferrin ( binding prord &n) 122
syphilis 95, 102. m HP?. 377-85 toxoid 261 ErarufcMlruiEiuci 5^, 441
congenial 3R 5 EincatnIiniE 262 Eransfarnruiut er-cMth factor- bera
endemic 1. 85 tetanus nconanmirn 259 (TGR 3ii
latent 380 tetrathionate broth 32 CransknC hypt mmj ahLdLiicrrtkj
nonvenercaJ 380,. 383
neuro 383, 3M
T even phases 462
Thajra^Martin medium 223, 2?- 5. 228 .
infancy 194 ^ ^
fraisaJaticHS inhibition prorein {TIFjl
primary 379 Theobald-Smich phenomenon 1M 454
scLoisdirv 37V
“
d
'
thermal death point 22 (ransplantadon , Lmmunolog) 176^-78 1
fixation (RPCF) lest 382 thymk hypoplajja (DigHKgePi; tBepnBmft 2ML 377
Rl*R t«c 3B1 syndrome ) 15.1, 1M carjuenjim 377, 3B6
trkhosporon beigcllii 60
tacaribe virus complex 4*l7 569 h
nigra 6 )2 triphenyJ tetrazoljum chloride I I' lt- ).
unpat 468 472 ?
pedis ( athlete's toot) 614, 616 test 277
tarshas nbedium 352 vtnitEtkir ( »e pityriasis triple ujiEifljeia (vsKone ) 218 , 26 L 143
T cells (T lymphocytes ) 118 120 .. .
vehicular) 612 fl
Eri|k rUgvc iron 'SR LT medium 46
2 L 122± 152, 1 S 5 , 4£i 4E5 tissue cultures 438 4Z ^ ^
Copyrighted materia
656 i TEXTBOOK OF MICROBIOLOGY >
trombiui ];i ilh vector hi embryonated eggs 439
tsutsugamushi disease 416 biological 65 tissue culture 439
tuberculin allergy 111 mechanical hi defective 4-39
{infection) type hypersensitivity 366 vcilbnc iliae 266 fixed 536
tubewulopMftifl 356, 1. 59, 360 venkatFanian-ramakrishnan ( VR) general properties 43ft 43
tuftsin deficiency 158 medium 306 . geneties uf 443
tularemia 3.24,. 331 VETO ceQ line 441, 444 growth Ln cell culture 439
I L.ICRI : ^ r necrc isihi JaCEcrf
yi . WOCytOWA ( car vErnlminJ 275 , 2 79 hemaBRturination of 434. Ml
< L- iK - lM!rtin ) 12fc. 327, BO , Hi verruca vulgaris ( human WJJESI 162 incomplete 439
futltcjpoat 468 verruga peruana 420 morphology nf 431 33 -
—
,
turlock virus 522 vesicular sranantis vmis 454. 522 oncogenic 574 Si
TWAH. ( Taiwan acute respiratofy) rcskukrnruj 522, 534 535 h spread in the body 45D 51 -
strain 423 v (actor 133 vaccines 453-. 457
TWE-IKN- Rft Jii viable count 19* 606 vista 566
lYndallLuEttm {see ^ i ^ rillscjciiiit ) Vi tut Eigen 69, 346. 464. 466 Vcsges-Proskausi ( VP) test 45273 .
typhoid fever (see enteric fever) vibrio 50S - -UH 3Q7,
typhoid vaccine alginolvticus 317 volutin granules. (Babca-Emst
injectable ( lyphim-Vi) 222 chnlene 3lii granules) H
live ( typhoid) 3G2 classification 36Z Von Magnus phenomenon 438, 506
typhus fever S3, 99, 295, 416 20 el tor 308-15 ,
- -
varicella zoster ( V Z ) 473-ftO classification and nomenclature of X
w»lt 469 71, 4Z5 846 xenografts {formerly heEcaeigrafti) 177
validation 4 Cultivation of 438 xenopsylJa
VTlRL antigen 379 animal Lnocubriuri 439 asoa 323
i
j monte eria
* INDEX 657
dicupiE 32Z
X factor 333h 33B
X- linked aguuiiigkibuKjHfflntt 152
,
X ]inked l}TnphopraliferatiYe
“
syndrome 4S 5
xeroderma 589
xytose 116. 334, 40S
¥
yaba vim & 468., 575.. 576
yaws (framboena ) 377* 385
yarn jjOlj, 610
yeisc - ILkc fyi gi 610
^
ydlaw Fewr (yellow jack) 43BP 439 p
z
Zichl-Ncd ^ ni sraiming technique 10,
17, 44, 351, 357,
Zinssers unioariui hypothesis 92
zona lomerukiKL, cells of 172
^
tortt uf inhibition 628, 629
%&m phannaHfl 24
J
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