Microbial Ecology in Growing Animals

Microbial Ecology
in Growing Animals
Edited by
W.H. Holzapfel
Institute of Hygiene and Toxicology BFE, Karlsruhe, Germany
P.J. Naughton
Northern Ireland Centre for Food and Health, School of Biomedical Sciences,
University of Ulster, Coleraine, Co. Londonderry, United Kingdom
in Series
Biology of Growing Animals

Series Editors
S.G. Pierzynowski
Department of Cell and Organism Biology, Lund University, Lund, Sweden
R. Zabielski
Department of Physiological Sciences, Warsaw Agricultural University
Warsaw, Poland
The Kielanowski Institute of Animal Physiology and Nutrition PAS
Jablonna n / Warsaw, Poland
Technical Editor
E. Salek
The Kielanowski Institute of Animal Physiology and Nutrition PAS
Jablonna n / Warsaw, Poland
Edinburgh • London • New York • Oxford • Philadelphia •

St Louis • Sydney • Toronto
2005
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First published 2005
ISBN 0 444 509 267
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Keynotes I
Genomic and proteomix projects have a profound impact on biological sciences and
the biotech-tech world. Nonetheless, we should be aware that probably nothing “new”
has happened in biology since Watson and Crick’s discovery—prions are something
new, but they are controversial. We are thus, in essence, constantly exploiting the dis-
covery of the structure of DNA, but with unbelievable speed and effectiveness. It
appears that the development of biological sciences coupled with genomic/proteomix
fields is occuring at a disproportionately fast rate in comparison with other biological
disciplines, provided that these disciplines are still in existence. In addition to strict
scientific problems, genomic/proteomix biology raises questions of an existential and
philosophical origin. Does nature produce more genes and more protein than needed,
or does it only take the chance to produce new proteins when it’s necessary to produce
them? Do we produce proteins before needing them? Or, more theologically, is biol-
ogy predestined to produce exclusively programmed proteins and nothing more, and
the genome is only waiting for the right signals? Or, maybe more cynically, we simply
do not know what these proteins are for? Definitely, they are for something and we
need to explore it. This “for something” leads us to another question. Do the particular
molecules/atoms taking part in these life mysteries get a different value?
Stefan G. Pierzynowski, prof.
vi
Keynotes II
The volume “Microbial Ecology in Growing Animals” is mostly, but not solely,
about the ecology of microorganisms living in the gastrointestinal tract of young
growing animals. This book is released two years after the corresponding volume
regarding the development of gut function, and entitled “Biology of the Intestine in
Growing Animals”. The Editors of the present volume faced a very ambitious task to
provide the reader with a new and comprehensive knowledge on the biology of the
gastrointestinal microorganisms simultaneously with describing a labyrinth of inter-
actions between them and the host. Furthermore in the early postnatal life the colo-
nization of the gut is just initiated, thus the changes are more complex and dramatic
than in the adults with balanced ecosystem. This of course makes all the story even
more complicated and difficult to put in plain words. Therefore we would like to
deeply thank the Volume Editors, William H. Holzapfel and Patrick J. Naughton,
and all Authors for their efforts since in our opinion they made a great job, and
supplied the academic society with a valuable and “must have” book.
Series Editors
vii
Preface
MICROBIAL ECOLOGY IN GROWING ANIMALS

This book “Microbial Ecology in Growing Animals”, is the second volume in the
Elsevier book series entitled Biology of Growing Animals. In individual chapters,
recent developments in our knowledge of the role of microorganisms in the gastro-
intestinal tract are reflected, whilst new approaches towards improving and stabilising
animal health are also addressed. The book discusses the interactions between the
animal host and the microbial population associated with the gastrointestinal tract.
No one publication can adequately describe the numerous interactions which occur in
the gastrointestinal tract of the growing animal and the myriad differences which exist
between these in different animals. This book attempts to draw together in one volume a
collection of work representing different areas of scientific research which, while distinct
in their own right, are presented here under the unifying theme of microbial ecology in
its relation to and interaction with the animal gastrointestinal tract.
Even though the complexity of the intestinal micro-ecology was recognised long
ago, investigations have thus far been limited to a few major bacterial groups,
considered to be dominating, and to pathogens, both in relation to concomitant finan-
cial losses in the production animal, and with regard to the food infection chain.
Thanks to recent developments, including improved microbiological detection and
sampling techniques, and the application of molecular tools to monitor the presence
of specific strains in the intestine, our knowledge has increased rapidly in recent
years. This book reflects on these developments, and addresses new approaches
towards improving and/or stabilising animal health. Special emphasis is also placed
on probiotics and the use of selected bacterial strains as vehicles for delivery of
biologically active compounds to the mucosa. Colonisation, development and
succession, as well as the normal microbial population of the mucosal surface in the
healthy animal, are addressed. Extensive information is provided on diverse and
ix
x Preface
dominating bacterial populations of different animal types. Reference is also made

to those microbial groups considered to be of special benefit to the health and
immune protection of the (young) animal. The development and application of models
of the gastrointestinal tract provide a solid basis for studying gut microbial inter-
actions, whilst molecular approaches and the use of molecular tools to monitor the
presence of specific strains in the intestine is treated in a comprehensive manner.
W.H. Holzapfel and P.J. Naughton
Volume Editors
Acknowledgements
The editors wish to thank all of the authors for their outstanding contributions to the
book. We also thank Ewa Salek for her assistance with technical editing. Thanks
also go to the Series Editors, Stefan G. Pierzynowski and Romuald Zabielski, for the
invitation and opportunity to put together this book. We sincerely thank the institu-
tions providing patronage and financial support, including Federal Research Centre
for Nutrition, Institute of Hygiene and Toxicology BFE (Germany), Northern Ireland
Centre for Food and Health, School of Biomedical Sciences, University of Ulster
(United Kingdom), Lund University (Sweden), The State Committee for Scientific
Research (KBN) – International Network Project, SPUB-M-MSN (Poland), The
Kïelanowski Institute of Animal Physiology and Nutrition, Polish Academy of
Sciences (Poland) and SGPlus (Sweden).
Volume Editors
xi
Contributors
Aschfalk A. – Section of Arctic Veterinary Medicine, Department of Food Safety

and Infection Biology, The Norwegian School of Veterinary Science, NO-9292
Tromsø, Norway
Beeckmans S. – Laboratory of Protein Chemistry, Institute of Molecular Biology
and Biotechnology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels,
Belgium
Birkbeck T.H. – Division of Infection and Immunity, Institute of Biomedical
and Life Sciences, University of Glasgow, Joseph Black Building, Glasgow
G12 8QQ, UK
Blaut M. – German Institute of Human Nutrition, Gastrointestinal Microbiology,
Arthur-Scheunert-Allee 114-116, D-14558 Bergholz-Rehbrücke, Germany
Bomba A. – University of Veterinary Medicine, Komenského 73, 041 81 Kosšice,
Slovak Republic
Ellis A.E. – Marine Laboratory, Victoria Road, Aberdeen AB11 9DB, UK
Fekete P.Zs. – Veterinary Medical Research Institute of the Hungarian Academy of
Sciences, 1143 Budapest, Hungária krt. 21, Hungary
Franz C.M.A.P. – Federal Research Centre for Nutrition, Institute of
Biotechnology and Molecular Biology, D-76131 Karlsruhe, Germany
Fuller R. – 59 Ryeish Green, Three Mile Cross, Reading RG7 1ES, UK
Gancarčíková S. – University of Veterinary Medicine, Komenského 73, 041 81
Košice, Slovak Republic
Gram L. – Danish Institute for Fisheries Research, Department of Seafood
Research, Søltofts Plads, c/o Technical University of Denmark bldg. 221, DK-
2800 Kgs. Lyngby, Denmark
Grant G. – Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen
AB21 9SB, UK
Havenith C.E.G. – Toegepast Natuurkundig Onderzoek (TNO) Prevention and
Health, Department of Infection and Immunology, Special Programme Infectious
Diseases, Post Box 2215, NL-2301 CE Leiden, The Netherlands
xiii
xiv Contributors
Holzapfel W.H. – Institute of Hygiene and Toxicology BFE, D-76131 Karlsruhe,

Germany
Katouli M. – Faculty of Science, University of the Sunshine Coast, Maroochydore,
Queensland 4558, Australia
Klein G. – Institute for Food Science, School of Veterinary Medicine Hannover,
Bischofsholer Damm 15, D-30173 Hannover, Germany
Kluciński W. – Department of Clinical Sciences, Faculty of Veterinary Medicine,
Warsaw Agricultural University, Ciszewskiego 8, 02-786 Warsaw, Poland
Kremer S.H.A. – Toegepast Natuurkundig Onderzoek (TNO) Prevention and
La Ragione R.M. – Department of Bacterial Diseases, Veterinary Laboratories Agency
(Weybridge), Woodham Lane, Addlestone, New Haw, Surrey KT15 3NB, UK
Mackie R.I. – Department of Animal Sciences, University of Illinois at Urbana
Champaign, Urbana, Illinois 61801, USA
Mathiesen S.D. – Section of Arctic Veterinary Medicine, Department of Food
Safety and Infection Biology, The Norwegian School of Veterinary Science,
NO-9292 Tromsø, Norway
Medina M. – Research Centre for Lactobacilli (CERELA), Chacabuco 145,
RA-4000 San Miguel de Tucumán, Argentina
Michalowski T. – The Kielanowski Institute of Animal Physiology and
Nutrition, Polish Academy of Sciences, Instytucka 3, 05-110 Jablonna near
Warsaw, Poland
Minekus M. – TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ, Zeist,
The Netherlands
Mudroňová D. – University of Veterinary Medicine, Komenského 73, 041 81
Kosšice, Slovak Republic
Nagy B. – Veterinary Medical Research Institute of the Hungarian Academy of
Naughton P.J. – Northern Ireland Centre for Food and Health, School of
Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Co.
Londonderry BT52 1SA, UK
Nemcová R. – University of Veterinary Medicine, Komenského 73, 041 81 Kosšice,
Slovak Republic
Newell D.G. – Department of Bacterial Diseases, Veterinary Laboratories Agency
(Weybridge), Woodham Lane, Addlestone, New Haw, Surrey KT15 3NB, UK
Niemialtowski M. – Immunology Laboratory, Division of Virology, Mycology and
Immunology, Department of Preclinical Sciences, Warsaw Agricultural
University, Grochowska 272, PL-03-849 Warsaw, Poland
Perdigón G. – Research Centre for Lactobacilli (CERELA), Chacabuco 145,
RA-4000 San Miguel de Tucumán, Argentina; Immunology Department Faculty
of Biochemistry, National Tucumán University, Argentina
xv Contributors
Pouwels P.H. – Toegepast Natuurkundig Onderzoek (TNO) Prevention and Health,

Department of Infection and Immunology, Special Programme Infectious
Ringø E. – Section of Arctic Veterinary Medicine, Department of Food Safety and
Infection Biology, The Norwegian School of Veterinary Science, NO-9292
Tromsø, Norway
Schillinger U. – Institute of Hygiene and Toxicology BFE, D-76131 Karlsruhe,
Germany
Schollenberger A. – Immunology Laboratory, Division of Virology, Mycology and
Immunology, Department of Preclinical Sciences, Warsaw Agricultural
University, Grochowska 272, PL-03-849 Warsaw, Poland
Seegers J.F.M.L. – Toegepast Natuurkundig Onderzoek (TNO) Prevention and
Sundset M.A. – Department of Arctic Biology and Institute of Medical Biology,
University of Tromsø, NO-9037 Tromsø, Norway
Schwiertz A. – German Institute of Human Nutrition, Gastrointestinal
Microbiology, Arthur-Scheunert-Allee 114-116, D-14558 Bergholz-Rehbrücke,
Germany
Tóth I. – Veterinary Medical Research Institute of the Hungarian Academy of
Van Driessche E. – Laboratory of Protein Chemistry, Institute of Molecular
Biology and Biotechnology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050
Brussels, Belgium
Wallgren P. – Department of Ruminant and Porcine Diseases, National Veterinary
Institute, Uppsala S-751 89, Sweden
Woodward M.J. – Department of Bacterial Diseases, Veterinary Laboratories
Agency (Weybridge), Woodham Lane, Addlestone, New Haw, Surrey
KT15 3NB, UK
Zentek J. – Institute of Nutrition, University of Veterinary Medicine, Vienna A-
1210, Vienna, Veterinärplatz 1, Austria
1 Development of the digestive tract of
gnotobiotic animals
A. Bomba, R. Nemcová and S. Gancarčíková
University of Veterinary Medicine,

Komenského 73, 041 81 Košice, Slovak Republic
Gnotobiology is the science of gnotobiotic animals. Such animals have a precisely

defined microflora, and have proved to be very useful models in studying the physi-
ology of the digestive tract. They mainly enable observation of the role of micro-
organisms in the process of functional and morphological development of the
digestive tract. Experiments on gnotobiotic lambs demonstrate that the functions of
the rumen, and the stability of the ecosystem, depend on the complexity and diver-
sity of the microflora. Gnotobiotic lambs have considerably shorter rumen papillae
than in conventional lambs. In germ-free animals, the normal structure and morphol-
ogy of the gut are altered in ways that emphasize the importance of an animal’s inter-
action with its indigenous microbial flora in establishing its defences against
microbial invasion, while, at the same time, adequate host nutrition is maintained by
normal alimentary tract function. The overall mass of the small intestine in germ-free
piglets is decreased, and its surface area is smaller, whereas the villi of the small intes-
tine are unusually uniform in shape and are slender, with crypts that are shorter and
less populated than in the respective conventional control animals. Further studies with
gnotobiotic animals should clarify the role of the host microecosystem in the physiol-
ogy of the alimentary tract, and the pathophysiology of gastrointestinal diseases.
1. INTRODUCTION
Quality nutrition and optimum development of the digestive tract are essential
for proper growth, high production and a good state of health of livestock.
Underdevelopment of the digestive tract of the young is a predisposing factor for
diseases and disturbances which negatively influence the economic effectiveness
of livestock husbandry. Diseases of the gastrointestinal tract can be considered to be
Microbial Ecology in Growing Animals

W.H. Holzapfel and P.J. Naughton (Eds.)
3 © 2005 Elsevier Limited. All rights reserved.
4 A. Bomba, R. Nemcová and S. Gancarčíková
the most important health and economic problem when rearing young livestock,
since they may cause extremely high losses as a consequence of morbidity, mortal-
ity, costs of treatment and weight loss. At an early age, diseases debilitate the
animal organism and cause delays in development, which can subsequently become
evident in further health problems and productivity decrease. For this reason, it
is extremely important to ensure the optimum development of the digestive tract
of young animals. Recent research provides extensive possibilities to carry out thor-
ough studies and to acquire new knowledge on the physiological and functional
development of the gastrointestinal tract of animals. Management of gnotobiotic
techniques and the use of gnotobiotic animals for experimental purposes have
substantially influenced the methodologic approach of scientists to the topic.
Microflora is of great importance in the development of the digestive tract. The use
of gnotobiotic animals in experiments has enabled the study of the role of micro-
organisms in the process of morphological and functional development of the diges-
tive tract. Gnotobiology has enabled scientists to gain new information that has
enriched the theoretical knowledge of developmental physiology of the digestive
tract and at the same time supported targeted manipulation of the development of
the gastrointestinal tract in young livestock.
2. CONTRIBUTION OF GNOTOBIOTIC TECHNIQUES AND

GNOTOBIOTIC ANIMALS TO RESEARCH INTO THE
PHYSIOLOGY AND PATHOPHYSIOLOGY OF ANIMALS
Gnotobiology is the science of gnotobiotic animals. Such animals possess a precisely
defined microflora (Duškin et al., 1983; Coates and Gustafsson, 1984). As a science,
gnotobiology emerged from the need to study the role of microflora in the living
processes of macroorganisms. After scientists revealed that microorganisms coloniz-
ing the macroorganism take an active part in many important processes of life, the
conception arose that the life of the microorganism was impossible without the active
involvement of microorganisms. Initial experiments proved that organisms can also
live in germ-free conditions. These experiments also showed the prospect of a rather
extensive use of germ-free animals in studies of microorganism interactions, so rela-
tions between microflora and the macroorganism are investigated to clarify the role
of microflora in the physiological and pathological processes of the macroorganism.
Gnotobiology has passed several stages of development. Initially, mainly the
technology of obtaining and rearing germ-free animals developed. Managing the
technology of obtaining germ-free animals and their biological characterization
established the basis for gnotobiotic animals to be widely used in scientific studies.
The possibility of standardizing gnotobiotic experimental models from the micro-
biological viewpoint presents an extraordinary benefit to experiments with gnotobiotic
animals since exact and comparable results can be achieved. Finally, gnotobiotic
methods have found use in medical and agricultural practice as well.
The digestive tract of gnotobiotic animals 5
Gnotobiotic animals typically display remarkable morphological and physiologi-

cal properties resulting from a total or partial absence of microflora. In the first phase,
changes occur in those organ systems which come into direct contact with the
microflora. Primary morphological deviations develop in the digestive tract and the
lymph organs (Kruml et al., 1969); later, secondary changes occur in the blood-making
system, the liver and other organs. In gnotobiotic animals, morphological changes are
accompanied by physiological changes of which digestive processes and immune
reaction changes are the most typical (Abrams and Bishop, 1967; Havell et al., 1970).
The possibility of exact control of the microflora in gnotobiotic animals laid the
foundations for using the latter in research in several branches of science and in
studies into the importance of microflora in the physiology and pathology of living
organisms. Gnotobiotic animals are an important contribution to studies into the
physiology and pathology of the digestive tract.
Gnotobiotic animals are extremely suitable for immunological research. Germ-
free animals that have not come into contact with any antigen, present an optimum
model for studies into the primary immune response. Germ-free animals are of
invaluable importance in research into the role of antigens in the ontogenesis of the
immune system and the role of the thyroid gland in the immune response (Wilson
et al., 1964). The use of gnotobiotic animals has enabled scientists to gain valuable
knowledge on cell and humoral immunity (Talafantová et al., 1989) and on the
immune response to various pathogens (Saif et al., 1996; Herich et al., 1999).
Gnotobiotic animals have also enabled scientists to gain new knowledge of the
physiology of the cardiovascular system (Gordon et al., 1963), the liver (Wostman
et al., 1983), the kidneys (Lev et al., 1970) and the endocrine system (Ukai and
Mitsuma, 1978). Germ-free animals have become a suitable experimental model for
studies into nitrogen and carbohydrate metabolism (Combe, 1973), and the metab-
olism of vitamins, minerals, fatty and bile acids and cholesterol (Coates et al., 1965).
Gnotobiotic animals are frequently employed in medical research. Experiments
in such animals help to clarify the role of gut microflora in the process of carcino-
genesis (Drasar and Hill, 1974; Narushima et al., 1998). They have also proved
suitable in studies into the role of microflora in the metabolism of different
substances and their pharmacological or toxic effects. Experiments with germ-
free rats helped to explain the toxicity of cycasine. For cycasine toxicity, the
presence of bacteria and bacterial glycosidases metabolizing cycasine to toxic
aglycon-methylazoxymethanol proved to be requisite (Laguer and Spatz, 1975).
Gnotobiotic techniques are also employed in radiopathology (Mandel et al., 1980),
where they were used to confirm the role of microorganisms in the pathogenesis of
radiation disease and the gastrointestinal syndrome, and in the mechanism of action
of radioprotective substances. These studies showed that microorganisms were
involved in the postradiation syndrome, both directly by damaging the anatomical
structure and physiology of the organism, and indirectly through the effects of
microbial metabolites. In comparison to conventional animals, germ-free ones
display increased resistance to irradiation (Mandel et al., 1979). In infectiology,

gnotobiotic animals help to disclose the role of microorganisms and their interrela-
tions in the etiology and pathogenesis of infectious diseases (Wilson et al., 1986;
Rogers et al., 1987a,b; Hodgson et al., 1989; Saif et al., 1996; Ellis et al., 1999).
3. THE USE OF GNOTOBIOTIC ANIMALS IN STUDIES INTO THE

FUNCTIONAL AND MORPHOLOGICAL DEVELOPMENT OF
THE DIGESTIVE TRACT
Gnotobiotic animals are a very useful model in studying the physiology of the diges-
tive tract, and enable the observation of the role of microorganisms during func-
tional and morphological development of the digestive tract. In ruminants, they
present the optimum model of developmental physiology of the rumen. The gnoto-
biotic young of ruminants can be used to observe the development of the rumen
ecosystem and its interrelations, to study the relations between rumen microflora,
microfauna and the macroorganism as well as to determine the effects of rumen
metabolism upon intermediary metabolism. The rumen wall is an important element
of rumen and intermediary metabolism, its epithelium being the connection site
between rumen and intermediary metabolism (Kalachnyuk et al., 1987) as well as
being capable of absorption, synthesis and secretion. The length of rumen papillae
is positively correlated with body growth. Rumen fermentation microflora catabo-
lizes soluble carbohydrates; in this process, volatile fatty acids (VFAs) are the major
final products of rumen fermentation. VFAs present the chemical stimulus of devel-
opment of the rumen epithelium. They stimulate the epithelial metabolism of the
rumen and support the structural development and resorption activity of the rumen.
The growth rate of rumen papillae depends on the amount of VFA produced
(Ørskov, 1985). In this way, the rumen microflora directly affects the development
of the rumen epithelium and the level of intermediary metabolism through the action
of rumen fermentation and its final metabolites – the volatile fatty acids. Fonty et al.
(1983a,b, 1988) used meroxenic lambs to assess whether the complexity and origin
of rumen microflora influenced VFA concentrations and composition. They also
strived to determine the minimum quantitative and qualitative composition of rumen
microflora that was required to enable rumen colonization by cellulolytic bacteria
and protozoa. Fonty et al. (1991) also studied the role of rumen microflora in the
development of the rumen ecosystem and the functional development of the rumen
at an early age. Bomba et al. (1995) used gnotobiotic equipment to study the devel-
opment of rumen fermentation in lambs from birth up to 7 weeks of age in relation
to the complexity of the digestive tract ecosystem. In gnotobiotic lambs, coloniza-
tion of the individual gut segments by lactobacilli and the inhibitory effects of
Lactobacillus casei on the adhesion of enterotoxigenic Escherichia coli K 99 to the
intestinal wall were also subjected to examination (Bomba et al., 1994, 1997).
Soares et al. (1970) and Lysons et al. (1976a,b) compared several parameters of
morphological and functional development in germ-free, gnotobiotic and conven-

tional lambs.
Monogastric gnotobiotic animals were also used to study the functional and mor-
phological development of the digestive tract. Nemcová et al. (1997) and Bomba et al.
(1994) studied the colonization ability of selected strains of lactobacilli in the small
intestine of gnotobiotic piglets. Studies also focused on the effects of lactobacilli on
intestinal metabolism during the first 3 weeks of life (Bomba et al., 1998), and upon
organic acid levels in the mucosal film and the contents of the small intestine (Bomba
et al., 1996a). Gnotobiotic animals present the ideal model to determine bacterial inter-
actions in the digestive tract. Bomba et al. (1996b, 1999) observed the interactions of
lactobacilli and enterotoxinogenic E. coli in the intestinal tract of gnotobiotic piglets.
In experiments on gnotobiotic animals, studies focused on the effects of microflora
upon morphology (Gordon and Pesti, 1971), motility (Gustafsson and Norman, 1969)
and secretion and absorption in the digestive tract (Yokota and Coates, 1982).
Gnotobiotic animals were also used to clarify the role of intestinal mucosa (Loesche,
1968) and pancreatic enzymes (Genell et al., 1977).
4. DEVELOPMENT OF THE DIGESTIVE TRACT IN

THE GNOTOBIOTIC RUMINANT YOUNG
In ruminants, the rumen is of major importance from the viewpoint of alimentary
tract development. The importance of the rumen increases with the age of the indi-
vidual, the growing intake of dry fodder, weaning, and transition to plant nutrition.
Weaning is conditioned by full functional development of the rumen. Digestion in
the rumen is a complex system of interactive processes, which include microflora,
feed and the animal (Demeyer et al., 1986). In the course of the anatomical devel-
opment of the forestomach, striking morphological changes become manifest in the
increased volume of the individual parts of the forestomach, and in the typical
phases of wall formation. In this process, the muscle tissue of the wall, the mucosa
and villi and the resorptive tissues are formed. This phase of development is basi-
cally influenced by mechanical and chemical stimuli, which arise from the ingested
feed. Functional and morphological development are closely connected. With the
onset of rumination and functioning of the abomaso-ruminal cycle, colonization
of the forestomach parts by bacteria and protozoa, increased metabolic activity and
the resorptive capacity of the forestomach are the criteria of functional development.
In addition to the aforementioned endogenous and exogenous factors of food intake
regulation, blood composition and increasing enzyme production in the digestive
tract exert their influence upon the functional development of the forestomach and
spleen system (Bergner and Ketz, 1975). Simultaneously with the ongoing morpho-
logical and functional development, the forestomach is colonized by bacteria and
protozoa, which are of decisive importance in the biochemical processes in this part
of the digestive tract. In this way, bacteria and protozoa influence the development
of the forestomach. Gnotobiotic animals present the optimum model for studies into
the role of microflora in the development of the digestive tract in young ruminants.
As the intake of dry feeds increases and the anatomical and functional development
of the rumen proceeds, the level of rumen metabolism increases as well.
4.1. Morphological development of the digestive tract in

gnotobiotic lambs
Soares et al. (1970) studied the morphological development of gnotobiotic lambs
obtained by hysterotomy or hysterectomy of ewes. The gnotobiotic lambs in the
experiment were not inoculated at all; the control group was reared under conven-
tional conditions.
At the age of 8 weeks, the body weight of a conventional and a gnotobiotic lamb
was 10.82 and 9.09 kg, respectively. There was a marked reduction in the total stom-
ach and reticulorumen weights in gnotobiotic lambs as compared to conventional
lambs. At the age of 8 weeks, reticulorumen weight in conventional and gnotobiotic
lambs receiving identical diets was 322 and only 93 g, respectively. Reticulorumen
weight in conventional lambs amounted to 74.5% of forestomach weight and 2.97%
of body weight, whereas in gnotobiotic lambs the respective proportions reached
58.1 and 1.02%.
Conventional lambs developed reticulorumens which seemed to be normal with
regard to age and size. In gnotobiotic lambs, however, reticuloruminal development
at 8 weeks of age, only approximated that of 2- to 3-week-old conventional animals.
Inspection of the papillary development revealed virtually no growth of the papillae
in the rumens of gnotobiotic lambs. The ruminal lining was thinner and pink in
colour, and the rudimentary papillae were not more than 1 mm high and were
rounded in appearance. In comparison, the ruminal lining of conventional lambs on
a sterile diet was black, thus indicating a parakeratotic condition, while that of
conventional lambs on a conventional diet revealed the normal greyish-green colour.
In conventional lambs, papillary development was normal. The papillae measured
about 5 mm in height and were finger-like in shape. Alexander and Lysons (1971)
compared the relative thinness of the walls of the gastrointestinal tract in gnotobiotic
lambs to that in conventional lambs and found the size of the rumen, reticulum and
omasum to be similar at similar ages. Lysons et al. (1971) reported gnotobiotic lambs
inoculated with a culture of 8 anaerobic rumen bacteria to grow more intensively than
germ-free lambs; their rumen papillae were better developed, too.
Lysons et al. (1976a) studied the morphological differences in the alimentary tract
of gnotobiotic and conventional lambs. The main gross differences were: a) the thick-
ness of the wall of the reticulorumen and the intestines, b) the consistency of the con-
tents, and c) the development of the papillae in the forestomachs. No differences were
observed between the individual categories of lambs with respect to wall thickness of
the oesophagus, abomasum, caecum or intestines, nor in the consistency of the

contents, except for the contents of the large intestines, which were softer in gnoto-
biotic animals. The overall size of the reticulorumen including the contents relative
to body weight was similar in gnotobiotic and conventional lambs. However, macro-
scopically, the muscles of the rumen in gnotobiotic lambs seemed to be poorly devel-
oped. The position of the rumen pillars in gnotobotic lambs was less obvious from
the outside and they had less effect in maintaining the shape of the rumen.
Histologically there was a marked hypoplasia of the external muscular layers
in the rumen of gnotobiotic lambs, that is, a reduction in the number and size of
the smooth muscle fibrils. The muscle bundles were small, shrunken and widely
separated from each other. The individual cells were small; they had dark pyknotic
nuclei and did not stain well with eosin.
Macroscopically, the inside of the rumen wall of uninoculated gnotobiotic lambs
was pinkish or greyish brown in colour and covered with low rugae rather than
papillae though some short papillae with bulbous extremities were present in the
anterior sac. There seemed to have been little or no development from the neonatal
stage on. Uninoculated gnotobiotic lambs and gnotobiotic lambs inoculated with
one bacterial species (Bacteroides ruminicola) had considerably shorter rumen
papillae than in conventional lambs.
Microscopically, the thinness of the reticular wall in uninoculated gnotobiotic
lambs was not as marked as that of the rumen. Histologically, however, the outer
muscular layers were hypoplastic. The fibrils of the muscularis mucosae, although
not apparently reduced in number, were poorly stained and had shrunken nuclei. The
lamina propria was much reduced in thickness in comparison with that of a normal
reticulum. Macroscopically, the papillae in the reticulum were poorly developed and
the mucosal folds less developed in the uninoculated gnotobiotic lambs. There was
no marked difference between gnotobiotic and conventional lambs concerning the
consistency of the ruminal and reticular contents.
The large papillae in the omasal groove near the reticulo-omasal orifice were
apparently well developed in gnotobiotic lambs but the papillae on the leaves of the
omasum were smaller in uninoculated gnotobiotic lambs than in inoculated and
conventional lambs. The consistency of the contents in gnotobiotic lambs was
similar to that in conventional lambs.
Macroscopically, the walls of the small intestines of gnotobiotic lambs appeared
to be thinner than those of conventional lambs. In the large intestine the difference
was less striking. Histologically, the longitudinal and circular muscle layers of
the small intestine in gnotobiotic lambs were not obviously thinner than those in
conventional lambs but there seemed to be some hypoplasia of the mucosal layer
(Lysons et al., 1976a). As figs 1 and 2 indicate, histological examination revealed
better development of rumen mucosa in conventional lamb in comparison to gnoto-
biotic lamb at 7 weeks of age (Žitňan, 2001, personal communication).
Fig. 1. Histological section of rumen mucosa in conventional lamb at 7 weeks of age (Žitňan, 2001,
personal communication).
4.2. Physiological development of the digestive tract in gnotobiotic lambs

The functional development of the rumen also depends on the complexity of its
microflora. In conventional animals it is difficult to study the specific role of
microorganisms and their interactions because of the complexity of the microbial
ecosystem of the rumen. In order to understand the digestive mechanisms involved
in the rumen, microbial components need to be simplified by using animals with a
reduced number of bacterial and protozoan species (Fonty et al., 1983a).
Rumination was observed in gnotobiotic lambs, but it occurred only occasionally
and much less frequently than in conventional animals. Inoculated gnotobiotic
lambs did not appear to ruminate more frequently than their uninoculated gnotobi-
otic fellows, but the conventionalized lambs ruminated normally within 6 weeks
after inoculation (Lysons et al., 1976a).
Bomba et al. (1995) observed the development of rumen fermentation in
conventional and gnotobiotic lambs from birth to 7 weeks of age. Conventional
lambs with a complex microflora did not receive any inoculum. The inoculum
of gnotobiotic lambs contained Streptococcus bovis, Prevoxella ruminicola,
Fig. 2. Histological section of rumen mucosa in gnotobiotic lamb at 7 weeks of age (Žitňan, 2001, personal
communication).
Butyrivibrio fibrisolvens and Selenomonas ruminantium at a concentration of 106

each. In both groups of lambs rumen fluid pH proved to be rather stable throughout
the observation period. The values of pH ranged within 6.5–6.8 and 7.1–7.4 in the
conventional and gnotobiotic groups, respectively. When compared to conventional
lambs, the pH of the rumen contents in gnotobiotic lambs was increased throughout
the investigated period, the differences being significant (P < 0.01) at 7 weeks of age
(conventional lambs 6.7, gnotobiotic lambs 7.4).
Comparison to gnotobiotic lambs revealed total volatile fatty acid (VFA)
concentrations in conventional lambs to be higher throughout the observation
period, the differences being significant at 4 and 5 weeks of age (P < 0.05 and P <
0.001, respectively). In conventional lambs, total VFA levels manifested an increas-
ing tendency between weeks 4 and 7 of age and reached their maximum at 7 weeks
(57 mmol l−1) whereas in gnotobiotic lambs the range was narrow (24.3–
30.1 mmol l−1) and the peak occurred at 6 weeks of age. In gnotobiotic lambs signi-
ficantly increased molar proportions of acetic acid were observed whereas in con-
ventional lambs the molar proportions of propionic acid proved to be significantly
increased. The molar proportions of butyric and valeric acids were increased in
conventional lambs but the group differences were not significant. In gnotobiotic
lambs no isoacids were found. Alpha amylase (E.C.3.2.1.1.) activity of the rumen
contents was significantly increased in gnotobiotic lambs between weeks 2 and 6
of age whereas cellulase (endoglucanase E.C.3.2.1.4. and cellobiohydrolase
E.C.3.2.1.91.) activity was significantly increased in 4-week-old conventional
lambs. Over the whole period of milk nutrition no significant differences in urease
(E.C.3.5.1.5.) activity of the rumen contents were observed in the groups examined.
The above study compared the level of rumen fermentation in conventionally
reared lambs and in lambs with an extremely reduced and defined microflora, the
latter enabled demonstration of the role of the complexity of the rumen ecosystem
in the functional development of the rumen at an early age. The results obtained
indicated that the complexity of rumen microflora significantly influenced the
development of rumen fermentation both from the quantitative and the qualitative
viewpoint. Fonty et al. (1988) observed the level of rumen fermentation in gnotobi-
otic lambs inoculated with 182, 106, 32 and 16 non-cellulolytic strains isolated
from the rumen. Volatile fatty acid levels rather differed from one lamb group to the
other. The more complex the inoculum administered to the animals was, the higher
were the VFA levels observed. In animals inoculated with 182 strains the VFA con-
centration was similar to that measured in conventional lambs fed the same diet
(approximately 80 mmol l−1 after feeding). In lambs inoculated with only 16 strains
there was almost no fermentation (30 mmol l−1 of VFA). These results demonstrate
that the functions of the rumen and the stability of the ecosystem depend on the
complexity and diversity of the microflora. In the light of present knowledge it is not
possible to determine accurately the composition of the minimum flora enabling
rumen development and function.
Soares et al. (1970) reported ruminal fluids from gnotobiotic lambs to contain
markedly less total VFA than those from conventional lambs. Lysons et al. (1976b)
dosed five gnotobiotic lambs with different combinations of 11 species of rumen
bacteria. Two of the species could not be reisolated but the remainder established
readily in the rumen and the viable counts of most of the individual species were
comparable to those in normal sheep, however, VFA levels were decreased and in four
of the lambs the proportion of butyric and propionic acids was higher and lower than
in normal sheep, respectively. Cellulolytic, ureolytic and methanogenic activities
appeared to be taking place and lactate-utilizing bacteria appeared to reverse the accu-
mulation of lactate, which resulted from the activity of lactate-producing bacteria.
Fonty et al. (1991) studied the development of rumen digestive functions in
lambs placed in sterile isolators at 1, 4, 8 or 9 days of age in order to define the role
of the bacterial species that colonize the rumen just after birth. The values of the
main rumen digestive parameters (pH, VFA levels, ammonia, lactic acid) in these
lambs were close to those observed in the conventional controls. Likewise, digestive
utilization of dry matter and starch was comparable in the isolated and control
animals but digestibility of crude cellulose was higher in isolated lambs which
harboured Fibrobacter succinogenes as the major cellulolytic bacterial species.
These results suggest that rumen flora of the very young lamb play an essential role
in the establishment of the rumen ecosystem and in the setting up of the digestive
functions. Those bacterial species that colonize the rumen immediately after birth
when this organ is not yet active, contribute a biotype favouring the establishment of
cellulolytic strains and the set-up of digestive processes that affect both degradation
of the lignocellulose-rich feeds and fermentation of the resulting soluble compounds.
Ecological factors controlling the establishment of cellulolytic bacteria and ciliate
protozoa in the lamb rumen were studied in meroxenic lambs (Fonty et al., 1983a).
The results obtained in this study suggest that establishment of cellulolytic bacteria
and protozoa requires an abundant and complex flora and is favoured by early inocu-
lation of the animals. The difficulty in establishing cellulolytic bacteria in the rumen
of animals with a limited flora is probably linked to the very high and strict nutritional
requirements of such organisms (Bryant, 1973). Creation of conditions necessary for
the establishment of cellulolytic bacteria probably depends on a number of very
complex requirements. These requirements are not necessarily provided by the
dominant bacteria of the flora, which are nonetheless generally thought to play the
main role in the rumen.
All the above-mentioned results point to the extremely important role microflora
plays in the development of the rumen. There is a good relationship between the devel-
opment of rumen function and flora complexity. The presence of a simple flora
cannot assure the digestive function as properly as a complex flora can (Fonty et al.,
1983b). The fact that early inoculation of animals is a factor favouring fermentation
and digestive activities in the rumen is probably related to the action of bacteria on the
development of papillae, rumen mucosa and the digestive tract (Lysons et al., 1976a).
A complex microflora presents a requisite condition of optimum development of the

alimentary tract in ruminants.
5. DEVELOPMENT OF THE GASTROINTESTINAL TRACT OF

GNOTOBIOTIC PIGLETS
The period immediately after birth is probably the most critical one in the whole life
of the animal. In this period significant growth, morphological changes and matura-
tion of the gastrointestinal tract take place. Prior to birth the alimentary tract is
exposed to substances from the ingested amniotic fluid, which seems to be of impor-
tance to its development (Trahair and Harding, 1992). The colostrum, however,
differs from the amniotic fluid by the density of nutrients, and having high
immunoglobulin, enzyme, hormone, growth factor and neuroendocrine peptide
levels (Koldovský and Thornburg, 1989). Widdowson and Crabb (1976) were the
first to demonstrate the effect of the colostrum upon alimentary tract development
by comparing piglets suckling colostrum to watered animals. In this way high
levels of several hormones and growth promoting peptides like insulin, cortisol,
epidermal growth factor (EGF) and insulin-type growth factor I (IGF-I) were stated
in the maternal colostrum. It was proved that colostral growth factors play an impor-
tant role in the postnatal development of the digestive tract of newborn young. From
this point of view, gnotobiotic piglets are a suitable model for studies into the devel-
opment of the digestive tract.
5.1. Morphological development of the digestive tract in gnotobiotic

suckling pigs
In germ-free animals, the normal structure and morphology of the gut are altered in
ways that emphasize the importance of an animal’s interaction with its indigenous
microbial flora in establishing its defences against microbial invasion while, at the
same time, adequate host nutrition is maintained by normal alimentary tract func-
tion (Heneghan, 1965). However, the overall mass of the small intestine in each
germ-free species is decreased, and its surface area is smaller, whereas the villi of
the small intestine are unusually uniform in shape and slender, with crypts which
are shorter and less populated than in the respective conventional control animals
(Meslin et al., 1973).
The structure of the small intestine is a very sensitive indicator of the shift from
the germ-free state into a state in which contact of the mucosa with pathogenic or
non-pathogenic microorganisms occurs (Kruml et al., 1969). The lamina propria is
much thinner in germ-free animals (Abrams, 1969). The mucosal villi of the small
intestine of germ-free piglets are very fine and they have a small amount of axial
stroma with low cellularity. The vilus/crypt cell ratio is always higher in germ-free
pigs than in conventional pigs which indicates that less proliferating tissue is
required to keep the germ-free mucosa intact (Heneghan, 1979). In general, manual
stereological morphometric techniques tended to confirm these morphological
trends (Heneghan et al., 1979). The lymph follicles, which are already present at
birth, increase only very slowly in size during postnatal ontogenesis. The increase
in the number of large pyroninophilic cells in the follicles is likewise only moder-
ate. No germinal centres were found within a 68-day observation period. In con-
ventional piglets, in addition to the large primary follicles, germinal centres of
varying size were already found on the 12th day. Small quantities of cells of the
plasmocyte series were also demonstrated. The mucosal villi of such animals were
more cellular and contained numerous lymphocytes, which in many places formed
large aggregates expanding the villous space (Kruml et al., 1969).
During the first 5–6 weeks of postnatal life, the epithelium of the small intestine
in germ-free pigs has a particular appearance. In epithelial cells great vacuoles are
found, thus forming the “water transparent” cells. As a rule, enterocytes of this type
are seen in pig fetuses and in newborn piglets but in conventional environment they
change within a few days after birth. The “ageing” or “senescent” enterocytes in
germ-free pigs are obviously a consequence of the prolonged life span of epithelial
cells which is caused by the lower mitotic rate of stem cells in Lieberkuhn’s crypts.
The life span of epithelial cells was found to be 96 h in conventional pigs but 200 h
in germ-free pigs. However, the “senescent” enterocytes were stated to be working
well in the transport of nutrients across the mucous membrane. It is of advantage
in rearing germ-free pigs that these animals do not exhibit an enlarged caecum
(megacaecum) and colon, as can be seen in many species of germ-free mammals,
particularly rodents (Mandel and Trávniček, 1987).
5.2. Intestinal metabolism in gnotobiotic pigs

Bomba et al. (1998) studied the intestinal metabolism in two groups of gnotobiotic pigs
(one non-inoculated and one inoculated only with Lactobacillus casei subsp. casei)
during the first 3 weeks of life. The Lactobacillus casei subsp. casei counts in
the jejunal and ileal contents of inoculated gnotobiotic piglets ranged from 8.37 to
9.87 log 10 ml−1 during the entire period of investigation whereas the numbers of
Lactobacillus casei subsp. casei adhering to the jejunal and ileal mucous membrane
were significantly lower (P < 0.05) ranging from 5.63 to 6.06 log 10 cm−2. The num-
bers of lactobacilli adhering to the jejunal and ileal mucosa and found in the jejunal and
ileal contents were comparable to the data obtained in conventional and gnotobiotic
piglets by other authors (Pollmann et al., 1980; Sarra et al., 1991; Tortuero et al., 1995).
At the age of 1 and 3 weeks, the actual acidity of the jejunal contents of gnoto-
biotic piglets inoculated with Lactobacillus casei subsp. casei was significantly
lower (P < 0.05 and P < 0.01) in comparison with that in non-inoculated animals (see
tables 1 and 2, respectively). The pH value of the ileal contents of inoculated piglets
was also lower, however, the differences were not significant. Žitňan et al. (2001)
Table 1. The influence of continuous application of Lactobacillus casei on colonization and

actual acidity in the jejunum and ileum in 1-week-old gnotobiotic piglets
Lactobacilli (content) Lactobacilli (mucosa)

Intestine Group (log 10 ml−1) (log 10 cm−2) pH
Jejunum O 0 0 7.49 ± 0.22*

L 8.39 ± 0.07 5.63 ± 0.41 5.63 ± 0.31
Ileum O 0 0 8.63 ± 0.10
L 8.38 ± 0.07 5.69 ± 0.65 6.43 ± 0.83
* P < 0.05.
Group L: inoculated with Lactobacillus casei.
Group O: non-inoculated.
observed the pH of the jejunal and ileal contents in conventional suckling piglets at
an identical age. When comparing the actual acidity of the individual small intestinal
segments it can be stated that the pH of the jejunal contents in non-inoculated
gnotobiotic piglets aged 1 and 3 weeks (7.49 and 7.12, respectively) was significantly
increased when compared to values recorded in conventional piglets of the same age
(6.23 and 6.19, respectively). In contrast, the pH of the jejunal contents in gnotobiotic
piglets inoculated with Lactobacillus casei subsp. casei was moderately lower (5.63
and 5.84, respectively). The actual acidity of the ileum in non-inoculated gnotobiotic
piglets (8.63 and 8.35, respectively) as compared to the conventional ones (6.27 and
6.79, respectively) was even more significantly increased than that of the jejunum.
In gnotobiotic piglets inoculated with lactobacilli, ileal pH (6.43 and 7.30, respec-
tively) was slightly increased when compared to that in conventional piglets.
Morphological changes in the digestive tract are also influenced by volatile fatty
acids (Goodlad et al., 1989), which play an important role in bacterial interactions of
the alimentary tract. The ability to generate organic acids, particularly lactic and
acetic acids, presents one of the mechanisms by which lactobacilli perform their
inhibitory effect upon pathogens (Piard and Desmazeaud, 1991). With decreasing
pH values, the inhibitory activity of the above acids increases (Daly et al., 1972),
their molecular form being toxic for bacteria. The increased toxicity of acetic acid is
Table 2. The influence of continuous application of Lactobacillus casei on colonization and

actual acidity in the jejunum and ileum in 3-week-old gnotobiotic piglets
Lactobacilli (content) Lactobacilli (mucosa)

Intestine Group (log 10 ml−1) (log 10 cm−2) pH
Jejunum O 0 0 7.12 ± 0.06**

L 8.81 ± 0.33 5.75 ± 0.52 5.84 ± 0.06
Ileum O 0 0 8.35 ± 0.69
L 9.87 ± 0.59 6.06 ± 0.36 7.30 ± 0.44
** P < 0.01.
Group L: inoculated with Lactobacillus casei.
Group O: non-inoculated.
attributed to its higher pKa in comparison to lactic acid. Increased lactic acid levels
intensify the toxicity of acetic acid (Adams and Hall, 1988).
Comparison of lactic acid levels in the jejunal and ileal contents of gnotobiotic
piglets (Bomba et al., 1998) and conventional suckling piglets (Žitňan et al., 2001)
at 1 week of age, revealed the highest levels in conventional animals (27.50 and
26.90 mmol l−1, respectively) and in Lactobacillus casei inoculated gnotobiotic
piglets (26.60 and 14.20 mmol l−1, respectively). The lowest levels of lactic acid in the
jejunal and ileal contents were seen in non-inoculated gnotobiotic piglets (4.40 and
6.45 mmol l−1, respectively). At the age of 3 weeks, lactic acid levels in the jejunum and
ileum reached maximum values in Lactobacillus casei inoculated gnotobiotic piglets
(33.15 mmol l−1) and in conventional piglets (15.52 mmol l−1), respectively. At 1 week
of age, maximum acetic acid levels in the jejunal and ileal contents were stated in con-
ventional piglets (33.05 and 21.81 mmol l−1, respectively), the respective levels in
Lactobacillus casei inoculated and non-inoculated gnotobiotic piglets were somewhat
lower (11.80 and 11.85 mmol l−1 vs 13.15 and 3.90 mmol l−1). At 3 weeks of age, max-
imum acetic acid levels were observed in the jejunal and ileal contents of conventional
piglets (10.17 and 25.9 mmol l−1, respectively) and Lactobacillus casei inoculated
gnotobiotic piglets (3.9 and 11.00 mmol l−1, respectively). These results show that the
complexity of the intestinal microflora affects the production of the investigated organic
acids in the alimentary tract of piglets.
Bomba et al. (1996a) investigated the effect of the inoculation of three
Lactobacillus strains upon lactic, acetic, acetoacetic and propionic acid levels in the
mucosal film and ileal contents of gnotobiotic pigs. In the jejunum of inoculated
animals, the mucosal film revealed significantly increased levels of lactic, propionic
and acetoacetic acids when compared to the contents (25.3 vs 10.8 mmol l−1, 18.5 vs
5 mmol l−1, and 29.7 vs 11.2 mmol l−1, respectively) as well as non-significantly
increased acetic acid levels (11.0 vs 5.8 mmol l−1). In the ileum of gnotobiotic pigs,
propionic acid levels in the mucosal film were significantly higher than those in the
contents (21.2 vs 9.5 mmol l−1). In comparison to the contents, the increased lactic,
acetic and acetoacetic acid levels in the film proved to be non-significant. The above
results suggest that significantly increased levels of the lactobacilli-produced
organic acids in the intestinal mucosal film may present an efficient barrier to inhibit
the adherence of digestive tract pathogens to intestinal mucosa.
6. FUTURE PERSPECTIVES
In the future, gnotobiotic research will be involved in many different fields of biol-
ogy, nutrition and medicine. It can be suggested that gnotobiology will be part of
mainstream modern ecology, environmental toxicology, molecular immunology,
modern clinical medicine, investigations of genetically modified microorganisms
and modern food research. In the field of digestive tract physiology, gnotobiotic
research will be aimed at gastrointestinal ecosystem interactions. Despite a lot of
knowledge obtained, the mode of action of probiotic microorganisms upon digestive

tract pathogens has not yet been explained. In order to enhance the efficacy of pro-
biotics it is necessary to obtain additional important knowledge on the mechanisms
mediating their effect in the digestive tract. Gathering knowledge in the given fields
will support the development of more effective probiotic products that will con-
tribute to increased health and a more effective prevention of alimentary tract
diseases in both humans and animals.
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2 Metabolism and population dynamics of
the intestinal microflora in the growing pig
M. Katoulia and P. Wallgrenb

aFacultyof Science, University of the Sunshine Coast, Maroochydore, Queensland
4558, Australia
bDepartment of Ruminant and Porcine Diseases, National Veterinary Institute,
Uppsala S-751 89, Sweden
The intestinal flora of pigs contains several hundred microbial species, mostly strict
anaerobes. A great amount of these bacteria reside in the large intestine, which in
adult pigs consists of mainly Gram-positive bacteria such as cocci, lactobacilli,
eubacteria and clostridia. The composition of the intestinal microflora is a result of
the interaction between the microorganisms that colonize the gut and the intestinal
physiology of the pigs. The initial inoculum is usually derived from the sow at the
time of birth and the climax flora is developed through a gradual process in which
there is a shift in relative abundance of various microorganisms, especially through-
out the first month of the pig’s life. While a part of this microflora is constantly
present in the gut (resident flora), some microorganisms have a short residence and
dynamically change the composition of the microflora. The turnover of this flora
(also known as the transient flora) in the gut depends on both the composition of the
resident flora and the degree of contamination of ingested food and other sources
which in traditional indoor farming include the sow’s skin and the pen’s environ-
ment. The stability and diversity of this flora has a tremendous role in maintaining
the health status of the pigs, especially during the suckling and post-weaning period.
Most investigations of the intestinal flora in pigs focus on classical and/or molecu-
lar methods, aiming to isolate, enumerate and/or qualitatively identify different
bacterial groups. Other recent studies that measure the metabolic capability and
functional status of the intestinal microflora in pigs have added knowledge about the
composition and dynamics of the gut flora, especially in pre- and post-weaning pigs.
1. INTRODUCTION
The intestinal microflora of pigs comprises hundreds of bacterial species most of
which are residing in the lower part of the gastrointestinal tract. This flora develops

22 M. Katouli and P. Wallgren
through a process of ecological succession and plays a tremendous role in the state
of health and disease of pigs, especially during suckling and post-weaning periods.
Among the important factors in this process is the influence of the interaction
between the microorganisms that contaminate the animal, diet regime and food
composition, immunological status of the pig and environmental factors on the
intestinal physiology. Several studies have tried to identify the types of bacteria
colonizing the intestine of growing pigs. Most of these studies utilize selective
media, which lead to enumeration of few particular bacterial groups. The problems
associated with culture-based techniques are yet exacerbated in anaerobic habitats.
Using conventional techniques of culturing and identification, only about 30−40
separate species have been generally recovered from any individual animal. Recent
development of more refined molecular techniques has opened new windows of
opportunity to study unculturable bacterial components of the alimentary tract or of
members of the intestinal microflora that cannot tolerate exposure to oxygen.
In addition to this new and promising approach, recent in vitro methods focus
on measuring the metabolic activities of the major intestinal flora. These methods,
alone or in combination, have been extensively used to investigate the population
structure and the functional status of the intestinal flora in growing pigs.
In this chapter we discuss the available information on population dynamics of
the intestinal flora in growing pigs, and address factors involved in changes of this
flora during different stages of the animal’s life and in health and disease.
2. PIG HUSBANDRY
Despite the fact that adult pigs may weigh over 300 kg, they only weigh between 1 and
2 kg at birth. A sow normally gives birth to a litter of around 10 piglets and, in accor-
dance with modern agricultural systems, piglets are allowed to suckle their dam for a
comparably short period. Many countries practise weaning when piglets are around
3 weeks old. However, the length of the suckling period varies somewhat between
countries and rearing systems. Thus, it could be summarized that piglets in modern sys-
tems have access to their dam and her milk for a period ranging from 2 to 7 weeks.
From the time of weaning until the weight of approximately 25 kg, piglets are referred
to as weaners or weaning pigs. From then, and until slaughter, pigs are denoted fatten-
ers or finishing pigs. Market weight varies over the world but is commonly around
100 kg live weight. The age at slaughter varies with health status, breed, feed intensity
and rearing system, but fatteners generally are around half a year when slaughtered.
The breeding stock is often primarily selected soon after birth in terms of
prospective gilts and boars. After a secondary selection at puberty, the selected gilts
are mated at approximately 7 months of age. After a pregnancy period of 116 days
they deliver their first litter at the age of 11 months. After that time they may deliver
slightly more than two litters annually. In modern husbandry, sows could give birth
to up to 10 litters, but they are generally replaced far earlier.
Intestinal microflora in the growing pig 23
3. PHYSIOLOGY AND ANATOMY OF THE GASTROINTESTINAL

TRACT OF PIGS
The digestive tract of the newborn piglet is specialized on a diet comprising milk.
Therefore, a high lactase activity is present in the small intestine (Hampson and
Kidder, 1986), at least for as long as milk comprises the dominant feed source. Other
organs important for the digestive system, such as the pancreas (Kelly et al., 1991a),
are not quite active during intensive suckling. Owing to the abrupt change from milk
to cereal consumption at weaning, the lactase activity rapidly declines during the
post-weaning period (Hampson and Kidder, 1986). Instead, α-amylase is increas-
ingly produced by saliva, stimulated by chewing. Further, the pancreas becomes
active at weaning, and starts to excrete pancreatic juice (Kelly et al., 1991a,b).
The sow’s milk constitutes a compact food, and the intestine (especially the large
intestine) is comparably small during the suckling period (Kelly et al., 1991a). At
weaning, the domesticated piglets are offered cereals instead of milk. As a conse-
quence, the stomach and the intestine rapidly increase in size (Kelly et al., 1991a).
Despite this, the ability to absorb nutrients might decrease due to a reduction in the
height of the intestinal villi during the post-weaning period (Hampson, 1986), result-
ing in a decreased total area of the surface of the intestinal lumen. The diet might also
influence the size of the intestine during the subsequent rearing of pigs. Fibre-rich feed
sources are correlated to an enlargement of both stomach and large intestine (Anugwa
et al., 1989). The latter is rather expected, because fermentation as well as absorption
of electrolytes and fluids takes place in the large intestine. However, a lower capacity
to absorb water during the first 2 weeks following weaning makes recently weaned
piglets vulnerable to loss of fluid from the intestine (van Beers-Schreurs et al., 1998),
and may possibly contribute to outbreaks of post-weaning diarrhoea (see below).
4. DIET REGIMES AND ALTERATIONS OF FOOD

COMPOSITIONS DURING THE GROWTH OF PIGS
It is of decisive importance that the newborn piglet consumes colostrum, not only to
get energy, but also to obtain immunoglobulins, since the porcine placenta does not
allow transfer of passive immunity from the sow. Therefore, the intestine of the
piglet allows digestion of macromolecules during the first 24−36 h of life. At far-
rowing, the colostrum comprises around 160 g (16%) protein per litre, which rap-
idly declines. Twenty-four hours later the protein content of the milk is around 6%.
During the first day post-farrowing the lactose content increases from 3 to 5%. In
contrast, the fat content is rather stable around 5.5−6.5% (Klobasa et al., 1987).
The young piglet is continuously dependent on milk until weaning. The compo-
sition of the milk varies somewhat over the suckling period, but generally comprises
5.0−6.5% protein, 5.5−6.5% lactose and 5.5−6.5% fat (Klobasa et al., 1987). The
milk also contains IgA, which may protect the piglet from enteric diseases by act-
ing locally in the gut.
The sow eagerly offers her milk to the litter during their first week of life.
However, sucking milk is generally initiated by the constantly hungry offspring
from the second week of life onwards (Algers, 1993). To protect herself from catab-
olism, the wild sow copes with this situation by avoiding contact with her litter
during a great part of the day. Thereby, wild piglets are weaned in a gradual process.
The access to milk will continuously be reduced in comparison to the energy
required, and the piglets are forced to successively search for alternative energy
sources. The final weaning takes place at around 16 weeks of age (Jensen and
Recen, 1985), and at that age the piglets are well adapted to other foodstuffs than
milk. Further, they are well developed with respect to immune functions at that age
(Joling et al., 1994).
A domesticated sow shares pen with her offspring during the suckling period. She
is thereby denied the ability to shun the litter and, as piglets prefer milk as a source
for energy, she will be intensively suckled. In order to protect domesticated sows
from their hungry brood, piglets in modern agricultural systems are weaned between
2 and 7 weeks of age. However, the early weaning system is chiefly employed to
improve production, i.e. to increase the number of piglets produced per sow per year.
Generally, this weaning is effected by removal of the dam from the offspring. As a
consequence, the domesticated piglet will experience weaning at an unexpected point
of time. There is a potential risk to develop disease at weaning due to:
1. an abrupt change of the feed composition where the diet is switched from
milk-based to solid-based feed, mainly cereals. This change also includes a
sudden withdrawal of the protective IgA that is also present in the milk;
2. a poorly developed immune system. In this context it is relevant to point out
that piglets aged 5−6 weeks are not fully developed with respect to immune
functions, and that piglets aged 2−3 weeks are even more immature
(Wallgren et al., 1998);
3. the social alterations at weaning, which contribute to a long-lasting unpleasant
situation for the piglet at weaning.
To prevent disturbances at weaning (and at other occasions), so-called growth
promoters have generally been added to the feed of growing pigs for decades.
The term “growth promoter” in this context refers to low dose administrations
of antimicrobials (i.e. antibiotics or chemotherapeutics). Recently such a routine
administration of antimicrobials to animal feed has been questioned, both from ethical
and from ecological and medical perspectives. For instance, a ban for routine in-feed
medication was effected in Sweden during 1986 (Swedish statute-book; SFS 1985:
295, Stockholm, Sweden). According to that act, antimicrobials may only be incor-
porated in animal feed for the purpose of preventing, alleviating or curing disease, i.e.
not for growth or yield promoting purposes. The European Communities (EC) have
followed this example regarding 8 out of 12 permitted substances during 1999 (Council
directive 70/524/EEC on Feed additives, EC, Brussels, Belgium), and the remaining
substances are to be discussed (COM 2002, 153: final, EC, Brussels, Belgium).
In this chapter it is assumed that antibiotics are not added to the food for growth
promotion.
The pigs will never again experience such a dramatic alteration of feeding habits
as they do at weaning. From that time they are offered feed based on cereals. The
cereal-based feed may be supplemented with protein-rich sources, such as fishmeal
and soybeans. Also bone meal and meat meal have been used as a protein source.
However, the present discussion concerning transmissible spongiforme encephalitis
(TSE) makes the future of the abattoir waste as protein source for meat producing
animals less clear. High protein levels in the feedstuff are known to stimulate growth.
However, protein may also provoke the enteric flora, which might lead to diarrhoea
(Newport, 1980; Shone et al., 1988; van der Peet-Schwering and van der Binnendijk,
2000). Predigestion of proteins, for instance casein, is proven to decrease the risk of
developing diarrhoea (Miller et al., 1984). Aiming to reduce feed provocation with-
out reducing the growth, feed proteins can therefore, to some extent, be substituted
with pure amino acids (Inborr and Suomi, 1988). In spite of this, proteins will always
be an important source of nitrogen because purified amino acids are expensive.
Historically the pigs’ feed has been served as a dry feed, and this is still the most
prevalent feed for weaners. Liquid feeds on the other hand, are becoming more
popular. They were initially introduced aiming to get rid of whey at cheese produc-
tion. However, as the access to whey is limited, liquid feeds based on water are
being used extensively. To avoid uncontrolled growth of bacteria in liquid feeds,
their pH should be below 4.
5. INDIGENOUS INTESTINAL MICROFLORA OF PIGS

AND ITS IMPORTANCE
The gastrointestinal (GI) tract of pigs is a dynamic ecosystem consisting of
microbes that colonize the gut and become established in the intestine (indigenous
or autochthonous) and those that are simply passing through (transient or allochtho-
nous). The normal microflora (also known as normal microbiota) develops as a
result of the influence of the intestinal ecophysiology, and the interaction between
the microorganisms that colonize the gut (Drasar and Barrow, 1985). It is believed
that the initial inoculum is usually derived from the sow at the time of birth (Drasar
and Hill, 1974; Savage, 1977). The climax flora is different in different animal
species and alters as the host ages.
The predominant microorganisms are anaerobes, which require special cultivation
techniques, involving rigorous exclusion of oxygen. In this habitat, the anaerobic
bacteria outnumber the aerobes by a factor of at least 3 to 5 log10. The obligate and
facultative anaerobic bacteria are of diverse genera and range over a wide spectrum
of taxonomic species. Implantation of bacteria in the GI-tract occurs by an elaborate
process of ecological succession in which the composition of microflora constantly
changes (table 1). Organisms which dominate the intestine early in this process, are
Table 1. The density* (log10 /g fresh weight contents) of microorganisms in various sections of the
gastrointestinal tract of pigs
Stomach Duodenum Ileum Caecum Rectum
Lactobacilli 7−8 6–7 7−8 8−9 6−9

Coliforms 5−6 4−5 6−7 7−8 6−8
Enterococci 0−7 0−6 3−8 4−8 5−8
Cl. perfringens Nil Nil 0−7 5−6 0−6
Bacteroides Nil Nil 0−7 5−8 5−10
Total anaerobes 5−6 5−8 7−9 9−11 9−10
Yeast 0−7 0−7 0−7 5−7 5−7
*The density of bacteria may alter with age. For details, see the references.
suppressed by other groups of microorganisms, which are in turn suppressed by new

groups and so forth until a balanced ecosystem is established dominated by anaero-
bic species (Lee and Gemmell, 1972; Savage, 1977; Varel and Pond, 1985). This
orderly ecological succession makes the pig’s intestine a complex milieu of mixed
bacterial populations, which is suggested to contain between 400 and 500 species of
bacteria (Moore and Holdeman, 1974; Drasar and Barrow, 1985). The population
size of each bacterial species is regulated by the whole ecosystem. Various micro-
organisms are completely eliminated from the digestive tract as a result of this effect.
5.1. Interference and protection

The indigenous intestinal microflora is known to be of substantial benefit to the host
(Hentages et al., 1985; Wilson and Freter, 1986; Wilson et al., 1988). It is generally
agreed that this flora serves as one of the major defence mechanisms that protects the
host’s body against colonization by invading bacteria, an effect which is referred to
as “colonization resistance” (van der Waaij, 1979; Finegold et al., 1983; Tancrede,
1992; Rolfe, 1997). This effect is postulated to be due to the competition for attach-
ment sites, nutrients and production of antimicrobial substances such as bacteriocins,
defensins and volatile fatty acids. This function of the flora, although of great impor-
tance to the host, has yet to be fully exploited in veterinary practice.
The pathogenic bacteria may colonize the host either by expressing specific
bacterial virulence factors which may overcome the colonization resistance, or by
taking advantage of an already reduced colonization resistance, such as that induced
by antibiotic treatment. For instance, it has been shown that the normal flora is
suppressed during antibiotic treatment and that this suppression is often correlated
to simultaneous colonization and overgrowth of potentially pathogenic bacteria
(Gorbach et al., 1987; Tannock, 1995). Both bacterial interactions and host defence
mechanisms are important weapons against colonization by pathogenic bacteria.
Data from experimental models reinforce conclusions about the efficacy of using
even some members of normal flora as biotherapeutic agents (probiotics) (Axelsson
et al., 1989; Gorbach, 1990; Fuller, 1992). For instance, some members of lacto-
bacilli have been successfully used as a preventive measure against colonization of
pathogens (Gorbach et al., 1987; Lidbeck and Nord, 1993; Salminen and Arvilommi,
2001). It should be noted, however, that not all members of the intestinal microflora
are beneficial to the host.
6. METHODS OF ANALYSING INTESTINAL MICROFLORA

Under normal conditions, with an intact immune system and normal ecology of the
gut, a high diversity of bacterial species is observed in the intestine. Most of these
bacteria are permanent inhabitants of the GI-tract. They are mostly strict anaerobes
and difficult to cultivate. Traditional cultivation techniques separate about 30−40
species from any individual (Moore and Holdeman, 1974; Drasar and Barrow,
1985). Yet, owing to the complexity of this flora, not all of them can be fully inves-
tigated. In this section we will briefly evaluate some of the available quantitative and
qualitative methods for sampling in order to understand the present limitations for
analysing the intestinal microfloras in animals.
6.1. Quantitative analyses

Of the various techniques used to study the intestinal microflora, most deal with the
investigation of physiological capabilities of specific species of bacteria (Lee and
Gemmell, 1972; Moore and Holdeman, 1974; Daniel et al., 1987). These techniques
require initial isolation steps, and do not represent the entire intestinal bacterial
flora. The total microscopic count of faecal samples together with viable counts of
the numerically most important groups of microorganisms, may suffice for some
samples of the intestinal contents. Such procedures, apart from problems of diluting
faecal samples under a reduced condition (Meynell and Meynell, 1970), require a
number of selective media. A wide range of selective media has been used to
estimate the number of easily recognized groups of intestinal microflora such as
coliforms, staphylococci, streptococci, yeast and lactobacilli. For strict anaerobes
such as Bacteroides, Fusobacterium, Clostridium, Eubacterium, etc., highly enriched
media containing antibiotics such as neomycin, kanamycin and/or vancomycin to
prevent growth of Gram-negative facultative anaerobes are used. It should be
noticed, however, that these media are not always highly selective and lactobacilli
frequently grow well on them, especially when strict anaerobes are present in small
numbers (Drasar and Barrow, 1985). Some of these anaerobes are extremely sensi-
tive to oxygen, dying within 10 min after exposure to air, which adds to the techni-
cal problems in culturing the intestinal microflora. In addition, the pure culture
condition is not a natural state of bacteria in a community, and characterization of
bacteria chosen for study under these circumstances may not be of great ecological
importance.
6.2. Qualitative analyses

Qualitative analysis of the intestinal flora has also been used to estimate microbial
activity of the GI-tract. For instance, in vitro systems have been used to assess the fer-
mentation capacity of colonic microflora by measuring the ability of this complex
ecosystem to metabolize specific carbohydrate(s) (Ehle et al., 1982; Edwards et al.,
1985; McBurney et al., 1985; Wyatt and Horn, 1988), as well as the metabolites
evolved by the sugar fermentation including the gas (Clarke, 1977; Smith and Bryant,
1979; Cummings, 1984; Ross and Shaffer, 1989). Since studying all microbial types
present in the GI-tract is virtually impossible, a convenient way would be to study the
metabolic activities of all or selected groups of bacteria by assessing, in vitro, their
capacity to metabolize a number of substrates (Clarke, 1977). Again, owing to the
complexity of the intestinal flora, study of the metabolic activities would be facili-
tated if rapid and multiple assay methods were used (Katouli et al., 1997a).
Still, one complementary way to study a complex flora is to investigate what the
microbes have done during their presence in the gut. Over the years, long series of
biochemical and microbial transformation processes have been studied in materials
from germ-free animals and their conventional counterparts (Midtvedt, 1999; Norin
and Midtvedt, 2000). As a result, a complementary way to study the metabolic
capacity of the intestinal microflora has been established to evaluate what the
microbes can do and/or what the microbes have done. With a slight travesty of the
terms initially used by Claude Bernhard, the mammalian organisms, or the host side
of the ecosystem, can be defined as Milieu interior (MI), and the non-host side as
the Milieu exterior (ME). MI plus ME together are referred to as Milieu total (MT)
(Midtvedt, 1985). A simple equation of MT minus MI gives ME or “what the
microbes have done”. The approach for such studies is investigating mammals with-
out any normal microflora, i.e. germ-free animals, thereby establishing the functions
of the microorganisms per se. When various microorganisms are associated with
these animals, their influence on host-derived structures and functions can easily
be studied. These findings have been described as germ-free animal characteristics
(GAC) and microflora-associated characteristics (MAC) (Norin and Midtvedt,
2000). MAC is defined as the recording of any anatomical structure, or physiologi-
cal or biochemical function in an animal that has been influenced by the microflora.
When the microbes that actually influence the parameter under study are absent, as
in germ-free animals, this particular recording is defined as GAC.
6.3. New methods to analyse the intestinal microflora

6.3.1. Application of nucleotide probes
The use of molecular probes to characterize the intestinal microflora has recently
been the centre of attention by many investigators. Using the most refined molecu-
lar methods together with the cultural-based methods to describe the natural
communities of the gut, have clearly shown the extent of the unknown microbial
diversity of the gut (Raskin et al., 1995). These methods are mainly based on the use
of oligonucleotide probes complementary to conserved tracts of the 16S rRNA of
phylogenetically defined groups of bacteria.
Using 11 DNA oligonucleotide probes targeting the small sub-unit rRNA of major
microbial groups, Lin and co-workers (Lin et al., 1997) have successfully quantified
several phylogenetically defined groups of methanogens and sulphate-reducing
bacteria of the GI-tracts of various domestic animals. This technique has also been
used to assess and analyse fibre-digesting bacteria of the gut (Stahl et al., 1988;
Lin et al., 1994; Lin and Stahl, 1995).
Apart from the high specificity and accuracy, another advantage of these methods
for detecting defined groups of bacteria is that the faecal samples can be frozen on
dry ice and stored at −80°C immediately after sampling until they are processed. One
should, however, realize that not all laboratories have equipment to utilize rRNA
techniques since the probes should be synthesized, purified by high-performance
liquid chromatography (HPLC) and labelled. Besides, high numbers of probes are
required to fully quantify different microbial groups of the gut.
6.3.2. Metabolic fingerprinting

Competition for nutrients in a mixed bacterial population depends, to a great extent,
on the population size and degree of affinities of each bacterial species to the avail-
able substrates. Two similar bacterial populations normally yield similar patterns of
metabolic activities upon utilization of similar substrates. Any changes in the popu-
lation size or type of bacteria in a sample would be reflected in the overall metabolic
fingerprint of that population. Therefore, measuring the metabolic activities of a
bacterial population will not only yield the metabolic potential of that flora, but will
also help to identify changes in functional status of that flora. Characterization of
certain microbial populations of the gut on the basis of their metabolic activities has
also been used to define the effect of environmental factors or nutritional status on
the natural structure of microbial populations (Rowe et al., 1979; Edwards et al.,
1985; McBurney et al., 1985; MacFarlande et al., 1992). Changes in the pattern of
substrate utilization have then been correlated to the environmental parameters that
regulate microbial populations/communities.
Katouli et al. (1997a) evaluated a microplate-based fingerprinting system (PhPlate
system) for characterizing and measuring the metabolic capacity of mixed bacterial
populations. This system is based on interval measurements of the colour changes
generated by an indicator caused by bacterial utilization of different sole carbon
sources and production or consumption of acids in microtitre plates (Möllby et al.,
1993). The bacterial strains chosen for this evaluation and their concentration in the
synthesized mixtures represented those commonly found in the colon of man and
animals (Katouli et al., 1997a). This simple approach successfully yielded metabolic
fingerprints that varied among samples. The results, however, should be interpreted
with care since these workers found that exclusion or addition of different bacterial
types did not cause a change in the resultant function of a microbial community on
some occasions. They also examined the suitability of the PhPlates system to detect
changes in the composition and function of the intestinal microflora in pigs (Katouli
et al., 1997b). The system proved to be efficient in detecting changes in the compo-
sition and metabolic function of the intestinal flora of the animals during different
nutritional and pathophysiological statuses. Among the useful information that they
obtained from such biochemical fingerprints, was the capacity of a given flora to fer-
ment different carbohydrates, an ability that they referred to as fermentative capacity
(FC) since most tests used in their system were carbohydrates. These workers also
concluded that several factors might contribute to the FC-value of a given microflora.
A flora with numerous but similar bacterial strains normally yields higher FC-values
than a flora with fewer strains. On the other hand, a flora with a few but metaboli-
cally more active strains, capable of fermenting a vast number of carbon sources, also
yields a high FC-value. The differences in types and numbers of the utilized carbo-
hydrates can also be used to compare different microflora (Katouli et al., 1992).
7. IN VIVO MODELS AND SAMPLE COLLECTION STRATEGIES

Most of our present knowledge about the composition of the intestinal flora has
come from animal studies. Faeces comprise the final phase of the intestinal flora. As
the consistency of the faeces reflects the status of the intestine, faecal samples are
assumed to represent the intestinal flora. Indeed, a major problem when studying the
intestinal microflora, is obtaining samples which truly represent parts of the GI-tract
that are normally inaccessible. For instance, samples from gastric, small intestinal
and colonic contents can only be obtained through a peroral or nasal tube, abdomi-
nal surgery, excised appendices, or the use of open-ended tubes. Withdrawal of the
contents at various levels by a magnetically guided tube has also been used (Wilson,
1974), but this method only affords a sample of the organisms that are free in the
lumen. Therefore, microorganisms that are attached to the villi or other parts of the
surface, which often are present in large numbers may be left out.
Samples from different parts of the intestinal content can be obtained by remov-
ing the relative portion of the alimentary tract while the animal is anaesthetized or
in abattoirs and immediately after the animal is slaughtered. The latter has been used
extensively for analysis of the caecum and colon contents of the rumen (Stewart and
Bryant, 1988; Lin et al., 1997). A way to scrutinize the enteric bacterial populations
in vivo would be to surgically insert cannulas at strategic spots of the intestine, and
to collect samples via these fistulas. Surgical insertions of cannulas have previously
been used in pigs, mainly to study the utilization of feed (Sauer et al., 1983;
Rainbard et al., 1984; Johansen and Bach Knudsen, 1994). One location often used
has been the ileo-caecal ostium, representing the transition from the small to the
large intestine (van Leeuwen et al., 1991). An advantage of using this method is that
a fistula can remain in place for a long period, and courses of events can be followed
in vivo at correct spots of the intestine. The surgical insertion of such a fistula at the
ileo-caecal ostium has recently been shown not to affect the intestinal coliform flora
by itself, not even close to the surgery (Högberg et al., 2001).
For obvious ethical reasons associated with these routes of sampling described
above, many investigators prefer to analyse bacterial flora found in faecal samples or
to collect rectal samples. Although the rectal bacterial flora differ from those located
in anterior parts of the intestine (Zoric et al., 2001), they share certain properties. For
instance, the diversity among coliform populations collected from different sites of
the intestinal tract in healthy pigs, has been shown to be equally high (Zoric et al.,
2001). In the pig, the ampulla of the rectum generally contains ingesta that can easily
be collected by swabs or specula. However, in newborn piglets, collection of rectal
samples may be obstructed because the anus is small and the ampulla may be empty
for long periods during the first week of life.
8. IN VITRO MODELS
Development of multistage reactors to simulate the gastrointestinal microbial ecosys-
tems has opened a new window to investigate the fermentation fluxes and products
(e.g. volatile fatty acids, enzymatic activities and head space gases) of this complex
system. These reactors are normally designed to simulate both the small and large
intestine. For instance, Molly and co-workers (1994) have developed a reactor, in
which the small intestine is simulated by a two-step “fill and draw” chamber and
the large intestine by a three-step reactor. These workers have used this system
to compare the composition and activity of microbial flora grown under various
concentrations and combinations of carbon sources such as arabinogalacton, xylan,
pectin, dextrin and starch with those described in the literature. The supply of differ-
ent media or enzymes at each stage of the reactor, to support microbial communities
resembling those of the GI-tract, is an additional advantage of such a system.
Construction of such bioreactors to simulate the GI-tract may be of high value
for monitoring microbial community structures during biological processes. These
in vitro models may be used for comparisons of microbial population changes
over time, and for assessing the diversity of microbial communities under certain
conditions. However, the input of host-derived substances and osmotic conditions
and redox-potential differences are very difficult to mimic within these systems.
9. MICROFLORA OF DIFFERENT REGIONS OF

THE ALIMENTARY TRACT OF PIGS
Development of the intestinal flora in pigs takes place through an ecological
process. During this process of succession, organisms which are dominant at the
early stage of life, are suppressed by other groups of microorganisms, which are
in turn also suppressed and so forth. This process will continue until a stable and
complex flora dominated by anaerobic species is established in the gut.

Unfortunately, due to the complexity of this flora, it is impossible to measure both
quantitatively and qualitatively all types of microorganisms present and this imposes
a great restriction in assessing changes in the composition of the intestinal flora of
the animal at any given time.
Since the beginning of the 20th century, an increasing number of investigators
have been engaged in studying the intestinal microorganisms in pigs. Many of these
studies comprise extensive quantitative and qualitative analyses of the intestinal flora
of conventional pigs at varying ages. The normal flora most studied include
Escherichia coli and other coliform bacteria, streptococci, and lactobacilli
(Komarew, 1940; Quinn et al., 1953a,b; Briggs et al., 1954) and, in some cases, the
total number of aerobic and anaerobic bacteria and yeasts (Willingale and Briggs,
1955; Horvath, 1957; Wilbur, 1959). Some later studies also report findings of
Bacteroides and Veillonella (Smith and Crabb, 1961; Smith and Jones, 1963; Smith,
1965a) and Clostridium perfringens (Månsson and Olsson, 1961; Van der Heyde and
Henderickx, 1964). Some of these workers even compared the bacterial flora in fae-
ces with that in the caecum (Briggs et al., 1954) or observed variation in the total
faecal counts between individual pigs and between days for one animal. In the case
of caecal samples, it appeared that the variations in the total count were small and of
the same order as in faeces. Using more selective media and rigorous techniques to
exclude oxygen, Kovacs et al. (1972) investigated variation in microflora of different
gut segments of pigs. These workers found that the bacteriological status of the stom-
ach, and small and large intestines, is strongly contrasted, as would be expected from
the anatomical and physiological differences of these functional units. Among the
four segments of the small intestine studied, the duodenum contained reduced num-
bers of all bacterial flora studied (except coliforms) compared to the stomach. These
workers suggested that the inhibitory factors operative in the duodenum affect
coliforms1 the least, compared to other groups such as streptococci, lactobacilli and
clostridia. On the basis of these and many other detailed studies it has been concluded
that certain groups of bacteria such as E. coli, streptococci and clostridia are among
the early groups of microorganisms that colonize the stomach within few hours after
birth; all obtained from the dam and the immediate surroundings (Savage, 1977;
Ducluzeau, 1983; Drasar and Barrow, 1985). Using a biochemical fingerprinting
method to measure the stability and diversity of coliforms and enterococcal flora of
rearing pigs, Kühn et al. (1995) established a population similarity model and used
this to measure similarity among the coliform and enterococcal populations of piglets
in a litter and their dam. These workers found that all studied piglets acquired a
diverse coliform and enterococci bacterial flora during the first day of life, which,
although common among the piglets, was different from that of their sow. Both the
enterococcal and coliform floras from different piglets were more similar to each
1 The term “coliforms” is used to avoid incorrect citation of the literature (see box).
The term coliforms has been traditionally used to refer to Escherichia coli (E. coli)-like bacteria
(coli-form) since these bacteria could be readily isolated from faecal materials of warm-blooded
animals. During the early 1900s, the technology was not available to easily distinguish E. coli from
other coliforms and therefore most of the coliforms recovered from human and animal faeces
were assumed to reflect the presence of E. coli. As a result, the term “coliforms” was considered to
be equivalent to E. coli. It is now known that coliform bacteria comprise of at least four genera
of the family Enterobacteriaceae that can all ferment lactose. These genera are Escherichia,
Klebsiella, Enterobacter and Citrobacter and collectively they represent only 1% of the total
bacterial populations in human and animal faeces. Among coliforms, however, E. coli represents
the majority of the population (90−95%). During the early 1950s, although more specific tests were
developed to easily identify E. coli from the rest of coliforms, the use of “faecal coliforms” was so
commonplace that the term was not dropped in favour of E. coli.
other during the suckling period than after weaning. These workers also found that
the enterococcal flora in the piglets was more persistent than the coliforms.
Pigs are continuously exposed to microorganisms from their surrounding environ-
ment. Microorganisms that pass through the GI-tract (transient microflora) may be
found in the luminal contents or in faeces (Savage, 1977). However, it is highly likely
that most of them are eradicated by host factors such as acids in the stomach, or bile
in the upper small intestine. In contrast, resident microflora represent microorganisms
that colonize different regions of the GI-tract. The type and number of these organisms
in these regions are highly variable. For instance, Lactobacillus was shown to repre-
sent 67% of the bacterial population of the non-secreting stomach region in healthy
unweaned pigs (McGillivery and Cranwell, 1992). Their level ranges between 107
and 108 CFU per gram caecal content in suckling piglets (Jonsson, 1986). The
Lactobacillus species that are most frequently isolated from the stomach, intestine and
faeces of healthy piglets are L. fermentum (Fuller et al., 1978), L. acidophilus and
L. delbrueckii (Mäyrä-Mäkinen et al., 1983). The majority of these isolates can attach
to epithelial cells of the small intestine. In addition to lactobacilli, other bacterial
groups have been isolated from the non-secreting part of the stomach. These include
Streptococcus (Fuller et al., 1978), Eubacterium, E. coli, Bifidobacterium,
Staphylococcus, Clostridium and Bacteroides (McGillivery and Cranwell, 1992).
However, their population size in the stomach is much smaller than in the large intes-
tine (McAllister et al., 1979). The viable cells of lactobacilli are continuously being
released from the non-secreting region, together with desquamated epithelial cells
and thereby inoculate the stomach and intestinal luminal content (Fuller et al., 1978;
Jonsson and Conway, 1992). Using a combination of protein profile analysis and
colony morphology, Henriksson et al. (1995) characterized the lactobacilli colonizing
various regions of the porcine GI-tract and detected several different groups of lacto-
bacillus. Specific groups of lactobacilli were associated with, and often unique for the
stomach, jejunal, caecal, and colonic regions of the GI-tract. These workers also found
that there were major differences between population densities of the gastric mucosal
Lactobacillus population of individual pigs.
Digesta transferred from the stomach to the duodenum are subjected to a dramatic
environmental change, mainly due to the introduction of host factors such as bile,
enzymes and bicarbonate (Drasar and Barrow, 1985). Compared to the large intestine,
this region is less densely populated by microorganisms, partly due to the high flow
rate of the luminal content through the small intestine. Lactobacilli are the dominant
species in the piglet’s small intestine, while E. coli, Clostridium, Bacteroides and
oxygen tolerant anaerobes are also present in large numbers. In addition low levels
(100−1000-folds) of Streptococcus, Enterococcus and Staphylococcus have been
detected on the mucosa of the small intestine (McAllister et al., 1979). The densities
of the small intestinal microflora tend to increase in the distal part. In piglets, this is a
major site for colonization by certain diarrhoegenic E. coli strains. The large intestine,
on the other hand, is the major site of microbial activities in the digestive tract of the
healthy pig, and the slowly moving digesta contains the most dense microbial popu-
lation of the entire GI-tract. The large intestine includes the caecum and the colon.
The pig is a monogastric herbivore with a relatively large caecum and colon.
Consequently, the transient time through this region is considerable, allowing large
populations of bacteria to be accumulated in the large intestine. Obligate anaerobes
dominate the microflora of this region and increase in number from the ileum to the
spiral colon (Cranwell, 1990). It is generally acknowledged that between 1011 and 1012
CFU bacteria are present per gram dry weight of the colon contents (Onderdonk,
1999; Borriello, 2002). This figure for facultative anaerobes such as Bacteroides and
clostridia can be as high as 109 CFU per gram (Smith, 1965b; Drasar, 1974; Salanitro
et al., 1977; McAllister et al., 1979).
10. DYNAMICS OF THE INTESTINAL MICROFLORA IN

HEALTHY CONVENTIONAL PIGS
Detailed identification of different bacterial species in the pig’s intestinal tract is an
extremely laborious and lengthy process. For this reason, studies investigating the
dynamics of the intestinal flora focus on methods that can yield information on
the functional status of the intestinal flora. These methods include measuring the
fermentative capacity (FC) of the microflora, which evaluates the amount of sugar
metabolized by the faecal microflora and the metabolites evolved (McBurney et al.,
1985), and testing the reaction of the whole or part of the gut flora against a relatively
high number of substrates (Clarke, 1977). Using a combination of 48 substrates,
Katouli and co-workers examined the pattern of metabolic response of the intestinal
flora of healthy pigs and compared that with diseased pigs (Katouli et al., 1997b).
They found that the response of the animal’s intestinal microflora to different carbo-
hydrates varied among individual piglets at different sampling occasions. Similar
results have also been reported by others (Edwards et al., 1985). Despite these indi-
vidual differences, the overall FC-values in most stages of animal life were similar
among piglets (Katouli et al., 1997b). These workers also showed that piglets receive
a high proportion of their intestinal microflora from their dams during the first few
days of their life. However, despite the close contact with their dams, they develop
intestinal floras that are very different from the sow’s flora. This suggested that the
milk-based diet in piglets would yield a flora that is different from the initial “birth
flora” derived from sows. This finding is supported by the fact that microfloras of
piglets during the suckling period show more similarity to each other than during the
post-weaning and fattening period.
The post-weaning period in piglets is associated with a dietary shift from milk to
solid food, which will be replaced with a high-energy fattening diet when piglets are
allocated into fattening stables. This dietary shift will result in substitution and/or
establishment of new microflora in the pig intestine. It has been shown that the
intestinal flora of pigs during the fattening period is more diverse than that of the
suckling period. This might be due to the fact that in fattening stables, pigs from
different pens may be mixed together and a direct contact between adjacent pens is
established. As a result, pigs are exposed to more diverse bacterial species (Katouli
et al., 1997b). The fermentative capacity of the intestinal flora, which is normally
high during the suckling period, decreases during post-weaning and fattening
periods, indicating that organisms dominating the pigs’ intestine very early in life
are able to utilize more diverse carbon sources than those dominating the animal
during post-weaning and fattening periods (fig. 1).
10.1. Intestinal microflora of sows

Several studies have attempted to determine the type of bacteria that are present in the
intestinal tract of sows. Such efforts, using selective plate media, have led to the
enumeration of only bacterial groups such as lactobacilli, streptococci, Bacteroides,
E. coli and C. perfringens (Rall et al., 1970; Terada et al., 1976; Salnitro et al., 1977).
These studies have clearly shown that Gram-negative anaerobic species of Bacteroides,
Fig. 1. Fermentative capacity (FC) - values of the whole intestinal flora of conventional pigs during the
suckling, post-weaning and fattening periods. FC - values are the mean of four pigs and their standard errors.
W = Weaning. F = Pigs were transferred to the fattening stable.
Veillonella, Fusobacterium and Peptostreptococcus can be isolated from different

segments of the intestinal tract (Aalbaek, 1972; Mitsuoka et al., 1974). Other groups
using strict anaerobic methods have isolated streptococci, Eubacterium species,
Clostridium species and Propionibacterium acnes as the predominant flora in adult
pigs (Salnitro et al., 1977). Kühn and co-workers measured phenotypic diversity and
stability of the intestinal coliforms (Kühn et al., 1993) and enterococcal floras (Kühn
et al., 1995) in piglets during their first 3 and 5 months of age, respectively, and
compared the results with those of their sows. They found that the diversity of
bacterial flora of the sows was higher than in most of the piglets during the first week
of the pig’s life. In a more comprehensive study, Katouli et al. (1997b) investigated
similarities between biochemical fingerprints of the whole intestinal microflora of
sows and their offspring. They found that the sow’s flora had a considerably lower
FC-value than those of the piglets at the time of birth, which remained so over the
entire suckling period. The fact that the bacterial floras of sows had lower FC-values
(even lower than those of pigs at the end of the fattening period) suggests that the loss
of fermentative capacity will continue as the animal ages (see fig. 1).
10.2. Intestinal microflora of piglets during the suckling period

As mentioned before, the piglet intestine is sterile at birth (Kenworthy and Crabb,
1963). Piglets receive their initial microflora from the sow’s teats and skin as well
as maternal faeces (Arbuckle, 1968; Berschinger et al., 1988). In fact, it has been
reported that piglets eat considerable amounts of their sow’s faeces during the suck-
ling period (Sansom and Gleed, 1981). Studies carried out during the early 1960s by
Smith and Crabb (1961) and Kenworthy and Crabb (1963), and more recently by
Melin et al. (1997) and Katouli et al. (1995), have shown that despite differences in
genetics and feeding strategies, healthy piglets reared in different environments
develop a very comparable intestinal flora. During the early life when diet consists
of mainly milk and the species-specific differentiation of the intestinal tract is low,
several bacterial groups increase and decrease in a similar way. For instance, Melin
et al. (1997) have shown that the number of faecal coliforms, E. coli, enterococci
and C. perfringens decrease over the first 9 weeks of the piglet’s life. C. perfringens
even reaches undetectable levels after 3 weeks.
During the first week of life the coliforms and enterococci in the piglets’ intestines
may differ considerably from that of the dam, suggesting that these floras are coming
from sources other than sows (Katouli et al., 1995; Kühn et al., 1995). However, this
may contribute very little to the overall similarity between the gut microflora of the
sows and their offspring. The diversity of these floras in piglets is very high already
from the first week and remains so during the suckling period. The fact that piglets
housed together develop highly similar floras during the first week and onwards, also
confirms the environmental nature of these floras among litter and pen-mates (Katouli
et al., 1999). The early differences among coliform and enterococci floras between
piglets and sows will gradually decrease during the suckling period so that before
weaning these specific floras of littermates are fairly similar to each other and to that
of their dams (Melin et al., 1997; Katouli et al., 1999).
The intestinal colonization of E. coli in piglets comprises successive waves of
different strains. Most of these strains are transient bacteria, the tenure of which
varies between a few days to 2 weeks. On the other hand, most resident E. coli
strains colonize the intestine of young piglets during the suckling period. Katouli et al.
(1995) have shown that during this period each piglet may carry more than one type
of resident strain in their gut. In a herd or stable, several piglets may be colonized
by the same resident strains, which indicates that both strain and host specificity,
especially during the suckling period, are important for colonization and persistence
of E. coli in piglets.
Comparison of metabolic fingerprints of the faecal samples from piglets and their
dams has shown that despite the difference in the E. coli floras, most members of the
intestinal flora in pigs are similar to those of their dams, suggesting that sows are the
initial source of most microflora for piglets. However, it seems that despite the close
contact of piglets with their dams during the suckling period, they will eventually
develop floras that might not be very similar to that of their sow (Katouli et al., 1997b).
10.3. Intestinal microflora of piglets following weaning including

effects of regroupings and movements
In modern agricultural systems, weaning is generally achieved by abruptly removing
the sow. However, these circumstances expose the piglets to a considerable amount of
stress that affects the immune system negatively (Blecha et al., 1985; Bailey et al.,
1992; Hessing et al., 1995; Wattrang et al., 1998). The stress and the sudden alteration
of diet also contribute to a disturbed enteric flora of the piglets during the post-
weaning period (Kühn et al., 1993, 1995; Katouli et al., 1995, 1997b; Melin et al.,
1997, 2000a). The diversity of the intestinal flora may decrease dramatically during
the first 3 days post-weaning among apparently healthy piglets. Since a high micro-
bial diversity of the gut is believed to protect the animals not only from intrinsic
microbes but also from microorganisms of external origin (Pielou, 1975; Kühn et al.,
1993), this points to a situation of potential danger due to a decreased colonization
resistance. It has been shown that the population size of bacteria is not altered during
this period (Melin et al., 1997), indicating that the decreased microbial diversity must
be achieved by proliferation of some strains. Melin et al. (1997) also showed that
the similarity of the intestinal flora among pen-mates decreases during this period. This
in turn points to the fact that while some strains proliferate in one pig, other strains
proliferate in other pigs. Thus, if a pig develops diarrhoea due to proliferation of a
pathogenic clone, there is an increased risk that also pen-mates will be diseased as
their colonization resistance is decreased due to the low diversity of the intestinal flora
at the actual time. In apparently healthy pigs the enteric microflora will again stabilize
between 2 to 3 weeks after weaning. However, if invaded by pathogenic strains of

E. coli, the intestinal flora may be disturbed for an even longer period (Melin et al.,
2000a).
Some pig herds practise mixing and moving of piglets at weaning. This will
potentially increase the risk of developing diarrhoea not only due to an increased
level of stress imposed on the piglets, but also because a larger number of pathogenic
strains (from more than one pen) have the chance of proliferating and invading the
vulnerable piglets (Katouli et al., 1999). It should be noted, however, that the
increased number of strains might also increase the piglets’ colonization resistance.
Therefore, the mixing practice may not always be detrimental to pigs especially if it
is done under proper hygienic management. Under high hygienic and management
standards, the disturbed normal flora will be restored in around 2−3 weeks and pigs
regain a high microbial diversity and a high similarity between microbial populations
within groups (Katouli et al., 1999). Analysis of the intestinal microflora of pigs after
weaning has shown that the post-weaning coliform populations differ from those
of the suckling period (Katouli et al., 1997b). Despite this, the overall similarity
between intestinal populations of pen-mates may remain high (Katouli et al., 1997b).
10.4. Intestinal microflora of pigs during the fattening period

Pigs are generally transferred from weaning facilities to the fattening enterprises at
the weight of approximately 25 kg, corresponding to an age of 10−14 weeks. This
transfer may provoke the pigs in a similar way as weaning (Wallgren et al., 1993),
and the provocations increase if the animals are transported and regrouped (Lund
et al., 1998). However, pigs are more immunologically (Wallgren et al., 1998) and
physiologically mature during this transfer than at weaning. Consequently, the effect
on the enteric flora because of this transfer is much less evident than at weaning
(Katouli et al., 1995). If healthy, the fattening pigs show a high diversity of the enteric
flora throughout the fattening period (Kühn et al., 1995). However, transient
microbes are continuously present, and the similarity of the intestinal flora between
pigs at this stage may be considerably lower than at the suckling or post-weaning
periods (Katouli et al., 1995; Kühn et al., 1995).
As mentioned before, an alteration of the composition of the intestinal popula-
tions takes place when pigs are allocated into fattening units and mixed with other
pigs. The intestinal populations may differ considerably between pigs, mainly due
to the fact that pigs are exposed to the diverse bacterial species, a situation which is
normally expected in stables of mixed pigs. The overall fermentative capacity of the
flora of the animals during this period is far less than during the suckling period.
This loss of fermentative capacity is a gradual process but will be accelerated dur-
ing the late post-weaning period (Katouli et al., 1997b). Changes in the composition
and fermentative capacity of the intestinal flora of pigs after weaning and after allo-
cation of pigs into the fattening stables, coincide with the dietary shift from milk to
solid food and further to a high-energy fattening diet (see fig. 1).
11. INTESTINAL MICROFLORA OF SPECIFIC

PATHOGEN FREE PIGS
Specific pathogen free (SPF) pigs are declared free from a defined number of
microorganisms pathogenic to pigs. However, it should be noticed that these pigs are
not reared under germ-free conditions. Diseases such as salmonellosis and swine
dysentery (induced by Brachyspira hyodysenteriae) are not present, but microor-
ganisms such as E. coli are in reality impossible to avoid. As feed and straw are
often of similar sources as those offered to conventional pigs, the intestinal
microflora is virtually the same for SPF pigs as for conventional pigs. Indeed, when
comparing intestinal microflora obtained from SPF pigs with those obtained from
conventional pigs they basically share a similar composition throughout their life.
On the other hand, owing to the absence of certain pathogenic microbes and pre-
cautions undertaken to avoid introduction of infections, development of clinical
diarrhoea is rarely seen in SPF herds.
We have recently studied the biochemical fingerprints and fermentative capacity
of the whole and/or selected intestinal microflora of SPF pigs during weaning, post-
weaning and the fattening period (Katouli et al., unpublished data). We found that, as
in conventional pigs, the fermentative capacity of the SPF pigs also decreased as the
pigs grew older and that there was a decrease in the fermentative capacity values of
the intestinal flora immediately after weaning and after the pigs were transferred to
the fattening stable (fig. 2). However, we also found that both SPF piglets and their
sows had much higher FC-values than their conventional counterparts during the first
week of life. These values, however, dropped to a level close to what we have
normally obtained from conventional pigs during this period (see fig. 2).
Interestingly, the FC-values of sows reached the same level as those of piglets at the
time of weaning. Similar patterns were basically observed among selected groups of
normal flora in SPF piglets except for Lactobacillus flora. The fermentative capacity
Fig. 2. FC - values of the whole intestinal microflora of specific pathogen free (SPF) pigs during suckling,
post-weaning and fattening periods. FC - values are the mean of four pigs and their standard errors.
W = Weaning. F = Pigs were transferred to the fattening stable.
Fig. 3. FC - values of streptococcal (a), coliform (b) and lactobacilli (c) populations of SPF pigs during the
suckling, post-weaning and fattening periods. W = Weaning.
of this flora, which was high during the first 4 weeks of weaning, showed a dramatic
decrease just before weaning, reaching its minimum level at the time of weaning (fig. 3).
12. ALTERATIONS OF THE INTESTINAL MICROFLORA

OWING TO STRESS AND DISEASE
Physiological stresses and disease especially during suckling and early post-weaning,
are a major concern within piglet production, and disturbances in the composition
of the intestinal microflora constitute the greatest problem (Cutler et al., 1999).
It is believed that changes in the stability of the intestinal flora will result in the
development of a low diversity of the flora making the animal susceptible to
gastrointestinal diseases. These factors and their effect on the health status of grow-
ing pigs are discussed below.
12.1. Intestinal coliforms during the suckling and post-weaning periods

As described earlier, piglets rapidly develop a highly diverse intestinal coliform flora.
Unless diarrhoea develops, this flora will remain stable until weaning and the intestinal
coliform populations of pen-mates are fairly similar, indicating a high colonization
resistance of the piglets within the pen.
Introduction of weaning, more or less, leads to a collapse of the intestinal coliform
population. At this time the piglets are highly vulnerable to disease and if they are
exposed to a low pathogen load, because of good herd management, they may be able
to resist developing diarrhoea before the disturbed flora is completely recovered.
In healthy pigs, the coliform flora will remain stable throughout the weaning period.
At transfer to the fattening enterprises, a situation similar to weaning may occur.
However, as the pigs are growing older and their feed composition changes less dra-
matically, the intestinal coliform floras are restored faster following this allocation.
12.2. At-risk situations

The intestinal bacterial populations may be influenced by changes in the life of a pig.
Consequently, all adjustments should be defined as situations that may threaten the
stability of the enteric microflora. The younger the pig and the more dramatic the
alteration(s), the larger the risk will be. The influence of alterations of the intestinal
flora on the pig’s life has been thoroughly described earlier in this chapter. Examples
of induced at-risk situations are weaning, regrouping, transportation and alterations
in feed regiments. In addition, non-optimized management may provide some addi-
tional risk situations, such as high pathogen load, chill, draught and moisture.
12.3. Diarrhoea pre-weaning

Pre-weaned piglets are frequently infected with enteropathogens at two stages: as
newborn and at the age of 2−3 weeks. In systems effectuating weaning at the age
of 2−3 weeks, the latter stage coincides with the weaning and thereby could poss-
ibly be referred to as post-weaning diarrhoea. Factors such as immune defence,
indigenous flora, pH, food composition and environmental errors may influence the
defensive capacity of the animals at these occasions and therefore exposure
to enteropathogens may be hazardous. A number of host mechanisms have
evolved which protect the GI-tract from invading pathogens. E. coli, Salmonella and
B. hyodysenteriae are among the globally most economically important causes of
bacterial induced diarrhoea in piglets (Bergeland and Henry, 1982; Edfors-Lilja
and Wallgren, 1999; Straw et al., 1999).
The two most important pathogenic microorganisms that affect newborn piglets
are E. coli (summarized by Fairbrother, 1999) and C. perfringens (summarized by
Taylor, 1999). Both these microorganisms may induce neonatal diarrhoea in large
numbers of piglets and may become fatal. However, owing to the unifactorial cause
of these diseases, vaccination of sows and the subsequent transfer of their protective
immunity to the offspring via colostrum have effectively prevented outbreaks of dis-
eases caused by these species in newborn pigs. Other microorganisms associated with
diarrhoea in neonatal animals include Bacteroides fragilis, Campylobacter spp. and
Yersinia enterocolitica (Holland, 1990) and a number of viruses (Straw et al., 1999).
It should be noted, however, that the maternal immunity declines with increasing
age of the piglets (Saito et al., 1986; Fu et al., 1990; Wallgren et al., 1998). Therefore,
diarrhoea induced by the species mentioned above may occur at the age of 2−3 weeks
if the pathogen load of the environment is high enough. Furthermore, these species
may be found in association with large numbers of other potentially pathogenic
microbes (summarized by Straw et al., 1999). Virulent organisms such as the proto-
zoan Isospora suis as well as rotavirus and coronavirus, have frequently been corre-
lated to diarrhoea in suckling piglets (Glock, 1981). The number of microbes that
could potentially contribute to development of gastrointestinal disturbances increases
as the piglets grow. Consequently, diarrhoea among somewhat older piglets may well
reflect mixed infections, and can certainly be influenced by environmental conditions
and hygiene.
When diarrhoea is observed during the first days of life, the causative agent can
often be re-isolated in pure culture from faecal samples, and under such conditions the
correlation between infection and signs of disease is obvious. Somewhat older piglets
will receive a rather diverse enteric flora prior to infection. Kühn and co-workers (1993)
have shown that during an E. coli associated outbreak of diarrhoea, the diseased piglets
had a lower diversity of the intestinal coliforms, indicating that the pathogenic strain
had outgrown the others. While studying the diversity of coliform populations in
a group of pigs, we also noticed that pigs that received antibiotic during an outbreak
of diarrhoea showed a lower diversity of coliforms. This effect, however, was not
seen among all piglets. We also noticed that the piglets affected with diarrhoea did
not recover from the low coliform diversity until long after weaning (Katouli et al.,
unpublished data) (fig. 4).
12.4. Diarrhoea post-weaning

As described above, the enteric microflora is severely disturbed following weaning,
thereby paving the way for potentially pathogenic microbes. Toxin-producing
strains of E. coli (mainly serogroups O138, O139 and O141) associated with
oedema disease, may act as the main sources of post-weaning diarrhoea, a disease
that is often fatal for newly weaned piglets (Berschinger, 1999). On the other hand,
it should be mentioned that experimental challenge of healthy, newly weaned piglets
Fig. 4. A representative figure showing the effect of sulfametoxasol/trimethoprim (administered during

an outbreak of diarrhoea) on the diversity of coliforms in conventional pigs. Four pigs (P1 to P4) from four
litters were studied. Diversity of coliforms was measured as Simpson’s index of diversity after testing
randomly 40 coliforms from MacConkey plates.
W = Weaning. DO = Onset of the diarrhoeal outbreak in the herd. F = Pigs were transferred to a fattening stable.
with any of the above pathogens, in most cases might not result in a state of diar-
rhoea. This may be due to the lack of environmental factors necessary for complete
disturbance of the flora. For instance, Melin et al. (2000a) used a highly virulent
strain of E. coli to challenge a set of weaned piglets. The challenge strain belonged
to serogroup O149 and carried surface antigen K88, which confer adhesion to the
F4 receptor on the epithelial cells of the pigs. Furthermore, the strain was a potent
toxin producer (STa, STb and LT) and the challenged piglets all had receptors for
F4 (Edfors-Lilja et al., 1995) and the pigs were proven truly infected. Still, post-
weaning diarrhoea (PWD) was not achieved, indicating that although PWD is
strongly associated with E. coli, it should be considered as a multifactorial syndrome
rather than a specific infection. Indeed, these workers succeeded in inducing an
experimental PWD by exposing piglets to a cascade of different pathogenic strains
of E. coli (Melin et al., 2000b,c), possibly imitating conditions that could occur
post-weaning in a herd (see above).
Microbes should not be regarded as the sole cause of PWD in practical
pig production. The influence of pathogenic microorganisms can be amplified
by environmental stress such as chill, draught, moisture, etc. Further, insufficient
management may contribute to the development of PWD. The newly weaned pig
is poorly developed with respect to immune functions (Blecha et al., 1985; Bailey
et al., 1992; Wallgren et al., 1998; Wattrang et al., 1998) and therefore vulnerable to
infections.
Pigs affected by PWD will express a decreased diversity of the intestinal flora
during the course of the disease owing to the overgrowth of one or several bacterial
strains (Melin et al., 2000c). Because of the influence of the strain(s) causing
disease, the similarity between intestinal coliform populations of diseased pigs may
be larger than between apparently healthy pigs. However, this type of similarity does
not indicate any colonization resistance, as seen among suckling pigs (Katouli et al.,
1999). Instead, it is achieved by infection of several individuals by the same patho-
genic strain(s).
The balance of the intestinal microflora may be severely affected for as long as
4 weeks following an infection with coliforms at weaning, regardless of whether
clinical PWD has been developed or not (Melin et al., 2000a). Of course, this may
facilitate infections with other pathogenic microorganisms, such as Brachyspira
species or Salmonella.
Taken together, the weaning is a critical physiological period for the pig. It is
often accompanied by an abrupt multiplication of some strains of E. coli in the
digestive tract and may result in development of diarrhoea and/or oedema disease.
To avoid PWD, good management should be applied.
13. PRECAUTIONS AIMING AT STABILIZING

THE INTESTINAL MICROFLORA
The negative influence of environmental disturbances and infections on the intestinal
flora could possibly be reduced. Different strategies are discussed briefly below.
13.1. Feed composition

The food itself can be a provoking factor in causing disease and/or disturbance of the
gut flora of the pigs. As an optimized growth of pigs is of economical importance, feed
consumption and feed utilization are of great importance in modern pig husbandry.
Pig feed is often processed, i.e. pre-heated, and the digestive ability of the food is
facilitated. However, this normally leads to a reduced chewing and a shorter residence
period of food in the stomach. As a consequence, the digestive and bactericidal effects
of saliva and hydrochloric acid will be reduced and disturbances in the digestive tract
may be facilitated. By avoiding pre-heating of dry feed, the natural protective effect of
intestinal flora to infection will increase. An increased amount of fibre in the diet will
further stimulate the natural protection towards disease owing to a slower passage
through the gut (Heidelberg et al., 1984; Hampson and Kidder, 1986).
In a situation of increased risk, the food composition may have a big impact on
the clinical outcome with respect to enteric health in a herd. For instance, the provo-
cation of the abrupt change of food at weaning may be minimized by adding lactose
to the food, thereby resembling the milk from the sow to some extent. In this
context it should be mentioned that commercially available milk substitutes gener-
ally emanate from either cow milk, that will include proteins from foreign species,
or even from soya.
Protein, which is required to stimulate the growth of pigs, may also affect the
composition of the enteric flora, leading to diarrhoea (Newport, 1980; Shone et al.,
1988; van der Peet-Schwering and van der Binnendijk, 2000). In fact, some protein
sources, such as soya have actually been linked to outbreaks of diarrhoea (Jager
et al., 1986; Nabuurs, 1986). Predigestion of proteins is proven to decrease the risk
of developing diarrhoea (Miller et al., 1984) and feed proteins can therefore to some
extent be substituted with pure amino acids (Innborr and Suomi, 1988). However, as
purified amino acids are expensive, the proteins themselves form the most important
source of nitrogen in pig feed.
13.2. Antibiotics and feed additives

Antibiotics have commonly been used to control the enteric health and improve the
growth rate. This is certainly achieved by suppression of the intestinal bacterial
activity. Animals given antibiotics in the feed generally perform better than those
offered probiotics (Eidelsburger et al., 1992). However, it should be remembered
that intestinal treatments with antibiotics may affect the normal flora severely
(Barza et al., 1987; Thijm and van der Waaij, 1979; Hashimoto et al., 1996; Wilson
et al., 1996). Further, a continuous use of antibiotics will increase the risk of devel-
opment of bacterial resistance to antibiotics used (Linton et al., 1988; Aarestrup,
2000). Consequently, a permanent use of antimicrobial agents in feed ought to be
avoided and replaced with proper management systems and well-designed feed that
maintain and stabilize the intestinal flora. As a result, development of diseases
would be minimized and the number of medical treatments would be reduced.
13.3. Zinc oxide supplementation of the feed

Zinc is an essential component of several enzyme systems and plays an important
role in stabilizing membrane integrity. As epithelial cells are the first line of defence
against microbial invasion, zinc has a special role in resistance to infections. Zinc
in the form of zinc oxide (ZnO) has been successfully used to prevent outbreaks of
PWD (Holm, 1988; Holmgren, 1994). By adding a high concentration of the ZnO
to the feed, it has been possible to preserve the integrity of the coliform population
in weaned pigs (Melin et al., 1996; Katouli et al., 1999). This may partly explain the
protective effect of the ZnO against post-weaning diarrhoea as the colonization
resistance of the gut flora is preserved. No similar effect can be achieved if an equal
amount of zinc is given parenterally (Shell and Korneay, 1994). This calls for a local
effect of the ZnO in the intestine and, since a high concentration of zinc is required,
it is possibly toxic. Piglets given ZnO-supplemented feed may grow faster than non-
treated piglets close to weaning. However, a continuous feeding of high amounts of
ZnO in the food should be avoided, because pigs that were offered a feed with
2500 ppm ZnO for 4 weeks expressed signs of intoxication (Jensen-Waern et al.,
1998). Further, as most of the zinc oxide will pass through the pig’s intestine, the envi-
ronmental aspects must be considered. Katouli et al. (1999) found that loss of diver-
sity and disruption of the integrity of coliform flora in weaned piglets supplemented
with ZnO in their feed could be restored within 14 days post-weaning. On the basis
of this finding, these workers concluded that feeds supplemented with ZnO should
be restricted to only 2 weeks post-weaning in veterinary practice.
13.4. Probiotics
Using probiotics to stimulate the intestinal flora has been tempting, mainly due to
the facts that probiotics can be used to improve colonization resistance of the intes-
tinal flora and thereby potentially reduce the dependence on antibiotics in order
to prevent and/or treat bacterial infection of the gut in animals (Kyriakis, 1989;
Kyriakis et al., 1999). Studies on the suitability of the members of the intestinal flora
have suggested potential candidates such as Lactobacillus species (Toit et al., 1998),
and Bacillus cereus (Kirchgessner et al., 1993) as probiotics of microbial origin.
Acidifiers have also been suggested and used as probiotics. These include fumaric
acid, hydrochloric acid and sodium formate (Eidelsburger et al., 1992).
The existing reports on the use of probiotics in pigs range from positive effects
on enteric health and weight gain (Eidelsburger et al., 1992) to no effects at all
(McLeese et al., 1992). Also increased weight gains have been reported without any
visible positive effects with respect to intestinal health (Eidelsburger et al., 1992;
Kirchgessner et al., 1993). Presently, much work is focused on probiotics. However,
the variations in the results obtained may indicate an influence of the management
and environmental conditions. Therefore, great efforts should be made to scrutinize
the effects of probiotics under unbiased conditions. The effect of probiotics on the
health and well being of animals is discussed elsewhere in this book.

The control of diarrhoeal diseases still presents a challenge in pig husbandry. Recent
developments in management and production facilities, as well as availability of
potential vaccines, has reduced mortality associated with diarrhoea in piglets.
Changes in the composition and stability of the intestinal flora of piglets have been
shown to play an important role in the development of diarrhoea during the suckling
and early post-weaning periods. Factors such as stress, especially at weaning and
early post-weaning periods, are among the main causes of disruption to the integrity
of the intestinal flora. Approaches to challenge enteric diseases should include
establishing diverse intestinal floras in piglets during the suckling period and main-
taining the stability and diversity of this flora after weaning. While several methods,
including molecular-based techniques, are available to detect and identify uncultur-
able bacterial flora of the gut, there is a need for more advanced techniques to
measure the functional status of the normal flora in response to dietary feed and
environmental stress. The recent practice of withdrawing growth promoters in pigs
in some countries should be monitored with respect to the composition of the gut
flora and the development of enteric disease. Furthermore, there is a growing

interest in ecological agricultural practice and the farming of pigs outdoors. This
may have a significant impact on the composition of the intestinal flora, which may
differ from that of the traditional indoor microflora. Application of new methods
alone or in combination with classical methods can be used to identify the stability
and the impact of the outdoor flora on the general health of pigs.
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3 Rumen protozoa in the growing domestic
ruminant
T. Michal̄owski
The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of

Sciences, Instytucka 3, 05-110 Jab l̄ onna near Warsaw, Poland
This review summarizes the development and activity of ciliate protozoa in the
rumen of domestic ruminants during the first months of postnatal life. The appear-
ance and establishment of ciliate fauna in the rumen following both natural mouth-
to-mouth contact of newborn animals with their dams and/or other members of the
flock as well as by experimental introduction of the rumen fluid or digesta inoculum
to the rumen of ciliate-free lambs, calves and kids are considered in relation to diet,
age of the animal, type of management and the pH of the rumen digesta. Ciliates as
a factor affecting fermentation in the rumen and the growth of young ruminants are
discussed. The growth and functional maturation of the rumen as a fermenting
chamber is briefly described. The presented information also concerns the taxon-
omy of ciliates and their role in the digestion and fermentation of nutrients in the
rumen, in particular with respect to the degradation of structural carbohydrates in
plant cell walls.
1. INTRODUCTION
Although the rumen microbial ecosystem plays a crucial role in ruminant nutrition, the
abomasum is the only well-developed and functioning portion of the complex stomach
in newborn animals (McGilliard et al., 1965; Warner and Flatt, 1965; Hofmann, 1988;
Lyford, 1988). Thus, during the first days of postnatal life ruminants do not differ from
monogastric mammals when digestive processes are considered.
It is known that the growing domestic ruminant becomes dependent on fermen-
tation products by 8 weeks of age (Lyford, 1988). Thus a 2-month-long period is
necessary for the rumen to become a functional fermentation chamber. The growth
and functional maturation of the rumen tissues is accompanied by the development

© 2005 Elsevier Limited. All rights reserved. 54
Rumen protozoa in the domestic ruminant 55
of a microbial population comprising bacteria, fungi and protozoa, which appear in

the rumen in a defined sequence (Cheng et al., 1991; Dehority and Orpin, 1997).
The aim of this chapter is to present the findings of studies on the development of
ciliate fauna in domestic ruminants during the first months of postnatal life with
respect to the role of protozoa in the rumen metabolism and performance of the host
animals.
2. ROLE OF MICROORGANISMS IN RUMINANT NUTRITION

Despite their inability to manufacture fibrolytic enzymes, mammalian herbivores
have for over 35 million years been eating plant material containing large quantities
of cellulose and hemicellulose (Langer, 1988). All of these animals harbour symbi-
otic microbes in the alimentary tract and depend on microbial digestion and
fermentation to release the energy stored in the structural carbohydrates of plant cell
walls (Hungate, 1966; Janis, 1976; McBee, 1977; Van Soest, 1982; Owens and
Goetsch, 1988; Flint, 1997; Russell and Rychlik, 2001). Symbiotic microbes
colonize either the pregastric (foregut fermentors) or lower gut (hindgut fermentors)
portions of the digestive tract, which become capacious chambers (Janis, 1976;
McBee, 1977; Hume and Sakaguchi, 1991). Independently of their location, these
chambers are filled with large quantities of plant organic matter characterized by
long residence time (up to 47 h), high but constant temperature (~+40°C) and low
(−350 mV) redox potential (Clarke, 1977; Van Soest, 1982). Such conditions allow
anaerobic microorganisms to densely colonize this habitat.
Ruminants are the most specialized mammalian herbivores (McBee, 1977) and
the most dependent on the symbionts harboured in the rumen. The rumen is the largest
portion of the complex stomach and also of the digestive tract in adult ruminants
(Warner and Flatt, 1965; Lyford, 1988). Its primary function in ruminant ancestors
was, perhaps, to store the ingested plant feed (Janis, 1976). However, it has evolved
into the almost ideal fermentation chamber (Russell and Rychlik, 2001). The large
quantities of plant material filling the rumen and the very dense (over 1010 cells/g)
microbial population colonizing this habitat allow ruminants to meet up to 80% of
their energy requirements from the end products of the carbohydrate fermentation
(Owens and Goetsch, 1988; Russell and Rychlik, 2001).
The microbial pathways involved in the fermentation of structural carbohydrates
are similar in all herbivore mammals (Janis, 1976). In ruminants, fibrolytic microbes
digest cellulose and hemicellulose to soluble oligosaccharides that can also be
utilized by non-fibrolytic species (Flint and Forsberg, 1995). End products of sugar
fermentation are released from the cells of microbes mainly as acetic acid, propionic
acid and butyric acid. They are absorbed into the blood via the fermenting chamber
epithelium, transported to the tissues and cells of the animal body and used there in
the ATP generation processes, gluconeogenesis and milk fat synthesis (Van Soest,
1982; Fahey and Berger, 1988).
56 T. Michal̄owski
The beneficial role of the ruminal microbes results not only from providing the
host animals with energy yielding products. It has also been reported that over 90%
of the amino acids reaching the duodenum can originate from the microbial protein
synthesized in the rumen (Russell and Rychlik, 2001). Microbial protein synthesis
and utilization by the host is, however, a complex problem affected by different
factors. Two processes should at least be distinguished, gross and net synthesis. The
latter can be interpreted as a microbial protein or N incorporated into the cellular
protein of microbes reaching the duodenum and is the true measure of microbes as
a source of amino acids to ruminants (Demeyer and Van Nevel, 1986).
3. POSTNATAL GROWTH AND FUNCTIONAL MATURATION

OF THE RUMEN
Ruminants are characterized by the presence of a complex stomach consisting of
four chambers: the reticulum, rumen, omasum and abomasum (Hungate, 1966).
Only the last chamber, the abomasum, is lined with glandular mucosa and is
capable of producing digestive enzymes. The three other chambers are lined with
non-glandular mucosa (Hofmann, 1988) and fail to produce hydrolases. All three of
these compartments are termed forestomachs. In adult animals, the rumen is the
largest chamber of the complex stomach. It is functionally integrated with a much
smaller reticulum and they are often considered as a common compartment, i.e.
the reticulorumen (Hungate, 1966). The weight of the reticulorumen content can
exceed 100 kg in cattle and 15 kg in sheep. It contributes to 9–13% of total
body weight and to over 70% of the digesta weight in the digestive tract (Van
Soest, 1982).
In newborn calves and lambs, the abomasum is the largest and functionally most
developed organ whereas the rumen is small and flaccid and the papillae are
only rudimentary (Hofmann, 1988; Lyford, 1988). Rapid growth of the rumen is
observed by 8 weeks of age with a maximum between 4 and 8 weeks. During this
period the rumen approaches adult proportions and becomes a capacious chamber
(Lyford, 1988). The relative weight of content filling the rumen of 8-week-old lambs
and calves is still less than in adult ruminants (6.8% vs 9–13%) but at this age they
become dependent on fermentation products for maintenance and growth (Warner
and Flatt, 1965; Van Soest, 1982; Lyford, 1988). This suggests that the rumen
ecosystem can already function in 2-month-old domestic ruminants. In newborn
animals, the rumen mucosa is also poorly developed and its absorptive and meta-
bolic abilities are very low (McGilliard et al., 1965). These abilities increase during
the first weeks of postnatal life. Solid food and end products of carbohydrate
fermentation are necessary to accelerate the growth and functional development of
the rumen (Warner and Flatt, 1965). It was found that hay is a stimulating factor of
rumen tissue growth, while concentrates stimulate the growth of rumen papillae
(Lyford, 1988; Swan and Groenewald, 2000). The presence of volatile fatty acids
(VFA) is necessary to accelerate the functional development of the rumen mucosa

as measured by VFA absorption rate, rate of glucose and butyrate oxidation, or rate
of acetoacetate and lactate production (McGilliard et al., 1965; Lane and Jesse,
1997). More recently published results suggest that exposure of the rumen mucosa
to VFA at an early stage of development is required to induce the genes responsible
for metabolic development of the rumen (Lane et al., 2000). It is obvious that
microorganisms are the only VFA producers in the rumen (Hungate, 1966; Van
Soest, 1982). This shows the significant role of microbes in the functional matura-
tion of the rumen.
4. RUMEN PROTOZOA
The findings presented above show that ruminants depend on the rumen microbiota
starting from the early hours of their postnatal life. Ruminal microorganisms belong
to three different groups, i.e. bacteria, fungi and protozoa. Bacteria are most numer-
ous in the rumen. They belong also to the most intensively studied organisms
and there are excellent articles summarizing progress in the rumen bacteriology
(see Stewart et al., 1997, for review). Ruminal fungi belong to the class
Chytridiomycetales and family Neocallimastigaceae. Five genera and more than 20
species have been identified to date (Theodorou et al., 1996; Orpin and Joblin,
1997). The importance of fungi to the host results from their ability to degrade
lignocellulosic plant material. The relation between this group of microbes and
young ruminants is not well known.
Rumen protozoa are principally ciliates representing two morphologically and
physiologically different groups, i.e. Entodiniomorphids (according to older litera-
ture the Oligotrich or Spirotrich protozoa) and holotrichs. According to Williams
and Coleman (1992) and references therein both groups belong to the same subclass
Trichostomatia (Bütschli, 1889) and two different orders: Entodiniomorphida
(Reichenow in Doflein and Reichenow, 1929) and Vestibuliferida (Puytorac et al.,
1974). Ruminal entodiniomorphs (fig. 1) belong to the family Ophryoscolecidae.
Dogiel (1927) distinguished five genera among the ophryoscolecid ciliates:
Entodinium, Diplodinium, Epidinium, Ophryoscolex and Opistotrichum.
Additionally to this, the genus Diplodinium has been divided into four subgenera:
Anoplodinium, Eudiplodinium, Polyplastron and Ostracodinium. In fact, the genera
Cunhaja and Caloscolex belong also to the family Ophryoscolecidae (Dogiel,
1927). However, they have not been found in ruminants. Following subsequent
revisions summarized by Williams and Coleman (1992) particular genera have
been raised to the rank of subfamilies, i.e. Entodiniinae, Diplodiniinae, Epidiniinae,
Opistotrichinae and Ophryoscolecinae, and the subfamily Diplodiniinae has been
divided into 10 genera: Eodinium, Diplodinium, Eudiplodinium, Eremoplastron,
Ostracodinium, Metadinium, Diploplastron, Elytroplastron, Enoploplastron
and Polyplastron. According to the same revisions the subfamily Epidiniinae
58 T. Michal̄owski
Fig. 1. Microphotographs of some common

Entodiniomorphid ciliates. 1. Entodinium
caudatum; 2. Eudiplodinium maggii;
3. Epidinium ecaudatum f. caudatum;
4. Ophryoscolex caudatus.
comprises the genera Epidinium and Epiplastron, whereas the other subfamilies are
represented by single genera, i.e. Entodinium, Ophryoscolex and Opistotrichum. In
contrast to this Imai (1998) distinguished only three subfamilies: Entodiniinae,
Diplodiniinae and Ophryoscolecinae which have been already postulated earlier by
Lubinsky (1957b). The first two families do not differ from those mentioned above,
while the last comprises all five genera from the subfamilies Epidiniinae,
Opisthotrichinae, Ophryoscolcinae and Caloscolecinae.
The taxonomy of the second group is also not quite clear. According to Levine
et al. (1980) the ciliates routinely termed holotrichs belong to the subclass
Vestibuliferia and Gymnostomata, orders Trichostomatida and Prostomatida.
However, Lee et al. (1985) included them in the subclass Trichostomatia, orders
Vestibuliferida and Entodiniomorphida. The species most often present in the rumen
and also the best known belong to the family Isotrichidae, order Vestibuloferida (fig. 2).
Taxonomy of rumen ciliates is based on the morphology of their cells. Over 250
species belong to the family Ophryoscolecidae. They are the most numerous of all
protozoa in the rumen. Their numbers often exceed 106 cells/g rumen digesta. The
ruminal ophryoscolecids are characterized by a rigid pellicle and ciliature reduced
to only adoral (Entodinium spp.) or adoral and dorsal ciliary bands (the remaining
genera). Other organella of taxonomic significance are skeletal plates (not present in
Fig. 2. Microphotographs of the most

common species of the ruminal holotrichs.
1. Dasytricha ruminantium; 2. Isotricha
prostoma; 3. Isotricha intestinalis.
Entodinium, Eodinium and Diplodinium spp.), the number and positions of vacuoles,
the shape of the macronucleus and position of both the macro- and micronucleus as
well as the presence of spines and lobes and even the ciliate cell dimensions (to study
this problem in detail see Dogiel, 1927; Kofoid and MacLennan, 1930, 1932, 1933;
Lubinsky, 1957a,b; Noirot-Timothée, 1960; Latteur, 1969, 1970). It is noteworthy
that many taxonomists believe that ciliates classified as different species are in fact
simply different forms of the same species. This concern is especially relevant to the
small Entodinia (Latteur, 1968). The author of this chapter agrees with this opinion
because considerable differences in cell morphology can be observed between indi-
viduals of the same clone (unpublished). These observations need to be confirmed
by molecular studies such as 18S ribosomal gene sequencing. Initial sequences
have been reported from some species of ruminal entodiniomorphs, i.e. Entodinium
simplex, Entodinium caudatum, Eudiplodinium maggii, Polyplastron multivesiculatum,
Epidinium ecaudatum and Ophryoscolex purkynjei (Embley et al., 1995; Wright
and Lynn, 1997; Wright et al., 1997) and suggest that rumen protozoa represent a
monophyletic group.
In contrast to the entodiniomorphs, the cells of holotrich ciliates are flexible and
the ciliature of the most common species is almost complete (fig. 2). The concen-
tration of holotrichs in the rumen is in the range of 104/ml rumen fluid.
60 T. Michal̄owski
5. ROLE OF PROTOZOA IN FOOD CONVERSION

Ciliates from the family Ophryoscolecidae prefer particulate matter as food, while sol-
uble substances are rather poorly utilized (Michalowski, 1989; Williams, 1989). The
ciliates Eudiplodinium maggii, Polyplastron multivesiculatum, Epidinium ecaudatum
and some other large ophryoscolecids are able to digest and metabolize cellulose
(Coleman, 1985, 1986; Bonhomme et al., 1986; Dehority, 1993; Michalowski, 1997).
It was also found that the ciliates Eudiplodinium maggii and Epidinium ecaudatum syn-
thesize both β-endoglucanases and β-endoxylanases (Michalowski, 1997; Michalowski
et al., 2001b). The genes encoding for β-endoglucanase and xylanase were also cloned
from Polyplastron multivesiculatum and Epidinium ecaudatum (Selinger et al., 1996;
Devillard et al., 2000). These findings confirm the direct participation of some large
ophryoscolecids in fibre degradation in the rumen. Thus it is not surprising that 50–70%
of the fibrolytic activity in the rumen results from the presence of ciliates (Coleman,
1986; Michalowski and Harmeyer, 1998). It is also well known that Entodinia and
other small entodiniomorphid species prefer starch (Williams, 1989). Engulfed carbo-
hydrates are digested and fermented while acetic acid, propionic acid and butyric acid
are released as end products (Williams and Coleman, 1992; Michalowski, 1997). It was
found that 50% of the VFA produced in the rumen of sheep fed a hay-concentrate diet
was of ciliate origin and that ophryoscolecids were dominating in the rumen of exam-
ined animals (Michalowski, 1987). Thus entodiniomorphid protozoa can play an
important role in providing the host with energy stored in carbohydrates including
structural polysaccharides. In contrast with entodiniomorphs, rumen holotrichs do not
ingest fibrous material and readily utilize soluble compounds (Williams, 1989). Lactic
acid is an important end product of carbohydrate metabolism in these protozoa (Prins
and Van Hoven, 1977; Van Hoven and Prins, 1977).
The ciliates engulf and digest ruminal bacteria (Coleman, 1989). Owing to this,
the protozoa negatively influence the microbial protein supply at the duodenum of
the host. They also enhance ammonia production in the rumen. This sometimes
results in diminishing weight gain in ruminants maintained on a low quality diet
(Jouany et al., 1988). The reduction in bacterial N supply can only partially be com-
pensated by protozoal protein because ciliates are selectively retained in the rumen
(Weller and Pigrim, 1974; Michalowski et al., 1986). Thus, the importance of pro-
tozoa to ruminants seems to result from their digesting and fermenting activities in
the rumen rather than from their role as an amino acid source (John and Ulyatt,
1984; Michalowski, 1990).
6. ESTABLISHMENT OF CILIATES IN THE RUMEN OF

YOUNG RUMINANTS
Ruminants are born with a sterile digestive tract but invasion of microorganisms
begins immediately after birth. Colonization of the rumen by bacteria begins from
association of microaerophilic and ureolytic species with the rumen epithelium and
this is followed by initial development of the cellulolytic consortia as early as 2–4 days
after birth (Cheng et al., 1991). However, Fonty et al. (1984) observed very low
concentration of cellulolytic bacteria in 8-day-old lambs. According to Cheng et al.
(1991) rumen fungi were observed in the rumen on day 8–10 after birth and were
followed by protozoa. This shows that ciliates appear in the rumen as the last
member of the microbial ecosystem.
Rumen ciliates produce neither resistance forms nor cysts (Dehority and Orpin,
1997) and Becker and Hsiung demonstrated as early as 1929 that direct contact is
necessary to transfer these organisms between the host animals. It is obvious that
mouth-to-mouth contact is more probably between newborn ruminants and their
dams. However, successful transmission of protozoa to the rumen of lambs and/or
calves is rarely possible during the first 1–2 days of their postnatal life. Thus they can
remain ciliate-free for a long period if they become separated from their dams within
the mentioned period (Bryant et al., 1958; Eadie and Gill, 1971). Development of
microfauna in the rumen of young domestic ruminants as a result of natural animal
to animal transmission has been examined by several authors during the past 50 years
(table 1). Lengemann and Allen (1959) observed protozoa in the rumen of calves
within the first week after birth, whereas in flock reared lambs they appeared by 7–20
days (Fonty et al., 1984, 1988). On the other hand Eadie (1962) found quite definite
fauna in 21-day-old lambs, while Naga et al. (1969) and Fonty et al. (1984, 1988)
have reported that a 60- to 150-day period was necessary to establish a mixed type of
rumen fauna at the adult animal level in lambs as well as buffalo and cow calves.
Few studies have investigated the development of populations of particular
genera (table 2). The results obtained showed that Entodinia colonized the rumen
at first, while the establishment of higher ophryoscolcesids varied in relation to the
host and management system. In general, the ciliates from the genera Diplodinium,
Eudiplodinium, Ostracodinium and Polypalstron followed Entodinia while
Elytroplastron, Enoploplastron and Ophryoscolex became established distinctly later.
The establishment of holotrichs varied also in relation to the mentioned factors.
Table 1. Appearance of ciliates and establishment of mixed type fauna in the rumen of growing
domestic ruminants
Days, weeks or months after birth
Animals Appearance Establishment References
Cow calves Up to 7 days 4–11 weeks Lengemann and

Allen, 1959
29–132 days 80–150 days Naga et al., 1969
Buffalo calves 13–46 days 40–60 days
Lambs 9–14 days 21 days Eadie, 1962
15–20 days 2 months Fonty et al., 1984
7–50 days up to 100 days Fonty et al., 1988
62 T. Michal̄owski
Table 2. Establishment of ciliates from different genera in the rumen of calves and lambs (days
after birth)
Naga et al., 1969 Fonty et al., 1988
Cow calves Buffalo calves Lambs
Ciliate genera A B A B Flock reared Dam reared
Entodinium 29 46 13 18 7–10 13–20

Diplodinium 30 70 18 37 – –
Eudiplodinium 30 60 22 18 10–20 30
Ostracodinium 30 70 27 35 – –
Elytroplastron 68 132 39 46 – –
Enoploplastron – – – – – 100
Polyplastron – – – – 10–20 30
Epidinium – – – – 20 –
Ophryoscolex – – – – – 50
Isotricha 30 46 28 33 50 –
Dasytricha 47 130 22 27 – –
A = early weaned calves. B = late weaned calves.
For example, Isotricha spp. appeared as the last ciliate in flock reared lambs but they
followed Entodinia in early-weaned cow calves. It was also reported that an estab-
lished population of Polyplastron multivesiculatum disappeared after existence in
the rumen for several months owing to undefined causes (Fonty et al., 1984, 1988).
Such a phenomenon was also observed in the case of the same species as well as
ciliates from the genus Ophryoscolex inoculated into the rumen of ciliate-free
buffalo calves and lambs (Eadie, 1967; Naga et al., 1969).
Development of the ciliate fauna was also examined following the experimental
inoculation of young ruminants. Pounden and Hibbs (1948, 1949) observed numerous
ciliates in the rumen of 3- and 6-week-old calves. The animals were inoculated by pass-
ing pieces of cuds taken from cows into the mouths of calves on the 5th, 10th, 15th and
21st day after birth. Bryant et al. (1958) kept ciliate-free calves 13–15 weeks of age
together with faunated mature animals and this resulted in transmission of ciliates from
adult ruminants to the young. Entodinia were observed in 17–20-week-old calves and
were followed by ciliates from the genus Diplodinium and family Isotrichae, which
were found in 27- and 33–37-week-old animals, respectively. A similar sequence in the
appearance of ciliates was found in calves inoculated with the samples of rumen
content from a mature cow (Bryant and Small, 1960). Abou Akkada and El-Shazly
(1964) inoculated 5-week-old lambs with the rumen fluid from a mature sheep.
Entodinium and Isotricha spp. were the first ciliates to develop in almost all of
11 lambs. Ophryoscolex, Polyplastron and Diplodinium spp. were observed in small or
appreciable numbers not earlier than on day 11 after inoculation. The authors noted
difficulties in developing a population of Dasytricha ruminantium, which became
established in only three of 11 animals examined. Borhami et al. (1967) inoculated 2–3-
week-old buffalo calves by introducing samples of fresh rumen content taken from
mature faunated animals. The authors observed Entodinia and Diplodinia as quickly as
4 days after inoculation. Eudiplodinium and Isotricha spp. were found 2 days later,
whereas small or moderate numbers of Metadinium spp. were identified as late as
7 months after inoculation and only in three of 11 calves tested. Similarly Eadie (1967)
observed a very long lag phase between the inoculation and appearance of ciliates from
the genus Ophryoscolex in the rumen of lambs and kids. Naga et al. (1969) inoculated
newly born buffalo and cow calves with rumen contents of adult buffalo, cow
and sheep. Ciliates from the genus Entodinium were found first (4–40 days after
inoculation), while Diplodinium, Ostracodinium and Dasytricha last (68–87 days after
transferring). However, the time of appearance of protozoa depended on the origin of
the inoculum (results are summarized in table 3).
It can be concluded on the basis of the cited findings that independent of the form
of inoculation Entodinia becomes established first, often followed by other ento-
diniomorphids and/or large holotrichs. It is noteworthy that Entodinia appear to be
the first of the ciliates that appeared in ruminant ancestors (Lubinsky, 1957a,b).
7. FACTORS AFFECTING ESTABLISHMENT OF CILIATES

IN THE RUMEN
Different factors are considered to affect colonization of the rumen by protozoa.
Williams and Dinusson (1972) showed that the number of ciliates transferred from
one animal to another influences the length of the period between the inoculation
and establishment of the ciliate fauna in calves. Diet is also a factor affecting the
time taken for a population to develop. For example, no increase in ciliate numbers
was observed for a period of over 11 weeks when calves were fed a milk-containing
diet (Lengemann and Allen, 1959). On the other hand, a milk diet inhibited the
anatomical growth and functional development of the rumen (McGilliard et al.,
1965; Warner and Flatt, 1965). In contrast to that, Eadie (1962) observed a positive
effect of milk on the development of ciliate fauna even in the rumen of the early-
weaned calves. According to the same author, the acidity of the rumen digesta is a
crucial factor affecting the establishment of ciliates in young ruminants. At a
pH below 6.0 only a small number of ciliates could be detected. At a little above pH
6.0 species from the genus Entodinium were most frequent while at pH above 6.5,
mixed fauna including populations of large ophryoscolecids became established. If
the pH in the rumen of calves with developed mixed ciliate fauna dropped, large
ophryoscolecids disappeared first whereas Entodinia and Isotricha spp. were the
last. It is well documented that feeding concentrate resulted in a drop in pH, which
is accompanied by the disappearance of ciliates from the rumen. In contrast
to concentrate, roughage feed favoured a pH increase and thus stimulated both
the development and establishment of adult-type ciliate fauna in calves and
Table 3. Appearance of ciliates in the rumen of growing ciliate-free animals following experimental inoculation
References
Item 5 6 2 3 1 4 4 7 7
Animals Cow calves Cow calves Cow calves Cow calves Buffalo calves Lambs Kids Buffalo calves Cow calves
Inoculation age 5,10,15,21d 1,3,6w 13–15w 2–3w 18,26,49w 13–18w Newborn

Appearance of:
Undefined protozoa 3,6w
Entodinium 3–6w 17–20w >4d 7a,7b,7cd 40a,4b,4cd
Diplodinium 3–9w 27w >4–6d 14,19,7d 87,38,8d
Eudiplodinium >6d 14,×,17d 49,6d
Metadinium >28w
Ostracodinium 39,×,20d 87d
Polyplastron 7d* 8d*
Elytroplastron 58d
Ophryoscolex 51,61,104w 34,51w
Isotricha >6d 28,42,20d 42,22,8d
Dasytricha 6–9w 33–37w 59,7d 68,8d
d = days. w = weeks.
a,b,c = inoculated with rumen content of cow, buffalo and sheep, respectively.
* = disappeared after a few days.

× = not present in inoculum.
References: 1 = Borhami et al. (1967), 2 = Bryant and Small (1960), 3 = Bryant et al. (1958), 4 = Eadie (1967), 5 = Pounden and Hibbs (1948), 6 = Pounden and
Hibbs (1949), 7 = Naga et al. (1969).
lambs (Eadie, 1962). Other factors are the weaning systems and to some extent host
specificity (Naga et al., 1969). The development of ruminal bacteria cannot there-
fore be ruled out. Some relations between establishment of the bacterial flora and cil-
iate fauna in meroxenic as well as conventional and conventionalized lambs were
studied by Fonty et al. (1983, 1984, 1988). The authors found that a period longer
than 30 days was necessary to establish protozoa in germ-free reared lambs.
Conversely, ciliates required only 4 days to become established when the rumen was
colonized by bacteria prior to the isolation of the lambs. They concluded that a well-
developed and complex bacterial flora is necessary to establish protozoa in the
rumen. Indeed bacteria are necessary in the rumen at least to produce the environ-
mental conditions such as appropriate acidity and redox potential (Fonty et al.,
1983). Bacteria seem also to be the main source of amino acids for protozoa
(Coleman, 1989). On the other hand, Lengemann and Allen (1959) found that
addition of aureomycin to the diet favoured the establishment of protozoal fauna
in calves.
8. EFFECT OF PROTOZOA ON RUMEN METABOLISM

IN CALVES AND LAMBS
The presence of ciliates in the rumen of adult ruminants enhances the deamination
processes and results in an increase in ammonia concentrations in the rumen fluid
(Jouany et al., 1988). A similar effect was observed in lambs and calves independ-
ently of age or body weight (table 4). However, a tendency to reverse the relationship
was found by Demeyer et al. (1982) in lambs fed a diet based on alkali-treated straw.
The author is of the opinion that large doses of urea supplemented to the diets were
responsible for the observed enhancement in the level of ammonia in the rumen of
experimental animals.
In the majority of performed experiments the presence of ciliates in the rumen of
calves and lambs resulted in higher concentration of volatile fatty acids (VFA)
compared with ciliate-free animals (table 5). This suggests that ciliates positively affect
the metabolism of dietary carbohydrates. Establishment of protozoa also resulted in a
decrease in or at least a tendency to decrease the proportion of acetate with a simulta-
neous increase in the proportion of propionate. Conversely, the proportion of butyric
acid decreased or increased in relation to the authors and experiments. For example,
butyrate decreased to an undetectable level in lambs faunated with mixed-type ciliate
fauna (Abou Akkada and El-Shazly, 1964), whereas it increased by over 40% follow-
ing colonization of the rumen of ciliate-free lambs by Diplodinium sp. (Christiansen
et al., 1965). A similar reaction followed the establishment of Eudiplodinium maggii in
the rumen of defaunated adult sheep (Michalowski et al., 2001a).
The cited results show that independently of diet and age colonization of the
rumen habitat in growing calves and lambs by protozoa led to both an increase in
VFA concentration and a shift in the pattern of carbohydrate metabolism. It has been
already described earlier that volatile fatty acids have an important role in accelerating
Table 4. The effect of absence (–P) or presence (+P) of ciliates on ammonia concentration in the rumen fluid of growing domestic ruminants
Ammonia mg/100 ml
Animal Age or weight Diet –P +P Significance References
Lambs 4–5 months Cotton seed, rice bran mixture 2–3 3.5–7 n.d Abou Akkada and El-Shazly, 1964
5–8 months Concentrate mixture 2–6.5 1.5–11
by 24 weeks Not given 4.5 8.2 n.d Abou Akkada, 1965
29–38 kg Alfalfa hay, concentrate mixture 6.4 9.2–12.2 n.d Christiansen et al., 1965
Alfalfa hay 80%, concentrates 20% 6.5 9.6 n.d Luther et al., 1966
25 kg Alfalfa hay 20%, concentrates 80% 6.0 14.4
Alfalfa hay, concentrate 3.5–8.3 8–16 0.01 Klopfenstein et al., 1966
25–27 kg Concentrate, urea 2–8 4–15 0.01
26 weeks Dried grass 8.4–9.6 3.3–19.7 0.001 Eadie and Gill, 1971
Beet pulp, molasses, urea 22.1 29.6 0.01
20 kg Alkali-treated straw, molasses, urea 27.0 22.7 ns Demeyer et al., 1982
As above + tapioca 17.7 15.2 ns
14.4–21.9 kg Molasses, beef promol 10.9 13.3 ns Van Nevel et al., 1985
Buffalo calves Milk 10–28 10–16
2–18 weeks Milk, concentrate, molasses, rice bran 11–20 10–28 n.d Borhami et al., 1967
Milk, concentrate, molasses 7–18 11–29
n.d = not determined. ns = not significant.

Table 5. Total concentration (mmol/100 ml) of volatile fatty acids and molar proportions of
individual acids in the rumen of ciliate-free (–P) and faunated (+P) growing domestic ruminants
Total VFA Acetate Propionate Butyrate

Animals References
–P +P –P +P –P +P –P +P
Lambs 4–5 3–6.5 77.5 67.5 14.9 32.5 7.6 0 Abou Akkada and
3–8 5–9.5 75.9 68.5 21.1 24.9 1.2 0 El-Shazly, 1964
7.1 8.5 not determined Abou Akkada, 1965
6.3 7.6 62.3 54.8 21.4 24.6 15.2 17.4 Christiansen et al., 1965
7.0 7.9 50.8 47.8 30.8 30.7 15.5 18.1 Luther et al., 1966
7.4 7.4 38.7 38.7 26.0 34.0 30.5 23.8
5.7 7.3 77.3 71.0 14.6 19.9 7.8 9.1 Eadie and Gill, 1971
10.6 9.7 66.1 67.6 22.9 24.9 10.2 7.0 Demeyer et al., 1982
8.5 9.0 76.4 73.4 17.1 19.1 6.5 7.5
6.3 8.0 58.8 60.5 29.4 31.8 10.8 8.0
7.5 7.8 58.0 56.8 18.9 25.8 22.0 17.2 Van Nevel et al., 1985
Buffalo 2.5–5.5 1.9–6.5
calves 2.8–4.7 3.6–7.2 not determined Borhami et al., 1967
2.4–5.5 2.5–6.5
3.5–6.0 2.7–8.3
Diet and age or weight of animals are given in table 4.
the metabolic maturation of the rumen epithelium. The effect of protozoa on VFA
production (Michalowski, 1987) suggests that ciliates can be considered as a factor
positively affecting the functional maturation of the mucosa of the rumen.
9. INFLUENCE OF PROTOZOA ON GROWTH OF

YOUNG RUMINANTS
The effect of the presence of the ciliates in the rumen on the growth of calves and
lambs was examined by several authors (table 6). A number of the investigations
observed either a positive or a neutral influence of ciliates on weight gain of ani-
mals. However, a negative effect was also found. For example Bird et al. (1979)
found a negative growth rate in faunated lambs fed a diet composed of oaten chaff,
sugar, urea and minerals. It was also found that a fish meal supplement abolished
this effect independently of the supplementation level. Moreover, the growth rate of
faunated animals tended to be higher than that of ciliate-free when fish meal was
added at a proportion of 102 g/kg diet. Similarly Demeyer et al. (1982) and Van Nevel
et al. (1985) observed that the effect of ciliates on the growth rate of lambs depended
on the diet. Ciliates diminished the growth rate of young animals when their diet
was based on molasses and straw. Partial replacement of alkali-treated straw with
tapioca was the factor in increasing the daily gain of the faunated animals and
improved the food conversion efficiency.
The nutritional behaviour of ciliates and the chemical composition of feed appear to
be very significant factors when the effect of protozoa on the growth rate of ruminants
Table 6. The effect of absence (–P) or presence (+P) of ciliates in the rumen of growing domestic ruminants on live weight gain (g/day), daily feed intake (g) and feed
conversion (g/g gain)
Weight gain Feed intake Feed conversion

Animals Age or weight Diet –P +P –P +P –P +P References
Lambs 48–51 days Bereseem hay, concentrate mixture 92 122.8 not determined Abou Akkada and
El-Shazly, 1964
up to 24 weeks Concentrate? 91 116 not determined 12.3a 128.3 Abou Akkada, 1965
31 kg Alfalfa hay, concentrate mixture 191 136.6 not determined 1.9 73 Christiansen et al., 1965
7–13 weeks 221 95.7 270 100 not determined
14–21 weeks Dried grass (ad libitum) 227 121.8 843 106.8 not determined
Eadie and Gill, 1971
22–29 weeks 144 98.0 1171 103.7 not determined
36–59 weeks Dried grass, concentrates (restricted) 83 86.2 1014 98.6 not determined
21–21.7 kg Oaten chaff, sugar, urea 37 –31.1 454 82.2 37.3 –
21.2–22.4 kg As above + fish meal (3%) 133 56.4 693 93.1 5.3 160.4 Bird et al., 1979
21.8 kg As above + fish meal (7%) 159 91.8 685 96.4 4.4 106.8
22.5 kg As above + fish meal (10.2%) 154 116.2 735 101.4 49 85.7
Beet pulp, molasses, urea 213 84.9 857 101.6 4.1 119.8
20 kg Alkali-treated straw, molasses, urea 140 72.9 964 91.1 7.2 123.8 Demeyer et al., 1982
As above + tapioca 192 124.5 1895 93.2 10.2 74.2
16 kg Oaten chaff, sugar, urea 133.5 91.4 not determined 6.8 107.4 Bird and Leng, 1984
14.4–21.9 kg Molasses, beef promol (0–5 weeks) 125 70.4 1085 93.5 8.5 131.5
As above (5–9 weeks) 99 110.1 1189 98.1 11.7 83.8 Van Nevel et al., 1985
Cow 3–6 weeks Colostrum up to day 4, then hay 170 100 not determined Pounden and Hibbs, 1948
calves 66 days Milk + pasture 26%M 29%M not determined Pounden and Hibbs, 1949
up to 8 months Milk, alfalfa hay, grains 104 102.8 not determined Pounden and Hibbs, 1950
up to 17 weeks Hay, grains 520 66 1648 61.5 not determined Bryant and Small, 1960
Buffalo 2–18 weeks Milk 330 115.2 1017 100 3.1 87.1 Borhami et al., 1967
calves Milk, concentrate, molasses, rice bran 310 129.0 140.8 103.6 4.5 82.2
As above with different feeding periods 240 150.0 1250 108.6 5.2 71.2
Milk, concentrate, molasses 320 128.1 1450 91.4 4.6 69.6
Values concerning faunated animals are expressed as a percentage of those of the ciliate-free.
a = N retention (g/day).
%M = gain per month.

is considered. Ophryoscolecid ciliates dominate in the rumen. They readily ingest

starch and grow well in vitro on insoluble protein (Michalowski, 1989; Williams, 1989)
whereas straw, soluble sugars and urea are purely utilized. Engulfment and digestion of
bacteria presumably increases when the quantity of preferred nutrients is insufficient to
satisfy the nutritional requirement of protozoa. This results in diminishing of both the
microbial protein flow to the duodenum and the growth rate of the host. Conversely,
diets supporting the development of the ciliate population improve the gain of rumi-
nants and their food conversion efficiency (Abou Akkada and El-Shazly, 1964;
Christiansen et al., 1965; Borhami et al., 1967; Jouany et al., 1988).
Another factor affecting the role of ciliates appears to be the age of the young
growing ruminants. Eadie and Gill (1971) observed a positive effect of ciliates on
the growth of only 14–21-week-old lambs fed dried grass. No significant influence
was found in either younger or older animals obtaining the same feed.
10. EFFECT OF CILIATES ON BLOOD COMPONENTS

There are few studies on the blood components in faunated and ciliate-free lambs
and/or calves. Abou Akkada (1965) found that reducing sugars, non-protein N,
ammonia N and urea N were lower while haemoglobin and protein N were higher
in faunated lambs when compared with ciliate-free (table 7). Borhami et al. (1967)
measured the glucose concentration as well as the ammonia and urea nitrogen in
blood samples of buffalo calves starting from the second week after birth. They
observed a continuous decrease in blood sugar from 95–99 to 49–83 mg/100 ml
during the next 6 weeks in ciliate-free and 16 weeks in faunated calves. The values
Table 7. Some of the blood components in ciliate-free (–P) and faunated (+P) growing domestic
ruminants
Animals
Item –P +P References
Reducing sugars, mg/100 ml 84.2 60.8 Abou Akkada, 1965

68–73 46–67 Borhami et al., 1967
Haemoglobin, g/100 ml 11.2 13.3 Abou Akkada, 1965
Protein, mg N/100 ml 2.32 2.69 Abou Akkada, 1965
Free amino acids, mg/100 ml 14.2 10.2 Klopfenstein et al., 1966
Ammonia, mg N/100 ml 0.9 0.35 Abou Akkada, 1965
Urea, mg N/100 ml 15.6 10.1 Abou Akkada, 1965
6–8.5a 9–12.5a Klopfenstein et al., 1966
3.5–5.5b 3–4.5b Klopfenstein et al., 1966
Oleic acid, % 18.5 23.1 Klopfenstein et al., 1966
Linoleic acid, % 32.4 29.6 Klopfenstein et al., 1966
Other long chain acids, % 49.1 47.3 Klopfenstein et al., 1966
a wethers fed hay-concentrate diet.

b wethers fed concentrate diet.
70 T. Michal̄owski
noted at the end of the experiment showed, however, a similar relationship to those
cited above. On the other hand, no differences in ammonia and urea N levels were
observed there in relation to the presence or absence of ciliates. Blood plasma lipids
were examined by Klopfenstein et al. (1966). The authors found that faunation of
the rumen of 5-month-old wethers resulted in an increase in concentration of oleic
acid and in a decrease in that of linoleic acid. On the other hand, the plasma
concentration of urea was related to the diet rather than to the protozoa.
11. CONCLUSIONS
The rumen in newborn ruminants is anatomically and functionally immature. Its
postnatal growth is accompanied by development of the microbial ecosystem inside
this organ. Colonization of the rumen by protozoa can begin within the first week of
life of the host, but establishment of the adult type ciliate fauna followed rather its
anatomical and functional maturation. Development and maturation of ruminal
fauna depends on several factors among which the age of calves and lambs, direct
contact with faunated animals, access to solid food and numerous and complex
bacterial flora, responsible for appropriate pH and redox potential of the rumen
digesta, are presumably the most important. The role of ciliates in rumen metabo-
lism and in the performance of growing ruminants seems to depend on diet and age.

The literature concerning the appearance and establishment of the ciliate fauna in
the rumen of young domestic ruminants is not comprehensive and in the majority of
cases relatively old. On the other hand the objectives of these studies and methods
used by the authors were different. Thus the results were sometimes hardly compa-
rable. Because of this further studies of a comparative character would be of value.
Of importance would be experiments similar to the excellent studies of Dr Margaret
Eadie (Eadie, 1962, 1967) from the Rowett Research Institute (Aberdeen, UK) and
Dr Gerard Fonty with co-workers (Fonty et al., 1984, 1988) from INRA (Clermont-
Ferrand, France) to determine the sequence of the appearance and establishment of
ciliates in the rumen of calves, lambs and kids faunated by natural transmission from
their dams. Studies on the role of ciliates in development and functional maturation
of the rumen would also be of value. Jouany et al. (2002) have lately postulated a
possible role of undefined factor(s) of host animal origin which could affect the
establishment of some species of ciliates in the rumen of adult ruminants. Similar
studies on calves and lambs would be of special interest. Rumen protozoa seem also
to be a natural barrier against the pathogens (Newbold et al., 2001) and a factor
diminishing diarrhoea. These findings suggest that studies on the involvement of
ciliates in both the development of the immune system and the function of the lower
portions of gut during early postnatal life of domestic ruminants seem to be an
intriguing future challenge for physiologists and microbiologists.
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Ophryoscolex. Can. J. Zool. 75, 963–970.
Wright, A.-D.G., Dehority, B.A., Lynn, D.H., 1997. Phylogeny of the rumen ciliates Entodinium,
Epidinium and Polyplastron (Litostomata: Entodiniomorphida) inferred from small subunit ribosomal
RNA sequences. J. Euk. Microbiol. 44, 61–67.
4 Microbial ecology of the digestive tract in
reindeer: seasonal changes
S.D. Mathiesena, R.I. Mackieb, A. Aschfalka, E. Ringøa and

M.A. Sundsetc
a
Section of Arctic Veterinary Medicine, Department of Food Safety and Infection
Biology, The Norwegian School of Veterinary Science, NO-9292 Tromsø, Norway
b
Department of Animal Sciences, University of Illinois at Urbana-Champaign,
Urbana, Illinois 61801, USA
c
Department of Arctic Biology and Institute of Medical Biology, University of
Tromsø, NO-9037 Tromsø, Norway
Reindeer populations in the Arctic region are limited by the availability and the
utilization of the diet they eat. Data on seasonal changes in the gastrointestinal micro-
biota were reviewed in reindeer in northern Norway and in Svalbard reindeer.
Digestion in reindeer depends on a highly active anaerobic symbiotic rumen bacterial
population, ciliated protozoa and anaerobic fungi, compartmentalized in the rumen
fluid, plant solid digesta and epithelial mucosa. The distal fermentation chamber also
harbours anaerobic fermentative bacteria. Cultivation studies indicate that the number
and composition of the microorganisms change with the season and chemistry of the
forage consumed. The Svalbard reindeer have large populations of cellulolytic bacte-
ria in their rumen in winter making their digestive system highly suitable for energy
utilization of poor quality food through slow rumen breakdown and fermentation;
rumen cellulolysis, however, is more rapid in Norwegian reindeer in winter than in
Svalbard reindeer. Digestion of plant polysaccharides may be limited by the availabil-
ity of easily digestible energy and non-protein nitrogen available for microbial
synthesis. Reindeer, unlike domestic ruminants, are highly adaptable mixed feeders.
1. INTRODUCTION
This chapter reviews the existing literature on the gastrointestinal microbiota in two
different sub-species of reindeer: Svalbard reindeer (Rangifer tarandus plathyrynchus)
living on the high-Arctic archipelago of Svalbard (74–81°N) and Norwegian reindeer

76 S. D. Mathiesen et al.
Fig. 1. Reindeer appeared 15 million years ago and their circumpolar distribution in the northern
hemisphere currently comprises about 5 million individuals divided between seven sub-subspecies:
Eurasian tundra reindeer/Norwegian reindeer Rangifer tarandus tarandus (A); Eurasian forest reindeer
R. t. fennicus (B); Alaskan caribou R. t. granti (C); Woodland caribou R. t. caribou (D); barren-ground
caribou R. t. groenlandicus (E); Peary caribou R. t. pearyi (F); Svalbard reindeer R. t. platyrhynchus (G).
(Courtesy of Dr Nicholas J.C. Tyler, University of Tromsø, Norway.)
(Rangifer tarandus tarandus) semi-domesticated by the Saami people on mainland

Norway (69°N) (figs 1 and 2). Reindeer have a four-chambered stomach system con-
sisting of the reticulum, rumen, omasum and abomasum (fig. 3), just like other rumi-
nants. No major differences are observed in the gross anatomy of the gastrointestinal
tract in Svalbard reindeer and Norwegian reindeer (Westerling, 1975b; Staaland et al.,
1979; Sørmo, 1998; Utsi, 1998; Sørmo et al., 1999). However, Staaland et al. (1979),
emphasized the importance of the large distal fermentation chamber in mineral
absorption in Svalbard reindeer compared to Norwegian reindeer, as well as their short
intestines (fig. 1).
Microbial ecology of reindeer digestive tract 77
Fig. 2. Reindeer experience large seasonal changes in forage availability and quality. These photographs
show Norwegian reindeer in their natural environment in northern Norway and Svalbard reindeer feeding
on the high-Arctic desert of Svalbard winter and summer. (Photos: S.D. Mathiesen and M.A. Sundset.)
Fig. 3. Both Norwegian reindeer and Svalbard reindeer have been classified as intermediate feeders and no
major differences are observed in the gross anatomy of their gastrointestinal tract, although Svalbard reindeer
have a larger distal fermentation chamber. This figure shows a photograph of the gastrointestinal tract of
Norwegian reindeer. Scale bar = 10 cm. (Photo: S. D. Mathiesen.)
Our knowledge of the rumen metabolism has increased greatly since Aristotle
first described the multiple ruminant stomach (Mason, 1962). However, today’s
understanding of the ruminant gastrointestinal microbial ecosystem is primarily
based on studies of artificially fed domestic sheep and cattle (Hespell et al., 1997),
and only a few limited studies have been conducted on wild ruminants such as mule
deer (Pearson, 1969), elk (McBee et al., 1969), buffalo (Pearson, 1967) and camel
(Hungate et al., 1959). Hence, relatively little is known on wild ruminants and how
their gastrointestinal microbiota has developed through a long history of feeding on
natural pastures. Ruminant evolution began more than 30 million years ago during
the Eocene period under very different climatic conditions. Ruminants such as
moose (Alces alces) and roe deer (Capreolus carpools) belong to an older type of
ruminant, while bovine species first evolved when cellulosic grasses became abun-
dant later in the Miocene (Hofmann, 1989, 1999). Reindeer are classified as highly
adaptable feeders intermediate to roe deer and the bovine species and are one of 180
different ruminant species in the world. According to Randi et al. (1998) reindeer
developed about 15 million years ago. Their circumpolar distribution currently
comprises about 5 million individuals, divided between seven different living
sub-species (fig. 1) and spanning a latitudinal range of 50 −82°N (Banfield, 1961).
Throughout history, global environmental changes have had a powerful influence on
climatic conditions in the Arctic region and have thus limited plant availability and
growth. The last glaciation in northern Europe came to an end 10 000 −20 000 years
ago or even as long as 40 000 years ago on Svalbard which was later occupied by
reindeer (Salvigsen, 1979). Persistent climatic instabilities have always affected
indigenous herbivores, the availability of their forage plants and their ability to
utilize plant carbohydrates and proteins for maintenance energy, growth and repro-
duction. Likewise, growth and survival of reindeer in the circumpolar region is
strongly dependent on seasonal climatic factors. The Svalbard reindeer experience
extreme variations in daylength through the year. From mid-November until
mid-February the light intensity remains below civil twilight, while from April until
mid-August the sun never sets. Norwegian reindeer living in northern Norway expe-
rience two hours twilight daily for two months in mid-winter and an equally long
period of midnight sun in summer. It is assumed that these seasonal changes in
daylength have a substantial influence on the intrinsic seasonal physiology of these
animals, including appetite and reproduction (Stokkan et al., 1994). All northern
ruminants investigated so far show pronounced seasonal changes in appetite and
growth; voluntary food intake and rate of growth being high in summer and low in
winter (Ryg and Jacobsen, 1982; Larsen et al., 1985; Tyler, 1987; Nilssen et al.,
1994; Tyler et al., 1999).
Norwegian reindeer select and eat a variety of plants (table 1), including shrubs
(e.g. Empetrum spp., Loiseleuira procumbers, Vaccinium spp.), birch (Betula spp.),
willow (Salix spp.), grasses (e.g. Poa spp., Deschampsia spp.) and sedges (Carex
spp.) from 37 different plant families. In early spring reindeer also eat different
herbs (Rumex acetosa, Ranuculus repens and Alchemilla subcrenata) and grasses
(Festuca rubra, Poa pratensis, Agrostis capillaris, Deschampsia caespitosa and
Phleum alpinum) along the coast of northern Norway when snow still covers
the mountain vegetation. The metabolic demands of Arctic ruminants are high in
summer when body growth and appetite are high and much reduced in winter
(Nilssen et al., 1984, 1994; Fancy and White, 1985). This seasonal fluctuation in
Table 1. Mean ruminal (% of total) composition of main groups of dietary plants in Norwegian
reindeer and Svalbard reindeer in different seasons (Mathiesen, 1999)
Grasses Woody plants Lichens Mosses
Norwegian reindeer
Summer pasture 65 17 11 4
Winter pasture 21 36 35 7
Svalbard reindeer
Summer pasture 40 44 0 16
Winter pasture 35 55 0 10
growth represents a major adaptation to seasonal variation in the quality and avail-
ability of forage, which strongly influences the gastrointestinal microbiota and
metabolism (Mathiesen, 1999). Svalbard reindeer forage on the tundra or on polar
desert vegetation all year round (table 1). Their diet is generally dominated by
Saxifraga oppositifolia, but grasses are also eaten in both seasons. Also sedges (e.g.
Deschampsia spp., Dupontia spp., Poa spp., Carex spp., Luzula spp.), shrubs (e.g.
Dryas octopetala and Salix polaris), herbs (e.g. Saxifraga spp.) and mosses are
found in the rumen of Svalbard reindeer (Sørmo et al., 1999). Over thousands of
years these reindeer have removed the dietary lichens by grazing and trampling and
these therefore no longer form a significant part of their winter diet. Svalbard rein-
deer mobilize a large proportion of their energy and protein reserves in winter result-
ing in a substantial decline in carcass mass from 72 kg in summer to 46 kg in winter
(Tyler, 1987). However, in free-living Norwegian reindeer the live body mass
decreased from summer (69 kg) to winter (59 kg) but they suffered no net decline
in carcass mass in winter (Mathiesen et al., 2000).
2. RUMEN FERMENTATION AND MICROBIOLOGY

In contrast to domestic ruminants living in more stable nutritional and climatic envi-
ronments, the understanding of the basic digestive physiology of Arctic ungulates
has to be considered in the light of the large seasonal variations outlined above. Very
different gastrointestinal microfloras have evolved in the two reindeer populations
studied − most probably due to differences in environmental conditions between
Svalbard and northern Norway.
The gastrointestinal tract of newborn ruminants is colonized from birth. The
rumen in reindeer contains a complex consortia of microorganisms that live in a
mutualistic relationship with the host (Utsi, 1998; Sørmo, 1998; Mathiesen, 1999;
Olsen, 2000). The dominant populations of microorganisms consist of anaerobic
bacteria (Bacteria), methanogens (Archaea), ciliates and anaerobic fungi (Eucarya),
compartmentalized into different populations associated with the rumen fluid, feed
particles and the rumen wall. Microbial enzymatic breakdown of complex plant
components in the rumen results in fermentation products such as short chain fatty
acids (SCFA), CO2 and CH4.
Energy-rich SCFA including acetate, butyrate and propionate are absorbed
across the rumen wall and may support up to 70% of the daily energy requirements
of the host (Hungate, 1966; Annison and Armstrong, 1970). In domestic ruminants
the concentration and the size of the SCFA pool in the rumen is correlated to the rate
of production of SCFA and reflects forage quality (Leng and Brett, 1966; Leng
et al., 1968; Weston and Hogan, 1968). Few measurements of rumen fermentation
rates have been conducted in wild ruminants (White and Grau, 1975; White and
Staaland, 1983; Lechner-Doll et al., 1991; Boomker, 1995; Sørmo et al., 1997).
Lechner-Doll et al. (1990) observed that the rate of dilution, rumen fluid volume and
rate of absorption influenced concentration of SCFA in the rumen fluid far more
than the rate of production in African domestic ruminants. Likewise, ruminal SCFA
concentration only partly reflects the forage quality eaten in Arctic ruminants, as
large seasonal differences in food intake probably also influence ruminal retention
and rate of absorption in these animals (Sørmo et al., 1997). The SCFA production
rate does, however, seem to reflect the quality of the pasture. On Svalbard the for-
age quality is very high in summer, but almost negligible amounts of SCFA were
produced in the rumens in winter, reflecting the poor quality of the winter diet. In
contrast, the Norwegian reindeer maintain a high SCFA production in winter when
they eat significant amounts of lichens (Mathiesen et al., 1999). Furthermore, the
late summer pasture in northern Norway was regarded as moderate in terms of car-
bohydrate fermentation in the rumen compared to Svalbard reindeer. The seasonal
and sub-species differences in body composition in Svalbard and Norwegian rein-
deer were also reflected in differences in their rumen metabolism (Mathiesen et al.,
1999). Given the low rate of rumen SCFA production, and the poor in vitro dry
matter digestibility recorded in Svalbard reindeer, these animals seem to survive
the winter by optimizing the ruminal utilization of the low quality food and by
reducing their energy expenditure to a minimum.
When ruminants are exposed to starvation or abrupt dietary changes, the micro-
biota colonizing the different parts of the digestive tract changes. Climatic condi-
tions in the Arctic often result in natural periods of starvation for reindeer during
winter, as pastures may be blocked by ice or crusted snow for shorter or longer
periods of time. Starvation for 3−4 days reduces the numbers of culturable anaerobic
rumen bacteria in reindeer by as much as 99.7%, and also changes the composition
of the population and its ability to digest cellulose (Mathiesen et al., 1984b; Aagnes
et al., 1995; Olsen, 2000). Likewise, starvation has a pronounced effect on the
rumen ciliate population, which is decreased by 75% after 3 days starvation
(Mathiesen et al., 1984b). Furthermore, the ruminal dry matter content and the
concentration of SCFA decrease, while the ruminal pH increases to as much as 7.8
after 4 days starvation. An increased intake of snow and a maintained rumen volume
and turnover time (Aagnes and Mathiesen, 1994), may, however, contribute to
sustain the microbial inoculum during starvation.
As much as 13% of the daily digestible food intake in domestic ruminants may
be lost in the form of methane (Hungate, 1966). The density of viable methanogens
in Svalbard reindeer, however, seems to be low regardless of season (Mathiesen,
1999). Likewise, Øritsland (personal communication) was unable to detect methane
from the rumen gas phase in live Svalbard reindeer. However, in Norwegian
reindeer fed pelleted reindeer concentrate large methane production has been
recorded (Gotaas and Tyler, 1994). Microbial competition for hydrogen could be
high in the rumen of these reindeer and future investigations should include studies
of the relationship between acetogenic and methanogenic bacteria in Svalbard
reindeer. Reduced loss of methane can be of significant importance for the growth
of Svalbard reindeer if hydrogen produced by the rumen microorganisms is used to
produce more acetate, rather than methane.
Savage (1989) defined bacteria isolated from the digestive tract of endothermic
animals as either autochthonous (indigenous) or allochthonous (transient), and
presented a list of criteria for autochthony. The composition of the microflora is
greatly influenced by the passage rates of fluid and particles through the digestive
tract (Van Soest, 1994). But the diet and hence the substrate available for fermenta-
tion is also a very important factor influencing the composition and the numbers of
microorganisms in the rumen. The rumen microbiology of Arctic ruminants has only
been partly investigated due to the considerable time required to return rumen
samples to the laboratory from the remote areas in which these animals live.
Furthermore, specialized techniques are required for cultivation of these strict anaer-
obes and for many years only direct microscopic observations were made of fixed
samples of bacteria and protozoa from Arctic ungulates (Dehority, 1975a; Hobson
et al., 1976). Live bacteria were first isolated from the rumen of Alaskan reindeer by
Dehority (1975b). During the past 15 years anaerobic techniques for field use have
been developed to investigate viable anaerobic bacteria in wild sheep, reindeer and
muskoxen (Orpin et al., 1985, Aagnes and Mathiesen, 1995; Aagnes et al., 1995)
making it possible to investigate the microbial ecology of the gastrointestinal tract
of animals living in areas remote to the laboratory.
3. BACTERIA
3.1. Numbers and composition of the bacterial population in different
niches of the rumen
Seasonal changes in forage quality and availability on Svalbard affect both numbers
of viable bacteria in rumen fluid (table 2) and their composition. Bacterial species
known to utilize soluble carbohydrates dominated in summer and those that utilize
fibrous polysaccharides dominated in winter (table 3). The viable bacterial popula-
tion decreased by about 75% from summer to winter but the winter population was
Table 2. Mean reticulo-rumen bacterial numbers in rumen fluid from Norwegian and Svalbard
reindeer in different seasons (Orpin et al., 1985; Aagnes et al., 1995; Olsen et al., 1997; Mathiesen
and Orpin, unpublished data)
Bacterial cells × 108/ml fluid
Norwegian reindeer
Summer pasture 6.4–9.9
Winter pasture 5.2–25.0
Starved for 4 days 0.1
Fed pure lichen 39.0
Svalbard reindeer
Summer pasture 209
Summer fed concentrate 350
Winter pasture 36
still regarded as high, compared to domestic ruminants and was

probably influenced by the increased ruminal retention of fibrous plant particles in
winter. Cellulolytic bacteria might be important in the large rumen of Svalbard rein-
deer in winter, since as much as 35% of all bacteria isolated in winter were cellulolytic
(table 3), and, although not very efficient, were slowly degrading the fibrous food
eaten. The bacterial population in Norwegian reindeer seems to increase from summer
to winter, a finding which might be related to the increased lichen intake in these ani-
mals in winter increasing the availability of digestible energy in the rumen (table 2).
Table 3. Cellulolytic bacteria as per cent of total viable bacteria in the rumen fluid of different
ruminants (Hungate, 1966; Orpin et al., 1985; Aagnes et al., 1995; Olsen et al., 1997;
Mathiesen and Orpin, unpublished data)
Cellulolytic bacteria
(% of total population)
Norwegian reindeer
Summer pasture 3.4
Winter pasture 2.5
Svalbard reindeer
Summer pasture 15
Summer fed pellets 21
Winter pasture 35
Muskox
Ryøy, summer pasture 18
Sheep
Domestic 4–10
Seaweed-eating 0
Cattle
Domestic 15
Fig. 4. Electron microscopic ultrathin sections of grass and lichen particles from the rumen of Norwegian
reindeer in summer on South Georgia (A) (scale bar = 500 nm) and in winter in Norway (B) (scale bar = 1 mm).
Morphological types resembling Fibrobacter are shown associated with both grass and lichen, while bacteria
resembling B. fibrisolvens are shown between the lichen particles. (Transmission electron micrographs:
S. D. Mathiesen.)
No major differences have been observed in the morphology of bacteria that

adhere to grass particles and lichens (fig. 4). Aagnes et al. (1995) reported that the
rumen bacteria seem to break down the lichen from the inside and that the density
of bacteria close to the plant particles was ten times higher than in the rumen fluid.
Microorganisms that utilize plant structural polysaccharides as their energy source
achieve preferential access to their substrate by associating closely with plant parti-
cles entering the rumen, and at the same time extend their residence time in the
rumen to that of the least digestible part of the animals’ diet. The ability of bacteria
to adhere to plant material and break down cell walls is of primary importance and
appears to be an essential first stage in the digestive process in the rumen.
Bacteria adhering to the rumen wall are taxonomically distinct from bacteria in
the rumen fluid (Cheng et al., 1979). Ureolytic species adhering to the rumen wall
may play an important role in nitrogen recycling in ruminants such as reindeer by
providing ammonia to the bacteria in the rumen fluid used for protein synthesis.
Direct examination by scanning electron microscopy (SEM) of sites on the rumen
epithelium of Svalbard reindeer showed that only 30% of their epithelial surface was
covered in adherent bacteria in summer and only 10% in winter, compared to 75% in
fed cattle (K.-J. Cheng, personal communication). Many epithelial cells were
partially sloughed, particularly in the summer, which might be explained by the high
rate of rumen fermentation in summer and high rate of metabolism in rumen epithe-
lial cells where the SCFA are absorbed (White and Staaland, 1983). The adherent
bacterial population consisted largely of curved cells and cocci which resembled
Ruminococcus spp., by their possession of a condensed glycocalyx on the cell
surface (fig. 5). The low level of adhering bacteria in winter in Svalbard reindeer
corresponds with observations in starving cattle (K.-J. Cheng, personal communica-
tion). We have therefore to assume that the low population of adhering bacteria in the
Fig. 5. Rumen papilla in Svalbard reindeer

on summer pasture (A, scale bar = 10 μm) and
its epithelial cells with facultatively anaerobic
adhering bacteria (B, scale bar = 5 μm).
(Scanning electron micrographs: S.D.
Mathiesen.)
rumen of Svalbard reindeer in winter is caused by a low rate of urea diffusion across
the rumen wall and reduced rumen carbohydrate fermentation rate in winter.
3.2. Cellulose degradation

Ruminants are highly specialized users of plant polysaccharides as their source of
energy for growth. The microbial breakdown of structural polysaccharides is a slow
chemical process under anaerobic conditions and therefore a large delay chamber
such as the rumen is necessary to enable enzymatic breakdown. The cellulase
complexes secreted usually consist of three major types of enzymes which function
synergistically in the hydrolysis of cellulose. They are: endo-1,4-β-glucanase,
cellobiohydrolase, and β-glucosidase (Forsberg et al., 1997, 2000). The ruminal
digestion of plant cell wall polysaccharides such as cellulose, however, is limited by
the availability of non-protein nitrogen in the form of ammonia and also amino acids
and easily digestible carbohydrates in the rumen (Ørskov, 1992). Available nitrogen
and carbon, rather than the cellulose content of the plants or the number of
cellulolytic bacteria, may limit the rates of rumen cellulolysis and fermentation in
reindeer (Aagnes and Mathiesen, 1994, 1995; Aagnes et al., 1996; Norberg and
Mathiesen, 1998; Sørmo, 1998; Utsi, 1998). This is probably one reason for the
seasonal changes in the density of the viable bacterial population and the cellulolytic
bacterial population in the rumen of Svalbard reindeer (Sørmo et al., 1997, 1999).
This might also explain the efficient ruminal breakdown of cellulose in summer
compared to winter in Svalbard reindeer (Sørmo et al., 1998).
Strains of B. fibrisolvens from Svalbard reindeer and Norwegian reindeer on
South Georgia have been shown to solubilize cellulose, but it has not been possible
to isolate dominant bacteria with strong cellulolytic activity in the rumen of reindeer
that eat pure lichen (Aagnes et al., 1995; Olsen and Mathiesen, 1998). Likewise,
Orpin et al. (1985) in their study were unable to isolate cellulolytic bacteria from the
rumen of seaweed-eating sheep on Ronaldsay. On the other hand the in vitro break-
down of pure cellulose in rumen fluid from Norwegian reindeer in winter and in
rumen fluid from lichen-fed reindeer was high (Olsen et al., 1997). Recently, Olsen
and Mathiesen (1999) isolated a cellulolytic bacterial strain of B. fibrisolvens from
the rumen of lichen-fed reindeer on a specially developed lichen medium and this may
partly explain the high rate of cellulose breakdown observed in lichen-fed animals.
Likewise, Deutsch et al. (1998) showed that roe deer eat highly cellulosic forage
plants in winter but are almost incapable of digesting cellulose because their popu-
lations of cellulase-producing bacteria are reduced in winter. Roe deer are concen-
trate selector ruminant feeding type with short ruminal retention of plant fibres.
Bacteria that require a long time for cell division are easily washed out of the rumen.
However, the low ruminal cellulolysis in this species in winter might also be
explained by the low availability of metabolizable energy and of non-protein nitro-
gen to microbial synthesis in winter. In contrast, ruminal cellulolysis in Norwegian
reindeer was maintained at a high level in winter, which could be explained by
supported bacterial growth owing to the high intake of highly digestible lichens
(Olsen et al., 1997). Intake of digestible lichens might also influence the diffusion
gradient of urea across the rumen wall and the recycling of nitrogen supplying the
microbial environment in the rumen in winter with ammonia (Hove and Jacobsen,
1975). The high numbers of cellulolytic bacteria in the rumen of Svalbard reindeer
in winter represent an important digestive adaptation to the fibrous food eaten,
but due to reduced availability of non-protein nitrogen and digestible energy the
degradation is not as efficient as in rumen fluid from Norwegian reindeer offered
lichens in winter. The large rumen, high population of cellulolytic bacteria in
Svalbard reindeer and perhaps a long ruminal retention time, however, allow for a
higher degree of digestion of fibrous plants eaten in winter in these animals and
represent an adaptation to a winter diet without lichens.
3.3. Genetic studies of the rumen bacteria from reindeer

Rumen bacteria essential for the utilization of plant materials such as Butyrivibrio
fibrisolvens, Selenomonas spp., Streptococcus bovis and Lactobacillus spp. have all
been isolated from reindeer (Dehority, 1975a, 1986). Dehority (1986) concluded
that bacterial species isolated from the Alaskan reindeer were similar to those
widely found in domestic ruminants and no unique physiological or biochemical
characteristics were observed in the species studied. It remains unclear whether
Arctic ruminants have unique species of bacteria in their rumen. However, the
composition of rumen bacterial populations in Svalbard reindeer is very specialized
in relation to fibre digestion and nitrogen metabolism (Mathiesen et al., 1984a;
Orpin and Mathiesen, 1990). As much as 30% of the culturable rumen bacterial
population in Svalbard reindeer in winter consists of Butyrivibrio-like bacteria
(Orpin et al., 1985), some of which have been studied in more detail to describe
genes and expressed enzymes responsible for the symbiotic fibre degradation in
these animals. Hazelwood et al. (1990) first successfully cloned and expressed a
novel endo-β-1-4-glucanase gene from B. fibrisolvens A46 in Escherichia coli.
Other cellulase enzymes induced by substrate availability may be produced by
B. fibrisolvens A46 that are multifunctional (Orpin and Mathiesen, 1990). Such
enzymes have been described for Chytridiomycetes (Orpin and Joblin, 1997) and
would be of great benefit in the rumen of these animals, which feed on diets that
vary greatly in quality. A cellulolytic strain (S-89) of Bacillus spp. from Svalbard
reindeer was later transformed to kanamycin resistance with plasmid puB110, and
was inoculated into the rumen of Norwegian cattle. This bacterium did establish
in the rumen of cattle, but at very low levels and unfortunately without significance
for the rumen cellulose breakdown (Mathiesen and Orpin, unpublished data).
Some attempts to return laboratory bacteria to the rumen have shown them to have
difficulties readapting to the rumen condition. Likewise, cellulolytic B. fibrisolvens E14
from the Svalbard reindeer were inoculated into the rumen of sheep. Their survival was
investigated using a combined plasmid DNA probe (Orpin et al., 1987; Mathiesen,
Orpin and Blix, unpublished data). Up to 105 cells/ml rumen fluid could be detected
30 days after the inoculation of the E14. However, it is doubtful that bacterial strains of
about 105−106 have a significant effect on the overall ruminal metabolic activities.
The genus Butyrivibrio represents a diverse group of obligate anaerobic, curved
rod-shaped bacteria that produce large amounts of butyrate when fermenting carbo-
hydrates, and B. fibrisolvens is the major culturable cellulolytic bacterium in the
rumen of Svalbard reindeer in both summer and winter (Orpin et al., 1985). Two
B. fibrisolvens strains (ARD-22a and ARD-23c) isolated from reindeer in Alaska by
Dehority in 1975, were later examined for DNA relatedness with a total of 37
different bacterial isolates (all resembling B. fibrisolvens) from the rumen or caecum
of sheep, steer, goats and pigs (Mannarelli, 1988). The guanine-plus-cytosine (G + C)
base content of strains ARD-22a and ARD-23c was 42 and 43 mol%, respectively,
but a large variability was observed in the G − C base content (39−49 mol%)
between the different strains, indicating that the various isolates were in fact different
species. In a later study Mannarelli et al. (1990a,b) determined taxonomic relatedness
between different strains of gastrointestinal bacteria using DNA−DNA hybridization
stating that two other strains of B. fibrisolvens (ARD-27b and ARD-31a) from the
rumen of Alaskan reindeer do not exhibit DNA relatedness to any of the previously
studied strains (from other animals), although strains ARD-27b and ARD-31a are
related to each other. However, a more recent study by Forster et al. (1996) that
sequenced the complete 16S rDNA of four strains of B. fibrisolvens isolated from
the rumen of white-tailed deer (Odocoileus virginianus) in Canada showed a high
similarity (96.5−99.7%) with the 16S rDNA sequence of B. fibrisolvens 49 from steer
(Bryant and Small, 1956). Unlike B. fibrisolvens H17c (isolated from a domestic
ruminant), B. fibrisolvens E14 isolated from the rumen of Svalbard reindeer (Orpin
et al., 1985) is unable to synthesize methionine in vivo and therefore needs methionine
added to the medium (Nili and Brooker, 1995). Methionine is, metabolically, the
most expensive amino acid to produce (Old et al., 1991). Tests with intermediates of
the methionine biosynthetic pathway indicate that in strain E14 the final step from
homocysteine to methionine is blocked, probably due to the lack of methionine
synthetase (Nili and Brooker, 1997).
4. CILIATE PROTOZOA
The rumen ciliate protozoa population in reindeer was first described by Ebenlein
(1895). He compared reindeer from a zoological garden with domestic ruminants in
Germany and concluded that they were the same. Similar results were obtained by
Lubinsky (1958) who worked with caribou in northern Canada. Reindeer were
reported to have a unique ciliate fauna as demonstrated in Finnish reindeer
(Westerling, 1970). Svalbard reindeer and Finnish reindeer both show clear seasonal
changes in numbers and composition of the ciliate population (Westerling, 1970;
Orpin and Mathiesen, 1988, 1990). Dehority (1975b) reported that the rumen cili-
ates of semi-domesticated reindeer in Alaska were similar to those of the domestic
ruminants in the area, while a typical reindeer fauna was observed in wild caribou
and reindeer. In the rumen of Svalbard reindeer the density of rumen ciliates varied
from 105 cells per ml rumen fluid in summer to 104 cells per ml in winter and only
entodiniomorphid ciliates were present, while no holotrich ciliates were observed in
contrast to Norwegian and Finnish reindeer (Westerling, 1970; Orpin and
Mathiesen, 1988, 1990). Their absence in the Svalbard reindeer could be due to
starvation resulting from poor quality of the forage eaten in winter, since they
metabolize only soluble carbohydrates and the rumen content of Svalbard reindeer
was highly fibrous in winter (Williams and Coleman, 1988). Short ruminal retention
in summer is known from ruminants of the CS ruminant type and this would make
the establishment of holotrichs difficult in Svalbard reindeer because of the long
time needed for cell division. The major species of entodiniomorphs identified in
Svalbard reindeer were Entodinium simplex, E. triacum triacum, E. longinucleatum,
Polyplastron multivesiculatum, Eremoplastron bovis and Eudiplodinium maggi.
There is little evidence that the Entodinium spp. is important in fibre digestion in
ruminants utilizing principally starch and bacteria as their carbon source (Williams
and Coleman, 1988). E. maggi, Polyplastron multivesiculatum and Eremoplastron
bovis are all known to ingest plant particles and to contain cellulase (Coleman,
1985). Holotrich and entodiniomorphic ciliates were detected in Norwegian rein-
deer that had survived on South Georgia for almost 100 years without lichens.
Diplodinium rangiferi, Epidiumun ecaudatum gigas and Polyplastron arcticum
were identified in population densities of 105−106 cells per ml rumen fluid. Species
such as D. rangiferi were originally associated with reindeer eating lichens
(Westerling, 1970). Although there are differences between the protozoal fauna in var-
ious Arctic ruminants, currently available information does not suggest that the fauna
are unique to a given animal species or that they are essential for the digestion of
Arctic plants; rather, it appears that diet and isolation of the host are likely to be the
primary factors that have determined faunal composition. Dehority (1986) empha-
sized that several protozoal species are unique to Arctic ruminants, i.e. the rangifer-
type fauna. Ciliate protozoa, however, seem not to be essential for rumen digestion in
Fig. 6. Ciliate protozoa in the reindeer rumen.

A micrograph of a rumen entodininomorph
ciliate, Entodinium triacum triacum, from the
rumen of a Svalbard reindeer in summer (A)
(phase-contrast micrograph by S.D. Mathiesen)
and a ciliate Entodinium sp. in Norwegian
reindeer on natural winter pasture (B)
showing a ciliate that engulfs bacteria associated
with lichen particles (transmission electron
micrograph by M.A. Sundset; scale bar = 2 mm).
reindeer since rumen contents without ciliates have been observed in healthy reindeer
and muskoxen (Mathiesen, unpublished data) (fig. 6).
5. ANAEROBIC FUNGI
The anaerobic fungi commonly found in the rumen of large ruminants were first dis-
covered by Orpin (1974). Several species are known to exist but only one type has
been found in the rumen of Svalbard reindeer. This was a monocentric species with
polyflagellated zoospores, endogenous development and a branching rhizoidal system
characteristic of Neocallimastics spp. Multiflagellated zoospores of the rumen fungi
Neocallimastix frontalis (Chytridiomycetes) have been reported in Svalbard reindeer
by Orpin et al. (1985). This species utilizes a range of different polysaccharides
including cellulose and their hyphae may penetrate plant vascular tissue normally not
accessible to bacteria (Bauchop, 1979; Orpin and Letcher, 1979). Relatively low num-
bers of zoospores were demonstrated in Norwegian reindeer calves on winter pasture
(1.5 × 103 zoospores/ml rumen fluid) as compared to summer pasture (3.1 × 102
zoospores/ml) (Olsen, 2000), while higher numbers of zoospores were found in adult
Svalbard reindeer on winter pasture (up to 1 × 105 zoospores/ml) (Mathiesen, 1999).
Large populations of anaerobic fungi are normally associated with a high intake of
rough, fibrous diets in domestic ruminants (Bauchop, 1979) and the higher concen-
trations of zoospores in Svalbard reindeer on winter pasture may support this. Electron
microscopic ultrathin sections of plant particles from the rumen revealed fungi pene-
trating the plant cell wall (Mathiesen and Aagnes, 1990; Mathiesen and Utsi, unpub-
lished data) (fig. 7). Rumen anaerobic fungi were isolated from free-living and
artificially fed reindeer in northern Norway (fig. 7). These fungi are able to invade the
plant tissue to a greater extent than bacteria and ciliates and some fungi express very
Fig. 7. Scanning electron micrograph (A) of anaerobic rumen fungi in Norwegian reindeer on South Georgia
in summer, with sporangium (s) and rhizomes (r) invading a grass particle; rumen bacteria (b) are shown in
the background. The transmission electron micrograph (B) shows grass particles from the same animals with
chytridiomycetes hypha (arrow) invading the vascular bundle sheet (v) and penetrating the plant cell walls (p)
(scale bar = 2 μm). (Photographs: S.D. Mathiesen.)
efficient multifunctional polysaccharide degrading enzymes in reindeer (Orpin and

Joblin, 1997) and could contribute substantially to ruminal fibre breakdown. We have
not yet been able to associate anaerobic fungi with ruminal lichen particles in reindeer.
6. THE DISTAL FERMENTATION CHAMBER

Many species of ruminants evolved before the spread of grasses during the Miocene
epoch of the Tertiary and earlier ruminant types seem to have a limited capacity to
digest cellulose. According to Hofmann (1999) they selected mainly dicot plants and
of these primarily their plant cell content. Fermentation of such forage plants in the
distal fermentation chamber (DFC) might have been important in these early rumi-
nant types. The caecum and colon of “modern” ruminants contain microorganisms
capable of producing SCFA from plant material not digested in the rumen (Ulyatt
et al., 1975). Svalbard reindeer have a DFC similar in size to that of concentrate selec-
tor ruminant feeders, while DFCs in reindeer offered lichens in northern Norway are
small. In concentrate selectors or in intermediate feeders with short ruminal retention
time, plant material escapes from ruminal digestion, and digesta may be retained in
an enlarged DFC for a second fermentation. Under these circumstances a consider-
able proportion of potentially digestible fibre may escape microbial fermentation in
the rumen and enter the DFC (Hofmann, 1989). The ruminal particle size distribution
in Svalbard reindeer indicates a rapid release of small particles from their reticulo-
rumen which could influence the large DFC in these animals. The large DFC of
Svalbard reindeer was, however, correlated with a high hemicellulose content of the
reticulo-rumen (Sørmo et al., 1999). The acidic environment in the abomasum and
the presence of pepsin might change the configuration of plant hemicelluloses and
facilitate their digestion in the DFC (Van Soest, 1994).
The total culturable bacterial population isolated from caecal contents from
Svalbard reindeer was 8.9 × 108 cells/ml in summer and 1.5 × 108 cells/ml in winter
(Mathiesen et al., 1987). Of the dominant species of culturable bacteria, B. fibrisol-
vens represented 23% in summer and 18% in winter, while S. bovis represented 17%
in summer and 5% in winter. Prevotella ruminicola represented 10% of the dominant
culturable species in summer and as much as 26% in winter. As much as 10% of the
viable bacterial population was cellulolytic in summer, compared to 6% in winter, the
most abundant cellulolytic species being B. fibrisolvens representing 62% of the total
cellulolytic population in summer, and R. albus representing 80% of the cellulolytic
population in winter. The hindgut bacterial population has the ability to digest starch
and major structural carbohydrates that escape digestion in the rumen.
The very low pH recorded in this organ in reindeer eating lichen indicates that the
digestive function of the DFC is not yet understood. In contrast, in Svalbard reindeer
feeding in an area on Svalbard characterized as an Arctic desert, the relative size of the
DFC was small but SCFA from the DFC contributed 17% of the total SCFA produced,
indicating that fermentation in the DFC might be important when the substrate is avail-
able (Sørmo et al., 1997). In comparison, in sheep fed chopped alfalfa hay SCFA
production in the hindgut contributed 7% of the maintenance requirement (Hume,

1977), while in black-tailed deer on natural pasture it contributed only 1% (Allo et al.,
1973). Fibre-degrading bacteria have been isolated from the caecum of Svalbard rein-
deer in both winter and summer indicating that it is important for fibre digestion.
Furthermore, the caecum of Norwegian reindeer on South Georgia in summer
contained low concentrations of anaerobic bacteria with cellulolytic activity (Mathiesen
and Aagnes, 1990). The breakdown of cellulose in the DFC is a slow reaction and the
microbial population might therefore need additional energy and nitrogen to contribute
substantially to the daily energy requirements in these animals.
7. THE SMALL INTESTINE

The small intestine is about 22 m long in Norwegian reindeer and 18 m in Svalbard
reindeer of similar sex and age (Westerling, 1975a; Staaland et al., 1979). It is covered
with villi and microvilli, the epithelial cells being rapidly replaced with new cells
(Myklebust and Mayhew, 1997), providing a large surface area for the absorption of
microbial protein, water and minerals. The small intestinal pH increases from 5.9 in
the duodenum, to 6.1 in the jejunum and 7.5 in the ileum (Sørmo and Mathiesen,
1993; Sørmo et al., 1994).
Electron microscopic examinations of the gut represent important tools for inves-
tigating the microbial ecology of the gastrointestinal tract ecosystem and for deter-
mining the presence of autochthonous or allochthonous microbiota. However, very
few microscopic examinations of the small intestine have been conducted so far, and
Sørmo and Mathiesen (1993) failed to reveal any bacteria associated to the tip of the
microvilli or between the microvilli. This may be explained by the low numbers of bac-
teria residing in the small intestine of reindeer. Table 4 shows the anaerobic bacterial
population level in the small intestine of free-living reindeer on natural winter
pasture in Finnmark and in captive lichen-fed reindeer. In two of the animals fed
natural winter pasture, no detectable viable bacteria were found colonizing the
proximal part of the small intestine (Sørmo et al., 1994). The authors suggested that
this probably was due to antibacterial substances activated in the acidic environment
in the abomasum, inhibiting the establishment and growth of bacteria in the proximal
Table 4. Numbers of viable anaerobic and aerobic bacteria per gram mucosa in the proximal
and distal part of the small intestine of Norwegian reindeer (Sørmo and Mathiesen, 1993; Sørmo
et al., 1994; Sørmo, unpublished data)
Anaerobes Aerobes
Proximal part Distal part Proximal part Distal part
Norwegian reindeer
Winter pasture 0–2 600 70–40 000 0–2 000 20–74 000
Captive lichen-fed 700–42 000 650–72 000 nd nd
Fed RF-71, after 117 000–140 000 500 000–600 000 nd nd
3 days starvation
nd = not detected
small intestine. Organic acids, lipids, antibiotics and usnic acid known to occur in
lichens are potential agents inhibiting bacterial growth (Vartia, 1949, 1973; Lauterwein
et al., 1995; Huneck, 1999; Müller, 2001). Autochthonous microorganisms associated
closely with the small intestinal epithelium could be important for the health and
welfare of the host by limiting direct attachment or interaction of pathogenic bacteria
to the mucosa (Slomiany et al., 1994; Henderson et al., 1996). It is well known that the
indigenous microbiota plays a protective role against pathogenic bacteria colonizing the
mucosal surface or the microvilli (Hentges, 1992; Salmonen, 1996). Microorganisms
colonizing the small intestinal mucosa could by various mechanisms inhibit the absorp-
tion of water and salt from the intestine, causing diarrhoea and dehydration of the host.
Sørmo and Mathiesen (1993) showed that bacteria colonizing the small intestine of
reindeer were dominated by the lactic acid bacteria Streptococcus spp., 66.3% of the
total bacterial population when the animals were fed lichens ad libitum. The most
common of the streptococci resembled S. bovis and S. durans, but the authors did not
present any molecular data confirming this statement. In a later study, variable popula-
tion densities of streptococci, from nil to 50%, were reported to colonize the proximal
and distal parts of the small intestine of reindeer grazing on a natural winter pasture
(Sørmo et al., 1994). However, the importance of streptococci in the small intestine of
reindeer remains unknown as great variations between individuals seem to occur. In 36
out of 40 faecal samples from clinically healthy reindeer calves (90%), Enterococcus
spp. were isolated (Kemper et al., 2002). These organisms are common inhabitants and
opportunistic pathogens in the intestinal tract of animals (Carter and Cole, 1990).
Strains of lactobacilli have been isolated from the small intestine of lichen-fed
reindeer, but they seem to represent only a minor part of the microbiota as they
constitute only 0.9% of the bacterial population (Sørmo and Mathiesen, 1993).
Lactobacillus spp. have also been isolated from the small intestine of free-living
reindeer grazing on natural winter pasture (Sørmo et al., 1994), but this study also
concluded that lactobacilli represent only a minor part of the small intestinal micro-
biota. The authors showed that these lactobacilli were more tolerant to low pH than
the rumen bacteria described by Kandler and Weis (1986).
Great variations in the population level of Bacterioidaceae, from nil to 68.4%,
colonizing the proximal part of the small intestine were found in four reindeer graz-
ing on natural winter pasture (Sørmo et al., 1994). The proportion of Bacterioidaceae
found associated to the distal part of the small intestine was 2.4, 6.5, 11.5 and 21.4%
of the total bacterial population. Propionibacterium has been isolated from the prox-
imal part and the distal part of the small intestine of free-living reindeer, but only at
low population levels, as it accounts for only 2% of the viable bacteria (Sørmo et al.,
1994). Likewise, Eubacterium spp. has only been isolated from the distal part of the
small intestine of free-living reindeer (Sørmo et al., 1994). Also bacteria belonging
to the genus Ruminococcus have only sporadically been isolated from the small intes-
tine of reindeer, and then they have only been isolated from the distal part of the small
intestine (Sørmo et al., 1994).
8. ENTERIC PATHOGENIC BACTERIA

Similar to the situation in other wild or free-ranging animals, little information is avail-
able on the occurrence of pathogenic faecal bacteria and on the impact of enteric
diseases on production in growing reindeer and in reindeer in general. The few avail-
able reports describe outbreaks of disease in single affected individuals only, as
possibilities to do systematic studies on bacterial diseases in free-ranging reindeer
calves are restricted. One must consider that the predominant extensive form of rein-
deer husbandry, on vast areas that are covered for a long period of the year by ice and
snow, does not necessarily favour outbreaks of infectious disease (Skjenneberg and
Slagsvold, 1968), even though potential pathogenic bacteria may be found in the nat-
ural environment (Kapperud, 1981) and also in the intestinal tract of healthy reindeer
(Aschfalk et al., 1998; Kobayashi et al., 1999). Outbreaks of disease are often depend-
ent not only on the presence of the bacterial agent, but also on other factors that may
alter the balance in the intestinal tract. In an unpublished study by Dr Wenche Sørmo,
it was demonstrated that when reindeer were eating lichens, then starved for three
days and thereafter fed RF-71 (a pelleted concentrate diet), simulating an emergency
feeding situation, the animals developed diarrhoea. At that time, a population level
of 5 × 105 anaerobic bacteria per gram of small intestinal mucosa was found. Diarrhoea-
induced diseases in the small intestines of reindeer frequently occur during emergency
feeding with commercial available pellets in winter in Norway. Replacement of new
epithelial cells, and the numbers of lymphocytes in the intestinal cells could be
important factors in the protection against pathogenic bacteria, the development of a
beneficial small intestinal microbiota and the ability to absorb nutrients.
Once established in a herd, an infection may spread easily among individuals,
especially if reindeer are kept intensively, e.g. for artificial feeding during winter or
calf marking. Numerous infectious agents including bacteria, virus and protozoa
have been related to diarrhoea in young ruminants (Tzipori, 1981; Munoz et al.,
1996; Busato et al., 1998). Bacteria such as Clostridium perfringens, Escherichia
coli and Salmonella spp. are among the most important bacterial agents in causing
enteric and other diseases, as is known from domestic, young ruminants (Dubourguier
et al., 1978; Lintermans and Pohl, 1983; De Rycke et al., 1986; Alexander and
Buxton, 1994; Munoz et al., 1996; Steiner et al., 1997; Busato et al., 1998, 1999).
De Rycke et al. (1986) classified some of the bacterial agents as primary calf
enteropathogens, e.g. enterotoxigenic E. coli and Salmonella spp., other bacteria as
having a less expressed enteropathogenicity, such as enterotoxigenic Clostridium
perfringens, and others as agents that are not directly associated to diarrhoea, such as
Yersinia enterocolitica. Obviously, a certain impact of these enteric pathogens on
reindeer production cannot be excluded even though there are only a very few reports
on diseases and mortality caused by these bacteria in reindeer.
The presence of Clostridium spp. colonizing the small intestine of reindeer has
been reported in lichen-fed animals (Sørmo and Mathiesen, 1993) where 6.4%
of the bacterial population belonged to genus Clostridium, and in one individual

from free-living reindeer grazing on a natural winter pasture (Sørmo et al., 1994).
C. perfringens was reported in the intestine and in faecal samples associated to
diseased reindeer (Kummeneje and Bakken, 1973), as well as healthy, adult reindeer
(Aschfalk et al., 1998, 2001). In all these examinations, C. perfringens toxin type A
(alpha-toxin) was the only or most dominant one diagnosed. In addition, the gene
for the novel described β2-toxin and the gene encoding for enterotoxin were found
by Aschfalk et al. (2001, 2002a). Outbreaks of disease in reindeer were also reported
by Rehbinder and Nikander (1999), caused by C. perfringens type C (alpha- and
epsilon-toxin) and type D (alpha- and beta-toxin). Mortality in reindeer caused by
C. perfringens enterotoxemia was reported by Kummeneje and Bakken (1973) and
by Rehbinder and Nikander (1999). C. perfringens is known to cause important
gastrointestinal and enterotoxemic diseases in young ruminants, sheep and deer
(Alexander and Buxton, 1994; Songer, 1998). The virulence and pathogenicity of
this organism are closely related to the expression of different toxins (Petit et al.,
1999), and outbreaks of clostridial enteric diseases are combined with further
factors, such as type of nutrition and seasonal changes.
The first report on the occurrence of E. coli (O-group 55) in the intestine of rein-
deer associated to calf mortality was by Clausen et al. (1980). In their study on
lichen-fed reindeer, Sørmo and Mathiesen (1993) reported that E. coli contributed
25.5% of the viable bacterial population colonizing the small intestine. Recently,
this bacterium was isolated in all faeces samples from clinically healthy, young
reindeer calves (n = 40). By polymerase chain reaction (PCR) analysis, the genes for
eae and hly could be detected in two and seven of these isolates, respectively
(Kemper et al., 2002). STEC, shigatoxin-producing bacteria were detected by
Kobayashi et al. (1999) and Aschfalk et al. (2001) in reindeer. However, as PCR
analysis was done on mixed cultures, the evidence of shigatoxin-producing E. coli
was not given. Escherichia coli is recovered from a wide variety of diseases,
such as colibacillosis and colisepticaemia in all domesticated animals as a primary
or secondary pathogenic agent (Carter and Cole, 1990) and is of special importance
in causing diarrhoea in young ruminants (Alexander and Buxton, 1994; Munoz
et al., 1996). STEC have been isolated frequently from cattle (Busato et al., 1998),
lamb and kid faeces (Beutin et al., 1993; Munoz et al., 1996), but there are only
a few reports on the association between occurrence of STEC and disease in rumi-
nants. However, Busato et al. (1998) assume that STEC, or rather the free faecal
verotoxin in the faecal matter, possibly may be a most significant cause of calf
diarrhoea.
Salmonella spp. associated to mortality of reindeer calves was reported by
Kuronen et al. (1998). The screening of serum samples from 2000 clinically healthy,
slaughter reindeer from Norway, revealed a seroprevalence of 0.6% for Salmonella
spp. (Aschfalk et al., 2002b), originating assumingly from an infection following the
faecal–oral route. In Finland, a prevalence of 2.8% was detected, as also sera from
animals showing intestinal disorders were examined (Aschfalk and Denzin, 2000).
Salmonella serotypes are known to cause enteric diseases and septicaemia, in young
ruminants (Carter and Cole, 1990; Alexander and Buxton, 1994).
Yersinia enterocolitica was found in one faecal sample out of 40 (2.5%) young,
clinically healthy calves (Kemper et al., 2002). Disease in reindeer caused by
Yersinia sp. was reported by Rehbinder and Nikander (1999). Disease caused by
Yersinia sp. is considered one of the more important diseases of corralled deer and
is also reported to affect young individuals (Alexander and Buxton, 1994).
Other than being the primary cause of diseases, several entopathogenic agents
generally not considered to be major aetiologic agents of calf diarrhoea may,
however, play a role as preceding or synergistic infections in animals with inade-
quate passive immunity. These organisms can also gain importance in situations of
epidemic outbreaks of diarrhoea in herds with specific management problems
(Busato et al., 1998). As corralling of reindeer is getting more and more common in
reindeer husbandry, this intensification of reindeer production eventually leads to an
increased putative risk of outbreaks of infectious diseases, by different bacterial
agents, as is known from domesticated ruminants brought into intensive farming
conditions (Mackintosh, 1998).
9. FUTURE PERSPECTIVES: THE IMPLEMENTATION OF

NEW MOLECULAR TOOLS FOR STUDIES OF THE
MICROBIAL ECOLOGY IN THE GASTROINTESTINAL
TRACT OF REINDEER
Extensive studies of the rumen ecosystem using conventional anaerobic cultivation
methods have provided us with a rich knowledge of diverse types of gastrointestinal
bacteria. The cultivation-based techniques are, however, hampered with some obvi-
ous limitations. Direct counts of fixed, filtered rumen fluid from the Svalbard rein-
deer showed total population densities of 5.5 × 1010 in summer and 1.1 × 1010 in
winter, as compared to viable counts of 2.1 × 1010 and 0.36 × 1010, respectively
(Orpin et al., 1985). Hence, 62−67% of the total population was unable to grow on
the culture media employed. Typically, several of the large bacteria, such as
Oscillospira guilliermondii, Magnoovum eadii and Quinella ovalis, have never been
cultivated. Synergistic microorganisms depend on others and this may also limit
their ability to grow in pure cultures. Furthermore, culture methods not only are very
laborious and time consuming, but they also identify the bacterial isolates from their
phenotypic pattern – which will vary depending on a range of factors such as, e.g.
the expression of their genes and the artificial conditions in the laboratory. In fact, a
vast majority of the rumen microorganisms have not been isolated and still remain
to be characterized (Whitford et al., 1998; Tajima et al., 1999, 2000; Ramsak et al.,
2000; Kocherginskaya et al., 2001). The microflora of the rumen may be far more
diverse than earlier believed (Avgustin et al., 1997; Forster et al., 1997). It has
proved impossible to identify several of the strains of the viable bacteria isolated in
Svalbard reindeer, and Norwegian reindeer on South Georgia and in Norway, using
standard anaerobic microbiological techniques (Aagnes et al., 1993; Mathiesen and
Utsi, unpublished). To what extent unfamiliar bacterial strains contribute to the
rumen ecosystem is unknown. Therefore it is still possible that some bacterial
species in reindeer could be unique. Likewise, a range of unidentified bacterial
strains has been isolated from the small intestine of reindeer, most of which are
strictly anaerobic, motile Gram-positive large rods, single or pairs of irregular rods,
capable of utilizing glucose, maltose, sucrose, cellobiose and starch, which have not
yet been characterized (Sørmo et al., 1998). The population level of these bacteria
seems to dominate in the small intestine of some animals, while their population
level in other animals is lower than the detection level.
In recent years, molecular methods have been developed that in combination
with the traditional cultivation-based methods can be used to give a complete
description of the gastrointestinal ecosystem (e.g. Lin et al., 1997; Whitford et al.,
1998; Simpson et al., 1999, 2000; Tajima et al., 1999, 2001a,b; White et al., 1999).
The molecular methods are based on comparative analysis of rRNA sequences and
its encoding genes, retrieving the sequences directly from the gastrointestinal tract
samples by the help of PCR using highly conserved primer binding sites on the
16S rRNA genes. Oligonucleotide hybridization probes can be designed that target
and discriminate between broad phylogenetic groups such as Archaea, Bacteria
and Eucarya, or even specific strains, allowing studies of population structure
and dynamics in the gastrointestinal microbial ecosystem (Amann and Kühl, 1998;
Mackie et al., 2000).
We are currently working on a project to construct 16S rDNA clone libraries to
examine rumen bacterial diversity in reindeer on natural pastures in northern
Norway and on Svalbard (Olsen et al., 2002). The diversity of bacterial, archaeal
and eucaryal populations in each compartment of the digestive tract of reindeer will
also be determined for the first time using molecular microbial ecology techniques.
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129–138.
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5 Microbial ecology of the gastrointestinal
tract of the growing dog and cat
J. Zentek
Institute of Nutrition, University of Veterinary Medicine, Vienna A-1210 Vienna,

Veterinärplatz 1, Austria
Microbial colonization of the gastrointestinal tract of puppies and kittens starts after
birth, and the composition of the intestinal microflora approaches the spectrum in
adult dogs and cats during the first weeks of life. The colonization of the
gastrointestinal tract starts with an aerobic type of flora, mainly streptococci,
Escherichia coli and in puppies, staphylococci. Lactobacilli have been isolated after
the first week of life and a similar time schedule was observed for Bacteroides, which
colonize the lower intestinal tract from the second week of life. In contrast to other
species, the composition of the gut flora is characterized by relatively high numbers
of Clostridium perfringens and also lecithinase negative clostridia, probably reflect-
ing the carnivorous type of diet. Puppies may acquire gastric Helicobacter infection
from dams or can infect each other in early life. Clostridium difficile is neither for
dogs nor for cats an important enteric agent. The possibility of occasional human
infections by household dogs and cats needs further investigation. Young puppies and
kittens can be regarded as potential transmitters of Campylobacter spp., whereas
salmonellosis seems to occur rarely and there is no unequivocal link between the
occurrence of these potentially harmful bacteria and the occurrence of diarrhoea.
1. INTRODUCTION
Historically, dogs and cats were useful models for studying the intestinal flora in
humans. Some researchers were driven by their specific interests into the potential
health implications of zoonotic disease transmission. Extensive work was done from
the beginning of the 20th century, regarding the physiological and pathological
microbial colonization of the oral cavity, the stomach and the intestinal tract
(Oppmann, 2001). In contrast to that in other experimental animals, the microbial

104 J. Zentek
colonization of the feline and canine digestive tract has been studied less intensively
in the later years of the 20th century. This can be explained by the decreasing
significance of dogs and cats as models for human research and by the limited inter-
est of researchers in specific particularities of the intestinal flora in these species.
The microbial colonization of the gastrointestinal tract in newborn dogs and cats
begins immediately after the delivery from the sterile uterine environment. It is influ-
enced by maternal, environmental, and nutritional factors. After birth, pups are
normally fed exclusively on dam’s milk until the age of 3−4 weeks. At that time, the
energy requirements of the growing young are beginning to exceed the dam’s capac-
ity for milk production. The first diet introduced is normally milk-based, liquid food
that can be easily ingested and which is characterized by similar nutritional traits to
canine or feline milk. This type of feed is gradually changed to a more carnivorous
type of diet, based on such ingredients as animal proteins and fats, starch and low
amounts of dietary fibre. This shift of dietary habits is normally coinciding with
environmental changes and both may affect the composition of the intestinal
microflora, but specific data for this period are lacking.
2. MICROBIAL COLONIZATION OF THE ORAL CAVITY

The development of the microbial colonization of the oral cavity has not been stud-
ied in dogs and cats and no data are available for newborn kittens and puppies.
A comparison of human, canine and feline mouth flora (Rayan et al., 1991) demon-
strated that human oral flora contained the smallest number of bacteria followed
by dog and cat oral flora. In cats between 6 and 12 months of age the mean number
of viable bacteria from samples taken from gingival margins was log 10 (10.7) with
a range of 7 to 16 different species (Love et al., 1990). Of a total number of 150
isolates studied, 73% were obligate anaerobes. Of the facultatively anaerobic species,
Actinomyces (including Actinomyces viscosus, Actinomyces hordeovulneris and
Actinomyces denticolens) comprised 12%, Pasteurella multocida 9.3% and
Propionibacterium species 6%. Gram-negative bacilli belonging to the genera
Bacteroides and Fusobacterium represented 77% of the obligate anaerobes isolated.
Clostridium villosum comprised 10.1% of the obligatory anaerobic isolates,
Wolinella species made up 6.4%, while 4.6% were Peptostreptococcus anaerobius.
The most commonly isolated obligatory anaerobic species was Clostridium villosum
and the most commonly isolated facultatively anaerobic species was Pasteurella
multocida. Pasteurella spp. have been described early in the oral cavity of dogs and
cats (Bisgaard and Mutters, 1986). Bacteroides species were isolated from the
oral cavity of dogs and were also demonstrated in cats, in part associated with
diseases (Love et al., 1989, 1990, 1992b). By dot-blot hybridization assay pig-
mented asaccharolytic Bacteroides/Porphyromonas species were investigated.
Bacteroides salivosus was distinguished from other anaerobic species isolated from
normal and diseased mouths of cats (Love et al., 1992a). Bacteroides tectum and
Bacteroides fragilis were cultured from the oral cavity of cats (Love and Bailey, 1993).
Microbial ecology of the dog and cat 105
Bacteroides species constituted, as a proportion of all anaerobic isolates examined,

37.5% from normal gingiva and 27.7% from diseased gingiva in cats (Love et al.,
1989). Gingival scrapings of dogs were examined for the presence of CDC Groups
EF−4 bacteria. Fifty-nine EF−4 strains were isolated from 92% of 49 dogs. Among
the Group EF−4 bacteria, the majority of isolates belonged to the arginine-negative
(biovar “b”) Group EF−4 (42 strains recovered in 82% of dogs). Seventeen arginine-
positive strains (biovar “a”) were recovered from only 35% of dogs (Ganiere et al.,
1995). Occurrence of Gram-positive, catalase-negative, facultatively anaerobic cocci
was reported. Different sublines belonging to the genus Gemella were cultured,
among these Gemella haemolysans, Gemella bergeri, Gemella morbillorum and
Gemella sanguinis (Collins et al., 1999). Ureaplasma spp. have been isolated from
the oral cavities of cats and dogs and genomic relatedness was shown (Harasawa
et al., 1990a,b, 1993). Filamentous bacteria could be cultured from oral eosinophilic
granulomas of a cat (Russell et al., 1988). Capnocytophaga spp. have been isolated
from the oral flora of dogs and cats (Blanche et al., 1998) and are of importance as to
their infectious potential in dog bite wounds.
3. MICROBIAL COLONIZATION OF THE STOMACH

The stomach seems to be colonized by few bacteria in newborn dogs and cats, com-
pared to calves, lambs or piglets. Clostridium perfringens and Staphylococcus
aureus were the predominant bacteria in the stomach contents of puppies fed
mother’s milk, while Bacteroides spp. were not detectable over the suckling period.
Lactobacilli were found only in puppies older than 10 days (Smith, 1965).
Staphylococcus aureus may have originated from the bitches’ breast skin, for this
organism was isolated from this location and from the stomach contents of puppies
but it was not found in the faeces of the dams (table 1). In clinically healthy
Table 1. Microflora (log10/g chyme) in the stomach of puppies receiving dam’s milk (Smith, 1965)
106 J. Zentek
post-parturient bitches staphylococci were isolated from 30.3% of milk samples in

pure cultures and 6.8% in anacultures. Small numbers of bacteria were isolated in
most of the samples, but 67.4% showed moderate bacterial growth. According to
this study, there was no direct influence of lactiferous gland colonization on the
mortality of puppies (Kuhn et al., 1991).
Advance in age and the potential influence on the luminal gastrointestinal microflora
of beagle dogs was investigated by comparing dogs less than 12 months of age with
dogs more than 11 years of age (Benno et al., 1992). There was no clear tendency for
the bacterial counts to be higher in the older beagles compared to dogs below 1 year.
Lactobacilli, enterobacteria and streptococci were found in the gastric chyme of most
dogs, while other bacteria and also yeasts were determined only irregularly (table 2).
Gram-negative bacilli were found adhering to the gastric surface as well as within
epithelial cells in the stomach of a puppy (Wada et al., 1996). Puppies may acquire
gastric Helicobacter infection, as proven for Helicobacter salomonis, from dams
during lactation or puppies can infect each other in early life (Hanninen et al., 1998).
In kittens, Clostridium perfringens, Escherichia coli and streptococci were found
in the stomach contents of newborns in reasonably high numbers (table 3). In
kittens over 5 days old Gram-positive anaerobic rod-like bacteria formed the major
component of the chyme in the stomach. Lactobacilli were only found in 10-day-old
kittens in higher numbers. In contrast to puppies, Staphylococcus aureus was not
Table 2. Microflora of the stomach (log10/g chyme) in beagles less than 1 year old compared to
beagles more than 11 years old (Benno et al., 1992)
Table 3. Microflora (log10/g chyme) in the stomach of kittens receiving dam’s milk (Smith, 1965)
cultured from the chyme of any location of the gastrointestinal tract, and breast
swabs taken from the queens were negative, too.
The gastric microflora of felines was investigated in three unweaned kittens and in
adult cats fed a conventional or chemically defined, elemental ration (Osbaldiston and
Stowe, 1971). According to this study, enterococci and lactobacilli were the predominant
microflora. Streptococci, Escherichia coli, Clostridium spp. and Bacteroides spp. were
isolated from one out of three individuals (table 4). Dietary changes were not associated
Table 4. Microflora (log10 /g chyme) in the stomach chyme of kittens receiving dam’s milk and
adult cats fed a conventional or a chemically defined diet (Osbaldiston and Stowe, 1971)
108 J. Zentek
with significant changes in the bacterial populations or patterns of distribution within the
intestinal tract nor was there a clear difference between the kittens and the adult cats.
4. MICROBIAL COLONIZATION OF THE SMALL INTESTINE

In newborns the small intestine is sterile, but first colonization with bacteria occurs
during the few hours after birth. Within 12 hours, the upper (table 5) and the lower
(table 6) parts of the small intestine of canine puppies were colonized by strepto-
cocci, staphylococci and Clostridium perfringens and after a few days also by
Escherichia coli and lactobacilli. Bacteroides species were not identified as part of
the small intestinal microflora in this study (Smith, 1965).
The small intestinal microflora may contribute to infectious diseases in puppies.
Gram-positive bacilli in association with focal to diffuse necrosis of the superficial
portions of the villi, were observed in histological sections of specimens of small
intestine from all except one of 57 dogs from which parvovirus and Clostridium
perfringens had been identified. These findings indicate that Clostridium perfringens
frequently proliferates in dogs with parvovirus infection (Turk et al., 1992). Enteric
infection with an attaching and effacing Escherichia coli was diagnosed in a puppy
with diarrhoea. Characteristic lesions of bacterial attachment of the brush border of
the enterocytes were demonstrated by transmission electron microscopy. The
Escherichia coli strain isolated from the small intestine belonged to serotype
O49:H10, and a positive immunoperoxidase reaction was obtained on the bacteria
attached to the enterocytes with an anti-Escherichia coli O49 antiserum (Broes
et al., 1988). In puppies with a clinical history of gastrointestinal disease attaching
and effacing Escherichia coli (AEEC) or enterotoxigenic Escherichia coli (ETEC)
Table 5. Microflora (log10/g chyme) in the upper small intestine (duodenum) of puppies receiving
dam’s milk (Smith, 1965)
Table 6. Microflora (log10/g chyme) in the lower small intestine (ileum) of puppies receiving
infection has to be considered often with coinfection with other enteric pathogens
(Drolet et al., 1994). Campylobacter jejuni was inoculated experimentally into the
duodenum of 13 puppies (2 to 5 weeks old). All had positive faecal cultures for 1 to
10 days without clinical signs of disease (Boosinger and Dillon, 1992).
Different and in relation to the current standards inadequate methods have to be
taken into account when comparing the results of the studies in puppies and in older
dogs. Compared to the findings in puppies, a broader spectrum and higher counts of
intestinal bacteria were demonstrated in young dogs less than 1 year old and in dogs
at the age of over 11 years (table 7). The total counts and the numbers of lactobacilli,
Bacteroides, peptostreptococci, and bifidobacteria in the elderly animals were sig-
nificantly lower than those in younger dogs. The numbers of lecithinase-positive
clostridia (mainly Clostridium perfringens) and bacilli were significantly higher in
older dogs compared to dogs below 1 year.
Clostridium perfringens, Escherichia coli and streptococci were the organisms
that first colonized the alimentary tract of kittens (tables 8 and 9). Gram-positive
anaerobic rods formed the major component of the flora in kittens that were over
5 days old. Individual variations have to be taken into account, as there were high
numbers of lactobacilli in one 120-day-old kitten. Lactobacilli were only rarely
found in the other kittens and in lower numbers. Uchida et al. (1971) investigated
the intestinal microflora of kittens after weaning at the age of 12 weeks, when a diet
based on horse meat and milk was fed. They found Bacteroides, bifidobacteria, lac-
tobacilli, eubacteria, clostridia, streptococci, staphylococci, Enterobacteriaceae and
moulds as part of the duodenal and ileal microflora, indicating the shift in the com-
position of the small intestinal microflora depending on age and diet and the
expected establishment of anaerobic conditions in the gut.
Table 7. Duodenal and ileal microflora (log10 /g chyme) in beagles (n = 8) of different ages
(Benno et al., 1992)
Table 8. Microflora (log10/g chyme) in the upper small intestine (duodenum) of kittens receiving
Table 9. Microflora (log10/g chyme) in the lower small intestine (ileum) of kittens receiving dam’s
milk (Smith, 1965)
5. MICROBIAL COLONIZATION OF THE COLON AND THE RECTUM

Bacteria are much more numerous in the colon and rectum compared to the small
intestine in newborn puppies and kittens. The flora in puppies consists of
Clostridium perfringens, streptococci and staphylococci shortly after birth and this
is subsequently followed by Escherichia coli, lactobacilli and Bacteroides (tables 10
and 11). Bacteroides have been found from day 9 and are the dominating part of the
colonic microflora in older puppies (Smith, 1965). In another study in newborn pup-
pies, rectal swabs were investigated periodically from nine puppies from three
bitches over a period of 55 days after birth (Matsumoto et al., 1976). The bacterial
Table 10. Microflora (log10/g chyme) in the colon of puppies receiving dam’s milk (Smith, 1965)
112 J. Zentek
Table 11. Microflora (log10 /g chyme) in the rectum of puppies receiving dam’s milk
(Smith, 1965)
groups most frequently encountered after 6 hours post natum were staphylococci,
streptococci, Enterobacteriaceae and clostridia; lactobacilli, Bacteroides and bifi-
dobacteria were found later, although their time of appearance varied considerably
with individuals. After their appearance these organisms showed sharp fluctuations
in number. The total count of viable bacteria in the faecal samples was log 109/g
or more 24 h after birth. Streptococci and Enterobacteriaceae were predominant
up to 7 days of age. After that no definite groups were prevalent. Lactobacilli and
bifidobacteria, however, were predominant at 42 days of age and later. The bacter-
ial flora in puppies at this stage was identical with the one in adult dogs (Matsumoto
et al., 1976). The composition of the normal staphylococcal flora of bitches and their
litters in a breeding unit showed that Staphylococcus intermedius formed the
predominant staphylococcal isolate. Staphylococcus intermedius counts at the vagi-
nal vestibulum of the pregnant bitches were higher than at any other site sampled
and did not alter markedly until whelping when a decrease was observed.
Staphylococcus intermedius was not found at the anal site in any of the six bitches
and only transiently colonized some of the puppies (Allaker et al., 1992).
The impact of age on the gastrointestinal microflora of beagle dogs seems to be
more pronounced in the large intestine compared to the stomach or the small intestine
(Benno et al., 1992). The numbers of peptostreptococci and bifidobacteria in the large
bowel of the younger dogs were significantly higher than those in elderly dogs. Larger
populations of lactobacilli were found in the caecum and colon of dogs less than 1 year
old, whereas the numbers of Clostridium perfringens and streptococci increased in the
older population (table 12). The numbers of Bacteroides in the caecum and rectum and
eubacteria in the caecum and colon of the elderly dogs were lower compared to the
younger individuals. The incidence of lecithinase-negative clostridia increased in the
older dogs only in the rectum but not in the other locations of the large intestine.
Table 12. Large intestinal microflora (log10/g chyme) in beagles (n = 8) of different ages (Benno et al., 1992)
114 J. Zentek
Clostridium difficile was monitored during the first 10 weeks after birth in puppies
(Perrin et al., 1993). More than 90% of the puppies and 43% of the dams harboured
Clostridium difficile at least once in their faeces and 58% of the puppies carried toxigenic
Clostridium difficile at least once during the survey. In the puppies, Clostridium difficile
carriage rates ranging from 3.1 to 67.1% were observed. In comparison, the Clostridium
difficile carriage rate was 1.4% in a control group of healthy dogs more than 3 months
old. Discrepancies in the toxigenic phenotype of the Clostridium difficile strains isolated
in the same litter, showed that the newborn dogs were transiently infected with different
strains, and that the dam is often not the source of infection with Clostridium difficile. The
incidence of Clostridium difficile was 46% in faecal samples from healthy puppies with
toxigenic strains found in 61.5% of the healthy neonate dogs (Buogo et al., 1995). It
seems that, in contrast to the significance for man, Clostridium difficile is neither for dogs
nor for cats an important enteric agent. The possibility of occasional human infections by
household dogs and cats needs further investigation (Weber et al., 1989).
Dogs in a closed breeding unit were shown to be asymptomatic excretors of
Campylobacter at the age of 8 weeks. Increasing serum antibody levels, which were
correlated with the excretion of organisms, were demonstrated in the puppies and
serum antibodies were also demonstrated in adult dogs (Newton et al., 1988). In a
cross-sectional study carried out in Denmark, 72 healthy puppies and kittens were
sampled for faecal Campylobacter shedding by culture of rectal swab specimens. 29%
of the puppies were positive for Campylobacter spp., with a species distribution of 76%
Campylobacter jejuni, 5% Campylobacter coli and 19% Campylobacter upsaliensis.
Of the kittens examined, only two (5%) excreted Campylobacter spp.; both strains were
Campylobacter upsaliensis. Young puppies and kittens can be regarded as potential
transmitters of human-pathogenic Campylobacter spp., including Campylobacter
upsaliensis (Hald and Madsen, 1997). In another study, 32% of healthy puppies were
positive for Campylobacter spp. with a peak at the age of 8 weeks (Buogo et al., 1995).
Salmonellosis seems to occur very rarely in puppies. Infection with Salmonella
typhimurium induces haemorrhagic enteritis with discrete fibronecrotic areas (King,
1988). In bacteriological studies of 159 faecal and intestinal content samples from dogs
with diarrhoea, Escherichia coli (73 non-haemolytic) was found in 157 samples,
Klebsiella and Staphylococcus aureus were found in nine cases and Salmonella sp. in
one (Zschock et al., 1989). Other investigators determined Salmonella in 6.5% of
faecal samples of puppies (Buogo et al., 1995), but could not establish a link between
the incidence of Salmonella, Campylobacter or Clostridium difficile with episodes of
diarrhoea. In 11-day-old puppies with diarrhoea, a mixed growth of non-haemolytic
Escherichia coli and Enterococcus durans serotype 2 was isolated from the jejunum
with lesions that resembled those reported in natural and experimental Enterococcus
durans infections in foals and gnotobiotic pigs (Collins et al., 1988).
The development of the colonic microflora in kittens has been shown to be com-
parable to the findings in puppies. Clostridium perfringens, Escherichia coli and strep-
tococci were the first organisms to colonize the alimentary tract of kittens (table 13).
Table 13. Microflora (log10/g chyme) in the colon of kittens receiving dam’s milk (Smith, 1965)
Bacteroides, lactobacilli and Gram-positive obligatory anaerobic rods could be

detected within 2 days after birth (Smith, 1965). Bacteroides were found in the
colon but not in the anterior parts of the alimentary tract in kittens.
Enterococci, Enterobacter, Catenabacterium and, in one individual, yeasts, were
determined in the midcolon of kittens (table 14). The concentrations were compar-
able to the findings in adult cats (Osbaldiston and Stowe, 1971). The concentrations
Table 14. Microflora (log10 /g chyme) in the midcolon chyme of kittens receiving dam’s milk and
adult cats fed a conventional or a chemically defined diet (Osbaldiston and Stowe, 1971)
116 J. Zentek
of enterococci were greater than in the stomach or in the jejunum. Clostridia were
not detected in these three kittens. The caecal and faecal microflora of 3-month-old
kittens fed on horse meat and milk was dominated by Bacteroides, clostridia, eubac-
teria and streptococci and in lower numbers staphylococci, Enterobacteriaceae and
moulds. The total bacterial counts were 9.1−9.2 log10 /g and lactobacilli and bifido-
bacteria were found only infrequently (Uchida et al., 1971).
Dual infection by Clostridium piliforme and feline panleukopenia virus (FPLV)
was found in three kittens. Pathology was characterized by focal necrosis and
desquamation of epithelial cells with occasional neutrophile infiltration in the large
intestine. Large filamentous bacilli and spores were observed in the epithelium
(Ikegami et al., 1999). Salmonella typhimurium was isolated in kittens with intestinal
crypt necrosis, hepatic, splenic and lymph node inflammation and necrosis. All had
been vaccinated previously with a modified-live virus vaccine. Salmonellosis was
interpreted as a consequence of a mild immunosuppression induced by vaccination
(Foley et al., 1999). Enterococcus hirae was isolated from a 2-month-old female
Persian cat that had been showing episodes of anorexia and diarrhoea. Cocci were
located along the brush border of small intestinal villi, without significant inflamma-
tory infiltration. Similar bacteria were present within hepatic bile ducts and pancre-
atic ducts and were associated with suppurative inflammation and exfoliation of
epithelial cells. Faecal culture from an asymptomatic adult female from the same
cattery also yielded large numbers of Enterococcus hirae (Lapointe et al., 2000).
The current knowledge of the intestinal microecology of puppies and kittens and its
development is limited and further investigations are needed. Questions to be
addressed include the significance of the intestinal microflora for kitten and puppy
losses and their potential significance as carriers for human diseases. The character-
ization of the intestinal microecology should be studied by molecular biological
methods and will offer new insights into the significance of the gut bacteria for
current topics of high scientific interest, such as the development and the function of
the intestinal immune system, the aetiology and pathogenesis of chronic inflamma-
tory bowel disease, allergies and the potential effects of manipulation of the
microflora by probiotics or dietary means. Age effects on the intestinal microflora are
of increasing interest in dogs and cats and both species could be useful models for the
situation in humans, as to the age distribution with an increasing geriatric population.
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6 Molecular approaches in the study of
gut microecology
A. Schwiertz and M. Blaut
German Institute of Human Nutrition, Gastrointestinal Microbiology,

Arthur-Scheunert-Allee 114-116, D-14558 Bergholz-Rehbrücke, Germany
Advances in molecular biology have led to the development of a variety of culture-

independent approaches to describe bacterial communities. Most of the new strate-
gies, based on the analysis of DNA or RNA allow direct investigation of community
diversity, structure and phylogeny of microorganisms in the gastrointestinal tract.
This chapter will cover molecular approaches for studying the microbial flora, and
the molecular tools to monitor the presence of specific strains in the intestine.
Special emphasis will be on the advantages and disadvantages, respectively, of
various DNA- or RNA-based methods for the study of the microbiota in the
gastrointestinal tract of humans and animals.
1. INTRODUCTION
The bacterial flora of the gastrointestinal (GI) tract of humans and animals has been
studied more extensively than that of any other anatomical site. This may be due to
the high number of bacteria encountered in the intestine. The total number of
bacteria resident in the human gastrointestinal tract has been estimated to reach 1014
bacterial cells, thus outnumbering the total number of body cells (Savage, 1977).
The highest bacterial density is found in the distal colon. The composition differs
between and within animal species. In humans, there are differences in the compo-
sition of the flora which are influenced by age, diet, cultural conditions, and the use
of antibiotics. Any estimations on the number of bacterial species are mere guesses.
It can be undoubtedly stated that the microorganisms described so far, represent only
a small fraction of the species making up the natural microbial community. In
ruminants, the microbial community of the rumen consists of 1010 bacteria/ml,
106 protozoa/ml and 103 to 107 fungi/ml (Hespell et al., 1997). A wealth of data on

120 A. Schwiertz and M. Blaut
the identity and metabolic potential of the bacteria have been accumulated, but it has
become clear that a considerable proportion of bacteria has eluded cultivation and
description. This is due to the fact that, until recently, the study of the gut diversity
was restricted to the use of classical microbiological techniques, such as selective
enrichments, pure culture isolation, and most-probable-number estimates. However,
many bacteria have eluded cultivation because their specific growth requirements
are not known and media are often not truly selective or specific. Hence, it has been
difficult to obtain a realistic view of the microbial community in the gastrointestinal
tract. It has been estimated that only 15−58% of the human intestinal bacteria have
been cultured or detected yet (Langendijk et al., 1995; Wilson and Blitchington,
1996; Suau et al., 1999). The classical culture methods may lead to over- or under-
estimations of bacterial species and groups (Langendijk et al., 1995; Doré et al., 1998).
Therefore, it is not surprising that the lack of exact classification schemes and biases
introduced by culture-based techniques have resulted in an inaccurate description and
understanding of the microbial community of the GI-tract. The classification and
enumeration of the genus Eubacterium may serve as an example. In the human intes-
tinal tract, Eubacterium is the second most common genus (Finegold et al., 1974,
1983). Since the identification of Eubacterium species based on phenotypic traits
requires experience and is time-consuming, many studies involving the analysis of
human faecal flora composition have refrained from looking at this genus. Recent
studies indicate that the numerical importance of several Eubacterium species has
been overestimated (Schwiertz et al., 2000).
The detection of organisms or groups has to be based on targets which allow their
unequivocal identification. The morphological and physiological characteristics of
prokaryotes are simpler than those of eukaryotes. Therefore, an identification based
on phenotypic features is relatively difficult, time-consuming and requires experi-
enced personnel. To overcome these difficulties, the information contained in the
molecular sequences of their DNA, RNAs and proteins is increasingly used to infer
the relationships of microorganisms. Phylogenetic investigations targeting phyloge-
netic markers such as large subunit rRNA, elongation factors, and ATPases have
shown that 16S rRNA-based trees reflect the history of the corresponding organisms
globally (Woese, 1987; Gutell et al., 1994; Amann et al., 1995).
Molecular sequence analysis, particularly of rRNA, reflects the phylogenetic
interrelationships of microorganisms (Woese, 1987). It is possible to identify
microbes based solely on their ribosomal RNAs. Taxonomists have become inde-
pendent of culturing for identifying a microbial species. The numerous techniques
employed in the description of gut microbial diversity are depicted in fig. 1.
2. MOLECULAR TECHNIQUES
Nucleic acid-based approaches for the detection and characterization of microbial
populations and their function within the GI-tract allow the microbiologist to deduce
Molecular approaches in gut microecology 121
Fig. 1. Strategies for the characterization of microbial communities. Arrows show the interconnections of
methods, material and information in the study of microbial ecosystems. PCR, polymerase chain reaction;
RT-PCR, reverse transcriptase PCR; FISH, fluorescence-in situ-hybridization; STARFISH, substrate-tracking
audioradiographic FISH; DGGE, denaturing gradient gel electrophoresis; RFLP, restriction fragment length
polymorphism; RAPD, randomly amplified polymorphic DNA. See text for further discussion.
evolutionary linkages between the involved populations. Various culture-independent

methods have been developed to identify microorganisms in samples from the GI-
tract without prior cultivation. These include direct sequence analysis, sequencing of
extracted 5S, 16S and 23S rRNAs, and analysis of rRNA using reverse transcriptase
or cloned rRNA genes obtained by amplification using the polymerase chain reaction
(PCR). Further techniques such as denaturing gradient gel electrophoresis (DGGE),
temperature gradient gel electrophoresis (TGGE), dot-blot or slot-blot hybridization
and whole cell-in situ-hybridization, better known as fluorescence-in situ-hybridization
(FISH), have been applied to analyse the complex microbial flora of the gut.
We begin with a brief description of the molecular-based techniques used in
microbiology. Emphasis will be on the RNA-based methods, simply because RNA
(especially rRNA) is the most commonly employed target nucleic acid in environ-
mental microbiology.
2.1. RNA versus DNA

Over the past decade, important advances in molecular biology have led to the devel-
opment of culture-independent approaches in describing bacterial communities.
These new strategies, based on the analysis of DNA or RNA directly extracted from
environmental samples, circumvent the steps of isolation and culturing of bacteria.
It is important to distinguish between identification, quantification, and monitoring
for function and activity.
Fundamental to the use of the various techniques is the ability to define suitable
nucleic-acid sequences that identify a particular microorganism or gene. For this
purpose various oligonucleotide probes have been developed and used successfully.
In general, target nucleic acid sequences fall into the following categories:
1. DNA sequences that code for proteinaceous toxins
2. DNA sequences that code for antigens
3. Unique plasmid-borne DNA sequences
4. Intergenic spacer regions (ISR)
5. Ribosomal RNA (rRNA) sequences
6. Messenger RNA (mRNA) sequences
All these strategies employ the unique physical properties of DNA and RNA to
reassociate or hybridize. Hybridization is a term used to describe the specific comple-
mentary association due to hydrogen bonding, under experimental conditions, of
single-stranded nucleic acids. It should more exactly be referred to as “annealing”, as
this is the physical process responsible for the association: two complementary
sequences will form hydrogen bonds between their complementary bases (G to C, and
A to T or U) and form a stable double-stranded, anti-parallel “hybrid” helical molecule.
The various hybridization techniques (DNA–DNA, DNA–RNA) are similar in so far as
they are simple, fast, inexpensive and universally applicable. Essential to most is the
determination of an optimal hybridization temperature. For a theoretical background on
the development and application of nucleic acid probes see Stahl and Amann (1991).
DNA-based technologies are aimed at the specific detection of genes. However,
the copy number of a given gene on the bacterial genome is usually low. In contrast,
the copy number of the various RNAs, in particular of rRNA, is considerably higher.
Depending on the type of RNA (mRNA, rRNA) it can vary from 1000 to more than
50 000 copies in any living cell. Moreover, RNA levels may be an indirect reflec-
tion of the cell’s metabolic activity.
2.2. Isolation of nucleic acids

Most molecular techniques require the prior isolation of nucleic acids from a faecal
sample. Depending on the method to be used, e.g. hybridization, cloning, or amplifica-
tion, the purity of the nucleic acid is crucial. Several techniques require high-quality
RNA or DNA in high yield, free of the respective other nucleic acid. Since the amount
of DNA or RNA isolated from a given bacterial cell may be very small, many investi-
gators concentrate on combinations of direct lysis methods with subsequent PCR
amplification (Wang et al., 1996; Hengstler et al., 1998). However, extraction of RNA
from faeces requires special attention as RNAs are highly susceptible to degradation
by RNases during the extraction procedures.
Several methods are now in use for the extraction from faeces of either RNA
(Stahl et al., 1988; Doré et al., 1998) or DNA (Marmur, 1961; Wang et al., 1996;
Hengstler et al., 1998). All of the methods have in common that the cell wall has to
be disrupted to allow the extraction of the nucleic acids. The cell wall is disrupted by
mechanic, enzymatic or chemical methods followed by methods involving extraction

with phenol and chloroform and/or further preparations using caesium chloride
gradients or spin columns to isolate the nucleic acids. A critical step in the procedure
is the purification of nucleic acids in such a way that they can be used for further
analysis. This is not a trivial task since the humic compounds and complex polysac-
charides (Monteiro et al., 1997) may inhibit enzymes involved in further analytical
steps. Especially humic substances have been shown to interfere with enzymatic
digestion of DNA, PCR amplification of DNA (Steffan et al., 1988; Rochelle et al.,
1992; Porteous and Armstrong, 1991; Tebbe and Vahjen, 1993) and dot-blot
hybridization of DNA (Tijssen, 1993). In a recent study Alm and co-workers showed
that humic substances may affect RNA hybridization (Alm et al., 2000).
Considering these effects, it becomes clear why recent developments in nucleic
acid extraction have focused on strategies to remove humic compounds (Tsai and
Olson, 1992; Herrick et al., 1993; Moran et al., 1993).
2.3. DNA-based detection

The application of DNA-based techniques for the detection and identification of
microorganisms is well established. Whole genomic DNA of an organism is employed
in the pulse-field gel electrophoresis which extends the size range of resolution of DNA
molecules to many megabases. Treatment of plasmid-DNA or fragments of genomic
DNA with restriction enzymes may yield DNA molecules larger than 25 kb which are
poorly resolved by standard agarose gel electrophoresis. A method resolving larger
DNA molecules is the pulse-field gel electrophoresis (PFGE) developed by Schwartz
et al. (1982). In this method, the DNA molecules are applied to an agarose gel in which
the direction of the electric field keeps changing constantly. While the molecules fol-
low the electric field, they become trapped in the gel matrix every time the direction of
the electric field is altered. They cannot make any further progress through the gel until
they have reorientated themselves along the new axis of the electric field. The larger
the DNA molecules, the longer the time that is required for the reorientation. DNA
molecules whose reorientation times are less than the period of the electric pulse, will
therefore be separated according to size (fig. 2). The protocols for PFGE are now
routinely used in many laboratories working with DNA. In gut microbial ecology,
however, this technique has rarely been used. Nevertheless, a recent study described the
distribution of Salmonella in swine herds using PFGE (Letellier et al., 1999).
In gut microbial ecology, various hybridization and PCR techniques have found
a wide acceptance. These techniques will be discussed in more detail.
2.3.1. DNA hybridization

The first attempts to differentiate biochemically indistinguishable bacterial species
of the GI-tract by using cloned genomic fragments were performed by Kuritza and
Salyers (1985). Fragments of genomic DNA isolated from faecal samples were
Fig. 2. Scheme depicting various molecular methods for the differentiation of microorganisms. A: PFGE of
λ-DNA cut with HindIII. B: RAPD profiles were obtained with genomic DNA of Eubacterium rectale (1),
Bacteroides fragilis (2), Escherichia coli (3) and various strains of Eubacterium ramulus using the M13-core
as random primer (Simmering et al., 1999). C: RFLP/ARDRA profiles of amplified 16S rDNA from
Eubacterium dolichum (lanes 2, 4, and 6) and Fusobacterium mortiferum (lanes 1, 3, and 5) digested with
EcoRI, BamHI and XmnI (Schneider et al., 1999). M depicts the used marker lanes.
bound to filter supports and subsequently hybridized with an oligonucleotide probe

specific for Bacteroides vulgatus, to detect this organism. Differences in the appli-
cation of the DNA to the filter gave rise to the following designations: dot blotting,
slot blotting and touch blotting.
In dot blotting, DNA in solution is applied to the filter by applying small
volumes of this solution to small circular wells of a manifold. Slot blotting is
identical except that the wells are elongated to form a slot. In touch blotting, DNA
or whole organisms are applied manually to the filter. In all these methods it is
essential to denature the DNA prior to hybridization. Denaturation can be done
either before or after blotting to the filter. The detection of the target is done by
hybridization of a probe, which is complementary to the target sequence. These
probes are single strands of nucleic acid with the potential of carrying detectable
marker molecules (32P, DIG, Biotin). Radioactively labelled probes are often
preferred because of their sensitivity, but non-radioactive systems (DIG, Biotin)
have the advantage of being more convenient in field or clinical assays (Yamamoto
et al., 1992; Kaneko and Kurihara, 1997; Miyamoto and Itoh, 1999). Their longer
shelf life compared to 32P-labelled probes is another advantage. Furthermore,
non-radioactive probes eliminate radioactive hazards.
2.3.2. PCR-based methods

With the advent of the PCR, it became possible to amplify DNA molecules from
very low quantities. The ability to analyse PCR amplification products is a prereq-
uisite for rapid data acquisition on genomic organization and regulation. Essential
to all this is the nucleotide sequencing of the PCR products to confirm the specificity
of the amplicon, identify genetic variations (e.g., polymorphisms), identify
unknown genes and map these genes within the bacterial genome. Thus, the PCR
has opened a huge variety of new methodologies for the study of environmental
samples including the GI-tract. The rapid amplification of DNA with PCR has also
accelerated the sequencing of several bacterial genomes (http://www.ebi.ac.uk/
genomes/). Most of the hitherto completely sequenced bacterial genomes are from
pathogens. Of the bacteria for which the complete genome sequence is available,
only Escherichia coli is relevant for the GI-tract. Additional sequencing of bacterial
genomes of the more abundant species of the GI-tract, will help to redefine our view
of this ecosystem and help in fast and accurate descriptions.
One of the first PCR applications to faecal DNA, aimed to identify enterotoxi-
genic Escherichia coli in clinical specimens (Olive, 1989). Since then the PCR
technology has been used repeatedly for the detection of bacteria in faecal samples
(Kreader, 1995; Wang et al., 1996; Hengstler et al., 1998).
2.3.3. DNA fingerprinting

DNA-fingerprinting methods have been introduced for the characterization of
bacterial species or communities (Kimura et al., 1997; Bateup et al., 1998;
McBurney et al., 1999). The DNA-fingerprinting methods are based on the amplifi-
cation of one or several stretches of genomic DNA. The first such method was arbi-
trarily primed PCR (AP-PCR), better known as randomly amplified polymorphic
DNA (RAPD; Welsh and McClelland, 1990). With this method it is possible to cre-
ate a genomic fingerprint of a given species. Since random primers are used, it is not
known to which sites of the genome they bind. The generated amplification prod-
ucts often show size polymorphisms even within species. RAPD/AP-PCR analysis
offers the possibility of creating polymorphisms without any prior knowledge of
the DNA sequences of the organism investigated. The patterns produced are highly
polymorphic, allowing even discrimination between isolates of a species, if suffi-
cient numbers of primers are used. The method is fast and economic for screening
large numbers of samples. Strain-specific arrays of DNA fragments (fingerprints)
are generated by PCR amplification using arbitrary oligonucleotides to prime DNA
synthesis from genomic sites which they fortuitously match or almost match (fig. 2).
DNA amplified in this manner can be used to determine the relatedness of
species (Fanedl et al., 1998; Simmering et al., 1999) or for analysis of restriction
fragment length polymorphisms (RFLPs). An RFLP may be the result of length
mutation, and/or point mutation at a restriction enzyme cleavage site at a given
chromosomal location. RFLPs can be detected by analysing restriction digests of

genomic DNA through Southern hybridization. The probes used in RFLP analysis
can be generated from cloned genomic DNA, cDNA, or from specific DNA
segments amplified by PCR. Depending on the probe used, RFLPs can be used to
analyse variations in the ribosomal rDNA region or in repetitive and single-copy
sequences. DNA hybridization-based RFLP analysis requires the isolation of large
amounts of purified DNA. With PCR it becomes possible to analyse specific
sequences from small amounts of cells. The advantages of PCR-RFLP lie in its
speed, sensitivity and specificity. PCR can be performed on crude DNA extracts
with a pair of region-specific primers. Variation of the amplified fragment can be
further analysed by restriction enzyme digestion and electrophoretic separation.
This technique is widely accepted and used in the characterization of GI-tract iso-
lates (McIntosh et al., 1999; Ohkuma et al., 1999; Barcenilla et al., 2000). There are
several variations of this technique but not all have been applied to the analysis of
GI-tract organisms. The regions most commonly examined by PCR-RFLP are the
rDNA sequences. This method is termed amplified ribosomal DNA restriction
analysis (ARDRA) and has been used among others for the identification of lacto-
bacilli and Enterobacteriaceae (respectively, Giraffa et al., 1998; Di Giovanni et al.,
1999). The main limitation of this method lies in the choice of restriction enzymes,
which is crucial for obtaining a good resolution. An example of ARDRA/RFLP is
given in fig. 2.
T-RFLP or “terminal-RFLP” and length heterogeneity PCR (LH-PCR) have
been introduced recently (Suzuki et al., 1998). Bernhard and Field (2000) used
T-RFLP and LH-PCR to analyse faecal samples from cows and humans with respect
to the Bacteroides-Prevotella group and the genus Bifidobacterium. They reported
that host-specific patterns suggested a species composition difference in the
analysed bacterial populations. The patterns were highly reproducible within cows
and humans, respectively. Both methods recognize differences in the length of gene
fragments owing to insertions and deletions and the relative abundance of each
fragment. In order to track a bacterial species or group, it is essential to identify
a sequence (marker sequence) which is common to the bacterial species or group.
Such a marker sequence, e.g. 16S rDNA, can then be amplified by specific primers
for PCR and cut by appropriate restriction enzymes which will give a unique pattern
of the amplified marker sequence. Once a reliable identification pattern of this
marker sequence has been obtained, the sequence can be monitored easily and
quickly with fluorochrome-labelled primers. In addition, a database of identification
patterns can be generated for a given restriction enzyme, thus making it possible to
identify the bands by a profile to profile comparison.
Amplified fragment length polymorphism (AFLP) analysis (Vos et al., 1995) is
a modification of RFLP. RFLPs can be converted to AFLPs by ligating fluorescently
labelled adapters to the primers used for PCR amplification. AFLP has the potential
to detect large numbers of amplification products although AFLP, just like RFLP,
does not target specific areas of the genome. AFLP has been successfully employed
in the detection of Chlamydia psittaci strains (Boumedine and Rodolakis, 1998),
Salmonella enterica subsp. enterica (Lindstedt et al., 2000a), and Clostridium
perfringens (McLauchlin et al., 2000). By combining the detection of fluorescent
bands with a laser detection system, it is possible to obtain much more accurate and
faster results (Lindstedt et al., 2000b).
Ribosomal intergenic spacer analysis (RISA) uses the internal transcribed
spacers (ITS), which are non-coding regions of DNA sequence separating genes
coding for the ribosomal RNAs (Jensen et al., 1993). These ribosomal RNA (rRNA)
genes are highly conserved across taxa while the spacers in between them can be
subspecies-specific (Barry et al., 1991; Narayanan et al., 2001). The conservation of
the rRNA genes allows for easy access to the ITS regions with “versatile” primers for
PCR amplification. The variation in the spacers has proven useful for distinguishing
among a wide range of microorganisms. Using the ITS regions, Brookman et al.
(2000) examined the relationships within and between two genera of monocentric gut
fungi gathered from various geographical locations and host animals. Tannock et al.
(1999) demonstrated the usefulness of this technique for the identification of intestinal
Lactobacillus spp.
Fluorescent primers can be used in the amplification reactions to result in
fluorescent amplification products that, when separated by electrophoresis, yield
highly discriminative profiles. However, all these techniques have limited resolution
in identifying a specific phylogenetic group within a complex microbial community,
since they do not take advantage of the sequence information but only of restriction
sites. The drawback of spacer polymorphism analysis is that more than one PCR
product can result from a single organism because of different spacers. For exam-
ple, there are two kinds of spacers in Escherichia coli. The E. coli genome is known
to contain seven loci coding for ribosomal RNA (Kiss et al., 1977). In four of them,
the spacer region contains a single tRNAGlu gene. The other three loci have two
tRNA genes in this spacer region: tRNAIle and tRNAAla.
Another method that takes advantage of PCR amplification for identification of
bacteria, targets repetitive extragenic palindromic sequences (REP-PCR). REP-PCR
genomic fingerprinting makes use of DNA primers complementary to naturally
occurring, highly conserved, repetitive DNA sequences, present in multiple copies
in the genomes of most Gram-negative and several Gram-positive bacteria (Lupski
and Weinstock, 1992).
The PCR-based method of single-strand-conformation polymorphism (SSCP)
analysis which has not yet been employed for the description of gastrointestinal
communities, has been first used to generate genetic profiles of a freshwater
community (Lee et al., 1996). The method is based on the fact that under non-
denaturing conditions, single-stranded DNA has a folded structure which results
from intramolecular interactions and its nucleotide sequence. Prior to the separation
by electrophoresis, the amplified DNA is denaturated by heat and subsequently
separated by electrophoresis. The electrophoretic mobility of the single-stranded

DNA in a matrix is dependent on its length, molecular weight, and shape (Yap and
McGee, 1994). Therefore, in SSCP analysis, DNA fragments with the same size
but different sequences can be distinguished by electrophoresis, because of the
different mobilities due to their structure (Hayashi, 1991).
The fact that the described DNA-fingerprint techniques require prior cultivation
of the bacteria to be studied, is a major drawback of the methods described in this
section. They may therefore be regarded as less suitable for use in general popula-
tion descriptions.
2.3.4. DGGE/TGGE
In recent years denaturing gradient gel electrophoresis (DGGE) and temperature
gradient gel electrophoresis (TGGE) have gained increasing popularity among
microbiologists as molecular tools to compare the diversity of microbial communi-
ties and to monitor population dynamics (fig. 3). Both techniques take advantage of
the fact that the electrophoretic mobility of fragments increases, as part of the DNA
double helix unravels. This allows in principle the separation of DNA on the basis
of a difference in one single nucleotide (Sheffield et al., 1989). In brief, the
sequences of interest are amplified with PCR using an appropriate pair of primers.
One of these has a so-called “G + C-clamp” attached to the 5′ end. This “G + C-
clamp” prevents the two DNA strands from dissociating completely even under
highly denaturing conditions. Strand separation can be induced by an increase in
temperature (TGGE) or in the concentration of chemical denaturants such as for-
mamide or urea (DGGE). The vertical orientation of the denaturing temperature
facilitates the simultaneous screening of many samples. The obtained fragments can
Fig. 3. Principle of denaturing gradient gel electrophoresis (DGGE). DGGE patterns of PCR products
obtained using universal primers for the detection of bacteria species on total nucleic acids isolated from
faecal samples.
be recovered from the gels and used in further analyses such as sequencing. Another
possibility is the direct blotting of fragments to a filter followed by hybridization
experiments. However, some practical disadvantages have to be mentioned. In order
to generate the GC-clamp, which is indispensable for the stability of transitional
molecules, a relatively long primer must be used, and this may cause artefacts in the
annealing step of the PCR. In addition, the results of TGGE/DGGE may be affected
by the heteroduplex molecules formed during PCR. Those mismatched base pairs
are less stable under TGGE/DGGE conditions and can lead to unspecific bands
(Ruano and Kidd, 1992).
Several studies employed DGGE and TGGE, respectively, for the description
of gut diversity (Millar et al., 1996; Simpson et al., 1999; McCracken et al., 2001;
Walter et al., 2001). DGGE analysis performed on pigs’ faeces by Simpson et al.
(1999) revealed changes in bacterial populations with age, and differences between
individual animals and gut compartments. Furthermore, the comparison of amplified
faecal DNA from pigs of different age revealed several unique bands indicating the
presence of unique bacterial populations. Comparison of different gut compartments
demonstrated that bacterial populations within a single compartment showed the
highest similarity followed by adjacent compartments. For a review of TGGE/DGGE
application in microbial ecology, see Muyzer and Smalla (1998) and Muyzer (1999).
The majority of the above mentioned DNA-based approaches suffer from the dis-
advantage of not providing any phylogenetic information on the examined species.
2.4. RNA-based detections

RNA-based detection has become increasingly important in microbial ecology.
Although technically similar, the RNA approach has major advantages over the DNA
approach. The content of RNA molecules in any living cell is usually more than 1000
(Amann et al., 1995; Langendijk et al., 1995) thus making detection of an RNA
target much easier because no amplification procedure is necessary. Moreover, ribo-
somal RNA sequences, especially 16S rRNA, have been deposited in databases for a
large fraction of bacterial species (Van de Peer et al., 1996; Maidak et al., 2001).
The first analysis of microbial diversity in an ecosystem based on RNA techniques,
was done by Stahl and co-workers (1985). Since the information content of the 5S
rRNA examined in those studies is rather limited, Olsen et al. (1986) suggested an
approach based on the larger rRNA molecules. The 16S rRNA of a bacterium has an
average length of 1500 nucleotides, and the 23S rRNA of about 3000 nucleotides. The
rRNA molecules comprise highly conserved sequence domains interspersed with
more variable regions (Gutell et al., 1994; Van de Peer et al., 1996). The latter, also
called signature-sequence motifs, may be used for bacterial identification (Woese,
1987; Amman et al., 1995).
The principles of the method are based on sequence comparison of 16S rRNAs.
Since Woese and co-workers introduced the concept of comparative small subunit
rRNA sequence analysis for the elucidation of bacterial phylogeny (Woese, 1987),
this technique has found worldwide acceptance and application. In the meantime an
ever increasing data set of ribosomal RNA sequences is available. The phylogenetic
analysis of these data provides the basis for ongoing research and reconstruction of
bacterial systematics. Initially, discovering bacterial evolution had been the major
goal. Meanwhile, however, the applied aspects have gained more and more impor-
tance. Besides comparative sequencing of rRNA genes, specific rRNA targeted
probes and diagnostic PCR in combination with a variety of experimental tech-
niques are at the centre of interest.
Ribosomal RNA sequences are generally obtained either directly from rRNA
or from the encoding genes located at various positions in the bacterial genome,
i.e. rDNA. In practice, sequences of 16S rRNAs are generated by amplification of
bacterial 16S rRNA genes (16S rDNA) using universal primers and PCR. PCR
products are then integrated into vectors, and rDNA clone libraries are created. With
these techniques Suau et al. (1999) obtained 284 clones from human faecal DNA
and classified them into 82 molecular species. Three phylogenetic groups contained
95% of the clones: the Bacteroides group, the Clostridium coccoides group, and the
Clostridium leptum subgroup. The remaining clones were distributed among a
variety of phylogenetic clusters. Suau et al. (1999) reported that only 24% of the
recovered molecular species corresponded to described organisms. All of these were
established members of the dominant human faecal flora (e.g., Bacteroides theta-
iotaomicron, Fusobacterium prausnitzii, and Eubacterium rectale). However, the
majority of generated rDNA sequences (76%) did not correspond to known organ-
isms but to hitherto unknown species within human gut microflora. It can therefore
be stated that owing to the limitations of culture-based techniques, our knowledge
of the gut microflora composition is far from complete. In particular, it is believed
that a significant portion of the gut microbial diversity has not been cultivated yet.
This view is corroborated by several studies (Snel et al., 1995; Lawson et al., 1998;
Barcenilla et al., 2000).
A number of computer programs for the analysis of sequences and phylogenetic
relationships are available at various internet sites. All databases provide ribosomal
RNA-related data services, including online data analysis, rRNA derived phylo-
genetic trees, and aligned and annotated rRNA sequences. The most up-to-date
sequence datasets are currently available at:
Genbank (http://www.ncbi.nlm.nih.gov)
EMBL (http://www.ebi.ac.uk)
the Antwerp database (http://www.psb.ugent.be/rRNA/index.html)
the Ribosomal Database project (RDP; http://rdp.cme.msu.edu/html)
ARB (Strunk et al., 1998; http://www.mikro.biologie.tu-muenchen.de/)
In the last two, there are currently more than 20 000 aligned sequence entries,
thus making them the largest databases on bacterial phylogeny. The user is able
to integrate new sequences into already existing databases and to subsequently
generate trees. For further information on the theory of bacterial phylogeny, based
on comparative sequence analysis, see Ludwig et al. (1998).
Tools for the design of diagnostic oligonucleotide probes, integrated in these
programs, make it possible to construct a variety of cluster, group- or species-specific
oligonucleotide probes for the detection of microorganisms. Hitherto uncultured
species can be detected and even be visualized. Thus, the designed probes enable the
scientist to monitor the spatial arrangements and interactions of unknown species in
their natural environment. Using oligonucleotide probes ranging from the group or
genus level down to species-specific probes (nested approach) on different culture
media, those species can even be isolated and subsequently be tested for their
biochemical properties. Considerable effort has been made in the past few years to
generate new probes for the detection of gut microorganisms (Langendijk et al.,
1995; Kaneko and Kurihara, 1997; Doré et al., 1998; Simmering et al., 1999).
Furthermore, the project FAIR-CT97–3035, financed by the European Union, was
solely devoted to the development and application of molecular approaches assess-
ing the human gut flora in diet and health.
2.4.1. Hybridizations targeting ribosomal RNA

Both quantitative dot-blot hybridization and whole-cell hybridization have been
used for the analysis of intestinal bacteria. For quantitative dot-blot hybridization,
total RNA is extracted from the sample and bound to a filter support. The RNA is
subsequently labelled with oligonucleotide probes. Universal probes hybridize to
targets in the rRNA that have been conserved during evolution and therefore recog-
nize all bacteria. In contrast, specific oligonucleotides are designed in such a way
that they recognize, depending on the specificity, bacteria at various levels of the
phylogenetic hierarchy. The relative abundance of a bacterial population group is
then calculated by dividing the amount of a specific probe bound to a given sample
by the amount of hybridized universal probe referred to as rRNA index. With this
technique Sghir et al. (2000) were able to quantify several bacterial groups within
the human faecal flora. By applying six probes to faecal samples from human adults,
70% of the total 16S rRNA could be accounted for by the Bacteroides (37%), the
Clostridium leptum subgroup (16%) and the Clostridium coccoides group (14%).
Bifidobacterium and Lactobacillus groups made up less than 2% and the enteric
bacteria accounted for less than 1%. This study indicated that cultural-based
estimations of the main bacterial groups may lead to overestimations and under-
estimations, respectively. The ribosomal RNA-targeted hybridization probes applied
in this study were also applied in studying the gut microbial ecology of pigs (Boye
et al., 1998; Sghir et al., 1998), cattle (Forster et al., 1997; Kocan et al., 1998) and
sheep (Forster et al., 1997).
The analysis at the single-cell level provides a more detailed picture than dot-blot
hybridization because not only can the cell morphology of bacteria be determined,
but also their spatial distribution in situ. The first applications of in situ nucleic acid
hybridization used isotopically labelled probes that bind to the rRNAs of fixed and
intact cells. Organisms recognized by the probes were identified by audioradiography
(Giovannoni et al., 1988). However, the major drawback of microaudioradiography is
the requirement for a relatively long exposure time and the low resolution. The
introduction of fluorescently labelled probes was an important step forward to a
much faster and more accurate detection of target cells. Furthermore, the use of
several oligonucleotide probes, each labelled with a different fluorescent dye, allows
the simultaneous detection of several different organisms. In recent years, whole
cell-in situ-hybridization, better known as fluorescence-in situ-hybridization (FISH)
has become one of the most widely used tools in microbial gut ecology (Franks
et al., 1998; Simmering et al., 1999; Harmsen et al., 2000). The major advantage of
whole-cell hybridization for the detection of bacteria in contrast to other molecular
methods (see above), lies in their microscopic visualization. Quantification of
positively hybridized cells in faecal preparations is performed by means of visual
counting procedures. It is a time-consuming process which depends highly on
the skills and experience of the person performing the counting. Therefore, only
moderate levels of accuracy are reached (Langendijk et al., 1995). In order to over-
come this problem, automated microscopic counting has been introduced (Jansen
et al., 1999). This methodology is particularly useful when large numbers of faecal
samples need to be processed. This is the case in studies that investigate the influ-
ence of diet on the faecal flora. The principle of FISH is depicted in fig. 4.
It has to be emphasized, however, that the application of whole-cell hybridiza-
tion to faecal samples also has its limitations, the most significant one being the lack
Fig. 4. Principle of fluorescence-in situ-hybridization (FISH).

of sensitivity. This may be partly due to the fact that the number of rRNA targets
is lower in cells in their natural environment than in cells growing in pure cultures
under optimal conditions. Nutritional limitation and other competitive factors
influence the cellular ribosome content (Amann et al., 1995; Langendijk et al., 1995).
It is therefore not surprising that the fluorescence signal of cells in faecal samples is
lower than in pure cultures (Langendijk et al., 1995). In addition, unspecific fluo-
rescence may hamper the visualization of bacteria. Another limitation is the possible
inaccessibility of the target sequence, which may be due to the structure of the ribo-
some, which includes rRNA–rRNA and rRNA–ribosomal protein interactions
(Binder and Liu, 1998; Clemons et al., 1999). In a recent paper, Fuchs et al. (2000)
described the use of unlabelled helper oligonucleotides to improve the in situ acces-
sibility to the 16S rRNA of E. coli. The in situ identification of individual cells may
also be hindered by a limited cell wall permeability (Binder and Liu, 1998; Fegatella
et al., 1998). Some researchers employed lysozyme to improve the permeability of
the cell wall (Simmering et al., 1999; Schwiertz et al., 2000). However, the intensity
of the signal depends on the penetration of the probes across the cell periphery.
In this respect, carbocyanine dye-labelled (Cy3) probes are superior to biotinylated
probes (Alfreider et al., 1996; Simmering et al., 1999; Schwiertz et al., 2000). It has
been reported that treatment with chloramphenicol, an inhibitor of protein synthesis
and RNA degradation, can lead to an increase in the percentage of detectable cells
(Ouverney and Fuhrman, 1999).
Several technical developments have aimed to increase the sensitivity of FISH: one
of these is the Tyramide System Amplification (TSA®; NEN Research Products). It
combines horseradish peroxidase (HRP)-labelled, rRNA-targeted oligonucleotide
probes and the TSA® system. The TSA® amplification is based on the covalent binding
of radicalized fluorochrome-tyramide substrate molecules to electron-rich moieties,
such as tyrosines or tryptophans. HRP-containing cells give a very bright fluorescent
signal. Schonhuber et al. (1997) observed an increase of fluorescence with the TSA®
system. Although the TSA® yields bright fluorescent signals, the disadvantages should
be noted. Owing to the relatively large molecular size of the HRP-oligonucleotide
probe-complex, a penetration of the fixed bacterial cells is rather difficult. This is more
easily achieved with the smaller fluorescently labelled oligonucleotides.
Developments of new molecular approaches in the study of bacterial ecosystems
on the basis of RNA detection are numerous. Recently a new technique for the analy-
sis of aquatic samples has been introduced by Ouverney and Fuhrman (1999). This
technique can be applied easily to gastrointestinal ecology. This technique is termed
substrate-tracking autoradiographic fluorescent-in situ-hybridization (STARFISH)
which affords the detection of cells that take up a radioactively labelled substance,
and its distribution. When combined with 16S rRNA-targeted in situ hybridization,
bacteria metabolizing the labelled substrate can be identified. Further development of
this technique will help to get a better look at the gastrointestinal ecology and to
study for example the influence of diet on the microbial community.
2.4.2. Other RNA-based detection methods

Numerous techniques have been developed to monitor gene expression in bacterial
cells. These include coupled reverse transcription PCR amplification (RT-PCR) and
ribonuclease protection assays (RPA). Of these methods, RT-PCR is the most sensitive
and versatile. It can be used to determine the presence or absence of a transcript,
estimate expression levels and clone cDNA products without the necessity of
constructing and screening a cDNA library (Noda et al., 1999). In contrast, RPA is a
highly sensitive and specific method for the detection and quantification of specific
mRNAs. The assay was made possible by the discovery and characterization of DNA-
dependent RNA polymerases from the bacteriophages SP6, T7 and T3, and the
elucidation of their cognate promoter sequences. These polymerases are ideal for the
selective and specific synthesis of RNA probes from DNA templates, because these
polymerases exhibit a high degree of fidelity to their promoters, polymerize RNA at a
very high rate, efficiently transcribe long segments, and do not require high concentra-
tions of rNTPs. Thus, a cDNA fragment of interest can be subcloned into a plasmid that
contains bacteriophage promoters, and the construct can then be used as a template for
the synthesis of radiolabelled anti-sense RNA probes (fig. 5).
Fig. 5. Principle of the

ribosomal protection
assay (RPA).
Advances in molecular technology have led to improvements in the methods avail-
able for studies of the gut microbial ecology. One of the most recent developments,
which may have a future impact on microbial ecology, is the so-called molecular
beacons. Molecular beacons are oligonucleotide probes that report the presence of
specific nucleic acids in homogeneous solutions (Tyagi and Kramer, 1996). They are
useful in situations where it is either not possible or not desirable to isolate the
probe-target hybrids from an excess of the hybridization probes, such as in real-time
monitoring of PCRs in sealed tubes or in detection of RNAs within living cells.
Molecular beacons are hairpin-shaped molecules with an internally quenched fluo-
rophore, whose fluorescence is restored when they bind to a target nucleic acid (fig. 6).
They are designed in such a way that the loop portion of the molecule is a probe
sequence complementary to a target nucleic acid molecule. The stem is formed by
the annealing of the complementary arm sequences located at the ends of the
oligonucleotide. A fluorescent moiety is attached to the end of one arm and a
quenching moiety is attached to the end of the other arm. The stem keeps these two
moieties in close proximity to each other, causing the fluorescence of the
fluorophore to be quenched by energy transfer. Since the quencher moiety is a non-
fluorescent chromophore and emits the energy that it receives from the fluorophore
as heat, the probe does not fluoresce. When the probe encounters a target molecule,
it forms a hybrid that is longer and more stable than the stem and its rigidity and
length preclude the formation of the stem hybrid. Thus, the molecular beacon under-
goes a spontaneous conformational reorganization that forces the stem apart, and
causes the fluorophore and the quencher to move away from each other, leading to
the restoration of fluorescence, which can be detected. In order to detect multiple
targets in the same solution, molecular beacons can be made in many different
colours utilizing a broad range of fluorophores (Tyagi et al., 1998). DABCYL, a
Fig. 6. Operation of molecular

beacons.
non-fluorescent chromophore, serves as the universal quencher for any fluorophore

in molecular beacons. Owing to their loop sequence, the recognition of targets by
molecular beacons is so specific that single-nucleotide differences can be readily
detected. In combination with the biochip technology, molecular beacons might
become a powerful tool in molecular ecology.
Biochips enable DNA or RNA sequences to be quickly analysed. They allow
rapid and precise information on the genetic composition of a given sample. The
principle of analysing material with a biochip is simple: to the substrate material,
genetic material with a known structure is applied in periodic patterns over an area
about the size of a thumbnail. These prepared measuring points contain for example
specific 16S rRNA oligonucleotide probes for the detection of different bacterial
genera, groups and species. Treated faecal samples in an aqueous solution are then
applied onto the chip and complementary 16S rRNA sequences are allowed to
hybridize. A positive hybridization result can then be proven with the aid of markers
previously bound to the specific probe. The attached molecules of dye are irradiated
with laser light, emitting a characteristic wavelength. A camera detects this light and
an analysis software shows where the marked points are located, and arranges them.
For a fast and more reliable detection of bacterial groups, genera or species from
environmental samples, specific molecular beacons can be developed and put onto a
chip. Owing to the nature of the molecular beacons, a high specificity and a rather fast
execution of samples is permitted. The application of these concepts can bring similar
benefits of speed and reduced costs to research in gastrointestinal microbiology.
In conclusion, the opportunities for the discovery of new organisms and the
development of techniques based on microbial diversity are greater than ever before.
Utilizing all those tools in conjunction with the study of mechanisms and interac-
tions between the microorganism and the eukaryotic cell, will lead to a better insight
into the microbiology of the gastrointestinal tract of humans and animals.
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7 Models of the gastrointestinal tract to
study microbial interactions
M. Minekus
TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ, Zeist,
The Netherlands
In vitro models can be a useful tool to study microbial interactions. However, it is

important to include sufficient parameters to obtain the desired predictability, while
keeping the model as simple as possible. When using an in vitro model of the gas-
trointestinal tract (GIT), it is necessary to have information on the relevant para-
meters in vivo, such as GIT morphology, residence times, secretion rates and
composition of digestive juices, pH profiles and microflora composition. These
parameters may vary between species and between the growing and adult animal.
Different types of models are used to study the intestinal microflora. Batch models
are the simplest systems where microflora and substrate are incubated in a vessel for
a certain period. Continuous models are systems where the culture is regularly fed
with medium, while the pH is kept at a set point. Several of these vessels can be con-
nected to mimic different parts of the GIT. Dynamic systems include the interactions
between gastric emptying, gastric pH profiles, rates of secretion, water absorption,
removal of digestive products and microbial metabolites, and transit of the meal
through a separate part of the gut. The use of in vitro systems is limited by the
absence of interactions between microflora and the host, the availability of relevant
in vivo data, and analytical methods to analyse the microflora composition and its
metabolites.
1. INTRODUCTION
The feed, petfood and pharmaceutical industries need to develop and improve
products continuously in order to meet the increasing demands of the market.
The feed industry aims at knowledge about the efficacy of feed ingredients, improv-
ing the bio-availability of nutrients, introducing alternative protein, carbohydrate

Models of GIT for microbial interactions 143
and fat sources, and reducing the faecal output of environmental pollutants such as
phosphate. The petfood industry is following the trend in human nutrition to offer
functional foods with additional health-promoting characteristics. New feed addi-
tives and pharmaceutical products are developed to improve and maintain the health
status of the animals. These developments require studies to test the behaviour of
compounds in the gastrointestinal tract (GIT) in relation to their efficacy, digestibil-
ity, fermentation and their effect on the microflora. These studies are often per-
formed in laboratory animals. However, animal studies are hampered by costs,
ethical constraints, sampling problems and variation between individual animals.
As an alternative to animal studies, experiments are performed on in vitro models.
These models can be used to perform simplified experiments under uniform and
well-controlled conditions. However, simulating such a complex system as the GIT
carries the risk of oversimplification. The predictive value of a model generally
increases when more parameters are included. However, the inclusion of more
parameters leads to more complex model systems. Thus, a model system should
take into account all relevant parameters, while being as simple as possible.
Therefore, each type of in vitro model has its specific use and limitations.
2. THE DIGESTIVE SYSTEM: VARIATION BETWEEN

SPECIES AND AGE
Although the digestive system of vertebrates has a general layout (table 1), each
species has adapted its GIT to its specific eating behaviour and nutrients, resulting
in variation of anatomy and physiology (Chivers and Langer, 1994).
The diversity in GIT morphology is expressed by different shapes, relative sizes
and functions of the digestive compartments, leading to different residence times for
the digesta in the successive parts of the GIT. Even compartments with specialized
functions have been developed such as the crop and gizzard in birds and the
forestomachs in ruminants. This anatomical and physiological variation has resulted
in intestinal microbial populations that are specific for each species. Besides varia-
tion between species, also age related differences exist.
Table 1. General layout of the digestive tract
Compartment Function
Oral cavity Ingestion and pre-treatment of the meal

Stomach (crop in birds) Storage, particle reduction, peptic digestion,
acidification or fermentation
Small intestine Digestion and absorption of nutrients and water
Large intestine (cloaca in birds, some fish,
amphibians and reptiles) Fermentation of undigested materials,
absorption of water, fermentation products
and electrolytes
144 M. Minekus
Table 2. Gastrointestinal aspects and their effect on digestive processes
Aspects Effect
Morphology Transit of the meal, specific compartments

Motility
Secretion and concentration of Digestion and uptake of nutrients
digestive compounds
pH
Gut wall properties
Microflora composition Fermentation and bioconversion of compounds
Young animals may differ from adult animals with respect to concentrations of
secreted digestive compounds such as bile and enzymes. In addition, some young
animals, such as the preruminant calf, have a different GIT morphology in
comparison to the adult animal.
To study microbial interactions in the GIT with an in vitro model, one should
take into account the relevant conditions prevailing in the digestive system of the
animal of interest. These conditions might be different between species, but also
between the young and the adult animal. Important gastrointestinal aspects and their
effect on digestive processes are shown in table 2.
3. MODELS TO STUDY MICROBIAL INTERACTIONS

The behaviour of intestinal microflora is predominantly studied in models simu-
lating the rumen of ruminants (Davies, 1979; Sudweeks et al., 1979; Dong et al.,
1997), and the large intestine of monogastric animals (Rumney and Rowland, 1992;
Minekus et al., 1999). Only a few models take account of the microecology of the
stomach and the small intestine (Coutts et al., 1987; Molly et al., 1993).
Three different types of model systems have been described to perform experi-
ments with complex microbial populations, viz.: a) batch culture systems, b) (semi-)
continuous culture systems, and c) dynamic systems.
3.1. Batch cultures

Batch cultures are mainly used for simple, short-term experiments (1 to 2 days) and
only involve incubation of test material with faeces or colonic contents (Vince et al.,
1990; Barry et al., 1995; Van Hoeij et al., 1997). The experiments are generally
performed in closed vessels under anaerobic conditions, while the pH is maintained
at a preset level with a buffer or by a pH-stat. Mixing of the content is absent or done
with a stirrer (Batch, fig. 1). Their predictability is limited by the fact that the
microflora is not continuously fed and that there is no removal of metabolites
during incubation. Accumulation of metabolites might eventually influence the
metabolic activity of the microflora.
Fig. 1. Schematic presentation of different

reactor types used to simulate gastrointestinal
transit.
3.2. (Semi-)continuous cultures

Semi-continuous cultures need a more sophisticated set-up. The microflora is fed
while part of the content is removed intermittently from the fermentor to mimic the
passage of chyme through the simulated part of the GIT. Continuous cultures have
the same set-up as the semi-continuous cultures, except that they have a regular
feeding of the microflora and removal of contents. Mixing is achieved by an impel-
lor, thus resulting in a continuously stirred tank reactor (CSTR, fig. 1). Incubation
in a CSTR results in a steady-state situation where the growth rate of the micro-
organisms is determined by the dilution rate. To simulate consecutive parts of the
gut, different systems have been designed with two, three or five vessels in series
(fig. 2) (Miller and Wolin, 1981; Manning et al., 1987; Gibson et al., 1988; Allison
et al., 1989; Molly et al., 1993). These systems generally allow for growth of strictly
anaerobic microorganisms by flushing with anaerobic gas. The pH is measured
and controlled within the physiological range through addition of acid or alkali.
Both continuous and semi-continuous cultures have been used to study the micro-
ecology of the flora, degradation of undigested materials, enzyme activities and
production of interesting metabolites such as short chain fatty acids, gases and
toxic compounds.
A drawback of (semi)-continuous culture systems is that they operate under
steady-state conditions which means that the concentrations of metabolites are kept
within the physiological range by a limited amount of substrates in the influent and
the dilution rate (Edwards and Rowland, 1992). Substrate limitation, dilution and
product inhibition limit the amounts of microorganisms in these systems. Realistic
feeding, and removal of the metabolites and water without the microorganisms, are
146 M. Minekus
Fig. 2. Schematic presentation of the SHIME model. M, feed; P, pancreatic enzymes; I, stomach;
II, duodenum and jejunum; III, ileum; IV, caecum and ascending colon: V, transverse colon; VI, descending
colon; E, effluent.
prerequisites for maintaining the amounts of microorganisms as well as their

metabolites at physiological levels. Feeding and mixing of dense fibrous and vis-
cous materials is a common problem in large intestinal systems (Edwards and
Rowland, 1992).
3.3. Dynamic GIT systems

3.3.1. Dynamic conditions in the GIT
The model systems described so far are static models that do not simulate
the dynamic conditions to which a compound or a microorganism is exposed when
travelling through (transiting microflora) or colonizing (resident microflora) the
stomach and intestines. The importance of including dynamic interactions of para-
meters is demonstrated when studying the survival of microorganisms during transit
through the GIT. Gastric pH and bile concentration are the main determinants of
survival (Marteau et al., 1997). However, both gastric pH and bile concentration are
not constant in time. Gastric pH increases during ingestion of a meal and then
decreases, depending on the rate of gastric acid secretion and the buffer capacity of
the meal. The duodenal bile concentration shows an increasing and later decreasing
pattern due to emptying of the bile bladder after the start of a meal. Along the small
intestine, the bile concentration first increases due to water absorption and later
decreases due to absorption of bile in the distal part of the small intestine. Not only
Fig. 3. Exposure of a meal to successive steps of digestion in a static system (A) and in a dynamic system (B).
pH and bile, but also secretion of other digestive compounds are affected by the
meal and therefore are not constant in time.
Another aspect that determines the fate of microorganisms during gastrointestinal
passage is the time that they are exposed to unfavourable gastric pH values and bile
concentrations, which is determined by the pattern of gastric emptying and the small
intestinal transit time of the meal. Gastrointestinal passage is often mimicked by
sequential static exposure to gastric and small intestinal conditions (fig. 3A).
However, this is not a realistic situation since the meal is gradually emptied from the
stomach after which it travels through the intestines (Decuypere et al., 1986; fig. 3B).
The gastric pH and the gastric emptying of milk in a preruminant calf are
shown in fig. 4, as an example of the dynamic interaction between pH and gastric
emptying. The figure shows that, in this case, more than 50 per cent of the meal has
left the stomach while the pH is above 4.
Fig. 4. pH (solid line) and gastric emptying (dotted line) during the digestion of milk in a preruminant calf
(adapted from Caugant et al., 1992).
148 M. Minekus
Fig. 5. Schematic diagram of the multi-

compartmental model of the stomach and
small intestine: a, gastric compartment;
b, duodenal compartment; c, jejunal compart-
ment; d, ileal compartment; e, gastric secretion
pumps; f, pH electrode; g, peristaltic valve
pump; h, duodenal secretion pumps; i, dialysis
fluid; j, hollow-fibre device; k, collecting
vessel for ileal delivery.
The considerations described above, have led to the development of a multi-

compartmental dynamic computer controlled model (nicknamed TIM) that simu-
lates the dynamically changing conditions in the different parts of the digestive
tract (Minekus et al., 1995, 1999). The complete system consists of a gastric–small
intestinal system (TIM-1) (fig. 5), usually used for digestive studies and a large
intestinal system (TIM-2) (fig. 6), used for microbiological studies. The two systems
are – for practical reasons – constructed and used as separate systems.
3.3.2. The gastro–small intestinal system

The gastro–small intestinal model consists of four successive compartments (fig. 5),
simulating the stomach (a), duodenum (b), jejunum (c) and ileum (d). A meal can
be fed to the gastric compartment during a preset time. In the gastric compartment
gastric juice is added (e), while the pH is measured (f) and adjusted to follow a
Fig. 6. Schematic representation of TIM-2.

a, Peristaltic compartments; b, pH-electrode;
c, alkali pump; d, dialysis circuit with hollow
fibres; e, level sensor; f, “ileal effluent”
container; g, peristaltic valve pump for influent;
h, peristaltic value pump for effluent;
i, sampling-port; j, N2 gas inlet; k, gas
collection bag.
predetermined curve. The compartments consist of connected glass units with

flexible walls inside. The walls can be squeezed by varying the pressure on the
water. The chyme in each compartment is mixed by alternately squeezing the
flexible walls. To control the transit of the meal, the compartments are separated
by computer-regulated peristaltic valve pumps (g). Bile and pancreatic juice are
secreted into the duodenal compartment (h). The pH in each small intestinal
compartment is measured (f) and controlled through the addition of sodium hydro-
gen carbonate. Products of digestion and water are absorbed from the jejunal and
ileal compartments by pumping dialysis liquid (i) through hollow-fibre membrane
units with a molecular weight cut off of approximately 5000 (j). The chyme deliv-
ered from the ileal compartment (ileal delivery) is collected on ice in a vessel (k).
The system is controlled by a computer that can be programmed with a protocol
that contains formalized data on the dynamic digestive conditions of a specific
species having a specific meal. The protocol for the TIM-1 contains data on: i) the
gradual gastric emptying of the meal and the transit of the meal through the small
intestine; ii) the gastric pH profile which increases first due to the buffer capacity
of a meal and then decreases due to acid secretion; iii) the different pH values in
the small intestinal compartments; iv) the rate of gastric and duodenal secretions;
and v) the absorption of water.
The small intestinal system is used to study the digestion and availability for
absorption of nutrients and pharmaceuticals. It has also been used to study the
survival of transiting microorganisms (Marteau et al., 1993, 1997), the transfer of
genetic material from genetically modified foods to microorganisms (van der Vossen
and Havenaar, 1997; Gänzle et al., 1999), and as a pre-treatment for studies in the
large intestinal model.
3.3.3. The large intestinal system

The large intestinal system (fig. 6) consists of connected glass units (a), each with a
flexible wall inside. Peristaltic movements are achieved by changing the pressure on
the water. The computer controls the sequential squeezing of these walls, thus caus-
ing a peristaltic wave which forces the chyme to circulate through the loop-shaped
system. The tubular-shaped lumen of the system prevents “constipation”. The pH is
measured with a pH electrode (b) and controlled by the addition of a sodium
hydroxide solution (c). The dialysis liquid is pumped through hollow-fibre mem-
branes positioned in the lumen of the reactor (d), to maintain the appropriate con-
centrations of electrolytes and metabolites. The amount of chyme in the reactor is
monitored with a level sensor (e) and kept on a preset level by the absorption of
water with a pump in the dialysis circuit. The feeding medium (f) is mixed and kept
anaerobic with nitrogen, and is introduced into the reactor with the peristaltic valve
system (g) as described for the gastro-small intestinal system. A peristaltic valve
pump is also used to remove chyme from the reactor (h). Samples can be taken from
150 M. Minekus
a sampling port (i). The system is kept anaerobic by flushing with nitrogen (j).
Produced gas can be collected in a bag (k) or is allowed to leave through a water lock.
The large intestinal model is controlled to simulate the conditions for the large
intestine with a dense microflora of human or animal origin (Minekus et al., 1999).
This is achieved by combining the feeding of concentrated ileal effluent with
the removal of water and metabolites through a semi-permeable membrane. The
peristaltic movements allow adequate mixing and transport of the chyme. Several
units can be connected to mimic successive parts of the large intestine. The large
intestinal system is used to study the fermentation of carbohydrates, production of
toxic metabolites, transfer of genetic material from food or microorganisms to the
microflora and the efficacy of probiotic strains.
All the models described, including the TIM systems, have the general limitation
that it is not possible to maintain a microflora that is completely identical to the in vivo
situation. The conditions that affect the microflora in vivo are much too
complicated to be completely simulated in an in vitro system. The first problem is to
have an inoculum that is an exact copy of the site of interest in vivo. A faecal inocu-
lum is easy to obtain, is considered representative of the large intestinal microflora and
therefore is widely used (Rumney and Rowland, 1992). A better inoculum, but more
difficult to obtain, would be the intestinal content itself, e.g. from the proximal colon.
The second problem is the composition of the substrate medium. Ideally, the
standard medium composition should allow the growth and maintenance of a
microflora that is identical to the inoculum. However, in practice some shift in
microflora composition cannot be avoided.
The most important drawback of in vitro models is that they do not include the
considerable interactions between microflora and the host (Umesaki et al., 1997).
First, the immunological response of the host is a major determinant of the micro-
flora’s composition and cannot be included in any in vitro model. Second, there is
no gut wall to include gut wall mechanisms. Under normal conditions only those
microorganisms colonize the GIT which grow fast enough to resist the flow or those
that are associated with the mucus layer of the gut wall. Association to the gut wall
is regarded as a prerequisite for invading microorganisms to overcome their lag
phase in the new environment and to colonize the intestine (Beachey, 1980; Freter
et al., 1983). Recent research has revealed that indigenous microorganisms are able
to modify the specific attachment receptor and thus prevent colonization of the
pathogen (Bry et al., 1997; Umesaki et al., 1997). The body can react to pathogens
by increasing cell turnover to dispose of the pathogens attached to the cells.
Experiments in fermentors have shown that attachment to the glass wall of the
vessel is also necessary for microorganisms to overcome the colonization barrier.
Although the ecological principles might be similar, the mechanisms of adherence
in a fermentor are clearly different from those in vivo.
Although it is not feasible to study adherence directly in the culture vessel, some
interaction of microorganisms can be studied with cell cultures. The adherence of
Fig. 7. Caco-2 cells on a semi-permeable membrane (A) cultivated in cups (B).
microorganisms to enterocytes can be studied using a cultured monolayer of Caco-2

cells on a semi-permeable membrane (fig. 7A) (Naaber et al., 1996). The Caco-2
cells are cultivated in cups (fig. 7B) and incubated with a bacterial suspension. After
incubation, the non-adhered bacteria are washed from the cells. The adhered bacteria
are removed from the cells by sonification and enumerated on selective agar plates.
4. ANALYTICAL METHODS TO STUDY THE MICROFLORA

An advantage of in vitro models is that they allow easy sampling, which can only
be exploited when adequate analytical methods are available. Apart from test
compounds, analysis is generally focused on the microflora composition, enzyme
activities and the products of microbial activity.
The composition of the microflora is traditionally determined by enumeration on
(s)elective culture media. In addition to traditional culturing techniques, various
molecular techniques are possible to collect information on the composition of the
microflora of the gastrointestinal tract (Tannock, 2001). These techniques include
polymerase chain reaction (PCR), fluorescent in situ hybridization (FISH) (Langendijk
et al., 1995; Harmsen et al., 2000) and denaturing gradient gel electrophoresis (DGGE;
Walter et al., 2001). These molecular techniques are directed towards the ribosomal
RNA encoding regions of the bacterium as the scientific community recognizes these
regions as perfect markers for the taxonomic position of an organism.
By using the PCR method, individual species can be detected. The specificity of
this detection approach depends on the specificity of the DNA synthesis priming
sequences. These sequences can be selected in such a way that they recognize only
a single species. PCR is particularly useful for showing the presence or absence of
a specific organism. Presently, it is also possible to quantify the presence of the
specific organism by using, among others, so-called “real-time-PCR”.
FISH offers a good way of detection and quantification of specific bacteria for
which in situ probes are available. These probes target the specific sequence in the
ribosomal RNA that is present in each ribosome of the specific microorganism.
Since more than 100 copies of the ribosomal RNA are present in an active cell,
152 M. Minekus
enough fluorescent material is bound to allow fluorescence under a fluorescence

microscope. Specific bacteria can be counted in this way. The only disadvantage of
the technique is that it can only provide information on those bacteria for which
probes are defined.
To get a more holistic view of the microbial flora DGGE is a more suited
approach. By using DGGE ribosomal sequences of the various species are separated
by their difference in DNA sequence. In this DGGE system there exists an increas-
ing gradient of denaturing agent. When a segment of DNA enters a region where
part of the duplex is unstable, the conformation changes and the mobility decreases
dramatically. This technique allows the visualization of the heterogeneity of micro-
bial floras and as a consequence changes in the flora by a banding pattern and
changing pattern, respectively. Each band in the DGGE pattern represents an indi-
vidual species. Since the technique is targeting ribosomal RNA or DNA sequences,
individual bands can be further analysed by nucleotide sequence analysis in order
to get information on their species affiliation. There exists a large database with
nucleotide sequence information that is extremely useful for taxonomists and phy-
logeneticists. In proceeding this way, it can be analysed as to which organism is
involved in a change of flora composition.
Although microflora composition is an interesting aspect to study microbial inter-
actions, probably more relevant when studying health aspects are the compounds that
are produced by the microflora. These compounds can be products of normal fer-
mentation, such as short chain fatty acids, lactic acid and gases (mainly H2, CH4 and
CO2). Microbial enzyme activities such as those involving β-glucuronidase and
azoreductase may lead to the production of toxic compounds (Rowland et al., 1985;
Bingham et al., 1996). Analyses of specific metabolites are necessary to study muta-
genicity, bioconversion, antimicrobial activity, gene stability and gene transfer.
In vitro models can be a useful tool to study the microbial interactions in the gut.
However, their use is limited by the complexity of the microflora and their interac-
tions with the host. More insight in the physiological conditions of the target species
is necessary to determine the right conditions during the experiments. The amount
of distinguishable species present in the gut microflora has increased rapidly
through molecular techniques and will increase further through the use of tech-
niques such as DNA chip technology. In spite of the enormous metabolic activity of
the microflora, little is still known about the microbial metabolites and their effect
on the interactions between the species and the host. Analytical techniques such as
nuclear magnetic resonance (NMR) and gas or liquid chromatography coupled to
mass spectrometry (LC-MC, GC-MS) can be used to detect relevant microbial
products. With pattern recognition techniques, specific metabolites can be linked to
specific conditions, substrates, or microorganisms.
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8 Adhesins and receptors for colonization by
different pathotypes of Escherichia coli in
calves and young pigs1
B. Nagy, I. Tóth and P.Zs. Fekete
Veterinary Medical Research Institute of the Hungarian Academy of Sciences,

1143 Budapest, Hungária krt. 21, Hungary
Enteric diseases of pigs and calves owing to Escherichia coli typically appear
during the first few days (and weeks) of life. The so far recognized pathotypes of
E. coli involved are the enterotoxic E. coli (ETEC), verotoxic E. coli (VTEC),
enteropathogenic E. coli (EPEC), and necrotoxic E. coli (NTEC). The first step in
the pathogenesis of all these types is to adhere to the intestinal microvilli with or
without inducing morphological lesions and produce specific toxins acting locally
on enterocytes and/or absorbed into the bloodstream. This action is assured by
specific ligand (adhesins) and receptor interactions which are characteristic of
the pathotypes involved and may vary according to the animal species. The
host-adapted adhesin/receptor systems are targets of several preventive measures
including vaccines, receptor blocking and breeding for genetic resistance. They also
provide the basis for cross-species infections including zoonoses. Therefore they
deserve the attention of epidemiologists, research scientists and technologists.
Besides, they provide tools for increased understanding of molecular pathogenesis,
and offer excellent models for comparative studies of different disease entities of
enteric colibacillosis in humans.
1. INTRODUCTION
Enteric colibacillosis of pigs and calves has decreased as a devastating problem of
intensive animal farming during the past two decades but even today it represents
1For their research data in this chapter, the authors acknowledge support from the following grants: OTKA T034970
(to B. Nagy), OTKA T026150 (to I. Tóth) and OTKA A312 (to the VMRI), as well as FAIR3-CT96-1335 (NTEC in
farm animals).

158 B. Nagy, I. Tóth and P.Zs. Fekete
one of the major health issues in the pig or cattle industry of several developed and
of most less developed countries. The reasons for the decreased losses are primarily
the new diagnostic tools and vaccines that have been developed and widely used as
a result of intensive research efforts on enteric E. coli infections of animals during
the 1970s and 1980s. The major breakthroughs in the diagnosis and prevention of
enteric colibacillosis of growing pigs and calves were mainly due to our increased
understanding of colonization and adhesion mechanisms of these enteric pathogens
to the intestinal mucosae. In spite of these positive developments there is still room
for research in this area partly because of the obvious human implications (several
pathotypes of E. coli are also prevalent in humans), and partly because our knowl-
edge is still quite limited. This is especially true for the area of intestinal receptors
of bacterial adhesions.
The main pathotypes involved in enteric colibacillosis of pigs and calves are the
enterotoxigenic E. coli (ETEC), verotoxigenic E. coli (VTEC), enteropathogenic
E. coli (EPEC), and necrotoxigenic E. coli (NTEC).
This review aims to give a general overview of the virulence factors and their
genetic regulators in E. coli. Furthermore it aims to describe the most important
adhesins and their receptors playing a role in the pathogenesis of different pathotypes
of enteric E. coli. It also points out some of the areas where future research is needed.
As there is a lot of analogy in the regulation of virulence factors of the above
pathotypes and as the most abundant information is available on ETEC, we will use
ETEC as the “veterinarians’s horse” to describe the basic organization of virulence
genes. Therefore we will start with the description of adhesions and receptors with
the ETEC pathotype and will refer to them in the case of analogies at appropriate
sections of other pathotypes. We will be able to describe the practical applications
of present knowledge essentially also on ETEC. We have to admit that little infor-
mation is available on that aspect for EPEC or NTEC.
2. ENTEROTOXIGENIC E. COLI
In enteric E. coli infections, especially in enterotoxic E. coli (ETEC) infections of
different species, bacteria adhere to the small intestinal epithelial cells (overwhelm-
ingly in newborn or very young animals), thereby colonizing the gut. They also secrete
proteins or peptides (enterotoxins) which stimulate the small intestine for increased
water and electrolyte secretion and/or decreased fluid absorption. The ability of adhe-
sion of ETEC to intestinal epithelial cells is mainly due to the production of thin
(3–7 nm) proteinaceous surface appendages (fimbriae or pili) which can be morpho-
logically, biologically and antigenically different on various strains. Some of them
morphologically resemble the common fimbriae (“Type 1” fimbriae or pili) of E. coli
(Duguid et al., 1955). With the help of these adhesins (fimbriae), the bacteria are able
to attach themselves to the microvilli of small intestinal epithelial cells, thereby more
intensively transferring the enterotoxins to the target cells. There is no characteristic
Adhesins and receptors for E. coli 159
Fig. 1. Schematic presentation of cellular changes due to interaction of E. coli bacteria pathotypes
with intestinal epithelial cells: (a) ETEC/VTEC: no obvious change in cellular microvillus morphology.
(b) EPEC/EHEC: attaching to cell membrane and effacing of microvilli with pedestal formation.
(c) NTEC: multinucleation, induced by CNF; distension of the cell and nucleus induced by CDT.
histological or ultrastructural morphology of adhesion or colonization by ETEC.

The microvilli and the epithelial cells remain intact (fig. 1a).
2.1. Adhesins and other virulence factors in the pathogenesis of ETEC

According to our present understanding, the pathogenesis of enterotoxic colibacillosis
starts with the adhesin–ligand interaction on the small intestinal microvilli, resulting in
a strong but morphologically non-destructive attachment of bacteria to the microvilli.
Therefore the virulence characteristics of ETEC are strongly dependent on the produc-
tion of adhesins (fimbriae) and enterotoxins. In addition to adhesive and enterotoxic
virulence factors, pathogenesis due to ETEC infection also involves host factors among
which the most important ones are the receptors for adhesin (and/or enterotoxin).
Species specificity − which is a general characteristic of ETEC infections − is largely
due to the presence of specific receptors in only one (or in a limited spectrum of)
animal species. Several of these adhesive virulence factors of ETEC and some of their
receptors are known and will be discussed in detail below, but some of them are still
unknown. Future research in this area is clearly needed and could bring further under-
standing of pathogenesis, thereby it would contribute to more successful strategies in
the prevention and treatment of enteric enterotoxic colibacillosis due to ETEC.
The most common adhesive fimbriae of animal ETEC strains can be differenti-
ated as surface antigens such as K88 or K99, 987P or F41 or F107 and 2134P in pigs
and calves, also designated as F4, F5, F6, F41, or F18ab and F18ac, respectively
(Ørskov and Ørskov, 1983; Moon, 1990; Rippinger et al., 1995) (table 1).
Table 1. Adhesins and their receptors of different pathotypes of enteric E. coli in calves and
in young pigs
Pathotype Adhesin Gene/Operon Location Receptor
ETEC F4 (K88) fae plasmid glycoprotein / mucin

F5 (K99) fan plasmid glycolipid
F6 (987P) fas plasmid glycoprotein
F18 (F107) fed plasmid ?
F41 fimf41 chromosome ?
EPEC Bfp pili bfp plasmid ? PE (phosphatidylethanolamine)
(EHECa) Intimin eae chromosome Tir (bacterial protein)
NTEC P (Pap) pap chromosome α-dGal(1-4)-β-Galactose

fimbria sfa chromosome (glycolipids)
S fimbria α-sialyl(2,3) β-Galactose
Afimbrial AFA afa chromosome/ Dr blood group antigen

Adhesions plasmid
a EHEC does not have Bfp.
These fimbriae are characterized as straight, bent or kinked proteinaceous

appendages originating from the outer membrane of the bacterial cells. They have
various molecular weights (from 15 to 25 kDa). In general, fimbriae are composed of
“major” and “minor” subunit structures governed and assembled under the direction
of structural and accessory genes respectively. For adhesive fimbriae, the adhesive
function is often represented by molecules at the tip of the filaments. The ability of
fimbriae (pili) to agglutinate red blood cells of different species was recognized very
early (Elsinghorst and Weitz, 1994) and it has been used for classification along with
the effect of 0.5% D-mannose: MS = mannose sensitive (adhesion blocked by mannose)
or MR = mannose resistant adhesion. Among fimbriae of animal ETEC bacteria we
can recognize the following categories: MS haemagglutinating fimbriae (Type 1),
MR haemagglutinating fimbriae (K88, K99, F41), and MR non-haemagglutinating
fimbriae (987P, F18ab, F18ac). Adhesive fimbriae provide the necessary first step for
the enterotoxins to act efficiently.
Enterotoxins can be described as extracellular proteins or peptides (exotoxins)
which are able to exert their actions on the intestinal epithelium. ETEC strains
are characterized by the production of one or both of the following enterotoxin
categories (Sherman et al., 1983), all of which are plasmid regulated:
large molecular weight (88 kDa) heat-labile enterotoxins (LT);
small molecular weight (11–48 amino acid containing) heat-stable peptide
toxins (ST) resistant to 100°C for at least 15 min.
LT enterotoxins are produced predominantly by human and porcine ETEC, while
ST enterotoxins are produced by ETEC of human, porcine and bovine origin.
LT toxins have good antigenicity while ST toxins do not. LT toxins can be

divided into two antigenically and biologically distinct but structurally similar
groups: LTI and LTII. Within LTI are the LTh−I (human) and LTp−I (porcine) strains,
while within LTII two antigenic variants (LTIIa and LTIIb) can be distinguished
(O’Brien and Holmes, 1996).
ST toxins fall into two classes: STa and STb (also referred to as STI and STII,
respectively). STa toxins have variants which are STaH and STaP [indicating human
(H) or porcine (P) type of the STa enterotoxins]. STa toxins are further characterized
by method solubility and by the ability to induce small intestinal fluid secretion in
baby mice and to a lesser extent in weaned pigs. STb is not soluble in methanol and
does not react in baby mice, however it can induce small intestinal fluid secretion in
newborn and weaned pigs.
2.2 Expression and regulation of adhesive virulence factors of ETEC

Genetic regulation and byosynthesis of fimbrial adhesins is of course different
according to different fimbriae. There is, however, a general scheme under which
these operons are constructed and function. There are regulator elements that code
for transacting polypeptides involved in the biogenesis of the whole fimbria and
there are several structural genes encoding polypeptides that partly form major
structural units ensuring fimbrial (pilus) formation or minor fimbrial units ensuring
adhesive capacity and variant specificity (de Graaf, 1990). Changes in the gene
expression can be the result of a random genetic event (stochastic process), but
expression of virulence factors is usually linked more to environmental signals, such
as temperature, ion concentration, osmolarity, carbon source, Fe++, pH, O2 etc.
These signals can also be sensed by ETEC bacteria in order to more appropriately
accommodate the in vitro and in vivo environment (stereotypic response). Under
in vivo conditions some of the above factors can induce a whole cascade of virulence
functions, turning on different genes while turning off others at different steps of the
infectious process (for instance: invasion genes are turned on early in the infection
but are repressed once bacteria are within the host cell) (Finlay and Falkow, 1997).
For ETEC, and for some other pathotypes mentioned below much less is known
about regulation. Virulence factors are influenced by the above signals through the
“regulator elements”. Some of these control the fimbrial synthesis only, some others
control the expression of many unrelated genes and are therefore called “global
regulators”. Virulence genes of enteropathogenic strains of E. coli are mainly genes
“foreign” to E. coli and they can be controlled by several regulators. These regulators
are therefore a possible exciting area of research for ETEC in terms of pathogenesis
(in vivo functions) and diagnosis.
Expression and regulation of virulence determinants are also dependent on secre-
tion mechanisms: there are three general secretion pathways recognized in Gram-
negative bacteria that export virulence factors (I–III). Another group of bacterial
proteins (IV) mediate their own transport and are therefore called autotransporter
systems (Finlay and Falkow, 1997). It is also known that the secretion of STa and
STb involves an energy and secA-dependent (type II) conversion of the performed
toxins to the extracellular toxins (Kupersztoch et al., 1990; Yamanaka et al., 1997).
However, several steps of enterotoxin and adhesin production as related to secretion
systems have yet to be clarified. It should be noted that the maturation of virulence
proteins is also part of the different secretion and expression mechanisms, i.e.
formation of disulphide bonds within the periplasm (for cholera toxin and for LT).
2.3. Adhesins and receptors for ETEC of calves

Most of the ETEC strains responsible for diarrhoea of newborn calves are charac-
terized by K99 (F5) and F41 and by STaP enterotoxins. They usually belong to the
O8, O9, O20 and O101 serogroups and often produce an acidic polysaccharide type
of K(A) antigen (K25, K28, K30, K35), making the colonies of such strains more
compact and less transparent. It seems that such capsular polysaccharide antigens
enhance colonization induced by K99 (Isaacson et al., 1977; Hadad and Gyles,
1982). K99 and other fimbrial adhesins mediate attachment of the ETEC to the
small intestinal (mainly ileal) microvilli, thereby resisting removal and facilitating
colonization. Thus bacteria are able to efficiently transmit the STa that they typically
produce, which in turn induces extensive excretion and loss of water and
electrolytes, rapidly leading to dehydration. Other, less frequently occurring
adhesins are the so-called F17 (earlier known as FY and Att25) (Lintermans et al.,
1988). Adhesions mediated by these surface proteins are generally dependent on the
presence of glycoprotein or glycolipid receptors, which are abundantly present in
newborn calves and lambs. In the case of K99, for instance, the receptors are acidic
glycolipids (gangliosides) like N-glycolyl-GM3, which gradually decrease with age
(Runnels et al., 1980; Willemsen and de Graaf, 1993; Teneberg et al., 1994).
Although K99 and F41 are frequently produced simultaneously by bacteria of the
same ETEC strain, there are different receptors for K99 (sheep and horse haemag-
glutinin) and for F41 (guinea pig and human-A haemagglutinin). K99 and F41
also differ in their genetic regulation (K99 is regulated by a plasmid while F41 is
regulated by a chromosome). Both K99 and F41 as well as F17 can, however, also
adhere to the porcine small intestinal brush border and can induce porcine entero-
toxic colibacillosis. Receptors for these adhesins are of course different. K99
receptors are certain glycolipids (as mentioned above), F41 receptors are glyco-
proteins (i.e. glycophorin) (Brooks et al., 1989), while the receptors for F17
(FY/Att25) are on the sialyated mucus (Mouricout and Julien, 1987). It must be
mentioned that association of F17 (FY/Att25) with ETEC is not quite clear. Original
descriptions of F17+ E. coli reported enterotoxic activities (Pohl et al., 1986;
Lintermans et al., 1988). Studies in recent years revealed that F17 fimbrial adhesins
are somewhat heterogeneous and they form a so-called F17 family of fimbriae
(F17a, F17b, F17c, F17d and G fimbriae) based on their receptor specificities
(Le Bougouénec and Bertin, 1999). Another (non-fimbrial) surface protein (CS31A)
has also been associated with calf diarrhoea (Girardeau et al., 1988) but it is also
detected on septicaemic E. coli from calves, in contrast to K99 or F41. Interestingly,
CS31A is genetically related to K88 fimbria (known as a typical porcine adhesin)
(Girardeau et al., 1988). In fact, the N-terminal sequence of purified CS31A shows
a homologous protein of 26.77 kDa between CS31A, F41 and K88, indicating an
evolutionary relationship between these fimbrial and afimbrial adhesions (Girardeau
et al., 1991). More on the F17 fimbriae will be mentioned in the section on necro-
toxigenic E. coli (NTEC).
In connection with ETEC of calves there should also be a few words on ETEC
of goat kids and lambs. As mentioned above, lambs have a very similar clinical
diarrhoeal disease and similar strains of ETEC as calves. However, this seems much
less certain in goat kids. In general, it is true for both animal species that we have
much more limited information about their ETEC infections as compared to those
of calves. For instance, the adhesins F17 (FY/Att25) and CS31A detected on calf
diarrhoea strains have not been described so far for E. coli bacteria from lamb or
goat diarrhoea, but such isolates can be prevalent among septicaemic strains of
lambs and goat kids (Le Bougouénec and Bertin, 1999). Information about ETEC
infection in goats is even more limited. According to our earlier studies (Nagy et al.,
1984), infection by K99 + ETEC may also cause diarrhoea of young goat kids
in some herds but cryptosporidiosis and rotavirus infections seem to be the main
aetiological agents. This observation is supported by the experimental infection of
goat kids with K99 + ETEC strains and by successful prevention of diarrhoea by
the K99 vaccine (Contrepois et al., 1993). In contrast to ETEC, verotoxic E. coli
(VTEC) strains have been isolated more frequently from 1–2-month-old goat kids
with diarrhoea and they seem to be the major diarrhoeal agent of this age group
(Duhamel et al., 1992). More information is needed, however, about ETEC (and in
general about enteric E. coli) infection of goat kids and lambs.
2.4. Adhesins and receptors for ETEC of pigs

Enteric enterotoxic colibacillosis produces significant losses in two different age
groups of pigs: first among newborn pigs and later at the post-weaning age.
Aetiology, pathogenesis and epidemiology should be discussed separately for the
two age groups, but diagnosis, treatment and prevention have enough in common to
be described under one separate heading for pigs and calves.
E. coli strains of enterotoxic colibacillosis in suckling piglets are characterized
by one or the other of the K88 adhesins (in variants K88ab, K88ac, and K88ad) also
known as the (F4), by K99 (F5) or 987P (F6) adhesins and occasionally by the F41
(Vazquez et al., 1996), F165 (Fairbrother et al., 1986) or F42 adhesins (Sperandio
and da Silveira, 1993). Among these adhesins K88 (F4) and 987P (F6) are specific
for pigs, while K99 (F5) and F41 seem to have receptors in both pigs and calves.
ETEC strains possessing K88 (especially K88ac) are the most common cause of diar-
rhoea and they usually produce LT in addition to STaP or STb. K88 + ETEC are also
characterized by haemolysin production in vitro. ETEC strains carrying K99 and/or F41
or 987P produce only STaP and are non-haemolytic. While K88 + ETEC may represent
about 40 – 60% of the E. coli strains causing diarrhoea in piglets, the above non-K88
strains make up between 20 –30% (Woodward and Wray, 1990; Nagy, 1993). The
typical O serogroups for neonatal porcine ETEC infections are O8, O9, O20, O101,
O141, O147 and O157 representing both K88+ and non-K88 ETEC. In our experience,
the two groups (K88+ and non-K88) of ETEC have a somewhat different clinical pic-
ture: K88 strains cause more severe diarrhoea at a younger age (1–5 days) while non-
K88 strains give rise to milder diarrhoea with a later onset (approximately 4–14 days of
age). It should also be noted that the rotavirus infection often complicates neonatal coli-
bacillosis of pigs, especially in non-K88 ETEC infections at the second week of age.
Small intestinal receptors of adhesins are the other essential element of intestinal
colonization by ETEC. It has been shown that the receptors for K88 are glycoproteins
and the lack of production is a recessive trait. Thus, homozygous piglets are resistant
to K88 mediated adhesions, to colonization and to disease (Sellwood et al., 1975).
The genes responsible for production of intestinal receptors belong to the TF blood
group linkage group (Gibbons et al., 1977). Receptor functions seem to be dependent
on the “b”, “c” and “d” components, and in genetically resistant piglets the receptors
are usually absent for both of these components of the K88 variants (Hohmann and
Wilson, 1975; Bijlsma et al., 1982). In vitro adhesion tests have revealed a polymor-
phism of intestinal receptors for K88 and indicated that there are 5–6 different adhe-
sion patterns (A–F) among piglets according to the K88ab, K88ac and K88ad
variants (Bijlsma et al., 1982; Rapacz and Hasler-Rapacz, 1986; Billey et al., 1998).
Unfortunately, this phenomenon of genetically determined resistance could not gain
a wide practical application. It may, however, complicate epidemiological pictures,
by partially producing non-diarrhoeal homozygous recessive (ss) litters, and by
partially leaving heterozygous (Ss) piglets (which are born to resistant sows and
sensitive boars) without colostral immunity (such sows would not have acquired the
infection and could not produce specific antibodies in their colostrum). The practical
application of this knowledge is further complicated by the fact that the correlation
of the adhesion of K88 variants to the small intestinal brush borders with suscepti-
bility to colonization and diarrhoea may be lacking. This can be explained by the
findings of Francis et al. (1998), suggesting that the intestinal mucin-type glyco-
protein (IMTGP) is a biologically more relevant receptor for K88ab and K88ac as
compared to the so far widely accepted enterocyte brush border glycoprotein.
So far, no information is available about the genetic determination of receptors
for K99, F41 or 987P in pigs, but there are mice that are genetically resistant to col-
onization by K99 (Duchet-Suchaux et al., 1990). Future research on these areas of
mammalian genetics would clearly be needed.
The production of receptors also influences age-related resistance to the disease.

This is, however, manifested in different ways for different adhesins. Receptors for
K88 are abundant in newborn pigs and will decrease with age but remain relatively
stable throughout the weaning and post-weaning periods. Receptors for K99 gradu-
ally decrease with age (Runnels et al., 1980). In contrast, production of receptors for
987P, does in fact increase with age (Dean et al., 1989). This invariably leads to a
lower intensity of adhesion and colonization because the receptors are shed into
the lumen and block bacterial adhesion before contacting intestinal epithelial cells.
The ageing nature of receptors for F41 is unknown but data indicate that they may
be produced all through the weaning age in pigs (Nagy, unpublished results).
Post-weaning diarrhoea (PWD) is usually the most constant disease problem of
large-scale farms, especially of those that wean around 3–4 weeks of age. PWD starts
a few days after lacteal protection completely ceases, and pigs are placed in an envi-
ronment that is completely new from a technical, social and microbiological point
of view. It is widely accepted that specific serotypes and pathotypes of ETEC are
responsible for the major part of PWD. It is also without debate that the disease is a
highly complex one in which ETEC only plays a part (although an essential one). It
is frequently seen in almost all large-scale piggeries but it is one of the most difficult
diseases to reproduce experimentally. Diarrhoea and reduction of weight are only
part of the losses. Retarded growth, which usually follows diarrhoeal episodes
in weaned pigs, makes the losses even worse. The main cause of post-weaning
diarrhoea is the weaning itself. Only on this basis can we understand the aetiology
and pathogenesis more realistically and can we be more humble about our capacities
to bring real (economically feasible) improvement to this enigma. The ETEC strains
involved are most frequently of the O serogroup: O8, O141, O138, O147, O149,
O157, of which O149:K88 seems to be the predominant serotype in most countries
(Hampson, 1994). So far, all the typical PWD strains of ETEC are haemolytic,
although haemolysin does not play an essential part in the virulence of porcine ETEC
(Smith and Linggood, 1971).
The most frequent adhesive virulence factors of ETEC strains in the case of
PWD are K88 (mainly K88ac) fimbria. Furthermore, K99, 987P and F41 have also
been described on some PWD strains (Nakazawa et al., 1987; Nagy et al., 1990a,
1996a) but they seem to be rarely involved in diarrhoea at that age. Recently, a new
fimbrial adhesin has been recognized under the F18 designation.
The F18 fimbriae have been described under different names, and misunder-
standings are frequent in the use of the earlier names and new designations. During
the past few years, three new colonization factors or adhesive fimbriae have been
described for groups of E. coli involved in PWD or oedema disease: F107 on
oedema strains (Bertschinger et al., 1990), 2134P on ETEC strains (Nagy et al.,
1992b), and “8813” also on ETEC strains (Salajka et al., 1992). Additionally,
fimbriae of two ETEC strains of serogroup O141 have also been described (Kennan
and Monckton, 1990), although no data have been given on their adhesive or
pathogenetic significances. As a first attempt to clarify the relationships between

these factors, pili 2134P were compared to fimbriae F107 by means of polyclonal
and monoclonal antibodies. It was provisionally concluded that these two adhesins
were morphologically similar and shared a common antigenic determinant in addi-
tion to a type-specific one (Nagy et al., 1992a). These findings were confirmed
(Wittig et al., 1994) and it was suggested that the symbol “a” should be used for the
common determinant and the symbols “b” and “c” for the specific determinants of
F107 and 2134P respectively. Furthermore, Rippinger et al. (1995) investigated
the morphological, immunological, genetic and receptor-binding relatedness of
fimbriae F107 and 2134P, together with the colonization factor “8813”. Based on
earlier suggestions made by Ida and Ørskov (International Escherichia coli Centre,
Copenhagen, 1992) for a new F18 fimbria, it was shown that two serological
variants were determined and should be designated as follows: F18ab (for F107) and
F18ac (for 2134P and 8813) (Rippinger et al., 1995). The genetic relatedness of the
above family of F18 fimbriae was described by Imberechts et al. (1994), supporting
the above grouping, and adding the fimbriae of Kennan’s O141 strains (Kennan and
Monckton, 1990) to the group of F18ac. In a recent study, it was pointed out that
F18ab and F18ac fimbriae are biologically distinct: F18ab fimbriae are poorly
expressed both in vitro and in vivo. They are frequently linked with the production
of SLT-IIv (VTEC strains), while F18ac are more efficiently expressed both in vitro
and in vivo and they are more characteristic of ETEC strains (Nagy et al., 1997).
It should also be mentioned that some ETEC strains may produce multiple adhesins
such as K88, F18ac or K88, F41 or even K88, F18ac, and F41 (Nagy et al., 1996a).
It remains to be shown if such strains have a pathogenetic advantage over strains
with one kind of adhesin. It may also be questioned under what conditions there are
receptors for these rarely occurring adhesins (K99, 987P, F41) available in the right
amount on the small intestinal mucosae.
In weaned pigs receptors for K88 are produced, although to a somewhat reduced
extent, all through the weaning age, while receptors for the variants of F18 (F18ab
and F18ac) are increasingly produced up to the weaning age (Nagy et al., 1992a,
1997) and the fimbriae F18ac seem to have more receptors around the ileal Peyer’s
patches (Nagy et al., 1992a). The lack of receptors for F18ab and F18ac in newborn
pigs offers an explanation why these VTEC and ETEC strains (and why the oedema
disease itself) are only prevalent in weaned pigs.
Inherited resistance to PWD owing to production of intestinal receptors of
fimbria F18ab has also been investigated by oral inoculation of weaned pigs and by
in vitro adhesion tests (Bertschinger et al., 1993), and it seems that phenotypes
susceptible or resistant to F18 adhesion can be differentiated. Pigs with at least one
copy of a dominant allele for receptors are susceptible to colonization and in vitro
adhesion (which is similar to the K88 receptors). Additional genetic marker studies
localized the receptor gene on the porcine chromosome 6, closely linked to the gene
encoding for halothane sensitivity (Vogeli et al., 1994). It seems that the lack of
receptors will coincide with halothane (stress) sensitivity, making it difficult to
select and raise pigs without small intestinal receptors for F18 fimbria. Small intestinal
receptors for K88 and for F18 seem to be different, on the basis of comparative
in vitro studies (Nagy et al., 1997) and the different localization of their regulation
on the porcine chromosome (Gibbons et al., 1977; Vogeli et al., 1994).
Breeding pigs resistant to ETEC adhesion seems to be very difficult. First of all
it is difficult to select subdominant alleles of two different, independently inherited
traits (lack of receptors for K88 and F18) but we should also consider that the E. coli
bacteria are genetically much more flexible than their host. This would ultimately
lead to the emergence and proliferation of new ETEC pathotypes. Furthermore, we
should take into account the possible co-selection of unwanted traits (such as
halothane sensitivity).
3. VEROTOXIGENIC E. COLI IN PIGS AND CALVES

Verotoxigenic E. coli (VTEC) is one of the alternative names for E. coli bacteria
producing a cytotoxin detectable on Vero (African green monkey kidney) cell
culture (Konowalchuk et al., 1977) and sharing a number of properties with Shiga
toxin (O’Brien et al., 1977). Therefore they are also called “Shiga-like” toxin
producing E. coli (SLTEC). The toxins of this group are generally characterized by
the same or similar structure and a pathomechanism like that of the toxin of Shigella
dysenteriae (composed of one A and five B subunits). As a result of enteric infection
with VTEC strains, these toxins (VT1 and VT2) produce enteric (haemorrhagic colitis)
and systematic disease (haemolytic uraemic syndrome) in humans, practically only
enteric disease in calves (calf dysentery) and systemic disease in pigs (oedema disease).
The pathomechanisms of these toxins are characterized by a receptor-mediated
endocytosis of the A subunit of the VT, followed by a fusion in lysosomes and
release of the enzymatically active fragment A1, leading to inhibition of protein
synthesis and cell death.
Most of the VTEC (SLTEC) bacteria of ruminants and humans produce a
characteristic attachment and effacement (AE) type of microvillous degeneration
and bacterial adhesion (fig. 1b) (as will be described later in the section on EPEC
and EHEC). However, there are verotoxin-producing E. coli strains which do not
have the AE phenotype, and therefore do not produce such characteristic lesions but
possibly adhere to the brush border, leaving the microvilli intact. In order to differ-
entiate these verotoxic bacteria from those which also produce characteristic lesions
(and haemorrhagic colitis) in this chapter we refer to them as verotoxigenic E. coli
(VTEC). Such VTEC bacteria produce oedema disease in pigs and there are
some others that produce milder diarrhoea in humans and in calves (Wieler, 1996;
Mainil, 1999).
3.1. Adhesins of VTEC for calves and pigs

In calves most of the VTEC strains also have the attachment effacement capacity
(Gyles, 1994; Wieler, 1996) and could therefore be designated as enterohaemor-
rhagic E. coli (EHEC) (see below). The non-AE strains of VTEC of calves have not
been thoroughly studied for adhesins yet. Wieler (1996) demonstrated that bovine
VTEC strains without AE genes were relatively frequent among VT2 strains of
calves. Wieler has also demonstrated that many of these strains also adhered to
cultured Hep2 cells, and they were all negative for bundle-forming pili (Bfp)
characteristic of human EPEC. At this point it remains an interesting question how
these strains could colonize the bovine intestine and continue being shed into the
environment. It can be speculated whether such VTEC strains of calves have lost
their earlier capacity to produce AE lesions or they have not gained it yet, or they
have developed as a clonally different lineage.
In pigs at present the only VTEC known are the ones producing oedema disease.
They produce a variant of VT2 (VT2e). This toxin leaves the intestinal epithelial
cells without damage but enters the bloodstream, using receptors on red blood cells,
and damages the endothelial cells of the small blood vessels by inhibiting protein
synthesis. This leads to perivascular oedema and hyalinization in several organs and
to death. The only known adhesins of the porcine VTEC are the F18 fimbria (as
described above). Adhesion and colonization mediated by F18 do not cause charac-
teristic damage to the morphology of the intestinal cells, resembling the morphol-
ogy of adhesion by ETEC (Bertschinger et al., 1990; Nagy et al., 1997).
4. ENTEROPATHOGENIC E. COLI AND

ENTEROHAEMORRHAGIC E. COLI
Enteropathogenic Escherichia coli (EPEC) were first described in the 1940s and 1950s
as the causative agents of infantile diarrhoea, and are still a major cause of infant diar-
rhoea in the developing world. EPEC do not produce enterotoxins and are not invasive;
instead their virulence depends on causing characteristic intestinal histopathology
called attaching and effacing (AE), which can be observed in intestinal biopsy and
in vitro (Moon et al., 1983; Knutton et al., 1987). The AE phenotype is characterized
by effacement of microvilli and intimate adherence between the bacterium and the
epithelial cell membrane. The AE phenotype develops due to a specific signalling
pathway and the AE lesions are characterized by localized effacement of the brush
border of enterocytes with intimate bacterial attachment and pedestal formation
beneath the adherent bacteria. EPEC have a set of adhesins (reviewed by Nataro and
Kaper, 1998). Intimin is essential, but not enough for the pathogenesis of EPEC, and it
is encoded by a chromosome (Jerse et al., 1990). Most of the EPEC strains possess
a plasmid of about 60 Mda which promotes the adherence to cultured epithelial cells
in a localized adherence (LA) pattern. Early studies proved the importance of this
plasmid, named EAF (EPEC adherence factor) (Baldini et al., 1983). The EAF plas-
mid encodes a fimbrial adhesin called bundle-forming pilus (Bfp, Giron et al., 1991).
4.1. Intimin and translocated intimin receptor (Tir)

The first gene to be associated with the AE phenotype was the eae (for E. coli
attachment effacement) encoding intimin, a large molecular weight outer membrane
protein (Jerse et al., 1990; Jerse and Kaper, 1991). Subsequently, the eae gene was
shown to be part of a large chromosomal region of DNA that encodes all the neces-
sary determinants for the AE phenotype (McDaniel et al., 1995). This chromosomal
region is named the locus of the enterocyte effacement (LEE) pathogenicity island.
The LEE is responsible for the AE intestinal histopathological changes caused by
EPEC and enterohaemorrhagic E. coli (EHEC) and related animal pathogens, first of
all rabbit EPEC. The LEE is organized into three main parts (gene clusters).
The middle part of the LEE contains the eae and tir genes as well as the cesT
gene. The right side encodes for the proteins secreted via the type III secretory
system (espA, espB, espD, espF genes), while the left side encodes for the genes of
the type III secretory system itself (partly functioning like a molecular syringe).
Details of the functions of the three areas of the LEE follow.
On the middle part the eae codes for intimin (Eae), a 94–97 kDa outer membrane
protein that is an intestinal adherence factor to epithelial cells, and tir (translocated
intimin receptor) encodes the Tir, the intimin receptor protein (Kenny et al., 1997;
Deibel et al., 1998). E. coli eae genes have been cloned and sequenced from differ-
ent EPEC and EHEC strains isolated from humans and animals including calf
(Goffaux et al., 1997). Sequence comparisons of different eae genes revealed that
the N-terminal regions show high conservation but the C-terminal regions encoding
the last 280 amino acids are heterogeneous. The cell-binding activity of intimin is
localized at the C-terminal 280 amino acids of polypeptide “Int280” (Frankel et al.,
1995; Liu et al., 1999).
Immunological and genetic studies revealed the existence of pathotype-specific
intimin subtypes. Agin and Wolf (1997) identified three intimin types, α, β, γ,
Adu-Bobie et al. (1998a) detected four distinct subtypes of intimin, α, β, γ, δ, and
recently Oswald et al. (2000) characterized an additional new intimin variant,
intimin ε. Molecular studies revealed that these intimin types are pathotype (and
species) specific. Intimin α was specifically expressed by human EPEC strains
belonging to classical EPEC (clone 1) serotypes of O55:H6, O125:H, O127:H6,
O142:H6 and O142:H34 (Adu-Bobie et al., 1998b). Intimin β appears to be the
most ubiquitous type: it is associated with EPEC strains belonging to clone 2
(O26:H−, O111:H−, O111:H2, O142:H2, O119:H2, O1219:H6, and O128:H2) and
EHEC O26:H11; intimin β was detected in rabbit O15:H−, O26:H11, and O103:H2
strains; and this subtype was present in O26:H11 bovine strains as well (Oswald
et al., 2000). Intimin γ is associated mainly with human and cattle Shiga-like toxin
producing E. coli (SLTEC) strains including sorbitol-fermenting and sorbitol non-

fermenting EHEC O157:H7, O157:H− strains, and SLTEC strains of serotypes
O111:H8, O111:H−, O86:H40, O145:H−, and EPEC O55:H− and O55:H7 strains
also harbour intimin γ. Intimin δ was associated with human EPEC O86:H34
(Adu-Bobie et al., 1998a). Intimin ε was present in human and bovine EHEC strains
of serogroups O8, O11, O45, O103, O121 and O165 (Oswald et al., 2000).
The observation that different intimin subtypes are associated with different
pathogenic clones can explain why these strains colonize different segments of
the intestine in different host species. Tzipori et al. (1995) infected pigs with human
strains having different types of intimin and demonstrated that the intimin α-
producing strain caused AE lesions in both the large and the small intestine, while
the intimin γ-producing EHEC strain caused AE lesions only in the large intestine.
When the pigs were infected with an eaeγ − but eaeα+ EHEC recombinant strain, AE
lesions were observed in both the small and the large intestine (Tzipori et al., 1995).
Interestingly, in the case of EPEC, the receptor for bacterial adhesin (intimin) is
another protein of the same bacteria called translocated intimin receptor (Tir). Tir is
a bacterial protein that is translocated into the host cell via a type III secretion
system and upon entry into the eukaryotic cell it serves as the receptor for the
intimin. Initially it was believed that the intimin receptor protein is a mammalian
membrane protein that was originally called Hp90 (“host protein”) and that was
tyrosine phosphorylated in response to EPEC infection (Rosenshine et al., 1996).
The combined interactions between host kinases and EPEC proteins result in
additional host signalling events such as actin aggregation and polymerization
leading to the characteristic cellular pathology. In E. coli O157:H7 infection the Tir
protein has an analogous function, but it is not phosphorylated after translocating to
the eukaryotic cell (DeVinney et al., 1999).
As mentioned above, the LEE contains two additional main functional clusters:
on the right side of the LEE are the espA, espB, espD, espF genes, of which the first
three genes are necessary for the AE phenotype. The EspA is a structural protein and
a major component of a large organelle; it is transiently expressed on the bacterial
surface and interacts with the host cell during the early stage of AE lesion formation.
EspA forms a physical bridge between the bacterium and the infected eukaryotic cell
surface and is required for the translocation of EspB into infected epithelial cells, and
may contribute to bacterial adhesion as well (Knutton et al., 1998). EspB protein is
translocated into the host cell membrane by EspA and cytoplasm and serves as the
distal end of EspA filament, and it might have a function in the host signal transduc-
tion events. EspB promotes tyrosine phosphorylation of Tir and induction of inositol
phosphates and calcium fluxes. The increased calcium levels can induce cytoskeletal
rearrangements and activate calcium-dependent kinases resulting in morphological
changes including microvillus effacement and pedestal formation.
McNally et al. (2001) observed clear differences in the expression of LEE-encoded
factors between O157 strains, with the same stx+ eae+ genotype, isolated from
human disease cases and those isolated from asymptomatic cattle. All strains
produced a detectable amount of EspD when grown in tissue culture medium, but
in the case of the human O157 strains that amount was on average 90-fold higher
than for the bovine O157 strains. The level of secretion also correlated with the abil-
ity to form AE lesions on HeLa cells, and with only high-level protein secretors in
tissue culture medium exhibiting a localized adherence phenotype (McNally et al.,
2001). These data correlate with earlier findings based on the results of a compre-
hensive molecular analysis (Kim et al., 1999). That analysis revealed the existence
of two distinct lineages of E. coli O15:H7 in the United States. Human and bovine
isolates are non-randomly distributed among the lineages, suggesting that lineage II
strains may not readily cause disease or may not be transmitted efficiently to
humans from bovine sources. Alternatively, the distribution may reflect a loss of
characteristics in lineage II that are necessary for virulence in humans, perhaps as a
consequence of adaptation to the bovine environment.
On the left side of the LEE is a set of genes coding for the type III secretion
system itself. These genes share sequence homology with the type III secretion
systems of Yersinia enterocolitica, Shigella flexneri and Salmonella typhimurium
(reviewed by Mecsas and Strauss, 1996). The type III secretion systems are respon-
sible for secretion and translocation of different virulence determinant proteins such
as the espA, -B, -D, -F encoded proteins and the Tir protein.
4.2. EPEC adherence factor plasmid

The majority of EPEC strains possess a plasmid 50–70 MDa in size, named the EAF
(EPEC adherence factor) plasmid. These plasmids share extensive homology among
various EPEC strains. The typical EPEC strains associated with diarrhoea possess
EAF, while EPEC strains that do not have the EAF plasmid are referred to as
atypical EPEC (Nataro and Kaper, 1998). The importance of EAF was demonstrated
in vivo by Baldini et al. (1983) and a volunteer study revealed that EAF is essential
for the full virulence (Levine et al., 1985). Giron et al. (1991) identified an EAF
encoded adhesin, called bundle-forming pilus (BFP), which is a member of the
type IV pilus family. The expression of BFP was associated with localized
adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid
(Giron et al., 1991).
Barnett-Foster et al. (1999) demonstrated that phosphatidylethanolamine (PE)
serves as a receptor for EPEC and EHEC. These bacteria bind to PE specifically and
in a dose-dependent manner, and this binding was consistently observed whether the
lipid was immobilized on a thin-layer chromatography plate, in a microtitre well or
incorporated into a unilamellar vesicle suspended in aqueous solution. Bacterial
binding to two epithelial cell lines also correlated with the level of outer leaflet PE
and it was reduced following preincubation with anti-PE. The PE-binding pheno-
type of EPEC correlated with the bfp genotype of a number of clinical isolates.
4.3. EPEC (and EHEC) in pigs and calves

In calves the first description of attachment effacement (AE) lesions due to “atypi-
cal E. coli” was given in the UK by Chanter et al. (1984), in relation to natural cases
of “calf dysentery”. The E. coli O5 strain produced bloody diarrhoea and typical AE
lesions in gnotobiotic piglets (Chanter et al., 1986) and it turned out to be verotox-
igenic as well. Further observations in the United States indicated that verotoxigenic
and AE lesion-producing strains of E. coli O26 are a relatively frequent cause of calf
diarrhoea (Janke et al., 1990) and such strains have also been detected to cause nat-
ural infections in the UK (Gunning et al., 2001). On the basis of the fact that most of
the bovine AE lesion-producing strains also produce verotoxins, they could also be
named enterohaemorrhagic-like E. coli (or EHEC-like strains). As is well known,
the classical human EHEC strains O157:H:H7 and O157:NM are frequently carried
asymptomatically by calves and older cattle as well as by small ruminants and it is
usually among the least frequent serotype occurring in cattle, in contrast to humans
where O157 is the leading serogroup of EHEC (reviewed by Dean-Nystrom et al.,
1998). The EHEC strains causing bovine diseases (O26, O103, O111, O118 and
O157) can also be transmitted to humans and, thus, these strains have a serious
zoonotic potential. So far it seems that several bovine and human strains have beta
intimin (supporting the zoonotic significance of bovine EHEC).
Non-verotoxigenic AE E.coli seem to be relatively rare in calves, although they
can also produce watery diarrhoea (Pearson et al., 1989), and can be regarded as the
bovine EPEC. The intimin type of these bovine EPEC strains is usually also the beta
intimin (Oswald et al., 2000). At present there is no solid information available on
any additional adhesive factor of bovine EPEC or EHEC strains, although it seems
quite likely that there are some peculiarities in the adhesins of these strains as well.
EPEC strains of porcine origin were first detected by Janke et al. (1989) and
porcine EPEC infection was studied on newborn pigs by Helie et al. (1991) who
have shown colonization and typical AE lesions in the ileum and jejunum as early
as 12–24 h after infection with a porcine O45:K“E65” E. coli, while the caecum and
colon were colonized at 24–48 h post infection. This group demonstrated that
porcine EPEC have virulence characteristics similar to those of human strains
(Zhu et al., 1994, 1995) and, by using transposon mutagenesis, identified a porcine
attaching-effacing-associated (paa) factor associated with the presence of the eae
gene. Interestingly this paa was found in EHEC O157:H7 and in O26 strains and a
strong association was with the heat-labile enterotoxin (LT) gene (An et al., 1999).
Further studies have proven that the eae gene of porcine EPEC prototype E. coli
1930 (O45) strain was a member of the beta intimin group and showed the highest
similarity with the rabbit EPEC strains (An et al., 2000). Such strains may be pres-
ent in small numbers in the pig population not only in North America but also in
Europe as well (Osek, 2001). However, the overall significance of porcine EPEC
strains cannot be judged on the basis of the available data. It seems that further epi-
demiologic studies are needed to establish their significance in porcine enteric disease.
It is interesting to see that in pigs two genetic lineages have diverged: one is VTEC
(verotoxin production without AE lesion) and the other is EPEC (AE lesion without
verotoxin production). The first is responsible for oedema disease of weaned pigs
(with well-identified fimbrial adhesin) while the other may induce diarrhoea in pigs
(with beta intimin and paa antigen).
5. NECROTOXIGENIC E. COLI
Necrotoxigenic Escherichia coli (NTEC) are defined as E. coli strains producing a
large molecular weight toxin named cytotoxic necrotizing factor (CNF). NTEC are
associated with intestinal and extraintestinal diseases in animals and human beings
(DeRycke et al., 1999). CNF was first identified from children with enteritis by
Caprioli et al. (1983). The large monomeric protein toxin causes necrosis in rabbit
skin and induces formation of multinucleation and thick bundles of actin stress
fibres in HeLa, CHO and Vero cells (Caprioli et al., 1984) (fig. 1c). Two types of
CNF (CNF1 and CNF2) have been identified, each of them being genetically linked
to several other specific virulence markers (DeRycke and Plassiart, 1990). The
CNFs covalently modify Rho proteins (small GTPases) that regulate the physiology
of the cell cytoskeleton of mammalian cells, and lead to polymerization of actin
fibres (Oswald et al., 1994; Fiorentini et al., 1995). CNFs are encoded by a single
structural gene. The CNF1 operon is located on the chromosome (Falbo et al., 1992)
and CNF2 is determined by a conjugative plasmid (Oswald et al., 1994).
NTEC1 strains can be found in humans and in all species of domestic mammals
(DeRycke et al., 1999). The CNF1 operon is frequently associated with other virulence
factor genes and these genes constitute large chromosomal regions called pathogenic-
ity islands (PAIs, Hacker et al., 1997). One of these PAIs (PAI II) encodes CNF1, alpha-
haemolysin and P-fimbriae and it was first identified in a human uropathogenic E. coli
(UPEC) strain. This virulence gene pattern was reported in intestinal strains isolated
from suckling (Garabal et al., 1996; Dozois et al., 1997) and from weaned pigs (Tóth
et al., 2000), which may be explained by the unusual mobility of the PAIs.
NTEC2 strains have only been reported in ruminants (DeRycke et al., 1999).
In NTEC-2 strains, CNF2 is encoded by a virulence plasmid (pVir, Oswald et al.,
1994). pVir also codes for a new member of the cytolethal distending toxin family
(CDTIII, Peres et al., 1997) and for the F17b or F17c fimbrial adhesin that confers the
ability to adhere to calf intestinal villi (Oswald et al., 1994) and enter the bloodstream
(Van Bost et al., 2001). It is tempting to speculate that the large conjugative plasmid
(pVir) is also carrying a PAI containing the operons for CNF2, F17b, and CDTIII.
5.1. Adhesins and receptors of NTEC isolated from animals

Molecular epidemiological studies revealed that most of the human and animal
NTEC strains have different fimbrial (pap, sfa, f17) and afimbrial adhesin (afa)
genes. Mainil et al. (1999) reported that most NTEC1 extraintestinal calf isolates
hybridized with the PAP probe and additionally either with the SFA probe (37%) or
with the AFA probe (49%). In contrast, the NTEC2 isolates hybridized with the F17
probe (45%), with the AFA probe (19%), or with the F17 and AFA probes simulta-
neously (22%). In correlation with the CNF2 prototype strains E. coli S5 (Smith,
1974) and E. coli 1404 (Peres et al., 1997) all the 19 NTEC2 cattle isolates had a
virulence plasmid coding for CNF2 and most of them coded for fimbrial (F17) or
afimbrial (AFA) adhesins as well, for which the PCR results suggested the existence
of a new variant of AFA (Mainil et al., 1999).
Examining 32 herds for NTEC, it was found that CNF2 was more frequently
detected than CNF1 in the faecal samples of healthy cattle. CNF2-producing NTEC
strains were significantly more frequently isolated from calves (24%; 17 of 71)
than from cows (4%; 11 of 257). Reports confirmed that healthy calves are a reservoir
of NTEC producing CNF2 (Blanco et al., 1998), and NTEC that produced CNF2
may be part of the normal intestinal flora of cattle (Blanco et al., 1993). A sero-
epidemiological study revealed that the O groups of CNF2+ strains isolated from cows
(O2, O8, and O14) were different from those found in calves (O8-O75, O15, O55,
O86, O88, O115 and O147) (Blanco et al., 1998). Depending on the serotypes the
CNF1-producing strains isolated from human extraintestinal infection had different
adhesins (Blanco et al., 1994). These latter authors also suggested that extraintestinal
infections are caused by a limited number of virulent clones. CNF1 strains of serotypes
O2:K7:H− and O4:K12:H1 express P fimbriae, whereas CNF1 strains of serotypes
O2:K?:H1, O2:K1:H6 and O75:K95:H5 possess the adhesin responsible for the
so-called MRHA type III. In the following section the above mentioned P and S fim-
briae and the afimbrial adhesions (AFA) will be discussed in some detail. Information
on the F17 fimbrial family has partly been provided in the bovine ETEC section.
5.2. P (pap) fimbriae

Type P fimbriae are also named pyelonephritis-associated pili (pap) and have been
recognized as P blood-group-specific adhesins (Kallenius et al., 1981). They are
composed of a thin fibrillum (carrying the adhesin) at the proximal end of a more rigid
pilus rod 7 nm in diameter (Kuehn et al., 1992). P fimbriae are part of a family of
adhesive organelles that are characterized by an assembly machinery consisting of a
periplasmatic chaperone (PapD) and a pore-forming outer membrane (PapC) usher
protein (Hultgren et al., 1996). The 11 genes coding for functional P fimbrial adhesin
are clustered in an operon encoding the main component of the pilus rod (PapA) and
several minor fimbrial subunits (PapH; K; E; F), the PapG which is the adhesin and the
assembly machinery (PapC; D; J), and the two regulatory proteins (Pap J; B) (reviewed
by Hultgren et al., 1996). PapG adhesin located at the tip of the fimbriae binds to
the alpha-D-galactopyranosyl-(1–4)-beta-D-galactopyranose or Gal alpha (1–4)Gal
disaccharides (Kuehn et al., 1992), while the receptor for the P-related sequences (prs)
is the GalNAc-α-(1–3)-GalNAc which is related to fimbriae of serotype F13.
There are known alleles of PapG, referred to as classes I, II, and III. These
classes have different haemagglutination patterns. PapG I agglutinates only human
erythrocytes, PapG II agglutinates human erythrocytes very well and sheep erythro-
cytes only poorly, and PapG III agglutinates only sheep erythrocytes (reviewed by
Hultgren et al., 1996).
The pap operon is located on the bacterial chromosome mostly associated with
other virulence factor genes forming PAIs in uropathogenic E. coli strains. At least
four PAIs are present in the genome of UPEC 536 of O6:K15:H31 prototype, and
three of them encode different adhesions: PAI I and II carry genes for P fimbriae and
haemolysin, while PAI III encodes the S fimbrial adhesin. UPEC J96 of serotype
O4:K6 has two PAIs. One PAI carries virulence determinants pap and hlyI. The
second PAI encodes CNF1, hlyII and harbours prs (pap-related sequence) genes.
Because these islands represent a mechanism for spreading the pathogenicity factors
between strains belonging to the same and different species, the presence of classi-
cal UPEC specific adhesins in intestinal isolates such as NTEC1 strains is under-
standable (reviewed by Hacker et al., 1997).
5.3. S fimbriae
S fimbrial adhesins I and II (SfaI and SfaII), produced by extraintestinal Escherichia
coli pathogens that cause urinary tract infections (UTI, Hacker et al., 1985) and
newborn meningitis (NBM, Hacker et al., 1993), respectively, mediate bacterial
adherence to sialic acid-containing glycoprotein receptors (Moch et al., 1987) pres-
ent on host epithelial cells and on extracellular matrix. The S fimbrial adhesin (sfa)
determinant of E. coli comprises nine genes (Schmoll et al., 1990). Both SfaI and
SfaII adhesin complexes consist of four proteins: SfaA (16 kDa) is the major sub-
unit protein and the minor subunit proteins are SfaG (17 kDa), SfaS (15 kDa), and
SfaH (29 kDa).
Genetic and functional analysis of the sfa I complex conducted by Khan
et al. (2000) revealed that sialic acid-specific binding is mediated by the minor
subunit protein SfaI-S, which is located at the tip of the fimbriae. The SfaI-S was
the only minor protein gene which increased the degree of fimbriation and pro-
vided adhesion properties for a non-adhesive derivative K-12 strain which had
the sfaI-A major subunit gene but had neither the sfaI-G nor the sfaI-H gene.
sfaEF genes are part of the assembly and transport apparatus, while sfaC and sfaB
genes are regulators. The receptor of the S fimbrial adhesions is α-sialyl (2,3)-
β-galactose.
Although both the P and S fimbrial families are recognized as typical extra-
intestinal (mainly uropathogenic) adhesive virulence factors, the fact that they
can be detected relatively frequently on intestinal isolates, indicates that they may
have a role in the intestinal colonization of animals (including pigs and calves)
and humans.
5.4. Afimbrial adhesins

Afimbrial adhesins (AFA) are the first adhesin structures that are not associated with
fimbriae. They were observed for the first time on a uropathogenic E. coli strain
(Labigne-Roussel et al., 1984). At present at least eight different afa gene clusters are
known. The afaI gene cluster identified first from E. coli strains associated with urinary
and intestinal infections encodes AfaABCDE proteins and it is involved in adhesion to
epithelial cells and haemagglutination (Labigne-Roussel and Falkow, 1988). Three Afa
proteins, AfaB, AfaC and AfaE, are required for the mannose-resistant haemagglutina-
tion (MRHA) and for adherence to uroepithel cells (Labigne-Roussel et al., 1984;
Labigne-Roussel and Falkow, 1988). Among these three proteins AfaA and AfaF are
transcriptional regulators, AfaB functions as a chaperone, AfaC is an outer membrane
usher, AfaD is an invasin, and AfaE is the adhesin protein (Walz et al., 1985).
Immunological and DNA hybridization studies revealed the existence of at least
four afa operons encoding different adhesins in which the afaB, afaC, and afaD genes
are highly conserved but the afaE genes (encoding the adhesin proteins) are variable
(Labigne-Roussel and Falkow, 1988). All these Afa I–IV variants were identified in
human UPEC strains but later a hybridization and PCR analysis based study revealed
the existence of related sequences in pathogenic E. coli isolates of bovine and porcine
origin (Harel et al., 1991). Further studies suggested that these operons are different
from the afa operons of human isolates (Maiti et al., 1993; Mainil et al., 1997).
Lalioui et al. (1999) cloned and characterized afa-7 and afa-8 gene clusters
encoding afimbrial adhesins from diarrhoeagenic and septicaemic E. coli strains of
bovine origin. The AfaE-VII and AfaE-VIII adhesin proteins are genetically differ-
ent from the AfaE adhesins produced by human pathogenic strains, and they also
have different binding specificity. The AfaE adhesins of human pathogenic strains
mediate the MRHA of human erythrocytes and specific attachment to HeLa, uroepithel
cells and Caco-2 cells via recognition of the so-called decay-accelerating factor
(DAF) molecule as a receptor. AfaE-VII mediates MRHA of human, bovine and
porcine erythrocytes and the adhesion of bacteria to HeLa, Caco-2 and uroepithel
cell lines, and to MBDK bovine kidney cell line and does not bind to canine kidney.
AfaE-VII does not recognize the SCR-3 domain of DAF, which is the receptor of
the human AfaE adhesins (Nowicki et al., 1993). AfaE-VIII binds to different still
unidentified receptors. In vitro assays showed that it binds to uroepithel cells and to
canine kidney cell line, but does not bind to HeLa and Caco-2 cell lines. AfaE-VII
is slightly similar to fimbrial adhesin AAF/I produced by enteroaggregative E. coli
isolates and AfaE-VIII is very similar to the M agglutinin (Lalioui et al., 1999).
Further, the afaE-VIII gene is frequent and highly conserved among E. coli strains
isolated from calves, particularly in NTEC strains in association either with the cnf1
or the cnf2 gene. The fact that the afa-VIII gene cluster is located on the chromo-
some or on the plasmid suggests that it could be carried by a mobile element, facil-
itating its dissemination among bovine pathogenic E. coli strains.
6. PRACTICAL APPLICATIONS
The above information on basic mechanisms of pathogenesis of enteric E. coli infec-
tions of calves and pigs has led to practical applications mainly in the area of diag-
nosis and prevention of these diseases. Unfortunately, specific preventive measures
have only been worked out against ETEC infection of newborn calves and pigs as
discussed below.
6.1. Diagnosis of enteric E. coli infections

Diagnosis of ETEC infections requires the phenotypic detection of virulence factors
(adhesins, enterotoxins) using in vitro tests (slide or latex agglutination or ELISA)
in most cases (Thorns et al., 1989). Adhesive fimbriae can, however, be most effi-
ciently detected in vivo, by an immunofluorescent method using absorbed poly-
clonal or monoclonal antifimbrial antibodies (Isaacson et al., 1978). In contrast to
fimbriae, enterotoxins produced in vivo are much more difficult to detect. Therefore,
in early ETEC studies in vitro produced toxins could only be tested by biological
assays: ligated small intestinal segments (for all enterotoxins) or baby mouse assay
(for STa), followed by cell cultures (for LT), and later on by ELISA assays (for LT
and ST) (Czirok et al., 1992). Now, with the advent of molecular methods in the
diagnostic laboratories, the cumbersome biological assays can be replaced by so-
called gene probes: DNA hybridization and PCR (recently in a complex form) for
detecting the genes of different virulence characters (Mainil et al., 1990; Franck
et al., 1998; Tsen and Jian, 1998). The question can be raised, however, of whether
our chances to discover new adhesive and other virulence attributes will not be
limited if we disregard classical biological assays in the long run.
6.1.1. Diagnosis in calves

According to our present knowledge, the diagnosis of ETEC infection in calves is
greatly facilitated by the high frequency of K99 antigens on bovine ETEC. The pres-
ence of K99 can, however, be covered by the K(A) antigens. Besides, the produc-
tion of K99 may also be repressed by the presence of glucose, while for other strains
glucose may even enhance K99 production (Girardeau et al., 1982). Therefore, spe-
cial media such as Minimal Casein Agar with Isovitalex® added (MINCA-Is) are
required (Guinee et al., 1977) for the detection of K99 in vitro. Alternatively, the
immunostaining of small intestinal segments from calves that died as a result of
diarrhoea proved to be more efficient (Isaacson et al., 1978; Nagy and Nagy, 1982).
Monoclonal based latex reagents (Thorns et al., 1989, 1992) and DNA probes
(hybridization and PCR) that detect the above fimbrial genes are available for more
efficient diagnosis (Mainil et al., 1990) not only for ETEC but for other pathotypes
as well.
6.1.2. Diagnosis in pigs

Piglet diarrhoea is almost always accompanied by some type of non-commensal
E. coli infection at the suckling age and within the first 2 weeks after weaning.
Today, we already know of several types of porcine ETEC (although it seems that
other pathotypes can also complicate and partly induce diarrhoea in newborn and
especially in weaned pigs). Furthermore, it should be remembered that on the herd
level, diarrhoeal episodes are infrequently monocausal. The presence of one or
more types of ETEC (for example) can often be accompanied by rotaviruses,
caliciviruses, coccidia, or by the coronavirus of porcine epidemic diarrhoea (PED)
in both age groups but especially in weaned pigs (Hampson, 1994; Nagy et al.,
1996b). In this chapter only the diagnosis of infections due to known and established
types of ETEC will be discussed, which are in most cases the dominant elements of
sporadic diarrhoeal diseases on the herd level. Diagnosis of ETEC infection is based
on the detection of known virulence factors (and of the serogroup) of the suspected
ETEC. This would not necessarily require culturing of bacteria (see below), but the
need to determine antibiotic resistance patterns simultaneously makes culture and
test of bacterial attributes in vitro an accepted routine for diagnostic laboratories.
For cultures, usually small intestinal or faecal samples are available, from which
it is advisable to inoculate specific media (besides classical media) required for
preferential growth of some adhesins (such as MINCA-Is for K99, or Difco Blood
agar Base with sheep blood for 987P) (Guinee et al., 1977; Nagy et al., 1977). To
test if the isolates are ETEC, the fimbrial antigens K88, K99, F41 and 987P can be
detected by slide agglutination using specific absorbed sera or by latex agglutination
for which there are monoclonal antibody based kits available (Thorns et al., 1989,
1992). Adhesive fimbriae produced in vivo can be more efficiently detected by
testing small intestinal smears of diarrhoeal pigs using fluorescence antibody
assays. As there may be ETEC strains without known (or detectable) adhesive
virulence factors, it is advisable to perform tests for enterotoxins as well. LT and
STa toxins can be identified by ELISA or by latex agglutination; unfortunately no
such tests are available for STb. DNA probes (hybridization and PCR) are also in
use for in vitro detection of almost all known virulence genes of porcine ETEC
(Mainil et al., 1990; Nagy et al., 1990a; Franck et al., 1998).
Besides bacteriological results, there is almost always a need for differential
diagnostic investigations (such as virus detection) as well. Therefore, in the case of
weaning pigs it is strongly advised not to be content with a possible bacteriological
result detecting some types of ETEC (carrying K88 or F18 surface antigens), but it
is also necessary to consider other physiological, environmental, dietary and viral
factors that may sometimes be as important as the given ETEC bacteria themselves.
Therefore, differential diagnosis should frequently include the detection of rota- and
coronaviruses as well as spirochaetes and Salmonella (Hampson, 1994; Nagy et al.,
1996b). Culturing and/or immunofluorescent in vivo identification of ETEC strains
from the ilea of diarrhoeal pigs is the most effective and simplest way of making a
bacteriological diagnosis (as described for diarrhoea of newborns). The bacterio-

logical analysis of faecal samples for ETEC is more difficult because the bacteria
present in the faeces may not reflect the microbial status of the small intestine. There
are a variety of in vitro techniques that detect virulence factors (adhesins and
toxins) of ETEC including immunological and biological assays, molecular probes
(DNA hybridization and PCR) as mentioned above for newborn diarrhoea.
6.2. Prevention by vaccination (using adhesins as protective antigens)

ETEC infections can, and should, be prevented by several hygienic and management
techniques which are outside the scope of this chapter. Among these, the most
important factor, in the case of newborn animals, remains the early and sufficient
colostral supply. The protective value of colostrum against diarrhoeal diseases of the
offspring caused by ETEC can be increased essentially by maternal immunization.
For that purpose several vaccines are used mainly by parenteral application (which
can be adjuvanted by oral immunization). These vaccines contain the so-called
protective antigens (virulence factors – fimbrial adhesins with or without LT entero-
toxins). Vaccinations should usually take place in late pregnancy and can be
repeated as “reminder” vaccinations before each subsequent farrowing. As a result,
colostral antibodies would block virulence factors and propagation of bacteria in the
intestine. Similar effects can be expected in the case of passive immunization, i.e.
the oral application of polyclonal or monoclonal antibodies (Sherman et al., 1983).
Immune colostrum or specific antibodies can also be applied metaphylactically,
however, with much less success. Amongst the mechanisms of action described
above, the success of colostral vaccines depends largely upon matching the right
protective antigens with the pathogens present in a given animal population. Our
knowledge about the possible existing virulence factors is, however, still limited and
further improvements in this area are to be expected.
Vaccines against enterotoxic colibacillosis of calves or small ruminants contain
both K99 and F41 (Contrepois et al., 1978; Acres et al., 1979; Nagy, 1980). In coun-
tries where F17(FY/Att25) fimbriae are prevalent, vaccines should also contain the
F17(FY/Att25) antigens (Contrepois and Girardeau, 1985; Lintermans et al., 1988).
As ETEC infections of calves and small ruminants frequently occur simultaneously
with rotavirus infection, most of the vaccines used today contain bovine rotavirus
antigens as well (Bachmann et al., 1984; Köves et al., 1987). So far, no information
is available about a possible shift in fimbrial characteristics of ETEC in herds or areas
where K99 and/or F41 containing vaccines are used. There is evidence, however,
suggesting that the strongly reduced incidence of K99 and F17 may be explained
by the use of vaccines containing these antigens (Contrepois and Guillimin, 1984).
During the past decade, no new adhesins or toxins of calf or ruminant ETEC strains
were discovered, although it seems almost impossible that the adhesin (and toxin)
spectrum in these animal species is that limited all over the world.
Vaccinations against neonatal diarrhoea of pigs caused by ETEC have been very
successful especially since the most prevalent adhesins (K88, K99, 987P) and toxin
(LT) became standard components of the vaccines (Moon and Bunn, 1993). It seems
that LT could act not only as a protective antigen, but also as an oral adjuvant (Ahren
et al., 1998). Such vaccines are almost always used to provide maternal immunity
through immune colostrum to the offspring. This requires parenteral (or oral) appli-
cation of the above antigens well before farrowing. As a result, passively acquired
antibodies through colostrum will protect piglets for about a week against most
types of ETEC under normal farming conditions, provided that the piglets ingest
immune colostrum early enough and in an adequate quantity during the first 12 h of
life (before the sharp decline of their absorptive capacity for colostral immuno-
globulins). There have been several ways to improve the efficiency of maternal
parenteral vaccines against ETEC (Morein et al., 1984; Nagy et al., 1990b).
Some companies advise the use of “in-feed” vaccines (containing killed or live
bacteria) for sows or to combine them with parenteral vaccines. The results of Moon
et al. (1988) suggest that effective presentation of the protective antigens would
require the use of live oral vaccines for such purposes. Such oral vaccines, if
licensed, could efficiently stimulate the mucosa-associated lymphoid system
(GALT) so that secretory antibodies (especially SIgA) – which are protected from
digestion – could be produced and provide the firmest protection. Strong lactogenic
immunity mediated in this way lasts for about the first 10−14 days of life. It should
be noted that first farrowing gilts are less able to produce high levels of antibodies
whatever the route of immunization. The combination of “in-feed” and parenteral
vaccines can be recommended for first and second pregnant gilts as well (Moon
et al., 1988). It should be remembered, however, that licensing of live oral bacterial
vaccines for use in veterinary medicine, especially those produced by genetic engi-
neering, is difficult in most countries. Killed oral vaccines are, however, of limited
value. Live oral vaccines still represent a more controlled and more effective way
of specific immune prevention of neonatal diarrhoea as compared to the so-called
“feed back” (feeding of diarrhoeal faecal material to pregnant sows, as practised on
some farms). The use of recombinant Salmonella-vector vaccines expressing the
necessary adhesive epitopes could also come into question (Attridge et al., 1988;
Morona et al., 1994). Finally, it is hoped that more progress in the area of geneti-
cally engineered plants (containing the required antigens produced for feeding) will
be made in the future.
Vaccinations against post-weaning diarrhoea of pigs have not shown much
progress lately, although the theoretical basis is clear and the need is unquestionable.
In-feed vaccines containing heat-treated ETEC bacteria have not been consistently
effective and most have been removed from the market. Parenteral vaccination of
piglets before weaning is advised by some companies but its efficacy against PWD
has not been convincingly demonstrated. At present the most promising experiments
are in the area of live oral vaccines applied before weaning. Bertschinger et al.
(1979) demonstrated the efficacy of such a vaccine when a low-energy diet was also
given. Further experiments of this group provided evidence about the protection of
pigs against PWD and oedema disease by a live oral vaccine containing F18 fimbria.
A combined (live oral plus killed parenteral) vaccine against PWD also seems to be
successful in preventing losses (Alexa et al., 1995).
7. CONCLUDING REMARKS
Adhesion and colonization are the first (but not the only) functional prerequisites for
a mucosal bacterium to be pathogenic. The previous sections have shown the vast
genetic and phenotypic arsenal of adhesins of E. coli bacteria for successful colo-
nization of small intestinal mucosal surfaces in calves and young pigs. These
adhesins represent surface proteins, governed by specific operons and constructed
in ways according to the particular adhesin (with fimbrial or afimbrial structures).
Beside their structure, these adhesins can also be grouped according to their recep-
tors usually present on the intestinal mucosal epithelium (but also on red blood cells
of different animal species and humans) and on the urinary epithelium. Our knowl-
edge of the genetics and function of these adhesins has helped so far to reduce losses
due to enterotoxic colibacillosis of calves and young pigs and may bring further
success in the prevention of diseases due to other pathotypes (EHEC, EPEC and
NTEC), which at present seem to be a greater threat to human health. Our tools in
combating these losses are better and more specific diagnostic reagents (including
DNA-based diagnostic tests) and vaccinations (mainly using the proteinaceous
adhesins as protective antigens). The knowledge on genetics of receptors for
adhesins of different E. coli pathotypes and subtypes has raised great hopes for
breeding genetically resistant animals – in the case of newborn piglet diarrhoea
(receptors for K88) and in the case of weaned pig diarrhoea or oedema (receptors
for F18). As the classical selection in breeding would not be practical (disadvanta-
geous linkage groups with other important genes), it seems that the utilization of
these genes will have to await further technological developments.
Because E. coli is a highly flexible organism (acquiring new virulence characters or
masking the ones that may be disadvantageous for survival) (Mainil et al., 1987),
and because there are several kinds of infections (due to viruses and protozoa as
described above) and conditions that may predispose the host to colonization by
ETEC, thereby enhancing the chances for E. coli to utilize its pathogenic potential,
the protection of pigs and calves from pathogenic E. coli is a constant challenge for
farmers and veterinarians alike. As described in the previous sections, the knowl-
edge on adhesins and receptors for colonization by different pathotypes of E. coli
has been utilized quite extensively for diagnostic purposes (antifimbrial diagnostic
sera and reagents) and for the prevention of diarrhoeal diseases (mainly in the form
of killed maternal vaccines containing fimbrial antigens). In the future, further
applications can be expected in the development of live oral vaccines (to establish
more efficient local immunity in the intestine). This would imply using non-virulent
but adherent E. coli strains with apropriate adhesins for the species and age of the
target animal population. Furthermore – in spite of the difficulties described above –
progress may also be expected in the area of application of genetic resistance against
enterotoxic colibacillosis of pigs. Apart from direct practical applications, there are
further significant scientific developments and applications expected in the area
of neonatal biology and comparative human pathobacteriology. The most likely
areas for further advancements will be (and in some cases are) the applications of
real-time PCR and DNA chip technology in studying quantitative aspects of gene
expression and functional analysis of the genes discussed above. The results of these
studies will reveal more complex interactions between the pathogenic bacteria and
the host on the gene expression level.
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9 The farm animal as potential reservoir
of antibiotic resistant bacteria in the
food chain
G. Kleina and C.M.A.P. Franzb

aInstitute
for Food Quality and Food Safety, School of Veterinary Medicine
Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany
bFederal Research Centre for Nutrition, Institute of Biotechnology and
Molecular Biology, D-76131 Karlsruhe, Germany
Food and food animals are often associated with the transfer of antibiotic resistances
to humans. The cause of this is frequently ascribed to the use of antibiotics in ani-
mal husbandry. However, not only the rearing of animals, but also the slaughtering
and processing as well as the further preparation are important factors for the spread
of resistances via microorganisms. There are three basic routes for the transfer of
antibiotic resistances via food or food animals. The first is a direct contribution via
antibiotic residues or chemotherapeutics in foods. However, based on actual data,
this route is of negligible importance. The second route is by ingestion of commen-
sal bacteria that can transfer their antibiotic resistance genes to pathogens in the
human gastrointestinal tract. One example of these is the enterococci that are con-
sidered to be part of the normal flora of a variety of foodstuffs. Glycopeptide resist-
ant enterococci can transfer this resistance and thus are potential causative agents for
complications in human infections. Enterococci in foods can be glycopeptide resist-
ant but differ from clinical isolates in phenotypic as well as genotypic properties.
There is no direct correlation with glycopeptide resistant enterococci from food and
human disease but one should keep a close eye on their ability to transfer this kind
of resistance to other bacteria. The third route is the ingestion of already resistant,
real pathogens such as Campylobacter spp. and salmonellae. For this route, fluoro-
quinolone resistance is becoming increasingly important as this antibiotic is also
used in animal husbandry at therapeutic levels and a connection between the two

192 G. Klein and C.M.A.P. Franz
is possible. Of further importance is the detection of new resistance variants and the
occurrence of new resistant clones. Molecular biological techniques are well suited
for this. Possible preventative measures include the ban of antibiotics as growth
promoters and the success of such policies are currently being evaluated. In addition,
a ban or strong reduction of therapeutic antibiotics is also being considered; however,
this raises questions of therapeutic emergencies in animal husbandry (Bogaard and
Stobberingh, 2001) and a balanced solution must be found. Some possible avenues for
reaching such a balance include the classification of therapeutic agents and a represen-
tative monitoring system to determine the status quo, as well as new resistance devel-
opments. Finally, for selection of technologically used strains (e.g., enterococci) it is
important to determine their resistance profile as well as their potential for transfer.
1. INTRODUCTION
One of the most important and, ironically, accidental discoveries in medical history
was that of penicillin by Alexander Fleming in 1928. Penicillin and subsequently
discovered naturally occurring substances that killed bacteria were termed anti-
biotics, a definition that was later broadened to include chemically derived, syn-
thetic antibacterial drugs (Walsh, 2003). The antibiotic era started in the 1940s,
when penicillin saved countless lives of soldiers in World War II. The success of
penicillin in saving human life soon led to the discovery (natural antibiotics) and
development (synthetic antibiotics) of other antibiotics (Walsh, 2003).
It has been estimated that since the late 1940s mankind has released 109 – 1010 kg
of antimicrobial agents into the environment (Davies, 1996). We know all too well
that bacteria have survived this onslaught because of their ability to mutate rapidly
and, more importantly, to inherit, express and disseminate genetic material encoding
antibiotic resistance (Davies, 1996; Khachatourians, 1998; Teuber et al., 1999;
SCOPE, 2002; Walsh, 2003). The past two decades have seen a dramatic develop-
ment in infections of bacterial aetiology, i.e., the rise of antibiotic resistant bacteria
that once were susceptible to treatment and now have developed resistance to these
medications. This has led to an alarming increase in fatalities from gonorrhoea,
pneumonia, tuberculosis, meningitis, dysentery, septicaemia, endocarditis and other
infections. The reason for this is considered to be two-fold: 1) the remarkable genetic
plasticity of bacteria to develop resistance to antibiotics, and 2) the abuse and misuse
of antibiotics.
2. DEVELOPMENT OF BACTERIAL ANTIBIOTIC RESISTANCE

Essentially, antibiotic resistance is the result of genetic change in the microorgan-
ism, either by mutation (a chromosomal change) or by genetic transfer (acquisition
of extrachromosomal genes) (Walsh, 2003). The resulting resistances are often
referred to as intrinsic or acquired, respectively. New phenotypic traits of bacteria
Antibiotic resistant bacteria in the food chain 193
result from mutations occurring in their genetic material. Mutations result from a
change in one or more nucleotide basepairs in the chromosomal DNA. In the case
of spontaneous mutations, they occur naturally at a rate of about 10−7–10−11 per
generation and result from errors in DNA replication. Although this rate appears to
be low, it actually is quite the contrary, when considering that a bacterium such as
Escherichia (E.) coli can produce 20 generations in about 7 h. Induced mutations,
on the other hand, occur at a far greater frequency than spontaneous mutations and
are caused by external factors such as chemical agents (e.g., antibiotics), heat, or
irradiation (Madigan, 1997). Although induced mutations are considered random
events, the likelihood of mutation increases when the challenging agent is of limited
strength and is applied over a prolonged period. Once developed, a mutant trait is
subsequently passed on to all succeeding progeny.
It is now well known that extrachromosomal genetic material containing genes
which encode, for example, antibiotic resistances can be exchanged by bacteria.
Such extrachromosomal genetic material exists in the form of plasmids (covalently
closed, circular DNA molecules that reside in the chromosome and replicate
independently of the chromosome) or in the form of transposons. Transposons are
also mobile genetic elements that can translocate within or between larger pieces
of DNA such as plasmids or the chromosome. Transposons themselves often carry
all possible combinations of antibiotic resistance genes (Clewell, 1990; Teuber et al.,
1999). Plasmid mediated resistance is a tremendous clinical problem because it
concerns most bacterial species and they often mediate multidrug resistance and can
have a high rate of transfer. Thus, these days it is generally accepted that there is wide-
spread transfer of genes (also antibiotic resistance genes), i.e., either vertical transfer
(to progeny) as well as horizontal transfer (to other genera, species or strains).
Antibiotic usage creates a phenomenon known as selective pressure, in which
susceptible bacteria are killed and the more resistant bacteria survive. Because of the
ability of bacteria to multiply rapidly, those that survive can give rise to millions of
progeny, all containing the genes for resistance to the antibiotic. These resistance
genes may confer cross-resistance to other antibiotics and more resistance genes
may also be acquired. Bacterial strains that are resistant to three or more antibiotic
agents with different mechanisms of inhibition are defined as multidrug resistant.
3. ANTIBIOTIC USE AND RESISTANCE IN ANIMALS

The production of meat, milk and eggs has since World War II been characterized
by greater intensity (i.e., fewer but larger farms) and high scales of production
(McEwen and Fedorka-Cray, 2002) so that in modern agriculture it has attained
industrial dimensions (Teuber, 1999, 2001). Antimicrobials, including antibiotics,
are used in food animals to prevent or treat disease or to promote growth (table 1).
Therapeutic use is when animals are diseased and antibiotics are administered to
cure the infection. In food animal production, the afflicted individual may be
Table 1. Types of antimicrobials used in animals for food production
Type of Route or vehicle of Administration to

antimicrobial use Purpose administration individuals or groups
Therapeutic Therapy Injection, feed, water Individual or group

“Metaphylactic” Disease prophylaxis, Injection, feed, water Group
therapy
Prophylactic Disease prevention Feed Group
Subtherapeutic Growth promotion Feed Group
feed efficiency,
disease prophylaxis
Adapted from McEwen and Fedorka-Cray (1999).
treated, but it is often more efficient to treat entire groups by medication of feed and
water (McEwen and Fedorka-Cray, 2002). For farming of some animals (i.e., poul-
try and fish), mass medication is the only feasible means of treatment. Thus certain
mass-medication procedures, called “metaphylaxis”, aim to treat sick animals while
medicating others in the group to prevent disease (McEwen and Fedorka-Cray,
2002) (table 1).
Antimicrobials approved for use in the United States in food animals either for
treatment of various infections or for growth promotion are shown in table 2.
Table 2. Examples of antimicrobials used in food animals in the United States
Purpose Cattle Swine Poultry Fish
Treatment of Amoxicillin Amoxicillin Erythromycin Ormetoprim

infections Cephapirin Ampicillin Fluoroquinolone Sulfonamides
Erythromycin Chlortetracycline Gentamicin Oxytetracycline
Fluoroquinolone Gentamicin Neomycin
Gentamicin Lincomycin Penicillin
Novobiocin Sulfamethazine Spectinomycin
Penicillin Tiamulin Tetracyclines
Sulfanomides Tylosin Tylosin
Tilmicosin Virginiamycin
Tylosin
Growth and Bacitracin Asanilic acid Bambermycin
feed efficiency Chlortetracycline Bacitracin Bacitracin
Lasalocid Bambermycin Chlortetracycline
Monensin Chlortetracycline Penicillin
Oxytetracycline Erythromycin Tylosin
Penicillin Virginiamycin
Tiamulin
Tylosin
Virginiamycin
Adapted from McEwen and Fedorka-Cray (1999).

Growth promoters are usually administered in relatively low concentrations, rang-

ing from 2.5 to 125 mg/kg (ppm), depending on the antibiotic used and the species
treated (Visek, 1978; Jukes, 1986; McEwen and Fedorka-Cray, 2002). High energy
feed for meat and dairy cattle, sheep and goats may be supplemented with 35–100 mg
of bacitracin, chlortetracycline or erythromycin per head per day, or 7–140 g of
tylosin or neomycin per ton of feed. For swine, 2–500 g of bacitracin, chlortetra-
cycline, erythromycin, lincomycin, neomycin, oxytetracycline, penicillin, strepto-
mycin, tylosin or virginiamycin may be added to each ton of feed; the same agents
are used for poultry, but at 1–400 g per ton of feed (Khachatourians, 1998). In
Europe (i.e., the EU) and for meat intended for import to the EU these substances
are not allowed as growth promoters. Most substances have been banned in the EU
recently, with the exception of salinomycin (swine), avilamycin (swine), flavomycin
(laying hens, turkeys, swine, calves, cattle for meat production), and monensin (cat-
tle for meat production). There are efforts to prohibit the use of these substances in
total in the EU.
The scale of agricultural use of antibiotics in the US is about 100 to 1000 times
greater than that for human use (Feinman, 1998; Khachatourians, 1998; Levy, 1998;
Witte, 1998). Overall, the annual estimates for application of antibiotics in the US
agrifood industry are over 8 million kg for animals (Khachatourians, 1998). In 1997
in the EU 5400 tons of antimicrobials were used in human medicine, while 3494 tons
and 1599 tons were used in animal medicine and as growth promoters, respectively
(Ungemach, 1999). A breakdown of antimicrobials used for animals in the EU in
1997 is shown in table 3, from which it is clear that tetracycline was the most
intensely used antibiotic for animals in the EU at 2294 tons.
As a consequence of the agricultural use of antimicrobials, antimicrobial resist-
ance is widespread in the bacteria of farm animals (Rosdahl and Pedersen, 1998).
Especially the growing animal is at risk of harbouring resistant bacteria because of
the admission of antimicrobial feed additives and the therapeutic use of antibiotics
during the breeding period (Kamphues, 1999). Antibiotics fed particularly to young
animals to promote their growth have physiological effects on the intestinal wall, pas-
sage, intestinal flora and absorption, together resulting in a better absorption of feed.
Table 3. Antimicrobials used in animals in the EU in 1997
Antimicrobial Tons
Beta-lactams 322
Tetracycline 2294
Macrolides 424
Aminoglycosides 154
Fluoroquinolones 43
Trimethoprim/sulfonamide 75
Other 182
Adapted from Ungemach (1999).

Used on a subtherapeutic scale, they also have a preventative function with respect
to infections, resulting in less disease and diarrhoea (Hoogkamp-Korstanje, 1999).
In recent years most of the antimicrobial feed additives have been banned within
the EU to reduce the percentage of resistant bacteria in farm animals (European
Commission, 1999). The effect of this ban on antibiotic resistance within farm
animals and for human medicine is still under discussion (Boerlin et al., 2001).
In part, the effect has been undermined by the increased use of therapeutic agents
(Kamphues, 1999). The amount of antibiotic resistance is dependent not only from
the application of antimicrobials, but also from the bacteria present in the farm
animal. The most important bacteria comprise Salmonella, Campylobacter, entero-
cocci, E. coli and various specific animal pathogens (including streptococci, staphylo-
cocci and Pasteurella). The distribution of these species is dependent on the animal
species and the kind of animal husbandry system (extensive, intensive, etc.). The
nature of the antibiotic resistance is highly variable, i.e. resistance can occur against
all known antimicrobial substances and groups. The most important substance
groups for human medicine, which will be mentioned in more detail, are the
fluoroquinolones, the glycopeptides, aminoglycosides, macrolides, tetracycline and
beta-lactams.
Resistance mechanisms are manifold and include destruction of the antimicro-
bial inside or outside of the cell, efflux mechanisms, target alteration and alternative
metabolic pathways. The genetic information of the resistance can be located chro-
mosomally or on extrachromosomal elements (plasmids). Aspects of bacterial and
animal species, acquisition, spread and mechanisms of resistance, as well as the
sources of resistance, will be discussed with respect to the special situation in the
growing animal.
4. RESISTANCE IN FARM ANIMALS

The growing animal is of great importance with respect to antibiotic resistance,
especially as a production animal. Most animals used for meat production will be
slaughtered within their growing phase (cattle within approximately 18 months, pigs
within 6–7 months, poultry within 30–42 days). Therefore, the focus of this chapter
is on the main production animals as mentioned before (cattle, pig, poultry).
Owing to the treatment of neonatal E. coli infections and pneumonia in calves,
antimicrobial therapy is quite common in cattle (Anonymous, 1998). Antibiotic
growth promoters were also in use until the recent ban of the antimicrobial feed addi-
tives. Especially Salmonella and E. coli are reported to be resistant in cattle, with focus
on S. Typhimurium DT 104, a serovar with multiple resistances (ampicillin, tetracy-
cline, sulfanomides, streptomycin, chloramphenicol, fluoroquinolones) (Anonymous,
1998). Major antibiotic classes which are currently allowed for therapy in cattle and
calves in Europe comprise beta-lactams including cefazolines, quinolones, tetracy-
clines, sulfonamides, aminoglycosides and macrolides (Kluge and Ungemach, 2000).
In the US, various antimicrobials are fed to cattle. Monensin and lasalocid were
commonly used for growth promotion, whereas some producers used neomycin or
virginiamycin. Chlortetracycline, chlortetracycline-sulfamethazine, oxytetracycline
and tylosin were fed on feedlots for group therapy of animals. For individual animal
therapy about 50% of feedlots used tilmicosin, florfenicol and tetracyclines, while
antibiotics used less frequently by feedlots for individual animal therapy included
cephalosporins, penicillins, macrolides and fluoroquinolones. Approximately 41% of
feedlots administered antimicrobials such as tilmicosin, florfenicol and oxytetra-
cyclines for metaphylaxis (McEwen and Fedorka-Cray, 2002). On US dairy
farms, penicillins, cephalosporins, erythromycin and oxytetracycline are mainly
administered through intramammary infusion to treat mastitis and are routinely
administered to herds to prevent mastitis during non-lactating periods (McEwen and
Fedorka-Cray, 2002).
Campylobacter coli, C. jejuni, E. coli and Salmonella are the main targets for
antibiotic use in pigs. Substance classes in use for antibiotic therapy in Europe for
weaning pigs are the same as summarized for cattle. In the US, several antibiotics
(table 2) are used for growth promotion or disease prophylaxis in swine.
Antimicrobials such as ceftiofur, sulfonamides, tetracyclines and tiamulin are given
to treat and prevent pneumonia, an important problem among swine. Gentamicin,
apramicin, and neomycin are used to treat bacterial diarrhoea, caused by pathogens
such as E. coli and C. perfringens. Swine dysentery (Serpulina hyodysenteriae) and
ileitis (Lawsonia intracellularis) are other important diseases that may be treated
with antimicrobials such as lincomycin, tiamulin and macrolides (McEwen and
Fedorka-Cray, 2002).
The main concerns for bacterial infections in poultry production are C. jejuni
and Salmonella. In Europe, a variety of fluoroquinolones are currently in use for
antimicrobial therapy (e.g., enrofloxacin, difloxacin). Consequently, the increase
of fluoroquinolone resistant bacteria in poultry is an emerging problem. In the US,
broiler feed usually contains coccidiostats (e.g., ionophores, sulfonamides) while
other antimicrobials (e.g., bacitracin, bambermycin, chlortetracycline, penicillin,
virginiamycin and arsenic compounds) are approved for growth promotion and feed
efficiency (McEwen and Fedorka-Cray, 2002).
5. BACTERIAL SPECIES AND RESISTANCE

Monitoring systems for antibacterial resistance often focus on zoonotic bacteria,
commensals and animal pathogens. Within the European Union no system exists for
EU-wide monitoring (OIE, 2001); however, effort exists to establish such a system
for the main zoonotic pathogens (Salmonella, Campylobacter), commensals (ente-
rococci, E. coli) and animal pathogens (e.g., enteropathogenic E. coli, streptococci
etc.) (Caprioli et al., 2000). In the following discussion on the antibiotic resistance
of zoonotic pathogens and commensals special focus is on the situation in Germany.
However, the regional resistance rates can be used as an example for the main trends
in Europe as well as in industrialized countries.
5.1. Salmonella
Numerous research studies report on the antibiotic resistance of salmonellae.
However, coordinated monitoring programmes for the EU have not been established
so far. In Germany, a total of 65.9% of isolates from animals and food were resist-
ant or multiple-resistant against antibiotics (BfR, 2003). Especially, pigs and calves
are responsible for the overall high resistance rates. Up to 40% of the isolates
are multiply resistant, often with resistances against five antibiotics (ampicillin,
chloramphenicol, streptomycin-spectinomycin, sulfanomides, tetracycline) (BfR,
2003). Salmonella Typhimurium definite type DT 104 is the most often found type
among resistant isolates. Especially, the multiple-resistant strains (96%) possess
integrons and can thus be able to transfer resistance (BfR, 2003). S. Typhimurium
DT 104 initially emerged in cattle in 1988 in England and Wales and was subse-
quently found in meat and meat products. Human illness occurred as a result of
contact of humans with farm animals, or from consumption of meat (Khachatourians,
1998). The number of DT 104 isolates from humans in Britain increased from 259 to
3837 between 1990 and 1995 (Lee et al., 1994). The proportion of antibiotic resistant
Salmonella associated with human infections rose from 17 to 31% of isolates between
1979/80 and 1989/90 in the US, and the proportion of Salmonella isolates exhibiting
antibiotic and multidrug resistance to ampicillin, chloramphenicol, streptomycin,
sulfonamides and tetracyclines increased from 39 to 97% in the same period (Lee
et al., 1994). In 1990, 90% of all DT 104 isolates obtained from humans were multi-
drug resistant, including resistance to fluoroquinolones (Khachatourians, 1998). In
1997, an interagency workshop with representatives from Canada, the US, the UK and
the Netherlands reported a rise in the number of multidrug-resistant DT 104 isolates
and resistance to trimethoprim and fluoroquinolone was also reported (Angulo, 1997).
However, some countries, e.g. Sweden, show very low prevalence of resistant
Salmonella isolates (SVARM, 2000). These countries, especially the Scandinavian
countries, have traditionally low rates of Salmonella contamination in animal
husbandry and apply a strict regime to establish Salmonella free livestock.
5.2. Campylobacter
In human medicine, the antimicrobials used for treatment of severe Campylobacter
infections are fluoroquinolones and macrolides (Skirrow and Blaser, 2000).
However, the use of fluoroquinolone antibiotics in veterinary medicine has led to
the emergence of antibiotic resistant C. jejuni in human and chicken populations.
The prevalence for the instances of enrofloxacin resistant strains of Campylobacter
in poultry and in humans increased from 0 to 14% and from 0 to 11%, respectively
(Khachatourians, 1998; Endtz et al., 1991). Erythromycin resistant campylobacters

have been reported, but erythromycin resistance is found in E. coli isolates rather
than in C. jejuni isolates (Smith et al., 2000). Resistance to fluoroquinolones in
isolates from human clinical specimens has been reported to occur worldwide
(Talsma et al., 1999). In some regions, the increase in antimicrobial resistance has
been very rapid, e.g. in Spain the development of resistance in the past decade was
remarkable (Sáenz et al., 2000). In coincidence with the increasing occurrence of
resistance to fluoroquinolones in human clinical isolates, isolates from food animals
also show increasing resistance rates (Sáenz et al., 2000).
In Germany origin-specific resistance rates for Campylobacter spp. from pigs,
broilers and cattle could be demonstrated (Luber et al., 2003). Whereas isolates from
pigs were significantly more often resistant to erythromycin (37.9%) and tetra-
cycline (60.8%) than those from cattle or broilers, the latter were significantly more
often resistant to ampicillin (37.9%), nalidixic acid and ciprofloxacin (each 55.2%)
(Luber et al., 2003). Multiresistant strains occurred also among poultry isolates.
These high resistance rates indicate the importance of food animals as potential
sources for resistant strains. The origin-specific resistance rates shown above reflect
the differences in the use of antibiotics in different animal species.
5.3. Enterococci
The focus of research concerning antibiotic resistant enterococci in animals and
food is on glycopeptides, especially vancomycin and teicoplanin. These substances
are reserve antibiotics in human medicine for severe nosocomial infections with
enterococci (E. faecium and more often E. faecalis) and are therefore of primary
importance. Vancomycin has been used since the 1950s in human medicine and the
structurally similar glycopeptide antibiotic avoparcin has been used as a growth
promoter in farm animals in Europe since the 1980s. Depending on the livestock,
4–50 mg/kg of avoparcin was added to animal feed (Feed Additive Directive 70/524
of the EC) (Witte, 1997). In Europe, ergotropic (growth stimulatory) use of
avoparcin has been suspected to contribute to the rise of vancomycin resistant ente-
rococci (VRE) in hospitals, as VRE isolates can be transmitted to humans via the
food chain (Bates et al., 1994; Klare et al., 1995a,b; McDonald et al., 1997; Witte
1997). The problem of VRE in European hospitals and the supposed food transmis-
sion route for infection led to a ban of avoparcin as a growth promoter in the EU in
1997. The prevalence of VRE was reported in recent years to be up to 17% (Peters,
2003). After the ban of avoparcin, studies from Germany indicated that VRE could
not be isolated from cattle and food from cattle or pig meat (Peters, 2003; Peters
et al., 2003). Klare et al. (1999) showed that the incidence of VRE from frozen and
fresh poultry meats decreased two years after the avoparcin ban. Also in other coun-
tries, a decrease of resistance rates could be demonstrated. However, the effect of the
ban of the growth promoter avoparcin (a glycopeptide) is still under discussion
(Boerlin et al., 2001). In the US, the situation regarding development of VRE differs
from Europe, in that ergotropic use of avoparcin did not occur as this antibiotic was
not licensed for use in animal husbandry. The development of VRE in the US may
have occurred as a result of intensive use of vancomycin to treat hospital-associated
infections.
Enterococci are known to be intrinsically resistant to a number of antibiotics,
including cephalosporins, β-lactams, sulfonamides, and low levels of clindamycin
and aminoglycosides (Murray, 1990; Leclercq, 1997; Morrison et al., 1997).
Acquired resistance, based on acquisition of plasmids and transposons, has been
observed for chloramphenicol, erythromycin, high levels of clindamycin and amino-
glycosides, tetracycline, β-lactams (by β-lactamase or penicillinase), fluoro-
quinolones and glycopeptides (Murray, 1990; Moellering, 1991; Landman and
Quale, 1997; Leclercq, 1997; Morrison et al., 1997). Antibiotic resistant enterococci
are well known to occur in various beef, poultry or pork products and such strains
may be resistant to chloramphenicol, tetracycline, erythromycin or high levels of
aminoglycoside (Klein et al., 1998; Quednau et al., 1998; Teuber et al., 1999; Pavia
et al., 2000; Baumgartner et al., 2001). Enterococci with multiple resistances includ-
ing resistance to cephalosporins, macrolide and tetracycline classes of antimicro-
bials, as well as resistance to streptogramin quinopristin-dalfopristin were shown
to be associated with the poultry environment (Joseph et al., 2001). Enterococci
isolated from pig gastrointestinal sources in Spain, Denmark and Sweden also
showed resistances to erythromycin, chloramphenicol, tetracycline and amino-
glycosides, with higher levels of resistance observed for isolates stemming from
Spain and Denmark when compared to those from Sweden (Aarestrup et al., 2002).
Aarestrup et al. (2002) suggested that this effect was a reflection of the fact that
higher amounts of antibiotics were fed as growth promoters in Spain and Denmark,
when compared to Sweden.
5.4. E. coli
In Germany, 42% of investigated E. coli strains were resistant and 36% were multi-
ple resistant in isolations from cattle, pigs and poultry (BfR, 2003). Especially iso-
lates from poultry and pigs showed a high prevalence of resistant E. coli (61 and
59%, respectively). Of the poultry isolates, 33% were quinolone resistant with
13.5% being fluoroquinolone resistant (BfR, 2003). In total over 300 isolates have
been tested. However, representative studies sensu stricto have to include more
isolates from different regions in Germany. E. coli has been tested very intensely in
different countries and always isolates from animals as well from food were found
to be resistant to a variety of antibiotics in significant percentages (e.g., Lehn et al.,
1996; Trolldenier, 1996; Altieri and Massa, 1999; Mathew et al., 1999). These
resistances comprise tetracycline (up to 83%, Bensink et al., 1981), ciprofloxacin
(0–13%, Sáenz et al., 2001) or ampicillin (0–47%, Sáenz et al., 2001).
A specific subgroup of the E. coli population, the potential enterohaemorrhagic

E. coli (EHEC) bacteria with verocytotoxic E. coli-associated virulence factors
(VTEC-AFV), has also been tested. In agreement with data from Germany for cat-
tle, lamb and sheep as well as food (Klein and Bülte, 2003), EHEC and VTEC-AFV
seem to be less resistant to antimicrobials and they have a high percentage of
susceptible isolates compared to E. coli isolates in general (CDC, 2000). Data
concerning this subpopulation are very rare and studies are needed to elucidate the
possible development of antibiotic resistance in these potentially pathogenic agents
entering the food chain. This is not necessary for effective antibiotic therapy in
human medicine, because infections with EHEC bacteria are normally treated with-
out antibiotics. However, the existence of such resistances may be an indicator and
can give an overview on the distribution of resistances in food of animal origin.
6. RESISTANCE TRANSFER IN THE GROWING ANIMAL

In principle the resistance transfer and the acquisition of antibiotic resistance is the
result of one of the following steps (Berger-Bächi, 2001):
acquisition of resistant non-pathogenic bacteria and subsequent transfer of the
resistance to the autochthonous gut flora and/or to gut-associated pathogenic
bacteria (fig. 1A),
Fig. 1. Transfer mechanisms and routes of antibiotic resistances from the farm animal via food to the human
gastrointestinal tract. (A) Antibiotic resistance gene transfer from the non-pathogenic, physiological animal
and/or food microflora to pathogenic microorganisms of the human gastrointestinal tract (example:
vancomycin resistant enterococci (non-pathogenic); transfer mechanism: conjugation). *VRE: vancomycin
resistant enterococci. (B) Transfer of antibiotic-resistant pathogenic microorganisms from animal and/or food
to the human gastrointestinal tract (example: fluoroquinolone resistant Campylobacter spp.; no transfer of
resistance possible, but dissemination of resistant strains/clones).
Fig. 2. Transfer of antibiotic resistances. Elements and transfer routes influencing the amount of antibiotic
resistance in farm animals, food and human medicine.
acquisition of resistant pathogenic bacteria from the environment or by direct

contact (fig. 1B), or
spontaneous mutation under selective pressure or without selective pressure.
The transfer of the resistance is dependent on the nature of the resistance
mechanism. Resistance resulting from mutation events will be transferred by the
dissemination of the resistant strain itself, whereas transfer by conjugation results in
dissemination of the relevant resistance gene in different strains or even bacterial
species. Therefore the acquisition of resistant non-pathogenic bacteria leads only
to resistant pathogens if a transfer mechanism is available. An example for a non-
transferable resistance caused by a single or double mutation step is the quinolone
resistance in Campylobacter or Salmonella (Bachoual et al., 2001). The induction
of the resistance by quinolone treatment is described for Campylobacter in broiler
production (Jacobs-Reitsma et al., 1994). An example for a conjugational transfer of
resistance is the transfer of the vanA-related glycopeptide resistance in enterococci.
This transfer mechanism, especially for the conjugative transfer of plasmids, is wide-
spread amongst Gram-positive bacteria (Grohmann et al., 2003).
A simplified scheme of transfer routes and possible influence factors for the
transfer of antibiotic resistances from the farm animal to humans is given in fig. 2.
7. EFFECT OF ANTIMICROBIAL GROWTH PROMOTERS ON

ANTIBIOTIC RESISTANCE
Antimicrobial growth promoters (AGPs) are used to enhance the growth of young
animals in order to gain the slaughter weight at an early stage. The working princi-
ple of AGPs is not fully understood. Some effects may be caused by the prevention
of simple enteric infections and the reduction of the microbial population in the gut
in general (Kamphues, 1999). Furthermore, such effects may include metabolic
effects, improvement of digestion or absorption of certain nutrients, nutrient sparing
effects in which antibiotics may reduce the animal’s dietary requirements and
increased feed and/or water intake (Gersema and Helling, 1986). Unwanted side-
effects are the possible development of antibiotic resistances in enteric bacteria,
because some substances used as AGPs can induce cross-resistance to substances

also used in human medicine. A well documented case is the application of AGPs in
poultry and pigs and the effects on the glycopeptide resistance of enterococci.
However, not only the much quoted VRE example shows that feeding of antibiotics
at sub-inhibitory concentrations can cause problems for humans. For example, the use
of the streptothricin antibiotic nuorseothricin as a porcine growth promoter in the
former East Germany between 1983 and 1990 resulted in the development of an
antibiotic resistance transposon (Khachatourians, 1998; Witte, 1998; Aarestrup, 1999).
Two years after its inroduction, resistant E. coli isolates were found in pig guts, meat
products, as well as the intestines of pig farmers and their families, patients with
urinary tract infections and the general public (Witte, 1997; Khachatourians, 1998). As
this antibiotic has not been applied in human medicine, these observations showed
that growth promoting antibiotics do induce resistances in the animal population
which later disseminate via resistant enterobacteria into the human population
(Teuber, 1999).
In the Netherlands, water medication with the fluoroquinolone antibiotic
enrofloxacin in poultry production was followed by the emergence of fluoro-
quinolone resistant Campylobacter species among poultry and humans (Endtz et al.,
1991; Aarestrup, 1999).
For the safety of food of animal origin and for the prevention of severe infections
without antibiotic treatment options, the reduction of antibiotic resistance in animal
husbandry to a minimum is essential. On the other hand, antibiotic therapy in vet-
erinary medicine is indispensable for reasons of animal welfare, and the necessary
treatment of infections with zoonotic agents. A balance between both objectives
must be established. Therefore, in the future, representative monitoring systems for
the evaluation of the prevalence of antibiotic resistance in farm animals as well as
in food of animal origin are required. Target organisms should be representatives of
zoonotic bacteria, commensal bacteria and animal pathogens. These systems must
cover nationwide development and should be evaluated European-wide or on an
international level. Representative samples are crucial for the value of these exami-
nations. Another important point is that the methods for the determination of antibi-
otic resistance (MIC values are recommended worldwide) and also the breakpoints
should be standardized to enable a comparison of the results. Also important for
future investigation should be the molecular characterization of antibiotic resistance,
so as to detect new variants of resistance mechanisms or emerging resistance pat-
terns. The application of the DNA microarray technique may in future facilitate such
molecular characterization both quickly and accurately. With the information
obtained from monitoring programmes the prudent use of veterinary therapeutics is
possible and the need for restrictions or the substitution of therapy schemes are more
easily recognized and performed. However, antibiotic usage in animal husbandry is

not the only source for resistant strains in human medicine and even in many cases
is only a minor contribution. Therefore, this should not be the only step, but also
clinical therapy in humans should be critically evaluated. In any case, a reduction of the
amount of antibiotics used, whether in human or veterinary medicine, is a step forward
to reducing the level of antibiotic resistance and thus to a more reliable therapy.
Finally, it should be mentioned that reduction in antibiotic uses at subtherapeu-
tic levels will come with a price. As an example, increased production cost as a
result of abstaining from the use of antibiotics as growth promoters was reported for
pork production in Denmark. Apparently, 25% of the piglets suffered from diar-
rhoea. Piglets also grew slower, so that in the worst case it took 20 days longer for
a piglet to reach a weight of 30 kg. In 2000 it was calculated that abstaining from
subtherapeutic use of antibiotics in a production unit with 200 sows and a yearly
production of 5000 piglets would result in a cost increase of 5700 to 6600 DM
(about E 3000) (Verseput, 2000). Clearly, increases in production costs will increase
product price and consumers have to expect price increases for meat products as a
result of the discontinuation of the use of antibiotic growth promoters.
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10 Pathogenesis and the gastrointestinal tract
of growing fish
T.H. Birkbecka and E. Ringøb

aDivision of Infection and Immunity, Institute of Biomedical and
Life Sciences, University of Glasgow, Joseph Black Building,
Glasgow G12 8QQ, UK
bSection of Arctic Veterinary Medicine, Department of Food Safety and
Infection Biology, The Norwegian School of Veterinary Science,

NO-9292 Tromsø, Norway
A diverse range of bacteria and viruses is associated with diseases of fish. The skin,
lateral line, gills and gastrointestinal tract or a combination of these organs are sug-
gested to be infection routes. The purpose of this review is to present current knowl-
edge on adhesion, colonization and translocation of pathogenic agents in the
gastrointestinal tract of growing fish.
1. INTRODUCTION
As fish live in an aqueous environment, their external surfaces will be regularly
exposed to potential pathogens, and water taken into the gastrointestinal (GI) tract
during feeding can deliver them to the mucosal surfaces of this tract. Even in the
non-feeding early stages of development of marine fish larvae, drinking of water is
required for osmotic regulation (Tytler and Blaxter, 1988) and this provides an early
entry into the GI tract for bacteria. As in all animals, the GI tract is the route of
nutrient uptake and any perturbation by microbial action can be harmful. This is
particularly so in the early stages of fish larval development. In contrast to mam-
mals, where numerous bacterial and viral pathogens produce severe diarrhoeal
disease there are no directly equivalent pathogens known for fish. However, a
number of bacteria cause pathology in the gut of fish and this can be a route of sys-
temic infection in many instances, comparable to that of invasive enteropathogens
of mammals.

Pathogenesis and GIT of growing fish 209
The bacterial pathogens of major importance in aquaculture are, with few excep-
tions, Gram-negative microorganisms. Aeromonas salmonicida, A. hydrophila,
Vibrio anguillarum, V. salmonicida, V. viscosus (Moritella viscosa or M. marina)
and V. ordalii belong to the Vibrionaceae; Yersinia ruckeri, Edwardsiella ictaluri
and E. tarda are members of the Enterobacteriaceae, and Piscirickettsia salmonis is
a member of Pisciriickettsiaceae. Of the Gram-positive bacterial pathogens,
Renibacterium salmoninarum belongs to the Corynebacteriaceae, Carnobacterium
piscicola to the lactic acid bacteria, and others, e.g. Streptococcus iniae, S. difficile
and Lactococcus garviae, are Gram-positive cocci. Microbial pathogenicity has
been defined as the biochemical mechanisms whereby microorganisms cause
disease (Smith, 1990). Not all pathogens have an equal probability of causing infec-
tion and disease. In this review, the term infection will be used to describe success-
ful persistence or multiplication of a pathogen on or within the host, while disease
will be described as an infection which causes significant overt damage to the host.
Intensive fish production has increased the risk of infectious diseases all over the
world (Press and Lillehaug, 1995; Karunasagar and Karunasagar, 1999), but to pre-
vent microbial entry fish have various protective mechanisms, such as production of
mucus by goblet cells, the apical acidic microenvironment of the intestinal epithe-
lium, cell turnover, peristalsis, gastric acidity, lysozyme and antibacterial activity of
epidermal mucus. At the same time, pathogenic microorganisms have evolved
mechanisms to target the skin, gills or GI tract as points of entry. The three major
routes of infection are through: a) skin (Kawai et al., 1981; Muroga and De La Cruz,
1987; Kanno et al., 1990; Magarinos et al., 1995; Svendsen and Bøgvald, 1997;
Spanggard et al., 2001), b) gills (Baudin Laurencin and Germon, 1987; Hjeltnes
et al., 1987; Svendsen et al., 1999), and c) the GI tract (Sakai, 1979; Rose et al., 1989;
Chair et al., 1994; Olsson, 1995; Grisez et al., 1996; Olsson et al., 1996; Romalde
et al., 1996; Jöborn et al., 1997; Robertson et al., 2000; Lødemel et al., 2001).
Pathogenicity can be divided into four different phases: 1) the initial phase where
the pathogen enters the host’s environment, including the GI tract, 2) the exponential
phase where the pathogen adheres to and colonizes mucosal surfaces, replicates to suf-
ficient numbers and/or translocates into host enterocytes, 3) the stationary phase where
the pathogen replicates within the host and circumvents the host defence system; in this
phase the host is moribund and this can quickly be followed by 4) the death phase.
In order to adhere successfully, colonize and produce disease, the pathogen must
overcome the host defence system. It is well known that stress from environmental
factors, such as oxygen tension, water temperature and water salinity, are important
in increasing the susceptibility of fish to microbial pathogens. The water milieu can
also facilitate transmission of these pathogens.
The purpose of this review is to present information on 1) adhesion of bacteria to
mucosal surfaces, 2) protection against bacterial adhesion, 3) bacterial translocation,
4) invasion of host cells, 5) effect of diet in disease resistance and 6) data obtained
from endothermic animals which may have relevance to pathogenesis of fish.
210 T.H. Birkbeck and E. Ringø
2. ADHESION OF BACTERIA TO MUCOSAL SURFACES

2.1. General factors
A number of environmental factors determine whether bacteria can adhere to and colo-
nize the digestive tract of endothermic animals and these have been extensively reviewed
by Savage (1983). Among these are: 1) gastric acidity (Gilliland, 1979); 2) bile salts
(Floch et al., 1972); 3) peristalsis; 4) digestive enzymes (Marmur, 1961); 5) immune
response; and 6) indigenous microorganisms and the antibacterial compounds which
they produce. In order to replicate to a sufficient number to allow transmission to a new
susceptible host, a microbial pathogen must enter a host, find a unique niche, circum-
vent competing microbes and host defence barriers, and obtain nutrients from the host.
Adhesion of bacteria to surfaces such as epithelial cells involves four different
types of interaction, depending on the distance separating the bacteria from the
surface. Attraction is initially by van der Waal’s forces operating at distances greater
than 50 nm, but at closer distances electrostatic interactions become more signifi-
cant. As epithelial and bacterial cells are usually negatively charged, electrostatic
repulsion normally prevents closer association. In regions of lower ionic strength
closer interaction may occur allowing hydrophobic interaction and specific receptor–
ligand binding within circa 1 μm separation. This leads to strong binding between
bacteria and host cell surfaces (Fletcher, 1996).
2.2. Adhesins
Several bacterial surface components can be involved in specific binding to epithe-
lial cell ligands. The best characterized bacterial adhesins are the fimbriae (or pili)
which are widely distributed on Gram-negative bacteria (Smyth et al., 1996), but are
also found on some Gram-positive bacteria (Klemm et al., 1998). Although fimbriae
are the most widely used adhesins in Gram-negative bacteria, flagella, capsules, pro-
tein fibrils, outer membrane proteins (Gram-negative bacteria), surface proteins
(Gram-positive bacteria) and crystalline protein surface arrays can all be used as
adhesins (Henderson et al., 1999).
A range of fimbriae can be expressed by any one bacterial species; for example,
14 different types of fimbriae are known in Escherichia coli, more than one of which
can be expressed at the same time (Hacker, 1992; Klemm et al., 1998; Nataro and
Kaper, 1998). Type 1 fimbriae of E. coli are perhaps the best studied example and a
single cell may express over 500 fimbriae. Of approximately 7 nm in diameter and
1 μm in length, type 1 fimbriae are composed of about 1000 copies of the major
structural protein FimA, in a helical cylinder (Brinton, 1965) capped by the FimH
protein which recognizes mannose-containing receptors on the target eukaryotic
cell. Other minor proteins, FimF and FimG are involved in binding FimH to the
FimA helix and other genes in the Fim complex are required for assembly of the
fimbriae and translocation through the bacterial membranes (Krogfeld et al., 1990;
Klemm et al., 1998).
Despite their widespread occurrence in Gram-negative bacteria and their impor-

tance in the pathogenesis of numerous infections, fimbriae have not yet been proved
to be important in bacterial infections of fish. Saeed (1983) observed that E. ictaluri
was heavily piliated and suggested that this could be important in infection,
although this awaits further examination.
The crystalline protein array (S layer or A layer) of A. salmonicida renders the
surface of this bacterium extremely hydrophobic to the extent that bacteria in broth
cultures autoagglutinate and sediment rapidly when allowed to stand unshaken.
Loss of the S layer can occur spontaneously, or can be induced by culture at elevated
temperature or by Tn5 mutagenesis; in all cases the loss of the S layer is accompa-
nied by loss of virulence (Ishiguro et al., 1981; Belland and Trust, 1985; Trust, 1986)
and loss of adherence to macrophages (Trust et al., 1983).
Flagella are important adhesins for bacteria such as V. cholerae (Guentzel and
Berry, 1975; Richardson, 1991) and Campylobacter jejuni (Wassenaar et al., 1991;
Nachamkin et al., 1993). Although flagella have been shown to be important for the
virulence of V. anguillarum, this was not at the level of adhesion, as motility-
deficient mutants which had much reduced virulence had similar adhesion levels to
chinook salmon embryo (CHSE) cells as the wild-type organism (Ormonde et al.,
2000). However, chemotactic motility and active motility are important for virulence
in waterborne infections of fish (O’Toole et al., 1996, 1999; Ormonde et al., 2000).
Infectivity studies revealed that disruption of the flagellum and subsequent loss
of motility correlated with an approximate 500-fold decrease in virulence when fish
were inoculated by immersion in bacteria-containing water. Once the pathogens
have reached the mucosal surface, several options exist: depending on their intrin-
sic colonizing or invasive capacities, the nature of the toxin(s) they produce and their
ability to resist host defences.
2.3. Electron microscopy studies of adhesion of fish-pathogenic bacteria to

tissues of the GI tract
In a recent study, Knudsen et al. (1999) tested pathogenic and non-pathogenic bacte-
ria isolated from fish for their adhesion to cryosections from different mucosal sur-
faces of Atlantic salmon by immunohistochemistry. The majority of the bacteria
tested – V. anguillarum serotype O1, V. salmonicida, V. viscosus, Flexibacter maritimus,
“gut vibrios” and intestinal isolates of V. salmonicida – all adhered to mucus from the
pyloric caeca, foregut and hindgut. In contrast to these results, V. anguillarum
serotype O2 (O2a and O2b), did not adhere to mucus.
The past decade has seen an explosion of information on our understanding of
bacterial adhesion at both the molecular and genetic level of endothermic animals,
and electron microscopy has contributed significantly to this knowledge (Knutton,
1995). Although several papers have described pathogenesis in fish, few investiga-
tions have used transmission electron microscopy (TEM) and/or scanning electron
microscopy (SEM) to evaluate the effect of bacterial infection on morphology in the
GI tract of fish. The advantage of using SEM is that large areas of the mucosal and
cell surfaces can be examined rapidly for adherent bacteria. Adhesion can then be
assessed qualitatively or quantitatively. For quantitative analysis a defined number
of fields is selected at random, photographed, and bacterial adherence assessed to
give an adhesion index consisting of numbers of bacteria per unit area (Yamamoto
et al., 1991) or percentage of area colonized by bacteria (Knutton et al., 1991). The
resolution of SEM is rarely sufficient to obtain detailed information about the
mechanisms of adhesion, although it has proved useful to determine bacterial
adhesion/colonization of gut enterocytes of fish (Magarinos et al., 1996; Ringø
et al., 2001, 2002). Magarinos et al. (1996) demonstrated that Photobacterium
damselae (Pasteurella piscicida) strains adhered strongly to the intestines from sea
bream, sea bass and turbot in numbers ranging from 10 4 to 10 5 bacteria per gram of
intestine depending on the bacterial isolate and the fish species employed. These
results are clearly supported by scanning electron microscopy studies. Sometimes,
bacteria colonizing the GI tract had their luminal ends protruding above the levels
of the microvilli (figs. 1 and 2). Micrographs displayed clear differences in levels of
bacterial association over a small area, as some enterocytes were heavily colonized
while others had no associated bacteria. Ringø et al. (2001) showed that some ente-
rocytes were heavily colonized by bacteria when charr were fed dietary soybean oil,
whereas a different situation was observed when fish were fed dietary linseed oil
(Ringø et al., 2002). In the latter situation, most bacteria associated with enterocytes
were located at the apical brush border (fig. 3).
Fig. 1. Scanning electron micrograph of the

apical aspects of enterocytes in the midgut of
Arctic charr (Salvelinus alpinus L.) fed dietary
soybean oil. The borders between adjacent cells
are clearly visible, as microvilli which cover
the cell apex. The luminal ends of bacteria
located in the intestines between microvilli are
also visible (arrows). × 7500. After Ringø et al.
(2001).
Fig. 2. Scanning electron micrograph show-

ing cell apices in the hindgut of Arctic charr
(Salvelinus alpinus L.) fed dietary soybean oil.
Cell borders can be seen and all cells have
associated bacteria (arrows), although numbers
vary from cell to cell. Note the small spaces
(arrowheads) between microvilli. These may
represent the transit paths of more deeply
embedded bacteria, or they may be created
by bacterial loss. The latter may be an artefact
of tissue preparation or a consequence of
local bacterial cell division. × 5000. After
Ringø et al. (2001).
Fig. 3. Scanning electron micrograph show-

ing bacteria associated with enterocytes in the
hindgut of Arctic charr (Salvelinus alpinus L.)
fed dietary linseed oil. Associated bacteria
(arrows) are located at the apical brush border.
× 7500. After Ringø et al. (2002).
Fish pathogenic bacteria, such as V. salmonicida and V. anguillarum, have been

shown in vivo to adhere to the intestinal epithelium of fish larvae and promote severe
destruction of microvilli (Olafsen and Hansen, unpublished data). In contrast,
Lødemel et al. (2001) did not show any destruction of microvilli in the pyloric
caeca, midgut or hindgut regions of adult Arctic charr (Salvelinus alpinus L.)
infected by A. salmonicida subsp. salmonicida. SEM investigations of human intes-
tinal mucosa infected with enteropathogenic E. coli (EPEC) showed that EPEC
adhere intimately in microcolonies and cause gross alterations of the brush border
surface of infected enterocytes (Knutton et al., 1987, 1989; Knutton, 1995). These
characteristic “attaching-and-effacing” lesions are formed on epithelial cells in a
three-stage process. After initial adhesion, mediated by bundle-forming (type IV)
fimbriae, a type III secretory system is activated in E. coli allowing secretion of a
receptor (translocated intimin receptor) into the epithelial cell membrane which acts
as a receptor for the E. coli outer membrane protein intimin. This leads to reorgan-
ization of the cellular actin cytoskeleton and formation of the characteristic elevated
pedestal to which E. coli is bound.
2.4. Host cell ligands

A wide range of potential receptors is present on the eukaryotic cell membrane
involved in the normal cellular functions of transport, signal transduction and
cell–cell communication, and bacteria can bind to many of these molecules. In addi-
tion, proteins of the extracellular matrix, such as fibronectin, fibrinogen and colla-
gen, are receptor molecules for which specific adhesins have been characterized in
bacteria such as Staphylococcus aureus (Smeltzer et al., 1997). In glycoproteins, the
sugar residues commonly act as receptor ligands for fimbriae; binding of E. coli to
eukaryotic cells via type 1 or type 5 fimbriae is inhibited by mannose leading to the
conclusion that mannose-containing glycoproteins are cellular targets for binding by
this organism (Krogfeld et al., 1990). Other sugars, e.g. fucose and galactose have
been similarly identified as receptor targets for other types of fimbriae (Ofek and
Doyle, 1994). Similar work by Wang and Leung (2000) has shown that strains of
Vibrio anguillarum differ in the types of receptors used. Two invasive strains of the
organism, G/Virus/5(3) and 811218-5W adhered strongly to three different fish
tissue culture cell lines. Adherence of strain G/Virus/5(3), and of nine other vibrios,
was inhibited by galactose-containing sugars, but adherence by strain 811218-5W
was not affected by a range of sugars tested. As no fimbriae could be detected in
either strain it was concluded that non-fimbrial adhesins were involved in both cases
(Wang and Leung, 2000).
The ability of Photo. damselae subsp. piscicida to adhere to fish tissue culture
cell lines was inhibited by galactose and mannose but not fucose, indicating a pos-
sible glycoprotein target for adhesins of this organism (Magarinos et al., 1996).
However, prior treatment of bacteria with proteinase K did not affect their capacity
to bind to tissue culture cells and the Photo. damselae subsp. piscicida adhesin
remains unidentified.
2.5. Consequences of bacteria/ligand interactions

As noted above, many cell surface molecules are receptors involved in transmem-
brane signalling. For bacteria, interaction with eukaryotic cells can lead to altered
cell growth patterns, induction of adhesins, e.g. for enteropathogenic E. coli
(Donnenberg and Kaper, 1992), or secretion of proteins required for invasion,
e.g. for Yersinia (Cornelis and Wolf-Watz, 1997). For the eukaryotic cell, uptake of
bacteria may result in cytokine release (Wilson et al., 1998), morphology alteration
(enteropathogenic E. coli) (Nataro and Kaper, 1998) or intercellular adhesion
molecule synthesis may be stimulated (enteroinvasive E. coli).
3. PROTECTION AGAINST BACTERIAL ADHESION

3.1. Mucus
The internal surface of the host is the first defence barrier to infection. Intestinal
mucins secreted by specialized epithelial goblet cells located in the intestinal entero-
cytes form a viscous, hydrated blanket on the surface of the intestinal mucosa that
protects the delicate columnar epithelium. This is thought to be a vital component
of the intestinal mucosal barrier in prevention of colonization by pathogens in both
fish and endothermic animals (Florey, 1962; Forstner, 1978; Westerdahl et al., 1991;
Maxson et al., 1994; Henderson et al., 1999; Mims et al., 2000). Gastrointestinal
mucus is thought to have three major functions: 1) protection of the underlying
mucosa from chemical and physical damage, 2) lubrication of the mucosal surface,
and 3) to provide a barrier against entero-adherence of pathogenic organisms to the
underlying mucosal epithelium. Intestinal mucus is composed almost entirely of
water (90–95%) and the electrolyte composition is similar to plasma, accounting for
about 1% of the mucus weight. The remaining 4−10% is composed of high molec-
ular weight glycoproteins (mucins), consisting of a protein core with numerous
carbohydrate (fucose and galactose) side chains. Hydrolysis of intestinal mucus
material of rainbow trout liberated increased amounts of N-acetylgalactosamine and
N-acetylglucosamine (O’Toole et al., 1999), indicating that these carbohydrates
may be present as mucin-bound moieties in fish intestinal mucus as is the case for
mucus from other animal species (Roussel et al., 1988). The majority of intestinal
mucus-associated lipids in rainbow trout partitioned to the organic phase during
extraction with chloroform/methanol and this contained saturated and unsaturated
free fatty acids, phospholipids, bile acid, cholesterol, and monoglycerides and
diglycerides (O’Toole et al., 1999).
The mucous blanket is constantly renewed by the secretion of high molecular
weight glycoproteins from individual goblet cells throughout the epithelium.
Fig. 4. Light microscopic view of villi in the midgut from Arctic charr (Salvelinus alpinus L.) fed soybean
oil (A) post and (B) prior to challenge with Aeromonas salmonicida subsp. salmonicida. Note the substantial
more conspicuous goblet cells (arrows) along the villi of infected fish. After Lødemel et al. (2001).
Goblet cells differentiate in the lower portion of the crypts of both small and large
intestine and gradually migrate on to the villi or mucosal surface.
In an early study on histopathological changes caused by V. anguillarum,
Ransom et al. (1984) found large amounts of goblet (mucus producing) cells in the
anterior part of the GI tract of infected chum salmon. The first reaction of Arctic
charr (Salvelinus alpinus L.) infected by pathogenic bacteria (A. salmonicida) is to
slough off the infected mucus by increasing goblet cell production (fig. 4A) com-
pared to uninfected fish (fig. 4B). A similar reaction to that found in infected fish is
also observed in rabbits and rats infected by pathogenic bacteria (Enss et al., 1966;
Mantle et al., 1989, 1991), and this may be considered a normal host response to
particular intestinal infections (Mims et al., 2000).
Gastrointestinal mucus is rich in nutrients that organisms, including pathogens,
may utilize for growth (Blomberg et al., 1995; Wadolkowski et al., 1988). Many
endothermic studies have implicated growth in mucus as a critical factor for intes-
tinal colonization by pathogens and several outer membrane proteins are necessary
for establishment of an infection focus (Freter et al., 1983; Myhal et al., 1982;
Krivan et al., 1992; Burghoff et al., 1993). Olsson et al. (1992) suggested that the GI
tract is a site of colonization of V. anguillarum as the pathogen could utilize diluted
turbot (Scophthalmus maximus L.) intestinal mucus as its sole nutrient source. More
recently, Garcia et al. (1997) examined the ability of V. anguillarum to grow in
salmon intestinal mucus, which they concluded is an excellent growth medium for
this species. This is an important aspect of the pathogenesis of this organism.
3.2. Ultrastructural changes in enterocytes caused by dietary manipulation

Recently, Olsen et al. (1999, 2000) showed that extensive accumulation of lipid
droplets occurred in Arctic charr enterocytes when the fish were fed a diet contain-
ing linseed oil and this caused significant damage to the epithelium with focal loss
Fig. 5. Linseed-oil-fed Arctic charr

(Salvelinus alpinus L.). These enterocytes
are obviously damaged by the fat vacuoles
(droplets). Note especially cellular membrane
ruptures (arrows). Bar = 7 μm. After Olsen
et al. (1999).
of enterocytes and consequent loss of the epithelial barrier. Such damage (fig. 5) is
likely to be pathological and therefore detrimental to fish health. Rupture in the
membranous system may also represent a major microbial infection route for poten-
tially pathogenic bacteria provided they are present in sufficient numbers in the gut.
The prebiotic potential of dietary fibres is well known in endothermic animals
(Gibson, 1998), and may also have interesting applications in aquaculture. However,
a recent study clearly demonstrated that feeding Arctic charr a diet supplemented with
15% inulin led to the occurrence of a large number of spherical lamellar bodies in the
enterocytes of the pyloric caeca and the hindgut. These structures were not observed
when fish were fed 15% dextrin (Olsen et al., 2001). Feeding inulin had a destructive
effect on microvillus organization which may increase translocation of pathogenic
bacteria if they are present in relatively high concentrations in the GI tract.
3.3. Autochthonous bacteria and antagonistic activity

Savage (1983) defined bacteria isolated from the digestive tract as being either
indigenous (autochthonous) or transient (allochthonous) depending on whether or
not they are able to colonize epithelial surface of the digestive tract of the host
animal. Recently, Ringø and Birkbeck (1999) presented a list of criteria for testing
autochthony of bacteria from the GI tract of fish. These were that they should i) be
found in healthy animals, ii) colonize early stages and persist throughout life, iii) be
found in both free-living and hatchery-cultured fish, iv) grow anaerobically, and
v) be found associated with epithelial mucosa in the digestive tract. The presence of
an autochthonous microflora fitting the above criteria was demonstrated recently by
Ringø et al. (2002) in that bacteria in the gut were found closely associated with the
intestinal epithelium and between the microvilli. On the basis of this observation,
one might hypothesize that the autochthonous microflora of fish which is associated
closely with the intestinal epithelium forms a barrier serving as the first defence to
limit direct attachment or interaction of pathogenic bacteria with the mucosa as
reported for endothermic animals (van der Waaij et al., 1972; Snoeyenbos, 1979;
Tancrède, 1992). In fish, situations such as stress, antibiotic administration, or even

small dietary changes affect the GI tract microflora. Stability of this microflora is
important in natural resistance to infections produced by bacterial pathogens in the
intestinal tract. The existence of antibacterial substances produced by bacteria
isolated from the digestive tract of fish has been demonstrated in several studies
(Schrøder et al., 1980; Dopazo et al., 1988; Strøm, 1988; Westerdahl et al., 1991;
Olsson et al., 1992; Bergh, 1995; Sugita et al., 1996, 1997, 1998; Jöborn et al., 1997;
Gram et al., 1999; Ringø, 1999; Ringø et al., 2000). However, a recent study by Gram
et al. (2001) demonstrated that in vitro activity in well diffusion assays and broth cul-
tures cannot be used to predict a possible in vivo effect even if a reduction of in vivo
mortality was observed in another system (Gram et al., 1999). These studies under-
line the importance of developing and testing cultures for each specific combination
of different pathogens, different fish species and environment that might occur.
4. BACTERIAL INVASION AND TRANSLOCATION MECHANISMS

The indigenous intestinal flora is prevented from gaining access to other sites in the
body by a single epithelial cell layer on the mucosa. In endothermic animals the
M cells of the intestinal epithelium are specialized structures that may allow natural
entry of bacterial pathogens (Jones et al., 1995; Neutra et al., 1996; Vazques-Torres
and Fang, 2000). Information about the interactions between intracellular pathogenic
bacteria and M cells in fish is not available, however, and is a topic of further studies.
The mechanisms by which bacteria can translocate from the gut to appear in
other organs are an important phenomenon in the pathogenesis of “opportunistic”
infections by indigenous intestinal bacteria (Finlay and Falkow, 1997). Once inside
a host cell, pathogens have a limited number of ways to ensure their survival
whether remaining within a host vacuole or escaping into the cytoplasm.
In endothermic animals the primary defence mechanisms preventing indigenous
bacteria from translocating from the gastrointestinal tract are: a) a stable GI tract
microflora preventing bacterial overgrowth of certain indigenous bacteria or colo-
nization by more pathogenic exogenous bacteria, b) the host immune defences and
c) an intact mucosal barrier. More than one of these defence mechanisms can be
involved, depending upon the animal model or clinical situation. An example of this
is Lactobacillus casei, which can prevent E. coli infection in a neonatal rabbit model
and inhibits translocation of E. coli in an enterocyte cell culture model (Mattar et al.,
2001). However, in fish these defence mechanisms are not well understood.
The pathogenesis of V. cholerae infections in mammals is primarily a non-
invasive toxin-mediated gut infection but such infections have not been found in
fish. Translocation of intact Vibrio antigens and bacterial cells by endocytosis has
been reported in the gastrointestinal tract of fish larvae (Hansen and Olafsen, 1990,
1999; Hansen et al., 1992; Olafsen and Hansen, 1992; Grisez et al., 1996). However,
when discussing endocytosis, the development of the digestive tract is an important
Fig. 6. Transmission electron micrograph of

the apical regions of enterocytes in the pyloric
caecum of adult Arctic charr (Salvelinus
alpinus L.). Bacterial profiles are seen scattered
at different levels within the brush border from
the tips to bases of microvilli. In addition, one
bacterial profile (arrowhead) is seen to be
contained in an internalized, membrane-bound
endocytic vacuole. × 15000. After Ringø et al.
(2001).
factor to be considered. At the time of hatching, the digestive tract of fish is an undif-
ferentiated straight tube which is morphologically and physiologically less elaborate
than that of the adult (Govoni et al., 1986). However, endocytosis was demonstrated
in pyloric caeca (fig. 6), midgut (fig. 7) and hindgut (fig. 8) of adult Arctic charr
(Ringø et al., 2002).
Fig. 7. High power transmission electron

micrograph of the midgut of adult Arctic charr
(Salvelinus alpinus L.). The opposed surfaces
of two enterocytes are shown. Both cells have
appreciable numbers of bacterial profiles
between their microvilli. Note the internalized
bacterium in the subapical cytoplasm (arrow-
head). × 15000. After Ringø et al. (2001).
Fig. 8. Transmission electron micrograph

showing bacteria associated with the microvilli
of enterocytes in the hindgut of adult Arctic
charr (Salvelinus alpinus L.). Enterocytes in
this region show endocytic activity and are
characterized by large numbers of intracyto-
plasmic vacuoles (V) with contents of varying
electron density. An internalized bacterium is
discernible (arrowhead). × 15000. After Ringø
et al. (2002).
5. INVASION OF HOST CELLS

Entry into host cells is a specialized strategy for survival and multiplication utilized
by a number of pathogens which can exploit existing eukaryotic internalization
pathways (Finlay and Falkow, 1989, 1997; Sansonetti, 1993). Three general
mechanisms are recognized by which bacteria can invade epithelial cells. The most
common method, as employed by Yersinia, Shigella and Salmonella, is by inducing
rearrangement of the actin cytoskeleton of the epithelial cell. Enteropathogenic
Yersinia spp. induce uptake into endocytic vacuoles of epithelial cells following
close contact of the bacteria at many points to the cell surface (zippering). This
involves three adhesins – the invasin, Ail and YadA proteins – and interaction
between invasin and its cell-surface receptor, α5β1 integrin induces actin cytoskele-
ton rearrangement via a protein tyrosine kinase signalling system (Cornelius and
Wolf-Watz, 1997; Lloyd et al., 2001). Invasion by Shigella is dependent upon
possession of a 220 kb plasmid encoding 32 invasion-associated genes (Menard
et al., 1996), including those for a type III secretion system which directly secretes
Shigella proteins into the cytoplasm of the epithelial cell; this induces actin
cytoskeleton rearrangement and pseudopodia formation to internalize the bacterial
cell. Once internalized, lysis of the vesicle is mediated by a Shigella protein releas-
ing the organism into the cytoplasm where it can multiply and spread through the
cytoplasm propelled by an actin “tail” (Menard et al., 1996). Inhibitors of actin
polymerization, such as cytochalasin D, block entry of such pathogens into cells.
However, invasion of epithelial cells by Campylobacter jejuni is unaffected by
cytochalasin D but is sensitive to the microtubule depolymerizing drug colchicine,
indicating an actin-independent, microtubule-dependent pathway of entry (Russell

and Blake, 1994; Biswas et al., 2000). A direct invasive mode of entry is utilized by
rickettsiae, which bind to the phospholipid cell membrane and gains entry via
expression of phospholipase A (Silverman et al., 1992). For fish pathogens, exam-
ples are known of invasion of epithelial cells. Although none has been characterized
to the extent of human pathogens, current knowledge is summarized below.
5.1. Aeromonas
Aeromonas salmonicida is the causative agent of furunculosis, a disease which
caused very serious losses in European aquaculture in the early 1990s (Munro and
Hastings, 1993) and which had previously caused major epizootics in wild fish
(Mackie et al., 1930). All salmonid species are affected, but Atlantic salmon and
brook trout appear to be more susceptible to infection than rainbow trout or Pacific
salmon (Cipriano, 1983). The intestine has long been considered a route of infection
for A. salmonicida as Plehn (1911) found inflammation of the gut to be a common
characteristic of furunculosis. However, there is still debate about the route of entry
of this pathogen (Bernoth et al., 1997). The presence of A. salmonicida in the intes-
tine of Atlantic salmon has been demonstrated using an enzyme-linked immuno-
sorbent assay by Hiney et al. (1994), who suggested that the intestine could be the
primary location of A. salmonicida in stress-inducible infections. O’Brien et al.
(1994) also detected A. salmonicida in faeces using a species-specific DNA probe,
in conjunction with a polymerase chain reaction (PCR) assay. Although the organ-
ism can be detected in the intestinal tract in the above assays, McCarthy (1977)
failed to infect brown trout (Salmo trutta) either by administering food pellets
soaked in a culture of A. salmonicida, or by direct intubation into the stomach.
In the latter case, 105–106 A. salmonicida were recovered per ml homogenized stom-
ach within 12 h of introduction, no organisms could be recovered by 48 h and no
mortalities occurred, despite recovery of low numbers of organisms from
homogenates of kidney within 5 h. Despite failing to cause disease by the intestinal
route the organism killed five of six fish exposed for 5 days to an aqueous suspen-
sion of the bacteria (106 cells/ml).
In experimental infections of turbot (Scophthalmus maximus L.) and halibut
(Hippoglossus hippoglossus L.) yolk sac larvae with A. salmonicida subsp.
salmonicida, Bergh et al. (1997) failed to re-isolate the pathogen from halibut larvae,
but using immunohistochemical techniques showed the bacteria to be present in the
intestinal lumen of some turbot larvae, but not associated to mucus or gut microvilli.
A recent study by Lødemel et al. (2001) clearly demonstrated that A. salmonicida
subsp. salmonicida could be detected within enterocytes of the midgut of Arctic charr
(Salvelinus alpinus L.).
In summary, although A. salmonicida can be detected in the intestine of infected
fish there is still doubt that this is the principal route by which systemic infection
occurs, although translocation of organisms from stomach to kidney has been

demonstrated (McCarthy, 1977).
A range of freshwater fish are susceptible to motile aeromonad septicaemia
caused by A. hydrophila (Thune et al., 1993) and this organism is capable of binding
to collagen and fibronectin (Ascencio et al., 1991), and invading EPC (epitheliosum
papillosum of carp) tissue culture cells (Tan et al., 1998). Studies with inhibitors of
tyrosine kinase, protein kinase C and protein tyrosine phosphatase indicated that the
organism initiated a signalling cascade involving tyrosine kinase, leading to actin
microfilament reorganization involving actin “clouds” (Tan et al., 1998).
5.2. Edwardsiella
Two species of this genus, E. ictaluri and E. tarda are serious pathogens of fish
(Plumb, 1993), causing distinctly different diseases in a range of fish species.
Edwardsiella septicaemia, which affects warm water fish is widely distributed in
the environment and can cause severe losses in farmed catfish, Ictalurus punctatus
(Plumb, 1993). Although Darwish et al. (2000) found no histological lesions in the
intestine of catfish during experimentally induced infections, a different type of
study by Ling et al. (2000) employing green fluorescent protein (GFP)-labelled bac-
teria showed that 3 days after intramuscular injection of 1.2 × 10 5 E. tarda into blue
gorami approximately 10 6 bacteria were recovered from the intestine, although the
highest concentrations of bacteria were found in the muscle and liver. However,
E. tarda is not considered a pathogen with significant involvement of the gut in
infection. Nevertheless, it has a pronounced capacity to invade both human and fish
tissue culture cells (Janda et al., 1991; Ling et al., 2000). The invasion of both tissue
culture cell types by E. tarda was sensitive to cytochalasin D (Janda et al., 1991;
Ling et al., 2000), and in fish cells was also dependent on protein tyrosine kinase
activity (Ling et al., 2000).
The second species, E. ictaluri, causes enteric septicaemia of catfish which can
result in high mortalities. Two disease conditions are known with infection of brain
via the olfactory organ or the intestine (Shotts et al., 1986; Francis-Floyd et al.,
1987). Doses of 5 × 10 9 bacteria were intubated into the stomach of fingerling
catfish and within 2 weeks fish developed enteritis and other chronic lesions.
Horizontal transmission occurred to cohabiting fish, which also developed lesions
beginning in the intestine (Shotts et al., 1986). As yet, the invasion pathway from the
gut to other tissues and organs has not been established.
5.3. Photobacterium damselae subsp. piscicida

This organism was found to adhere to tissue sections of intestine from sea bream
Sparus aurata, sea bass Dicentrarchus labrax, and turbot Scophthalmus maximus at
concentrations of 10 4–10 5 per gram by Magarinos et al. (1996).
Evaluation of the invasive capacities of the Photo. damselae subsp. piscicida on

different poikilothermic cell lines indicated that according to the Janda index (Janda
et al., 1991), the strains studied were weakly or moderate invasive, with the number
of intracellular bacteria ranging from 101 to 103. Photo. damselae subsp. piscicida
was able to invade CHSE-214 tissue culture cells and to remain viable for at least
2 days inside the infected cells.
5.4. Piscirickettsia salmonis

Piscirickettsia salmonis is an obligate, intracellular, Gram-negative organism and
such fastidious bacteria have been increasingly detected as emerging pathogens in a
range of fish species in different geographic locations (Fryer and Mauel, 1997).
In 1990 it was recognized that the causative agent responsible for the loss of 1.5
million coho salmon in the previous year (Cvitanich et al., 1990, 1991; Fryer et al.,
1990; Branson and Diaz-Munoz, 1991; Garces et al., 1991) was a rickettsial agent
of a new genus and species (Fryer et al., 1992). Whereas the Rickettsiaceae are
members of the α-Proteobacteria, P. salmonis is assigned to the γ-Proteobacteria.
The disease was termed salmonid rickettsial septicaemia because of the systemic
nature of the disease (Cvitanich et al., 1991). Several organs were affected in dis-
eased fish, including the intestine, which was severely damaged with necrosis and
inflammation of the lamina propria and sloughing off of epithelial cells (Branson
and Diaz-Muoz, 1991). The route of infection was studied by Smith et al. (1999)
who investigated various routes as possible portals of entry for the pathogen.
Subcutaneous injection of P. salmonis (104 TCID50) resulted in 100% cumulative
mortality of fish by day 33 post injection. Application to the skin or gills of patches
soaked in P. salmonis (104.2 TCID50 per patch) resulted in 52 and 24% mortalities,
respectively, whereas 24 and 2% cumulative mortalities occurred following intestinal
or gastric intubation (104 TCID50 administered in both cases). The authors concluded
that rickettsia could infect the fish directly through the skin or gills and that the
intestinal route was not the normal route of infection.
5.5. Vibrio anguillarum

Vibrio anguillarum is an important pathogen of marine and estuarine fish species
and is the causative agent of vibriosis. This disease is one of the major bacterial dis-
eases affecting fish, as well as bivalves and crustaceans (Austin and Austin, 1999),
and vibriosis can cause substantial losses to the aquaculture industry. Vibriosis is
characterized by deep focal necrotizing myositis and subdermal haemorrhages, with
the intestine and rectum becoming swollen and filled with fluid (Horne et al., 1977;
Munn, 1977). The GI tract of fish appears to be a site of colonization and amplifi-
cation for pathogenic Vibrio species (Horne and Baxendale, 1983; Ransom et al.,
1984; Olsson et al., 1996), and Olsson et al. (1998) recently demonstrated that orally
ingested V. anguillarum can survive passage through the stomach of feeding juve-
nile turbot (Scophthalmus maximus L.). Vibrio anguillarum and V. ordalii have been
found primarily in the pyloric caeca and the intestinal tracts of three species of
Pacific salmon (chum salmon, Oncorhynchus keta; coho salmon, Oncorhynchus
kisutch; and chinook salmon, Oncorhynchus tshawytscha) (Ransom et al., 1984).
In addition, Olsson (1995) and Olsson et al. (1996) demonstrated that the GI tract
can serve as a portal of entry for V. anguillarum and it can utilize intestinal mucus
as its sole nutrient source (Olsson et al., 1992; Garcia et al., 1997). Although some
evidence indicates that V. anguillarum can invade fish either via the skin or the GI
tract, Grisez et al. (1996) showed that the organism is transported across the intes-
tinal epithelium by endocytosis. Chemotactic motility mediated by a single polar
sheathed flagellum is essential for virulence as bacteria deficient in this activity
were unable to infect fish when administered by immersion in bacteria-containing
water but were virulent when given by intraperitoneal injection (O’Toole et al.,
1996). These findings imply that V. anguillarum responds chemotactically to certain
fish-derived products in a manner that promotes the infection process prior to
penetration of the fish epithelium.
Recently, it was shown that V. anguillarum exhibited a stronger chemotactic
response towards intestinal mucus than towards skin (O’Toole et al., 1999). Of the
free amino acids identified in the intestinal mucus, glutamic acid, glutamine,
glycine, histidine, isoleucine, leucine, serine and threonine, and carbohydrates such
as fucose, glucose, mannose and xylose behaved as chemoattractants, while the lipid
components identified, bile acid, taurocholic acid and taurochenodeoxycholic acid
induced only a weak chemotactic response. A combination of all individual
chemoattractants identified from mucus reconstituted a high level of chemotactic
activity similar to that present in the intestinal mucus homogenate. On the basis of
these results, the authors proposed that multiple chemoattractants in rainbow trout
mucus indicated a strong relationship between chemotaxis and bacterial virulence.
The invasion mechanism of vibrios for fish cells has been investigated recently by
Wang et al. (1998) in a comparison of 24 isolates of seven different species. Thirteen
isolates were invasive for gruntfin (GF) and EPC tissue culture cell lines including all
five V. vulnificus and both V. harveyii isolates. Of the 11 V. anguillarum isolates
tested, three were invasive, two of which adhered strongly to EPC cells. Cytochalasin D
inhibited invasion by both strains although one was also sensitive to inhibition by vin-
cristin, a microtubule depolymerizing agent, indicating different routes of invasion for
the two strains. This difference was confirmed by the difference in response of the
strains to inhibitors of the signalling molecules protein kinase C and tyrosine kinase.
5.6. Streptococcosis
Streptococcosis is a septicaemic disease that affects freshwater and marine fish in
both farmed and wild populations. Among commercially important fish species, this
disease has been reported worldwide in yellowtail (Seriola spp.), eels (Anguilla
japonica), menhaden (Brevoortia patronus), striped mullet (Mugil cephalus),
striped bass (Morone saxatilis) and turbot. Romalde et al. (1996) demonstrated the
capacity of Enterococcus sp. to overcome adverse conditions in the stomach when
associated with food or faecal materials, since the pathogen was able to establish an
infective state and to produce mortalities after 16 to 20 days post ingestion.
6. VIRUSES
With the availability of effective vaccines against many major bacterial fish pathogens
(Gudding et al., 1997) agents such as infectious pancreatic necrosis (IPN) virus, infec-
tious salmon anaemia (ISA) virus, viral haemorrhagic septicaemia (VHS) virus,
infectious haematopoietic necrosis (IHN) virus and nodavirus have emerged as more
prominent threats to aquaculture. In mammals, the enteroviruses, rotaviruses, corona-
viruses and Norwalk virus group are important causes of diarrhoeal disease trans-
mitted by the faecal–oral route (Mims et al., 2000). For poliovirus the initial
replication in the GI tract can be followed by invasion of the bloodstream and pene-
tration of the blood–brain barrier to cause paralytic poliomyelitis (Mims et al., 2000).
In salmonids, IPN virus is a serious pathogen causing major losses in Atlantic
salmon aquaculture in Norway, Scotland and Chile (Smail and Munro, 2001). As its
name implies this virus causes significant necrosis of the pancreas in salmonids but
other organs, including the intestinal tract, may also be affected (Wolf, 1988).
Pathological changes in the intestinal tract have also been shown in larval sea bass
(Bonami et al., 1983) and larval halibut (Biering et al., 1994). In the latter study,
focal necrosis was observed in the intestinal tract with sloughing off of epithelial
cells, and the GI tract was considered the most likely route of entry and replication
for the virus (Bergh et al., 2002). However, there was no evidence of damage to the
pancreas in larval halibut.
Viral encephalopathy and retinopathy (VER), caused by nodaviruses, is a
recently recognized serious disease of Atlantic halibut which poses a serious threat
to larval culture of this fish (Grotmol et al., 1995, 1997; Munday and Nakai, 1997).
Although pathology is largely restricted to lesions in the brain, spinal chord and
retina (Grotmol et al., 1995), experimental infection models indicate that the intes-
tinal epithelium is the probable route of entry for this virus into the larval fish
(Grotmol et al., 1999). However, as with IPN virus, little is known of the pathogenic
mechanisms involved in invasion from the intestinal tract to the sites where signifi-
cant pathological damage is caused, and this awaits further investigation.
7. THE EFFECT OF DIET ON DISEASE RESISTANCE

Intensive fish production has increased the risk of infectious diseases. Therefore,
there is a growing need to find alternatives to antibiotic treatments for disease con-
trol, as indiscriminate use of antibiotics in many parts of the aquaculture industry
has led to the development of antibiotic resistance in bacteria. Nutritional status is

considered an important factor in determining disease resistance. The complex rela-
tionship between nutritional status, immune function and disease resistance has
been documented for higher vertebrates in several comprehensive reviews and
books (Gershwin et al., 1985; Chandra, 1988; Bendich and Chandra, 1990). The
influence of dietary factors on disease resistance in fish has been extensively
reviewed (Lall, 1988; Landolt, 1989; Blazer, 1992; Lall and Olivier, 1993; Waagbø,
1994; Olivier, 1997), and micronutrients such as vitamins have received particular
attention. Studies on the essential fatty acid, vitamin and trace element requirements
of several warm and cold water fish have demonstrated their integral role in the
maintenance of epithelial barriers of skin and the gastrointestinal tract. Although
there is some information on the relationship between disease resistance and dietary
lipid (Salte et al., 1988; Erdal et al., 1991; Obach et al., 1993; Waagbø et al., 1993;
Li et al., 1994; Bell et al., 1996; Thompson et al., 1996), there is a lack of informa-
tion about the functional role of dietary lipid on intestinal microbiota, their antago-
nism and disease resistance. However, a recent study showed clear differences in the
gut microbiota of fish fed different oils (post and prior to challenge) and the ability
of the gut microbiota to inhibit growth of three fish pathogens (A. salmonicida
subsp. salmonicida, V. anguillarum and V. salmonicida) (Ringø et al., 2002). Also,
Lødemel et al. (2001) clearly demonstrated that survival of Arctic charr after
challenge with A. salmonicida subsp. salmonicida was improved by dietary soybean
oil. These results are in agreement with those reported by Hardy (1997) that
replacement of dietary fish oil with plant- or animal-derived fats increases resistance
of catfish (Ictalurus punctatus) to disease caused by experimental challenge with
E. ictaluri.
Bacterial and viral diarrhoeal diseases are major causes of mortality and morbidity
in mammals but no equivalent diseases are recognized in fish, presumably because
dramatic fluid loss does not occur so readily in an aquatic environment. However,
the GI tract still presents a route of infection, especially for opportunistic bacteria
present on ingested food particles, and there is clear evidence for this as a route for
invasion to affect other organs and tissues.
The main reasons why studies on fish pathogenic bacteria have lagged behind
those of mammalian pathogens is because intensive aquaculture has developed quite
recently as a significant industry, several of the pathogens are novel, and there has
been a relatively small research effort in this field, in comparison with human and
veterinary medicine. The past decade has seen major developments in methodology
for studying microbial pathogenicity, and the techniques applied to human
pathogens are only now being applied to fish pathogens (O’Toole et al., 1996, 1999;
Tan et al., 1998; Ling et al., 2000; Mathew et al., 2001). Undoubtedly, the most
significant development in microbiology for 50 years has been the genome sequence
determination for many prokaryotes. Since the first complete sequence was pub-
lished in 1995, a total of 56 genome sequences have been completed to date and a
further 210 are in progress (see www.tigr.org and www.integratedgenomics.com).
Those in progress include genomes for the fish pathogens A. salmonicida and
P. salmonis and this will provide unique insights into the potential pathogenic mech-
anisms of these bacteria, including evolutionary distances between P. salmonis and
Rickettsia prowazekii and whether pseudogenes are also prevalent in P. salmonis
(Andersson et al., 1998; Andersson and Andersson, 1999).
Other methods, including the use of expressed markers such as green fluorescent
protein and laser confocal microscopy (e.g. Ling et al., 2000), will provide more
definitive analysis of pathways of invasion by pathogens taken up via the GI tract.
One area in which there is particular deficiency at present is in the nature of fish-
cell-surface colonization by bacteria and any downstream signalling which occurs.
This would be of considerable practical value in designing pathogen prevention
strategies using probiotic bacteria to prevent colonization by pathogens.
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11 Modelling of salmonellosis1
P.J. Naughtona and G. Grantb

aNorthern Ireland Centre for Food and Health, School of Biomedical Sciences,
University of Ulster, Cromore Road, Coleraine, Co. Londonderry BT52 1SA, UK
bRowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK
Salmonella continues to pose questions in terms of its pathogenicity and host speci-
ficity and also remains a key organism in the study of general infection mechanisms.
It elicits a disease that can vary from localized gut disorder to severe systemic
bacteremia, depending on the strain or serovar of the pathogen. In humans, the
prevalent form is a self-limiting infection confined primarily to the gastrointestinal
tract. The severity of the illness is, however, greatly influenced by factors such as
age, dietary history and health/immune status of the individual.
A plethora of animal models have been adopted to study salmonellosis. Few
appear to effectively model the overall infection in humans. In particular, there is
great variation in the levels of colonization, invasion and systemic spread that are
observed, as well as in the incidence of bacteremia and the effects of the pathogen
on long-term health. However, their use has allowed very detailed study of specific
aspects of pathogenesis.
Ideally the animal model chosen would be that which best exhibits the facet
of salmonellosis to be studied. However, other factors such as susceptibility to the
disease, the infective dose required, the ease of non-invasive monitoring or ready
availability of species-specific reagents often influence our choice. The mouse, rat
and pig are widely used. In this chapter, their strengths and weaknesses as models
of salmonellosis will be evaluated.
1. INTRODUCTION
Infections caused by Salmonella species continue to be a worldwide health problem.
They usually occur as a result of consumption of contaminated food or water and
1This work was supported in part by the Scottish Executive Environment and Rural Affairs Department (SEERAD).

236 P.J. Naughton and G. Grant
can manifest in a variety of disease states. These range from asymptomatic carrier
status through to localized gastroenteritis or to severe systemic infections that lead
to death (Buchwald and Blaser, 1984; Kotova et al., 1988; Bean and Griffin, 1990;
Darwin and Miller, 1999; Tsolis et al., 1999b; Kingsley and Baumler, 2000; Ohl and
Miller, 2001; Santos et al., 2001a). The nature and severity of the infection varies
according to bacterial strain and is also greatly influenced by host factors, such as
age and health status. The young, the elderly and immunocompromised individuals
are particularly susceptible.
For epidemiological purposes, the Salmonella can be placed into three
groups: 1) those that infect humans only e.g., S. typhi, 2) the host-adapted serovars,
some of which are human pathogens and may be contracted from foods, including
S. gallinarum (poultry), S. dublin (cattle), S. abortis-equi (horses), S. abortus-ovis
(sheep) and S. cholerasuis (swine) and 3) non-adapted serovars, such as S. enteritidis
and S. typhimurium, that are pathogenic for humans and other animals and encompass
most food-borne serovars.
In Europe and North America, the majority of salmonellosis cases are non-
typhoidal, mostly of the self-limiting gastroenteritis type and in the main caused by
S. typhimurium and S. enteritidis (Rampling, 1993; Mandal, 1994; Tauxe and Pavia,
1998; Mead et al., 1999). In contrast, typhoid-type diseases remain the predominant
forms in Asia, Africa and South America. These are due to S. typhi and S. paratyphi
A, B and C and continue to cause a high incidence of mortality in these regions
(Candy and Stephen, 1989; Mandal, 1994; Merican, 1997). The occurrence of
typhoid-like diseases is very low in Europe and North America but there is a tendency
for the levels to increase with time, possibly as a result of increased foreign travel
(Ryan et al., 1989; Mead et al., 1999).
2. SALMONELLA TYPHI
S. typhi colonizes the human gastrointestinal tract and adheres to and invades the
epithelium. It passes through into the subepithelium, where it is phagocytosed into
macrophages. It survives in these cells, rapidly spreads via the reticuloendothelial
system to internal tissues, such as liver and spleen and triggers chronic systemic
responses including onset of fever (Hornick et al., 1970; Mandal, 1994; Weinstein
et al., 1998; Santos et al., 2001a). Invasion appears to be primarily via the ileum,
where infiltration of mononuclear cells and thickening of the mucosa is evident soon
after infection. Furthermore, haemorrhage, ulceration and perforation occur in this
region of the gut in the longer term. The mesenteric lymph nodes, liver and spleen
enlarge and cellular dysfunction and lesioning develops in these tissues. Chronic
damage to the intestine appears, however, to be a primary cause of death (Hornick
et al., 1970; Weinstein et al., 1998; Santos et al., 2001a).
S. typhi is host-specific and elicits typhoid-like disorders only in humans and
chimpanzees (Pascopella et al., 1995; Weinstein et al., 1998). S. typhi does colonize
Modelling of salmonellosis 237
the gut of ex-germfree mice and enters Peyer’s patches (Collins and Carter, 1978).
However, it does not spread to other tissues or cause chronic infection (Collins and
Carter, 1978). S. typhi may be unable to proliferate, persist or cause damage in cells
of the murine Peyer’s patch (O’Brien, 1982; Kohbata et al., 1986; Pascopella et al.,
1995). The absence of a suitable animal model has severely hampered the study of
S. typhi infection.
3. SALMONELLA TYPHIMURIUM AND SALMONELLA ENTERITIDIS

S. typhimurium and S. enteritidis colonize the human gut, attach to and invade the
epithelium and pass into the subepithelium. They may translocate to the mesenteric
lymph nodes but, unlike S. typhi, they do not generally reach the liver and spleen.
Rapid clearance of the pathogens by macrophages is thought to limit the spread
beyond the level of the lymph nodes. Extensive infiltration of inflammatory cells
into the intestine is triggered by infection. There is also severe disruption of gut
structure and integrity, loss of fluid and electrolytes and onset of acute diarrhoea.
The disease is, however, self-limiting, usually clearing within 7 days (Old, 1990;
Tsolis et al., 1999a). Salmonella may nonetheless continue to be shed in faeces for
a significant period after the disappearance of clinical signs. Severe systemic infec-
tion is rare (2–5% of cases) in otherwise healthy individuals (Blaser and Feldman,
1981). It is, however, a significant risk for immunocompromised patients, the very
young and the elderly (Old, 1990; Tsolis et al., 1999a)
S. typhimurium and S. enteritidis are not host-specific and infect a wide range of
domesticated or wild animals (Old, 1990; Lax et al., 1995). In general, they cause a
self-limiting gastroenteritis or settle as a carrier population with no overt detrimen-
tal effects on the host. Chronic systemic infection is infrequent (Old, 1990; Lax
et al., 1995). One exception is the mouse, in which these serovars cause severe
systemic infection and death (Tsolis et al., 1999a).
4. GENERAL ASPECTS OF SALMONELLOSIS IN HUMANS

Salmonella spp. can thus cause two types of disease in humans: an acute gastro-
enteritis or systemic typhoid disease. While some non-specific symptoms in
Salmonella disease (e.g. nausea and abdominal pain) are shared, the clinical features
of Salmonella-induced diarrhoea and systemic typhoid infections are quite different.
For example, gastroenteritis may occur within 8−36 h after ingestion of contami-
nated food, whereas typhoid may follow after a period of 10−20 days. Diarrhoea
(which is usually watery, may be severe, and sometimes bloody) is the predominat-
ing feature of gastroenteritis, whereas in the case of adults constipation can occur in
the early clinical stages of typhoid.
Current perceptions are that gastroenteritis results from the interactions of
S. typhimurium or S. enteritidis and/or their products with the gut mucosa. Diarrhoea is
thought to be triggered by host-derived inflammatory products liberated as a result of

invasion of the intestine by the pathogen (Wallis et al., 1986; Worton et al., 1989;
Lodge et al., 1995) or by an enterotoxin or cell-free bioactive components in the
absence of invasion (Sandefur and Peterson, 1977; Finkelstein et al., 1983).
Certain aspects of pathogenesis are considered to be dose-dependent (Mintz et al.,
1994). High infective doses appear to provoke a more intense gastrointestinal response;
the onset of diarrhoea occurs earlier, vomiting is more common and stool frequency is
increased. Repeat exposure to Salmonella spp. also resulted in higher rates of vomiting
and hospitalization. However, clear relationships between inoculum, incubation periods
and other symptoms were not apparent (McCullough and Eisele, 1951).
Antibiotic treatment of gastroenteritis caused by Salmonella species has had lim-
ited success. It does not appear to reduce the duration or severity of illness and may
in fact prolong asymptomatic carriage (Jewes, 1987). Combined with the growing
resistance of Salmonella species to clinically important antibiotics (Lee et al., 1994),
this has meant that alternative therapeutic strategies to prevent or ameliorate salmo-
nellosis are becoming increasingly important.
Typhoid fever appears to be triggered by S. typhi organisms which translocate the
mucosa, survive within macrophages, multiply rapidly in systemic tissues and release
endotoxin which triggers the highly complex endotoxin-cascade (Aleekseev et al.,
1960; Santos et al., 2001a). Nonetheless, gut damage may be a primary cause of
death (Hornick et al., 1970; Weinstein et al., 1998; Santos et al., 2001a). Higher inci-
dences of infection and shorter incubation periods were reported for volunteers given
increasingly larger doses of S. typhi (Hornick et al., 1970). The clinical course, once
illness occurred, did not, however, appear to vary with infectious dose. Inoculum size
was therefore not a strong predictor of intensity, nor of duration of fever.
The dose of Salmonella required to initiate infection is not well defined and can
be variable. Bryan (1977) showed it to be approximately 10 4 colony forming units
(CFU) for S. typhi and more for other serotypes. However, lower doses (<10 3 CFU
salmonellae per gram of food) may also cause disease and outbreaks of entercolitis
in receptive individuals (Silliker, 1980). In addition, there are compelling data that
Salmonella infection can follow the ingestion of even very small numbers of bacteria
(<10 CFU) (Gill et al., 1983; Mintz et al., 1994).
The nature and severity of the infection caused by Salmonella spp. in humans
thus varies according to serovar, dose, virulence factors and host factors including age
and prior health or immune status. It follows, therefore, that the choice of organism/
host combination for experimental study of salmonellosis and precise definition of
the question(s) to be addressed, are of crucial importance.
5. SALMONELLOSIS IN THE RAT

Salmonella infection was first described in rats by Orskøv et al. (1928) and the
pathogenesis of the disease has been described by several investigators over the past
Fig. 1. Tissue distribution of S. enteritidis LA5

1 and 6 days post-infection (single oral dose of
≈108 CFU) of rats. (A) St, stomach; J, jejunum;
I, ileum; Ca, caecum; Co, colon. (B) MLN,
mesenteric lymph nodes; L, liver; Sp, spleen.
decades (Bakken and Vogelsang, 1950; Habermann and Williams, 1958; Böhme
et al., 1959; Maenza et al., 1970; Naughton et al., 1996, Havelaar et al., 2001).
S. enteritidis and S. typhimurium colonize the whole gastrointestinal tract of the rat
(fig. 1) and cause disruption of the small intestine epithelium and possibly the
caecal epithelium, induce epithelial cell hyperplasia and trigger infiltration by
inflammatory cells into the tissue. Invasion appears to be mainly via the ileum, high
numbers of Salmonella reach the mesenteric lymph nodes and moderate numbers
spread to and persist in the liver and spleen (fig. 1). In general, experimental rats
continue to grow, albeit slowly. Despite historical evidence suggesting that
Salmonella can be an effective rodenticide (Leslie, 1941; Threlfall et al., 1996),
recent studies indicate that death of experimental Hooded Lister rats owing to
Salmonella infection is rare, unless infective doses are very high or health or
immune status or gut integrity or function has been compromised by other factors
(Naughton et al., 1996, 2000; Islam et al., 2000; Havelaar et al., 2001; Takumi et al.,
2002). This ability to survive despite carriage of a significant Salmonella infection
would be consistent with the suspected role of rats in the spread of Salmonella
between poultry units (Davies and Wray, 1995).
Early work with rats concentrated on pathogenicity of Salmonella, the determi-
nation of infective dosage in specific pathogen free (SPF) albino Wistar rats
(Kampelmacher et al., 1968), the study of diarrhoea in Walter Reed strain albino rats
(Maenza et al., 1970), the comparison of Salmonella strains (Radoucheva et al.,
1994; Naughton et al., 1996) and evaluating the effects of nutrition on pathogenesis
in Sprague-Dawley rats (Omoike et al., 1990) and in Hooded Lister rats
(Guggenheim and Buechler, 1947; Naughton et al., 2000). Suprisingly, little work
has been done on the effects of Salmonella on rat ileal morphology (Naughton et al.,
1995; Havelaar et al., 2001) and in understanding the early events following entry
of Salmonella into the host. Morphologically, the lesions in the rat have been shown
to exhibit features evident in both severe human gastroenteritis with S. typhimurium
(Aleekseev et al., 1960) and human typhoid fever (Gay, 1918). Nonetheless, the
time scales are different. The timecourse of Salmonella entercolitis in the rat resem-
bles human gastroenteritis far more closely than it does typhoid fever.
Habermann and Williams (1958) stated that a Salmonella infection in rats could
take an acute or chronic form, or any degree of severity between the two extremes,
depending on the level of exposure (Bloomfield and Lew, 1942; Maenza et al.,
1970), virulence of the organism, host age and the dietary treatment (Guggenheim
and Buechler, 1947). Fasting overnight or prolonged protein or calorie intake defi-
ciency significantly increased susceptibility to infection (Guggenheim and
Buechler, 1947; Maenza et al., 1970; Omoike et al., 1990). Kampelmacher et al.
(1968) found that infection could occur at very low doses (e.g., 10 2 CFU) but with
considerable inter-individual variation. However, others found no persistent infec-
tion following a single dose of 10 3 CFU/ml of S. enteritidis (Naughton et al., 1996).
Colonization, invasion and persistence was, however, achieved at higher (up to
10 8 CFU) infective doses (Kampelmacher et al., 1968; Naughton et al., 1996).
Age had marked effects on the susceptibility of rats to oral Salmonella infection
(Kampelmacher et al., 1968). A persistent infection was evident in 4-day-old rats
given an infectious dose of 10 6 CFU/ml, but not in 20-day-old rats. However, an
infectious dose of 10 8 CFU/ml did cause a persistent infection in 100-day-old rats.
Kampelmacher and co-workers gave S. typhimurium, S. dublin and S. oranienburg
at varying doses to rats aged 4, 20 and 100 days in order to study persistence of the
organism in the faeces and the duration of the infection in the organs. It was
assumed the three variables – age, dosage and type of Salmonella – would influence
persistence. However, it was found to correlate directly only with age and dosage.
Repeated administration of Salmonella did not appear to lead to an increase in
persistence, although there was considerable inter-animal and inter-group variability
in the data. S. typhimurium, S. dublin and S. oranienburg showed unmistakable
differences in their ability to survive but different strains of the same type gave
almost identical results.
In experiments where the fate of intraperitoneally administered S. dublin was
tested in rats and mice, rats expressed greater cellular responses against the
pathogen and quickly suppressed or limited its growth. In contrast, the cellular
response of mice to the pathogen was poor. Later work by Veljanov et al. (1984)
showed that S. dublin caused systemic infection in mice but was avirulent in rats.
The avirulence of the bacteria for rats was related to the high phagocytic activity of
leucocytes against S. dublin (Veljanov et al., 1984). Bacterial growth in rat peri-
toneal fluid was attenuated and the microorganisms were found only at the site of
inoculation (Veljanov et al., 1984). This suggested that the systemic immune system
of the rat was more effective at suppression or clearance of salmonella than that of
the mouse (Havelaar et al., 2001; Takumi et al., 2002).
Only a limited number of studies have been done on the involvement of virulence
factors in salmonellosis of the Hooded Lister rat. Type 1 (SEF21) and other (SEF14,
SEF17, pef, lpf) fimbriae have transient roles in the early interactions of the
pathogen with the gut (Robertson, 2000; Naughton et al., 2001a,b; Robertson et al.,
2003). However, their presence does not appear to be a prerequisite for the occur-
rence of infection. Inability to produce functional flagella or flagellin significantly
reduces pathogenicity of Salmonella in the rat (Robertson, 2000; Robertson et al.,
2003). Despite this, aflagellate strains still cause a severe and persistent infection
suggesting that virulence factors in addition to flagella and fimbriae are involved in
the early interactions of Salmonella with the rat gut.
S. typhimurium and S. enteritidis in general cause a self-limiting gastroenteritis-
like disorder in rats. The infection is located primarily in the gut and associated
tissues and its severity is dependent on the infective dose, age and health and
dietary status. Salmonellosis in the rat thus has many features that are similar to the
gastroenteritis-type infection commonly elicited by these bacteria in humans and
domesticated animals. It is thus a useful model for the study of this disorder and the
effects of diet or environmental factors on pathogenicity.
6. SALMONELLOSIS IN THE MOUSE

Mouse strains vary greatly in their ability to deal with infection by S. enterica (table 1).
Balb/c or C57Bl/6 mice are very susceptible to S. typhimurium and S. enteritidis.
As little as 10 CFU of either of these serovars administered systemically or around
10 5 CFU given orally can cause death. In contrast, CBA and A/J mice are resistant
to S. typhimurium and S. enteritidis. With these mouse strains, ≥ 10 4 CFU given sys-
temically or ≥10 8 CFU orally are needed to cause a lethal infection. All mice appear,
however, to be resistant to S. typhi, even at inputs above 10 9 CFU orally (Collins and
Carter, 1978; O’Brien, 1982).
The resistance of mice to S. typhimurium has been linked to at least five differ-
ent gene loci. Most of these, including ity (Nramp-1) and lps, are important during
the early phases of infection whilst the others appear to be associated with late-
phase responses to infection (Mattsby-Baltzer et al., 1996; Pie et al., 1996;
Cannone-Hergaux et al., 1999; Forbes and Gros, 2001). Disablement of these genes
greatly increases susceptibility to infection. For example, the intraperitoneal LD50
for the resistant C3H/HeN mouse is around 10 3 CFU but is less than 10 CFU for the
Table 1. Comparison of LD50 values (CFU) of Salmonella enterica serovars Typhimurium and
Enterica reported for various strains of mice
S. typhimurium S. enteritidis
Route SC IV IP Oral Oral
Balb/c <10 20 <10 2 × 104−9 × 105 20−2 × 105

C57Bl/6 20 <10 <10 3 × 104−2 × 105
B10 <10 20
B10.D2 <10−50 <10
DBA/2 2 × 105 40
C3H/HeN 1 × 106 1 × 10 3−4 × 10 3 >1 × 107
C3H/HeJ 1 × 10 4
CD-1 2 × 10 6
CBA 1 × 107 1 × 10 3−1 × 10 4 >1 × 108 >1 × 10 8
A/J 2 × 106 2 × 10 4 1 × 10 4 −1 × 10 5
SC, subcutaneous injection; IV, intravenous injection; IP, intraperitoneal injection; Oral,
administered by gavage.
Based on data from: Carter and Collins, 1974; Plant and Glynn, 1974; Hormaeche, 1979; Eisenstein
et al., 1982; Lockman and Curtiss, 1990; Morrissey and Charrier, 1991; Baumler et al., 1996;
van der Velden et al., 1998; Lu et al., 1999; Mastroeni et al., 1999; Shea et al., 1999; Edwards et al.,
2000; Monack et al., 2000; Ikeda et al., 2001; Nicholson and Baumler, 2001; Schmitt et al.,
2001; van der Straaten et al., 2001.
C3H/HeJ mouse, a related strain that is LPS and TLR4 defective (table 1). Mouse
strains that are most susceptible to Salmonella, such as the Balb/c or C57Bl/6, are
defective in ity (Nramp-1) (Mattsby-Baltzer et al., 1996; Pie et al., 1996).
S. typhimurium colonizes the whole gastrointestinal tract of susceptible (itys) mice,
attaches to enterocytes and invades primarily through M cells of ileal Peyer’s patches
(Tannock et al., 1975; Jones et al., 1994; Darwin and Miller, 1999; Kingsley and
Baumler, 2000). There is proliferation of the pathogen within the Peyer’s patches,
destruction of M cells, infiltration of inflammatory cells into the gut and considerable
disruption to the structure and integrity of the intestine (Tannock et al., 1975; Jones
et al., 1994; Santos et al., 2001a). A recent study indicates that colonization of the
Peyer’s patches may be caspase-1 dependent since it is attenuated in casp-1−/− (Monack
et al., 2000). In the subepithelium, macrophages or dendritic cells capture
S. typhimurium. The pathogen can, however, survive within these cells and is carried to
the mesenteric lymph nodes, liver and spleen (Carter and Collins, 1974; Vazquez-
Torres et al., 1999; Isberg and Barnes, 2000; Resigno and Barrow, 2001). In susceptible
mice, very rapid proliferation of the pathogen occurs in the liver and spleen. Indeed,
bacterial numbers can increase more than ten-fold per day (Tsolis et al., 1999a). This
triggers an influx of polymorphonuclear cells into the tissues and leads to formation of
granulomata and development of hepato- and speno-megaly. Subsequently, when bac-
terial levels in the liver and spleen reach over 108 CFU/tissue, there is a breakout of the
pathogen and development of bacteraemia. Liver and spleen damage and dysfunction
appear to be the main cause of death in susceptible mice (Tsolis et al., 1999a).
S. typhimurium numbers in the liver and spleen of resistant (ityr) mice do not
increase in this uncontrolled manner. This is probably as a result of effective clear-
ance from the tissues, suppression of proliferation or limitation of systemic spread
as a result of ity (Nramp-1) expression in macrophages (Hormaeche, 1980; Lang
et al., 1997; Mastroeni et al., 1999; Cuellar-Mata et al., 2002).
Many virulence factors and their modes of action have been identified through
study of S. typhimurium infection in mice (Darwin and Miller, 1999; Galan, 2001;
Ohl and Miller, 2001). Fimbrial adhesins have been shown to be important in facil-
itating initial attachment of the pathogen to Peyer’s patch cells (Baumler et al.,
1996; van der Velden et al., 1998; Humphries et al., 2001). The type III secretion
system encoded on Salmonella pathogenicity island 1 (SPI1) and the array of bio-
active components it releases are essential for invasion of the epithelial layer and for
proliferation of the pathogen in the subepithelium (Galan, 2001; Ohl and Miller,
2001). Salmonella pathogenicity island 2 (SPI2), in particular its type III secretion
system and effector proteins, Salmonella pathogenicity island 3 (SPI3) and
Salmonella plasmid virulence (spv) genes are required for survival and proliferation
of the pathogen at systemic sites (Galan, 2001; Ohl and Miller, 2001).
Flagellin, the core protein of the flagella, has also been identified as a potent
trigger of cytokine release and inflammation (Eaves-Pyles et al., 2001; Gewirtz et al.,
2001; Ikeda et al., 2001). However, its role in S. typhimurium infection of mice
remains equivocal. Bacteria unable to express flagellin (FliC blocked or deleted) were
found to be less able to cause infection than wild-type counterparts (Carsiotis et al.,
1984; Schmitt et al., 2001). However, in other cases, the mutant strains appeared as
infective as wild-type strains (Lockman and Curtiss, 1990; Ikeda et al., 2001).
Colonization, invasion, systemic spread, persistence and proliferation by
S. typhimurium in the mouse occurs progressively, as a result of the integrated action
of the various virulence factors expressed by the bacterium. However, no one viru-
lence factor appears to be an absolute requirement for these processes to occur.
Whilst interference with SPI1 or SPI2 or deletion of multiple fimbriae greatly
reduces pathogenicity of S. typhimurium, the mutated strains can still cause a lethal
infection, albeit that the bacterial numbers required are significantly increased. This
would indicate the bacterium has the inherent capacity to compensate, at least in
part, for functional impairment or loss of individual virulence factors.
Few studies appear to have dealt with the effects of S. enteritidis in mice.
However, the susceptibility of various mouse strains to S. enteritidis (table 1) and the
nature of the infection it causes appear, at least in general, to be similar to those found
for S. typhimurium (Carter and Collins, 1974; Humphrey et al., 1996; Lu et al., 1999).
7. OTHER INFLUENCES ON PATHOGENICITY IN THE MOUSE

The severity of the infection caused by Salmonella can vary significantly when the
pathogen is administered orally to mice. This is, at least in part, due to actions of the
resident gut flora, which can significantly limit the ability of S. typhimurium to
colonize the gut and/or translocate and spread systemically (Miller and Bohnhoff,
1963; Que and Hentges, 1985). If the flora is disrupted or removed completely, this pro-
tection is lost. Thus, as little as 10 CFU S. typhimurium can be lethal to susceptible/
antibiotic-treated mice whereas at least 105 CFU is needed in conventional counter-
parts (Miller and Bohnhoff, 1963; Que and Hentges, 1985; Murray and Lee, 2000).
The level and diversity of the commensal flora can differ significantly between
batches of mice and even between individuals, depending on conditions under
which they have been bred and reared. There is thus likely to be an inherent vari-
ability in the susceptibility of mice to oral pathogen challenge. However, this prob-
lem applies equally to other animal models.
Re-infection through coprophagy and cross-contamination can also be a factor in
mouse studies. For ease of management and on animal welfare grounds, mice are
generally housed as groups in solid floored caging. As a result, infected mice
are exposed to and consume significant amounts of faeces and can therefore be
re-infected with the pathogen. This in itself may not be a problem. However, the
expression of key virulence factors by Salmonella and pathogenicity can be changed
dramatically due to passage through the gut and the overall virulence of a strain may
be greatly increased (Kampelmachar et al., 1968; Hormaeche, 1979; Old, 1990). The
responses observed in infected animals, in particular the intestine–pathogen inter-
action may therefore be greatly affected by these re-ingested bacteria. Group-housed
animals appear to succumb to Salmonella far more quickly than singly housed ones
(Kampelmachar et al., 1968).
Food intake and food quality significantly influence the susceptibility of animals
to Salmonella (Omoike et al., 1990; Peck et al., 1992). Mice are usually given free
access to food and it is difficult to control or monitor intake owing to group housing.
However, mice develop a strong hierarchy within groups, particularly strains such as
the Balb/c or C57BL. Even though food is freely available, dominated animals tend
to eat less. This in turn is likely to affect their susceptibility to infection.
Many studies on the roles of virulence compounds in Salmonella infection use
the LD50 dose as an index of pathogenicity. However, this may be a poor measure.
Salmonella produces a plethora of virulence factors and appears able to compensate,
at least partially, for loss of individual virulence factors. Equally, a dose that causes
death of the mouse is likely to trigger multiple tissue and systems dysfunction. Any
effects due to loss of a virulence compound may thus be swamped by general meta-
bolic responses to infection. An example of this may be with flagellin. In its puri-
fied form, flagellin has pronounced effects on the metabolism of mice (Eaves-Pyles
et al., 2001; Gewirtz et al., 2001). However, inability to express flagellin has limited
or no effect on the pathogenicity of S. typhimurium (Carsiotis et al., 1984; Lockman
and Curtiss, 1990; Ikeda et al., 2001; Schmitt et al., 2001). Studies that utilize lower
infective doses in combination with monitoring of tissue distribution and key
cellular responses in the gut and systemic tissues may be more revealing as to the
roles and impact of flagella or other virulence factors in the infection process.
8. MOUSE MODEL OF TYPHOID FEVER

S. typhimurium usually causes a self-limiting gastroenteritis-type infection in
humans and most domesticated animals. The lethal infection caused by the serovar
in susceptible (itys) mice is thus atypical. However, it has strong similarities to the
disease caused by S. typhi in humans and chimpanzees. In the absence of a direct
in vivo model for S. typhi infection, the S. typhimurium-infected mouse is therefore
widely accepted and used as a model to study typhoid-like disease in vivo (Tsolis
et al., 1999a; Ohl and Miller, 2001; Santos et al., 2001a). Use of the mouse model
of typhoid has enabled identification of the routes and mechanisms by which
S. typhi may colonize, invade and cause gut and systemic tissue dysfunction (Tsolis
et al., 1999a; Santos et al., 2001a; Ohl and Miller, 2001). In addition, it has high-
lighted a number of key bacterial genes that may be targeted in order to reduce vir-
ulence. This in turn has facilitated development of attenuated S. typhi strains for
possible use as live oral vaccines. The mouse model of typhoid has thus been essen-
tial for development of treatments of this disease and can continue to make a con-
tribution in the future.
The present mouse model of typhoid has, however, serious limitations. The incu-
bation period for typhoid-like disease in mice is much shorter than that for S. typhi-
infection in humans. In addition, the mode of lethality is different (Tsolis et al.,
1999a). For S. typhi infection, death appears to be linked primarily to intestinal dam-
age whereas liver and spleen dysfunction are the main causes in the mouse model.
In addition, S. typhi lacks a number of the factors known to be of particular impor-
tance for the infectivity of S. typhimurium (Woodward et al., 1989). As a result, most
of the potential live oral vaccines developed are Typhi strains with defects in
specific functional or regulatory genes rather than in those for key virulence factors.
Furthermore, although data from the mouse model suggest that live oral vaccines
should be most effective against S. typhi, killed vaccines actually appear to be more
protective in human subjects (Eisenstein, 1998; Pang, 1998). S. typhi infection clearly
involves additional factors or pathways that do not manifest in the mouse model of
typhoid. A major challenge for the future will be to identify these mechanisms. This
is unlikely to be achievable with the present model or possibly even in the absence of
S. typhi itself as the infective agent. At present no suitable alternatives exist. However,
development of transgenic or knockout mouse strains may in the future lead to a
model that exhibits more of the characteristics of typhoid-like infection.
9. INTERACTIONS IN THE MOUSE GUT

Although the infection caused by S. typhimurium in susceptible (itys) mice is atypi-
cal, it has generally been assumed that the nature of early interactions of the pathogen
with the gut would be fairly similar to those in other animal species. Bacterial
fimbriae are of particular importance for the initial interactions of S. typhimurium
with the gut epithelium, in particular with the M cells and for overall pathogenicity
in susceptible (itys) mice (Baumler et al., 1996; van der Velden et al., 1998;
Humphries et al., 2001; Santos et al., 2001a). However, in other animal models (rat
or chick), virulent S. enteritidis strains unable to express five fimbriae (SEF14,
SEF17, SEF21, pef and lpf), although possibly transiently disadvantaged, were able
to colonize, invade and persist in the long term as effectively as wild-type counter-
parts (Allen-Vercoe et al., 1999; Robertson, 2000; Robertson et al., 2003). These
contrasting findings might have been ascribed to differences between
S. typhimurium and S. enteritidis in their modes of action. However, the nature and
severity of the infection caused by both serovars appear very similar in mice (table 1;
Humphrey et al., 1996; Lu et al., 1999; Santos et al., 2001a,b). This is also the case
in rats (Naughton et al., 1996). The major difference is in the type of disease elicited
in the respective models. Whilst S. typhimurium and S. enteritidis cause a lethal
infection in mice, they elicit a self-limiting gastroenteritis-like infection in rats
(Naughton et al., 1996, 2000; Havelaar et al., 2001; Takumi et al., 2002). This may
suggest that the requirement and potential roles for particular surface appendages
vary according to the animal model or severity of disease. Since the infection
induced by S. typhimurium in susceptible (itys) mice is atypical, caution may there-
fore need to be applied in extrapolating from the S. typhimurium-mouse model to
likely effects of S. typhimurium and S. enteritidis in other species.
10. SALMONELLA IN PIGS

Salmonella infections in domestic pigs Sus scrofa are associated with significant
animal suffering and economic losses and pigs may indeed be a reservoir of infection
for humans (Berends et al., 1997). Pigs acquire a severe systemic disease at least
superficially similar to typhoid fever as a result of infection with S. cholerasuis (Gray
et al., 1996; Baumler et al., 1998, Anderson et al., 1998). In contrast, they do not
contract systemic disease when infected with S. typhi (Metcalf et al., 2000) or
S. typhimurium (Wilcock and Schwartz, 1992). Carriage of S. typhi in the porcine tonsil
has, however, been observed (Metcalf et al., 2000). This organ was previously noted
to be important in S. cholerasuis (Gray et al., 1996) and S. typhimurium infection of
pigs (Wood et al., 1989, 1991; Wood and Rose, 1991; Fedorka-Cray et al., 1995).
Natural infection of pigs with Salmonella typhimurium is associated with an
enteric disease (Wilcock and Schwartz, 1992), that is a major problem affecting grow-
ing pigs in several parts of the world (Reed et al., 1986; Grøndahl et al., 1998). The
mechanisms by which Salmonella typhimurium cause this, particularly scouring, are
poorly understood and have largely been extrapolated from work done in rodents.
Bacterial invasion is not correlated with the incidence or severity of diarrhoea (Wallis
et al., 1986, 1989). An inflammatory response with an influx of polymorphonuclear
leucocytes is needed to trigger scouring, since S. typhimurium fails to induce fluid
secretion in the absence of leucocyte influx (Giannella et al., 1975). Another virulence
factor seems to be an enterotoxin with a partial resemblance to cholera toxin (CT) and
Escherichia coli heat labile enterotoxin (LT) (Wallis et al., 1986; Chopra et al., 1991).
However, despite the efforts of many groups of investigators, a role for an enterotoxin
in S. typhimurium-induced fluid secretion has yet to be defined. Unlike CT and LT, the
S. typhimurium enterotoxin is not secreted (Wallis et al., 1986).
The digestive tract of the domestic pig has distinct structural and functional
similarities to that of humans (Kidder et al., 1961; Hill, 1970). In addition, experi-
mental pigs infected orally with S. cholerasuis or S. typhimurium exhibit diseases
that are almost identical to those reported in the field (Reed et al., 1986). Pigs there-
fore provide a good model for study of the potential effects of gut–microbe–food
interactions on colonization, invasion and persistence in humans by Salmonella spp.
11. SALMONELLA AND CALVES

Salmonellosis is a significant cause of ill health, poor growth or weight loss and
occasionally death in cattle. Many serotypes can cause infection but S. dublin and
S. typhimurium are the serovars most often associated with disease (Wray, 1991).
S. dublin is predominant in older animals, causing both enteric and severe systemic
infections (Wray, 1991; Wallis et al., 1995; Watson et al., 1995; Libby et al., 1997).
However, it can also be present in a chronic carrier state or may trigger abortion in
cows and heifers without otherwise eliciting other overt clinical signs of infection
(Sojka et al., 1974; Hall et al., 1979). Scouring is generally the major clinical sign
of S. dublin infection of calves. Nonetheless, in the long term, bacteraemia, fever
and severe systemic dysfunction can also develop, often in the absence of ongoing
gastroenteritis (Wray and Sojka, 1981; Wray, 1991).
S. typhimurium is the main cause of salmonellosis in calves. It causes acute gas-
troenteritis, with severe scouring developing around 12−48 h post-infection (Wray,
1991; Wallis et al., 1995; Santos et al., 2001a,b). At moderate infective doses
(approximately 106 CFU), the infection is self-limiting and usually clears by around
8 days. The disorder can be lethal at higher (up to 1010 CFU) infective doses, due
mainly to extensive intestinal lesions and dehydration due to extreme fluid loss
(Tsolis et al., 1999a; Santos et al., 2001a,b).
S. typhimurium colonizes the calf gastrointestinal tract, attaches to enterocytes
and invades the tissue (Santos et al., 2001a,b). Invasion may occur initially via
M cells of ileal Peyer’s patches but can be observed in all regions of the ileal epithe-
lium soon after infection. The pathogen translocates rapidly to the mesenteric lymph
nodes but, in most cases, only limited spread to systemic organs is evident, even
with very high infective doses (Kingsley and Baumler, 2000; Santos et al., 2001a,b).
Infection causes severe disruption of the epithelium and leads to loss of gut integrity.
There is infiltration of polymorphonuclear cells, potent inflammatory responses in
the tissue and extensive loss of fluid and electrolytes.
A number of virulence factors have been identified as potentially important in
S. typhimurium infection of calves (Wallis and Galyov, 2000; Wallis et al., 1999).
In particular, bioactive components associated with Salmonella pathogenicity
island 1 (SPI1) are crucial for triggering release of inflammatory cytokines, recruit-
ment of neutrophils, development of intestinal lesions and onset of scouring. The
flagellar secretory apparatus also appears to be required for maximal influx of neu-
trophils and fluid secretion in a bovine ligated intestinal loop model (Ikeda et al.,
2001; Schmitt et al., 2001). However, the exact mechanisms responsible for the
onset and severity of scouring and gut damage at high and moderate Salmonella
doses remain unclear (Tsolis et al., 1999a,b; Santos et al., 2001a,b).
Bioactive factors linked to other Salmonella pathogenicity islands appear less cru-
cial for S. typhimurium-linked gastrointestinal disease in calves (Tsolis et al., 1999a,b).
They do, however, have key roles in S. dublin infection, in particular systemic spread,
survival and proliferation of the pathogen (Wallis et al., 1999; Wood et al., 1998). As
with S. typhimurium, SPI1 appears to be important in the early responses of the gut to
S. dublin, but other SPIs, such as SPI5, may also have roles (Wood et al., 1998).
S. typhimurium infection of calves in vivo is used to model human gastroenteritis
in order to study pathogen–gut interactions during development of this disorder. The
intestinal loop in situ model has been particularly useful in short-term (up to 12 h)
study of inflammatory responses, neutrophil recruitment and fluid secretion provoked
by Salmonella and the likely roles of specific virulence components (Wallis et al.,
1995; Tsolis et al., 1999a; Santos et al., 2001a,b). In some circumstances, the models
in situ and in vivo may, however, give contrasting results, because they are dealing
with short- or long-term responses respectively. For example, neutrophil recruitment
and fluid secretion were attenuated when sopB-blocked mutants of S. typhimurium or
S. dublin were tested in ligated loops (Wood et al., 1998; Santos et al., 2001a,b).
Nonetheless, a sopB-blocked S. typhimurium mutant was as pathogenic as a wild-type
strain in vivo when administered at a dose of 1010 CFU (Tsolis et al., 1999a,b). This
may have arisen because the Salmonella could compensate for the loss of sopB and
was thus only transiently disadvantaged (Tsolis et al., 1999a; Santos et al., 2001a,b).
S. typhimurium causes a lethal infection if given in very high numbers to calves
but a self-limiting gastroenteritis if administered in moderate amounts (Tsolis et al.,
1999a; Santos et al., 2001a,b). In both cases, the infection is located primarily in
the intestine and mesenteric lymph nodes. However, the physiological responses and
gut disruption triggered by the lethal infective dose are far more severe and likely to
involve additional virulence components. Any changes in pathogenicity owing to
blocking of an individual virulence factor, such as sopB, may therefore be masked
when high infective doses of Salmonella are used. Study of the roles of individual
virulence factors may be better achieved with lower infective doses, where the
disease caused by the wild-type strain is of a self-limiting type.
12. HOST SPECIFICITY

The factors influencing Salmonella serotype host-specificity remain poorly defined,
although there is some evidence that bacterial survival within macrophages is
important (Kok et al., 2001; Santos et al., 2001a). S. typhimurium can persist in
significantly higher numbers than S. typhi in primary murine macrophages in vitro
(Pascopella et al., 1995; Schawn and Kopecko, 1997), which correlates with the
virulence of these serotypes for mice. Similarly S. typhi persists better in human
macrophages compared to murine macrophages in vitro, which again correlates with
virulence. However, the relative persistence of S. typhi and S. typhimurium in human
macrophages varies considerably between different studies (Vladoianu et al., 1990;
Alpuche-Aranda et al., 1995; Ishibashi and Arai, 1996). This variation may be
explained by non-quantifiable differences in the magnitude of macrophage lysis,
which can have a major effect on the interpretation of results from bacterial persist-
ence studies (Guilloteau et al., 1996; Sizemore et al., 1997).
13. DIET AND SALMONELLA INFECTION

Diarrhoea is highly prevalent among malnourished children (Scrimshaw, 1970;
Bovee-Oudenhoven et al., 1997). Furthermore, epidemiological studies have shown
that the duration and severity of diarrhoea increase with malnutrition (Chen et al.,
1981; Black et al., 1984). However, the relationship between infection and malnu-
trition is complex and its determinants remain poorly understood (Guggenheim and
Buechler, 1947; Maenza et al., 1970; Peck et al., 1992). Omoike et al. (1990)
showed that adherence of Salmonella to the intact mucosa in situ was consistently
higher in well-fed rats rather than malnourished Sprague-Dawley rats. Nonetheless,
the infection caused very marked cell destruction and lysis in tissues of malnour-
ished rats, whereas only limited histological changes were detected in mucosa from
the well-fed animals. Adherence of pathogen to the gut is clearly not the only factor
dictating the severity of the infection. It is likely that malnutrition severely impairs
intestinal cell metabolism and reduces the efficacy of the local and systemic immune
systems. This may greatly increase the permeability of the gut to the pathogen or its
products and predispose the malnourished animal to salmonellosis.
Rats on a low calcium milk diet had a significantly impaired colonization resist-
ance to S. enteritidis (Bovee-Oudenhoven et al., 1996). The barrier potential of the
gut was reduced and the pathogen was able to translocate in significant numbers
across the intestine to the systemic circulation. Biliary factors may have a significant
role in infection by S. enteritidis in the rat since diversion of bile from the intestine
lumen greatly reduced the ability of the pathogen to invade and spread systemically
(Islam et al., 2000). Newberne et al. (1968) observed a much higher mortality rate in
S. typhimurium-infected rats if they were fed diets deficient in copper or in protein.
The severity of infection tended to be greater in Charles River and Holtzman rats fed
a 12% casein diet than in those fed an 18% casein diet (Newberne et al., 1968).
Similarly, nitrogen losses by Salmonella-infected rats were higher if they were fed
low-protein diets instead of diets containing adequate amounts of protein (McGuire
et al., 1968). A net protein-catabolic response to an infection stress has also been
shown to occur in humans (Beisel, 1966). Muscle polysome profiles are also altered
when Salmonella-infected rats are given a low-protein diet (Young et al., 1968).
Faecal nitrogen outputs by rats were found to be much higher with S. typhimurium
infection than for S. enteritidis infection (Naughton et al., 1996). It has also been
shown that dietary plant lectins, such as GNA (from Galanthus nivalis, the snow-
drop) and Concanavlin A (ConA, from the Jack bean), can alter the course of
Salmonella infection (Naughton et al., 2000). The lectin GNA was shown to
decrease the numbers of Salmonella and ConA was shown to increase the numbers
of Salmonella in the small intestine for rats. Particular plant lectins may, therefore,
interfere with the association of type 1 fimbriae with the rat gastrointestinal tract
(Naughton et al., 2001a,b). The rat may have significant advantages as a model for
the study of dietary influences on susceptibility to and severity of salmonellosis.

The growth in the elderly human population in much of the developed world, the
high incidence of immune deficiency diseases worldwide and rising degrees of
antibiotic resistance amongst pathogens mean that novel strategies to prevent or
limit pathogenic infections are urgently required. A significant amount of research
will be required into diseases affecting the gastrointestinal tract. Ways will have to
be found to ameliorate the effects of old age or immunodeficiency on susceptibility
to infection. There will be increasing efforts to modify and supplement the diet in a
manner that will prevent or limit pathogenic infections, support gut function and
promote gut integrity and health. With the plethora of animal models available to us,
it belies researchers to choose wisely and address the correct questions.
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12 Bacterial colonization of avian mucosal
surfaces
R.M. La Ragione, D.G. Newell and M.J. Woodward
Department of Bacterial Diseases, Veterinary Laboratories Agency (Weybridge),

Woodham Lane, Addlestone, New Haw, Surrey KT15 3NB, UK
The skin and particularly the mucous membranes of humans and animals are heavily
colonized by indigenous bacteria and, in poultry, bacterial colonization of the
mucosal surfaces occurs within the first few hours of life (Gibbons, 1977). Neonatal
birds inhale or ingest the majority of the microorganisms that go on to colonize
and develop into the normal flora, a flora that contains many diverse populations of
bacteria. In healthy birds, the bacterial composition of the mucosal surfaces remains
relatively stable and many of these bacteria play an essential role in the development
and well being of the host. However, if the stability of the native flora is broken,
opportunistically pathogenic organisms are able to colonize which may lead to
potentially fatal septicaemia. Infectious disease, on the other hand, requires as a first
step, colonization of the host. Colonization for many pathogenic bacteria occurs ini-
tially on a mucosal or skin surface (Smith, 1972) and in competition with the native
flora that is considered to have a protective effect (Nurmi and Rantala, 1973).
Colonization of host tissues by commensal and pathogenic bacteria commonly
involves adhesin–receptor interaction to overcome host defence mechanisms such as
urination, desquamation and peristaltic propulsion through the intestinal tract,
although other factors also play a role in the establishment of infection. Aspects of
colonization are discussed along with the particular mechanisms employed by spe-
cific bacterial species. Additionally, the role of intervention strategies for the modi-
fication of the commensal microflora to control bacterial pathogens is discussed.
1. INTRODUCTION
Colonization of host tissues by commensal and pathogenic bacteria commonly
involves adhesin–receptor interaction to overcome host defence mechanisms such as

Colonization of avian mucosal surfaces 259
urination, desquamation and peristaltic propulsion through the intestinal tract,

although other factors also play a role in colonization. Motility may aid in penetra-
tion of mucus (Smyth, 1988). Lipopolysaccharides and capsule may contribute
to resistance to bile salts and protect against host iron-binding proteins and non-
specific serum factors such as complement (Mims, 1987; Woolcock, 1988).
Gastrointestinal surfaces are dynamic three-dimensional compartments. These
compartments are composed of mucilaginous glycoproteins, flowing on glycocalyx
overlaying the epithelial membrane that is convoluted into microvilli (Dyce et al.,
1987). The membranes themselves are presumably in a dynamic fluid state with
continual movement and transposition of their macromolecular constituents
(Savage, 1983). One of the many functions of the intestine is to prevent lumen bac-
teria and endotoxins from reaching systemic organs and tissues. Failure of the intes-
tinal barrier function results in the systemic spread of bacteria from the gut to
systemic organs, a process that is known as translocation (Allori et al., 2000). Berg
(1992, 1995, 1999) has defined several routes for bacterial translocation that may be
induced by disruption of the ecological equilibrium in the intestinal tract which
enables bacterial overgrowth, increased permeability of the mucosal barrier, includ-
ing disruption, and deficiencies in the host immune system. Interestingly, virulent
strains of certain enteric pathogens were also observed to associate with the intes-
tinal mucosa, whereas avirulent mutants did not (Drucker et al., 1967; Smith and
Halls, 1968; Bertschinger et al., 1972).
The intestinal mucosa undergoes a continuous renewal process whereby prolif-
erating cells differentiate predominantly to enterocytes that migrate up the villus and
are sloughed into the lumen from the villus tip (Wright and Alison, 1994). During
this migration process, the enterocytes acquire differentiated functions in terms of
digestion, absorption and mucin secretion (Imondi and Bird, 1966; Weiser, 1973;
Meddings et al., 1990; Traber et al., 1991; Ferraris et al., 1992; Thomson et al.,
1994). The normal flora of the gastrointestinal (GI) tract contains many diverse
populations of bacteria that play a role in the development and well being of the host
(Berg, 1990, 1996). This is an area of novel research and, by way of example,
Freitas et al. (2001) have shown that heat labile soluble factors of Bacteroides
thetaiotaomicron, a common gastrointestinal tract inhabitant, increase galactosyla-
tion of target cells. In poultry, bacterial colonization of the mucosal surfaces occurs
in the first few hours of life. The majority of the microorganisms are inhaled or
ingested by the neonatal birds. However, some bacterial pathogens including E. coli
and Salmonella may be acquired by transovarial infection and subsequently
the neonatal bird may hatch already infected. In healthy, adult animals, the bacterial
composition of the mucosal surfaces remains relatively stable. However, that stability
may be broken. Internal factors such as hormonal shifts (Havenaar and Huis in’t
Veld, 1992) and external factors such as feed deprivation, antibiotic administration,
obstruction and radiation (Tannock and Savage, 1974; Berg, 1981; Lenener et al.,
1984; Lizko et al., 1984; Deitch et al., 1990; Havenaar and Huis in’t Veld, 1992)
260 R.M. La Ragione, D.G. Newell and M.J. Woodward
may induce sudden flora changes. Although many of these quoted studies refer to
the mouse model, it is highly likely that the same events are also probable in avian
species. Under such circumstances, pathogenic and opportunistic pathogenic organ-
isms may be able to colonize, leading to localized lesions, bacteraemia and or even
septicaemia (Berg, 1999).
The bacterial composition of the gut microflora varies considerably from one
region of the GI tract to another. For example, in poultry the crop is lined with
Lactobacilli, which form a close association with the epithelium and are involved in
fermentation processes (Barnes et al., 1972, 1980; Fuller and Brooker, 1974).
Additionally, Lactobacilli are the predominant culturable flora in the crop and small
intestine, whereas obligate anaerobes dominate the caeca with Coliforms such as
E. coli found mainly in the large intestine. The natural microflora also varies with
age with a greater diversity of organisms found in older birds. However, it has been
shown that as soon as chicks hatch, even before feeding, several species of bacteria
may be found in the caeca (Barnes, 1982). Barnes et al. (1980) examined many
groups of commercial and experimental chickens and found that the microflora of
day-old chicks consisted of combinations of Streptococci, Coliforms or Clostridia.
Interestingly, Lactobacilli were never found in the caeca of day-old chicks whereas
various other organisms were isolated, some of which are not normally found in
adult birds. These included Pseudomonas aeruginosa and Proteus spp. From 3 days
of age, the caecal flora was shown to be remarkably consistent, and Lactobacilli
were present in high numbers together with Coliforms, Streptococci and Clostridia.
However, it was reported to be difficult to isolate any non-sporing anaerobes that
only established after the facultative aerobes such as E. coli had multiplied. It is pos-
tulated that the facultative anaerobes contribute to environmental changes in the GI
tract so that by 2 weeks of age the non-spore-forming anaerobes colonize to become
the predominant flora (Barnes and Impey, 1970).
2. AVIAN BACTERIAL PATHOGENS

2.1. Escherichia coli
Escherichia coli is the aetiological agent of numerous disease syndromes in poultry
including colisepticaemia, egg peritonitis, yolk sac infection and coligranuloma
(Jordan and Pattison, 1996). Interestingly, numerous serotypes of E. coli including
those implicated in disease are found as commensals on the mucosal surfaces
of clinically healthy birds. Colonization of the alimentary and respiratory tracts
with these serotypes occurs in the first few hours of life (fig. 1). The source of these
primary colonizers is commonly faecal contamination of housing and occasionally
transovarian infection. It has been demonstrated in experimental infection that
E. coli isolates of non-avian origin, such as human O157:H7 isolates, are also able to
colonize the avian host (Schoeni and Doyle, 1994; Best, personal communication).
Siccardi (1966) identified 74 of 154 (48%) of the known serotypes present in
Fig. 1. Scanning electron micrograph

(magnification × 3500) of chicken tracheal
tissue infected with E. coli O78:K80 (previously
published in La Ragione et al., 2000a).
commercial poultry and amongst these were serotypes O1:K1, O2:K1, O3, O6, O8,
O15, O18, O35, O71, O74, O78:K80, O87, O95, O103 and O109 which are recog-
nized avian pathogenic E. coli types. Of these O1:K1, O2:K1, O8, O35 and
O78:K80 are very common (Sojka and Carnaghan, 1961). However, clinical disease
only occurs when there are predisposing factors.
It has been shown that several bacterial factors influence colonization and
include, amongst others, surface antigens that may be elaborated under favourable
conditions (La Ragione et al., 2000a). It has also been shown that commensal
isolates not associated with disease are often less able to colonize to the same degree
as isolates directly associated with clinical disease. The vast majority of E. coli of
avian origin are capable of elaborating flagella and possibly three distinct morpho-
logical types of fimbriae, type 1, curli and P, respectively.
Type 1 fimbriae are the most commonly cited fimbriae on E. coli isolated from poul-
try irrespective of source (fig. 2). Additionally type 1 fimbriae have been shown to be
expressed in vivo in the chicken (Pourbakhsh et al., 1997). It has been hypothesized
that type 1 fimbriae recognize receptors at the epithelial surface and are involved
in intimate attachment to epithelial cells, a prerequisite for subsequent invasion.
Fig. 2. Transmission electron micrograph

(magnification × 525 000) of E. coli O78:K80
showing peritrichous type 1 fimbriae.
Type 1 fimbriae have also been shown to adhere to mucus thus aiding colonization
(Klemm, 1985; Mooi and De Graaf, 1985). It has been demonstrated experimentally
that type 1 fimbriae are important in adherence to avian gut and tracheal explant
tissue. Additionally, isogenic type 1 fimbriae mutants constructed in an avian E. coli
O78:K80, were reduced in their ability to adhere and invade cultured epithelial cells
and explant tissues (La Ragione et al., 2000a). Also, knockout mutants lacking type 1
fimbriae have been shown to be attenuated in terms of colonization, invasion and
persistence in the specific pathogen free (SPF) chick compared to the wild-type
parent strain (La Ragione et al., 2000b).
Curli fimbriae are thin, coiled filamentous structures found on the bacterial sur-
face of E. coli and Salmonella spp. (Olsen et al., 1989; Collinson et al., 1993) and
are commonly associated with avian E. coli clinical disease isolates. Curli mediate
bacterial binding to extracellular matrix and serum proteins such as fibronectin,
laminin, plasminogen and plasminogen activator protein (Olsen et al., 1989, 1993;
Arnqvist et al., 1992; Sjobring et al., 1994; Hammar et al., 1995). Also, curli have
been cited as important in attachment to avian gut tissue and in pathogenesis in
experimentally infected SPF chicks (La Ragione et al., 2000a,b). However, their
exact role in pathogenesis is unclear but it is possible that curli fimbriae mediate
aggregation of curliated bacterial cells and so enhance the number of bacteria colo-
nizing epithelial surfaces (Collinson et al., 1993).
P fimbriae have been cited as important in the pathogenesis of urinary tract infec-
tions in the mammalian host (Hagberg et al., 1983; Lindberg et al., 1984; Roberts
et al., 1989) but their role in colonization in the avian host is unclear. P fimbriae
do not appear to be involved in bacterial adherence to tracheal or pharyngeal cells
in vitro (Van den Bosch et al., 1993; Vidotto et al., 1997). However, by immuno-
fluorescence P fimbriae have been shown to be expressed in the lungs, air sacs
and internal organs of experimentally infected chickens (Pourbakhsh et al., 1997)
and this may indicate a role in the later stages of infection as well as in initial
colonization.
Many non-fimbrial adhesins (NFAs) have been described on E. coli that are elab-
orated by many diverse serotypes, some of which are found colonizing avian species
(Schmidt, 1994). These NFAs are defined as not showing any detectable ordered
structure on the bacterial surface as visualized by electron microscopy. However,
they may appear as amorphous masses and appear similar to capsule. The distribu-
tion of fimbrial operons among E. coli is assumed to be widespread and, therefore,
it is assumed that these are common mechanisms whereby both pathogenic and non-
pathogenic E. coli may colonize.
Flagella have been cited as important in colonization for many pathogens in a
number of hosts (Yancey et al., 1978; Montie et al., 1982; Attridge and Rowley,
1983; Smyth, 1988; Allen-Vercoe and Woodward, 1999a,b; Dibb-Fuller et al., 1999;
Lee et al., 1999). It is thought that flagella enable bacteria to swim through the
mucus sheet covering the mucosal surfaces and reach the underlining epithelium,
where flagella may act as an anchor, subsequently allowing fimbriae to attach

intimately (Smyth, 1988; La Ragione et al., 2000a). E. coli are regarded to be
monophasic, possessing a single flagella antigen type that may be phase on or phase
off. The regulatory mechanisms controlling expression are complex and linked with
environmental factors. Interestingly, only one serotype of E. coli, O148 which is
not regarded as an avian pathogenic serotype, has been described as biphasic,
namely possessing two antigenic types of flagella and therefore is similar to many
Salmonella serovars (Ratiner, 1999). Additionally, motility conferred by the flagella,
aids bacteria to move towards chemical stimuli such as food sources and away
from adverse environmental conditions (Macnab, 1987a,b). However, aflagellate
bacterial avian pathogens, such as S. gallinarum and S. pullorum (see below), have
been described which lends further support to the concept that colonization is
multifactorial.
2.2. Salmonella
S. enterica serotype Gallinarum biotypes Gallinarum and Pullorum cause disease in
many avian species. Both biotypes are antigenically very similar but lack flagella,
however, and cause two distinct systemic diseases – fowl typhoid and pullorum dis-
ease (Shivaprasad et al., 1997). Transmission is environmental, largely faecal–oral,
and transovarial with colonization starting in the distal ileum and then the caecum
although colonization of the gastrointestinal tract is considered to be poor by
comparison with other Salmonella serotypes (Barrow et al., 1987, 1994). Clinical
signs are inconsistent but rapid death in the very young and copious diarrhoea is
often noted. Mortality rates may be high and systemic spread and lesions in many
organs may be noted. Only Gallinarum infection may result in mortality in birds
aged 3 weeks or older. Prince and Garren (1966) described mortality and resistance
to infections by S. gallinarum in certain inbred lines of poultry, a finding that has
been confirmed in more recent studies (Bumsted and Barrow, 1993). Egg transmis-
sion and the cycle of S. pullorum infection has been well documented by Rettger and
Plastridge (1932) who indicated that those birds surviving infection often become
carriers that consistently contaminate the environment.
Disease caused by non-host-specific Salmonella infections is uncommon and is
usually seen in chicks, poults or ducklings under 2 weeks of age and rarely in birds
over 4 weeks of age. Clinical signs are not pathognomic and diagnosis relies on bac-
terial isolation. The serotypes of current concern for human health are Salmonella
enterica serotypes Typhimurium and Enteritidis, although others such as Hadar,
Heidelberg and Kentucky, amongst others, are associated with human illness mediated
by poultry as the vector. The main site for colonization by Salmonella in the intestinal
tract of chickens is the caecum, however, other sites may be colonized including the
crop (Fanelli et al., 1971; Snoeyenbos et al., 1978). Barrow et al. (1988) reported that
the majority of Salmonella organisms in the caecum were isolated from the lumen
rather than epithelial homogenates. Evidence has been gained to support the hypoth-
esis that the advantage of caecal colonization, an organ with a low flow rate of
contents, could be to provide an inoculum for fresh chyle when the caecum is
refilled after emptying (Hill and Smith, 1969; Barrow et al., 1988; Craven and
Williams, 1997). However, the number of Salmonellae associated with the epithe-
lium in the crop frequently exceeded the numbers in the lumenal contents (Craven
and Williams, 1997; Barrow et al., 1988).
Salmonellae produce a variety of putative virulence determinants encoded by up
to seven “pathogenicity islands” that are associated with all aspects of systemic
disease. For the avian species-specific serovar S. gallinarum, Jones et al. (2001)
showed that virulence in the chicken requires the Salmonella pathogenicity island
(PAI) 2 type III secretion system but not PAI 1. Other virulence determinants
include haemagglutinins, adhesins, invasins, fimbriae, exotoxins, endotoxins, type
III secretion systems and so forth (Duiguid et al., 1966; Formal, 1983; Freter, 1981;
Koo et al., 1984; Halula and Stocker; 1987; Galan and Curtiss, 1989; Baumler et al.,
2000). Apart from commonly shared fimbrial operons (see below), the virulence
plasmids of S. gallinarum, S. pullorum and S. dublin shared genes that with homol-
ogy K88 fimbrial genes and, for S. gallinarum, mutants in these genes resulted in
attenuation for all aspects of chicken pathogenesis (Rychlik et al., 1998). In the
mouse model, S. Typhimurium virulence is dependent on its ability to attach, invade
and multiply in the Peyer’s patches (Carter and Collins, 1974). Interestingly,
S. Typhimurium also attaches to cells of the intestinal epithelium (Finlay et al.,
1988; Galan and Curtiss, 1989). The role of specific fimbriae (LPF and PEF; see
below) is important for initial attachment to Peyer’s patches (Baumler et al., 1997,
2000). However, loss of flagella and loss of either mannose-sensitive or mannose-
resistant haemagglutination structures reduces the organism’s ability to adhere to or
invade epithelial cells in vitro (Curtiss et al., 1993). Interestingly, non-motile or non-
haemagglutinating mutants are fully virulent, following oral mouse challenge
whereas mutants deficient in both are attenuated (Carsiotis et al., 1984; Lockman
and Curtiss, 1990, 1992; Curtiss et al., 1993). S. pullorum and S. gallinarum
are fully virulent although lacking flagella. However, Holt and Chaubal (1997)
have demonstrated that these serovars are capable of expression of these organelles
in vivo in the chicken, which suggests tight regulation of this function and a prob-
able role in colonization.
Salmonella enterica serotype Enteritidis is able to elaborate monophasic flagella
and at least five morphological distinct fimbriae have been observed, although the
genome sequence indicates a genetic potential to express up to 12 discrete fimbriae
(Baumler, personal communication). The well characterized S. Enteritidis fimbriae
include SEF14 fimbriae that are serovar restricted to most group D Salmonellas
only, the curli orthologue SEF17, the type 1 orthologue SEF21, the long polar fim-
briae (LPF) and plasmid encoded fimbriae (PEF). In gut explant adhesion assays it
was observed that flagella were important in adhesion to avian proximal gut tissue,
but fimbriae were not (Allen-Vercoe and Woodward, 1999a; Dibb-Fuller et al.,
1999). In these studies it was observed that motility conferred by flagella and the
presence of the flagellum itself, were important in adhesion to avian gut tissue.
It may be hypothesized that the flagella either increase bacterial surface area for
initial contact or may help in overcoming repulsive forces. Interestingly, Jones et al.
(1992) reported that the direction of flagellar rotation affected the ability of
S. Typhimurium to invade cultured epithelial cells and, in addition flagella have
been implicated in assisting survival of S. Typhimurium within murine macrophages
(Weinstein et al., 1984). It has been observed that type 1 fimbriae of S. Typhimurium
are important in adhesion to gut epithelial cells (Takeuchi, 1967; Lockman and
Curtiss, 1992; Vidotto et al., 1997) and invasion (Ernst et al., 1990). In studies
conducted in mice and in 3-week-old chicks, Turner et al. (1998) used a signature
tagged mutagenesis approach to identify factors essential for persistence. LPS
O antigen amongst many other factors was found to be an important factor whereas
fimbriae were not identified by this approach. Several studies have demonstrated
that lipopolysaccharide (LPS) of S. Typhimurium plays a role in the colonization
of mice and chickens (Craven, 1994; Licht et al., 1996; Turner et al., 1998).
Interestingly, LPS endotoxin promotes translocation of bacteria from the gastro-
intestinal tract, at least in mouse studies (Deitch et al., 1987).
In vivo studies with S. Enteritidis showed that aflagellate mutants were attenu-
ated but the role of fimbriae was less clear although they did contribute to persist-
ence (Allen-Vercoe et al., 1999; Allen-Vercoe and Woodward, 1999b; Dibb-Fuller
and Woodward, 2000). In 5-day-old SPF chicks that have a gastrointestinal
microflora not dissimilar to that of an adult bird (Coloe et al., 1984), similar results
were observed. However, it had been reported that a natural microflora significantly
reduces the susceptibility of these animals to colonization by Salmonella spp.
(Barrow and Tucker, 1986; Berchieri and Barrow, 1990; Duchet-Suchaux et al.,
1995). Collectively, these studies suggest that motile flagella may contribute to gut
colonization whereas the contribution, if any, from fimbriae was too subtle to
observe in the models used. It must be borne in mind that although significant dif-
ferences were not seen between different ages of birds, only caecal contents were
examined and differences may have been observed if epithelial surfaces and whole
tissue were tested separately.
2.3. Campylobacter
Particular concern about poultry and poultry products as a source of human
food-borne illness has arisen in recent years with the S. Enteritidis epidemic of the
late 1980s and early 1990s, and the increasing awareness of Campylobacter jejuni.
Of the food-borne pathogens carried by poultry, Campylobacter, and, in particular
C. jejuni, is now recognized as the most significant in terms of human illness and
potential economic impact.
C. jejuni and C. coli colonize the gastrointestinal tract of most animals asympto-
matically (Shane, 1991; Hu and Kopecko, 2000) and are primarily associated with
disease in susceptible humans in the industrialized world (Van Vlient and Ketley,
2001). All members of the genus Campylobacter are motile, Gram-negative
microaerophilic, spiral, uniflagellate organisms (Skirrow and Benjamin, 1980) but
C. jejuni and its close relative C. coli are thermophilic, growing optimally at 42°C
(Genigeorgis, 1986) and appear to have evolved to colonize the avian GI tract.
Surveys show that 15−95% of broiler flocks are naturally infected (Blaser, 1982;
Newell and Wagenaar, 2000) and subsequent faecal contamination of carcasses
results in up to 75% of retail poultry product contamination. Campylobacter is rec-
ognized as the most significant in terms of human illness and potential economic
impact. It is estimated that there are 500 000 cases of Campylobacteriosis per
annum in the United Kingdom alone.
Chickens appear to acquire Campylobacters horizontally from their environ-
ment. Although oviducts can be colonized (Camarda et al., 2000), hatched chicks
are Campylobacter-negative. Flocks do not usually become positive until 2−3 weeks
of age (Newell and Wagenaar, 2000). Interestingly, experimental infection can be
established in chicks by oral dosing from day of hatch onwards (Ruiz-Palacios et al.,
1981). Once colonization occurs in one animal, it spreads rapidly throughout the
flock, reaching levels of up to 10 6 −10 7 CFU/g in the caecal contents (Corry and
Atabay, 2001). This colonization appears to have no adverse effects on the flock
health. Thus, in poultry, Campylobacters act as a highly successful GI tract com-
mensal. However, in ostrich chicks, C. jejuni is associated with enteritis and even
death (Newell, 2001). A severe disease “Vibrionic hepatitis” was prevalent in the
1950s and 1960s in chickens in North America and Europe, and may have been
caused by C. jejuni (Peckham, 1984).
The reason for the lag phase in colonization is intriguing. It occurs even in organ-
ically reared chicks surrounded by an environment heavily contaminated with
campylobacters suggesting that lack of exposure is not the explanation and indicat-
ing that the immature avian GI tract is refractory to infection. Newly hatched chicks
have circulating and mucosal antibodies directed against Campylobacter surface
antigens (Cawthraw et al., 1994) but experimentally infected chicks are susceptible
to infection (Cawthraw et al., 1996) suggesting that these antibodies are not protec-
tive. Alternative explanations include antimicrobial feed additives and competitive
members of the native flora. An understanding of the mechanism of the lag phase
may be an important factor in the development of targeted strategies for the control
of this organism.
Little is known about the nature and mechanism of avian GI tract colonization
by Campylobacters. Experimentally, the colonizing organisms are confined prima-
rily to the intestinal mucus layer in the crypts of the intestine and caecum (Beery
et al., 1988). There appears little close association of Campylobacters with intestinal
epithelial cells but Campylobacters have been recovered from extraintestinal sites
including the liver and spleen. This suggests that adherence (Ziprin et al., 1999) is a
rare or possibly intermittent event that may lead to invasion across the mucosa.
Motility is an essential factor for Campylobacter colonization of the intestinal
mucous layer (Newell et al., 1985; Lee et al., 1986; Szymanski et al., 1995). This
motility is conferred by polar flagella, and combined with their “cork-screw” form
allows them to efficiently penetrate the mucous layer. The possession of active
flagella (Newell et al., 1985; Guerry et al., 1991) and the ability to undergo chemo-
tactic motility (Yao et al., 1994; Penn, 2001) are well established prerequisites for
virulence in mammalian systems. However, in experimental chick models, motility
may not be so important (Wassenar et al., 1993) presumably because the primary
colonization site, the caecum, has restricted mucus flow and, once organisms have
reached this site, colonization can be persistent despite lack of motility.
Flagella also appear to play a role in invasion into host cells (Wassenar et al.,
1991; Penn, 2001), possibly reflecting some adhesive capacity to the host intestinal
cells (Newell et al., 1985). There are two tandem flagellin genes, flaA and flaB, and
mutants unable to express the products of these genes exhibit differences in motil-
ity and colonization, as well as invasive potential (Wassenar et al., 1991, 1994a;
Nuitjen et al., 1992; Penn, 2001). Several other putative adhesins have been described.
Early observations of fimbrial-like structures on C. jejuni (Doig et al., 1996) appear
to be artefactual (Gaynor et al., 2001). Experimental studies suggest that the outer-
membrane proteins (OMPs) and lipopolysaccharides (LPS) may play roles in
colonization (McSweegan and Walker, 1986; McSweegan et al., 1987; Fauchere et al.,
1989; Ziprin et al., 1999). There are a number of surface exposed antigens (PEBs)
which have been investigated as adhesins. Pei et al. (1998) showed that peb1A mutants
had reduced adherence in in vitro models and significantly reduced colonization in the
mouse but still colonize chicks. Interestingly, cadF (fibronectin-binding protein)
mutants were unable to colonize newly hatched chicks (Ziprin et al., 1999).
2.4. Spirochaetes
Avian intestinal spirochaetosis (AIS) is a poorly understood disease. Although it
was first described by Fantham in 1910, only in the past two decades have detailed
pathogenesis studies been undertaken (Dwars et al., 1989; Swayne et al., 1995). AIS
is defined as a sub-acute to chronic, non-septicaemic intestinal disorder character-
ized by variable clinical illness, morbidity and mortality (Swayne and McLaren,
1997). AIS has been reported in broilers, laying hens, pheasants, geese and ducks
(Swayne and McLaren, 1997; Webb et al., 1997). The spirochaetes that colonize the
intestinal tract of birds are a heterogeneous group of bacteria, with few character-
ized sufficiently for taxonomic identification. These pathogenic spirochaetes may be
an integral part of the autochthonous intestinal flora, or may be opportunistic colo-
nizers. Of the better characterized Spirochaetes, Brachyspira (Serpulina) pilosicoli
(Trott et al., 1996) colonizes the crypts of the lower intestinal tract of the pig, but
may extend to the small intestinal tract. More needs to be done in avian species
regarding colonization by these highly motile organisms, but all show polar attach-
ment to the mucosa. Trampel et al. (1994) studied natural AIS cases and showed by
dark-field and light microscopy that spirochaetes were located in the caeca.
Typically, the apical surfaces of caecal enterocytes were covered by a dense layer of
spirochaetes aligned parallel to each other and perpendicular to the mucosal surface.
Sueyoshi et al. (1986) orally inoculated Treponema hyodysenteriae into young
broiler chicks and the organisms were present both on the mucosal surface and in
the deep crypts of the caecum 7 and 14 days later. The numbers colonizing were
dose dependent but were readily observed by light microscopy. The caeca of chicks
infected with treponemes were atrophied and the lumen was filled with a white
watery fluid instead of digested feed. The lesions were primarily confined to the
caecum and comprised desquamation of epithelial cells, oedema, leucocytic infil-
tration and haemorrhage of the mucosa. This model was developed further by
Sueyoshi and Adachi (1990) as a surrogate model for swine dysentery. Trott and
Hampson (1998) dosed specific pathogen free chicks aged 1 day with strains of five
different species of intestinal spirochaete originally isolated from pigs or humans.
The findings in this model were similar to those described above with, by way of an
example, a virulent strain of Serpulina hyodysenteriae (WA 15) colonizing the
chicks well to cause retarded growth rate and histological changes, including caecal
atrophy, epithelial and goblet cell hyperplasia, and crypt elongation. Sagartz et al.
(1992) described necrotizing typhlocolitis in 13 juvenile common rheas (Rhea
americana) from three separate, geographically isolated Ohio flocks, with mortality
ranging from 25 to 80%. At post mortem examination, a diphtheritic membrane cov-
ered ulcerated caecal mucosa was observed. Caecal sections showed necrosis and
granulomatous-to-suppurative inflammation that extended into the submucosa and
often surrounded large eosinophilic colonies of bacteria. Warthin-Starry staining
showed these colonies to be composed of entangled spirochaetes that invaded the
submucosa and frequently were present transmurally. Muniappa and Duhamel
(1997) have identified a partially heat stable, subtilisin-like, serine protease in the
outer membrane of all spirochaetes. They suggest this factor may be essential for
survival in the intestinal environment and may indirectly contribute to intestinal
damage caused by pathogenic spirochaetes during association with the mucosal
surface of the host.
2.5. Clostridia
A number of species of Clostridium are normal inhabitants of the intestinal tract
of healthy chickens and can be recovered from the intestines of chicks from
approximately 1 week of age (Ficken and Wages, 1997). However, many, including
C. botulinum, C. colinum, C. septicum and C. perfringens cause disease in poultry.
The main mechanism of virulence is the production of toxins. Yet, other putative
virulence determinants such as the mechanisms of colonization have not been

studied in detail.
C. perfringens (mainly type A or type C toxin) is the aetiological agent of avian
necrotic enteritis (NE), which is a sporadic disease of 4-week or older chickens and
turkeys. NE was first described in 1930 by Bennetts and later by Parish (1961) and is
economically very important. The disease is initiated by a predisposing factor such as
poor husbandry practices, intestinal mucosal damage caused by high-fibre litter or
concurrent infection with Coccidia (Al-Sheikhly and Al-Saieg, 1980). Kimura et al.
(1976) investigated the bacteriological and histopathological changes in the caeca of
young chickens after infection with sporulated oocysts of Eimeria tenella. Lactobacilli
and Bifidobacteria showed a remarkable decrease in number on the fifth day after
infection when destruction of mucosa along with severe haemorrhaging was noticed.
Clostridium perfringens proliferated 5 days after infection following the decline of
Lactobacilli and Bifidobacteria. Proliferation of Clostridia was so intense that the
number was almost a million times greater than that of the uninfected chicken. Other
predominant bacteria, such as Bacteroidaceae, Catenabacteria and Peptostreptococci
showed only moderate and temporal decrease in number during the infection.
Colonization of the mucosa by Clostridia has not been described in detail largely
because the focus of study has been towards the toxins that mediate the necrosis. For
example, Al-Sheikhly and Truscott (1977) reproduced necrotic enteritis by intraduode-
nal infusion of chicks with Clostridium perfringens type A. Typical lesions of necrotic
enteritis, characterized by oedema in the lamina propria and desquamation of epithelial
cells, were seen as early as 5 h after infusion. Large numbers of Clostridia were seen
among these sloughed cells and clumps of clostridia were obvious among the necrotic
tissue. Thus it is recognized that C. perfringens can proliferate in the large intestine and
caeca with subsequent migration to the small intestine, or sites of mucosal damage
caused by coccidial infection that leads to further proliferation and production of toxin
that results in tissue damage and necrosis. In severe manifestations necrosis can extend
the entire width of the intestinal mucosa (Murakami et al., 1989) although acute catarrh
without necrosis is more common in the lower intestine. Histological examination of
tissues reveals Gram-positive bacilli that are often detected in the lamina propria and
frequently attached to cellular debris (Songer, 1997).
In a hamster model, Borriello and Barclay (1985) demonstrated that a non-toxigenic
strain of C. difficile adhered to gut mucosa and afforded some protection against toxi-
genic trains. It is proposed that the protection afforded by the non-toxigenic strains may
be due to competition for ecological niches. This approach could be investigated for NE
in chickens.
2.6. Mycoplasma
Mycoplasmas are highly fastidious microorganisms that differ from other eubacte-
ria in that they are significantly smaller and lack a cell wall. This characteristic
accounts for their “fried egg” colonial morphology and total insensitivity to antibi-
otics that have cell wall biosynthesis as a target (Kleven, 1998). Mycoplasmas are
normal inhabitants of the mucosal surfaces of poultry and gain entry into the host
through the respiratory tract or via the infected embryo. A number of serotypes of
Mycoplasma have been isolated from healthy poultry. However, M. gallisepticum
and M. synoviae cause disease in chickens, M. iowae in chickens and turkeys and
M. meleagridis solely in turkeys. Mycoplasmas cause a variety of clinical syndromes
in poultry including keratoconjunctivitis, chronic respiratory disease, embryonic
mortality, skeletal abnormalities, exudative synovitis, tendovaginitis, bursitis, salp-
ingitis and airsacculitis (Yoder, 1991). The severity and sometimes the appearance
of disease may be influenced by concomitant infection with other pathogens or
debilitating factors (Cullen et al., 1977).
As with many bacterial pathogens, a characteristic that permits Mycoplasma
to effectively establish infection and proliferate within the host is associated with
its capability of attachment to tissues, mainly epithelial linings of the oviducts
and respiratory tract (Fiorentin et al., 1998). Adherence to the surface receptors
of host cells by Mycoplasmas is generally mediated by adhesins expressed on
the cell surface (Kahane et al., 1985; Razin and Jacobs, 1992). At least three cytad-
herence proteins are involved in M. gallisepticum adherence and colonization
(Fiorentin et al., 1998). Keeler et al. (1996) cloned and sequenced the 3366-
nucleotide open reading frame, mgc1, that encodes a 150-kDa cytadhesin-like pro-
tein. The 1122-amino-acid protein, MGC1, has characteristics of a class I membrane
protein and has homology with the MgPa cytadhesin of M. genitalium and the P1
cytadhesin of M. pneumoniae. Hnatow et al. (1998) identified a second cytadhesin-
like protein, MGC2, and Boguslavsky et al. (2000) identified a third, PVP-A. The
encoding genes have been sequenced and all share homology one with another
and other related Mycoplasma adhesins. Variable expression, possibly phase-on
phase-off, for the cytadhesins is observed as is variability in their respective sizes.
Functional studies with attachment inhibition assays have confirmed their role
(Hnatow et al., 1998; Boguslavsky et al., 2000). These data suggest that there is a
family of cytadhesin genes conserved among pathogenic Mycoplasmas infecting
widely divergent hosts. Additionally, some Mycoplasmas possess surface organelles
that have been implicated as potential colonization factors. The first to be described
was the “bleb” of M. gallisepticum, a small organelle at one or both poles of the cell
(Maniloff et al., 1965).
2.7. Other pathogens

Avian tuberculosis caused by Mycobacterium avium was a common disease of
domestic fowl that is largely controlled by improved husbandry practices. Bird
density, poor hygiene and deep litter are regarded as influencing factors. The host
range is limited to domestic fowl in the main although some caged birds may be
affected also. Pigs and humans may also be affected by this pathogen. Initial infec-
tion is considered to be by entry of the organisms into the premises by unsanitary
practices. Many studies have focused upon Johne’s disease in ruminants and little
has been done on poultry. In the mouse model, M. avium was found to invade the
intestinal mucosa by interacting primarily with enterocytes and not with M cells
(Sangari et al., 2001). Thus, it may be assumed that colonization of the gastroin-
testinal tract is largely an invasive process. Once within host cells, tubercles develop
in the intestine and large numbers of bacteria may be excreted in the droppings to
spread contamination rapidly. The bacterium is hardy and may survive in the envi-
ronment for prolonged periods. Tubercles may develop in the reproductive tract but
egg transmission is not considered important (Thoen and Karlson, 1991).
Yersinia pseudotuberculosis is found commonly in domestic poultry, other avian
species and rodents. Again, poor hygiene and overcrowding are associated with the
spread of this agent that colonizes via the intestine or through breaks in the skin
(Rhoades and Rimler, 1991a). Many studies on other Yersinia spp. have identified
virulence factors including adhesins and secretory proteins essential for invasion.
Mecsas et al. (2001) used signature-tagged mutagenesis to isolate mutants of
Y. pseudotuberculosis that failed to survive in the caecum of mice after orogastric
inoculation; Yersinia pseudotuberculosis localizes to the distal ileum, caecum, and
proximal colon in this model. Several mutations were in operons encoding compo-
nents of the type III secretion system, including Yop proteins and O-antigen biosyn-
thesis; these mutants were also unable to invade epithelial cells as efficiently as
wild-type Y. pseudotuberculosis. This indicates that one or more Yops and LPS may
be necessary for colonization of the gastrointestinal tract.
Avian pasteurellosis is caused by a number of different bacteria that include
Y. pseudotuberculosis, Moraxella spp. and Pasteurella multocida. P. multocida is
the highly infectious cause of fowl cholera of which several pathogenic and highly
invasive serotypes have been defined. Access is gained to the host via broken skin
and nasal, conjunctival and intestinal routes. The organisms of this group are all very
susceptible to air drying and airborne routes are not considered important in their
spread (Rhoades and Rimler, 1991b).
Of the Gram-positive cocci, Staphylococcus aureus readily colonizes poultry
skin and the nasal passages and is considered ubiquitous. It is well established that
S. aureus possesses various bacterial adhesins that bind skin and various cell types
and that adhesion is mediated by fibronectin-binding protein (Fnbp), fibrinogen-
binding protein (Clf) and collagen-binding protein (Cna). Recent studies (Cho et al.,
2001; Massey et al., 2001) have proven that fibronectin and fibrinogen, but not col-
lagen, play major roles in the enhanced adherence of S. aureus to skin and cells.
It must be assumed they play similar roles in colonization of avian species. However,
the co-aggulase positive strains are opportunistic pathogens that are associated with
many clinical outcomes after translocation (e.g. having gained access to the bird via
broken skin). Arthritis, synovitis, spondylitis, other general abscesses, dermatitis,
septicaemia and yolk infection have been recorded frequently. Enterococcus

faecalis is a common inhabitant of the intestine, and may invade causing septi-
caemia and endocarditis, as are S. aureus, Streptococcus zooepidemicus and
Erysipelothrix insidosa (Skeeles, 1991; Wages, 1991). With regard to Enterococci,
adherence to intestinal, urinary tract epithelial cells and heart cells is by means of
adhesins expressed on the bacterial surface (Johnson, 1994). The expression of these
is growth phase dependent and is enhanced if the organisms produce a proteina-
ceous surface material that aggregates bacteria and that also facilitates plasmid
transfer (Johnson, 1994). Enterococci are capable of inducing platelet aggregation
and tissue factor-dependent fibrin production, which may be relevant to the patho-
genesis of enterococcal endocarditis (Johnson, 1994).
Erysipelothrix rhusiopathiae is a common pathogen of turkeys, but may also
affect other avian species. Faecal shedding is noted, as is frequent carrier status. The
source of infection is largely environmental and entry to the bird is via damaged
mucosal layers. The early work of Sadler and Corstvet (1965) demonstrated the
requirement for direct access to the host because oral, nasal and conjunctival inoc-
ulation with known pathogenic strains did not cause morbidity or mortality. The
closely related bacterial species Listeria monocytogenes causes sporadic outbreaks
with up to 40% mortality and which was first reported as a serious problem in the
poultry industry in the mid 1930s. The source was believed to be silage, especially
from maize, rye and grass, and it also affected sheep and cattle, both considered risk
factors in transmission. The infection is septicaemic and the bacterium is shed in
nasal exudate and faeces. With regard to mucosal colonization, little is known, espe-
cially in avian species. Bubert et al. (1992) showed that the p60 extracellular protein
of Listeria monocytogenes is required for adherence to and invasion of 3T6 mouse
fibroblasts but not for adherence to human epithelial Caco-2 cells. However, Cowart
et al. (1990) proposed that attachment of Listeria to eukaryotic cells occurs by inter-
action of the microbial alpha-D-galactose with that of a eukaryotic galactose receptor.
The role of these factors in the avian species is untested. Of concern is contamination
by this organism of the skin of processed carcasses (Gitter, 1976), especially as the
organism may multiply at typical domestic storage temperatures (Mead et al., 1990).
3. THE NATIVE COMMENSAL FLORA

The native commensal flora of avian species is highly complex with at least 200 dis-
tinct species either identified after isolation and classic bacteriology or partially
identified by genetic means. Direct analysis of the microbiota of GI tract contents
by 16S rRNA analysis (Suau et al., 1999) as well as by DNA analysis techniques
(Apajalahti et al., 1998) has been used to determine the species and relative num-
bers of types of bacteria in the GI tract. The early work of Barnes and Impey (1970)
demonstrated various distinct predominant classes of bacteria in the caeca of chick-
ens and turkeys. Animals of 5 weeks of age with what may be regarded as a mature
flora possessed Gram-negative non-spore-forming anaerobes, largely Bacteroides

spp., and Gram-positive non-spore-forming anaerobes with Bifidobacteria compris-
ing approximately 80% of the cultivable bacterial population.
Many of the species identified in the intestine also inhabit the oral cavity in mam-
mals and the adherence of these has been studied intensively. Leung et al. (1989)
demonstrated that Bacteroides intermedius possessed four distinct fimbriae and
whereas Bacteroides forsythus possesses an adherence factor, the BSP-A surface
antigen, that binds oral tissues and triggers host immune responses (Sharma et al.,
1998). Similar surface structures play a role in intestinal colonization. Brook and
Myhal (1991) demonstrated that piliated and capsulated strains of Bacteroides
fragilis adhered at least five times greater than the adherence of their non-piliated
and non-capsulated or capsulated only counterparts to the gastrointestinal mucosa.
However, the efficiency of adherence to oral epithelial cells and INT-407 cells varied
between 10 and 40 bacteria per host depending upon the source and strain of the
Bacteroides (Guzman et al., 1997). Also, Baba et al. (1992) showed that the balance
of the normal flora is readily perturbed by infections. In conventional chickens,
fewer Bacteroides vulgatus and Bifidobacterium thermophilum adhered to the
E. tenella-infected caeca than to the uninfected caeca.
The remaining flora comprise Peptostreptococcus, Prevotella, Ruminococcus,
Propionibacterium, Fusobacterium, Lactobacillus and Eubacterium, amongst many
other genera. Particular interest has focused on certain species because of their asso-
ciation with reducing colonization by avian pathogens and these will be discussed
in detail below.
3.1. Lactic acid bacteria

Large populations of Lactobacilli inhabit the digestive tracts of fowl. Interestingly,
some gastrointestinal strains of Lactobacilli have the ability to adhere to and colo-
nize the surface of stratified squamous epithelium in the oesophagus, crop and stom-
ach whereas other Lactobacillus strains appear to be inhabitants of the
gastrointestinal lumen only (Fuller and Turvey, 1971; Fuller and Brooker, 1974).
Lactobacilli, irrespective of origin and whether shed from epithelial surfaces or mul-
tiplying in ingested food, permeate all regions of the digestive tract in a number of
animals (Fuller et al., 1978; Tannock, 1987; Gusils et al., 1999a). Although it has
been suggested that several factors are associated with host specificity, one signifi-
cant speculation is that Lactobacilli adhere to epithelial surfaces by interactions
occurring between specific molecules on bacterial cells and on the gastrointestinal
surface of the host. Fuller (1975) reported the possible involvement of a carbohy-
drate in the adhesion process. Henriksson et al. (1991) reported that Lactobacillus
strains adhere to porcine stomach epithelium through proteinaceous components
located on the bacterial surface. Additionally, Savage (1969) demonstrated that
Lactobacilli colonized the surface of non-secreting keratinized epithelial cells in the
stomach of rats and mice, but not the surface of the secreting stomach epithelium.
Interestingly, Tannock (1990) mentioned that carbohydrate-specific molecules
(lectins) contribute to the adherence of Lactobacilli to epithelial surfaces and
Gusils et al. (1999b) found lectin-like structures in the external layers of L. animalis,
L. fermentum and L. cellobiosus. These molecules, present on the cell surfaces,
would favour adhesion to epithelial cells. Also, immuno-stimulating activity of cell
walls from L. casei CRL 431 can be induced by an adhesion phenomenon in which
lectin-like structures are involved (Morata de Ambrosini et al., 1988a,b). Feeding
diets containing Lactobacillus spp. has been shown to enhance the production of
anti-salmonella IgM antibodies and the function of T cells in newly hatched chicks
and poults (Dunham et al., 1993).
3.2. Bifidobacteria
Bifidobacteria are Gram-positive, anaerobic, non-motile, non-spore-forming,
fermentative rods which are normal inhabitants of the mammalian and avian
gastrointestinal tract (Van der Werf and Venema, 2001). It has been reported that
Bifidobacteria have a positive effect on the hosts’ health status and that humans with
higher numbers of Bifidobacteria are less at risk from diarrhoea (Albert et al., 1978;
Saavedra et al., 1994; Heinig and Dewey, 1996). A total of 32 species of
Bifidobacteria have been identified in the genus Bifidobacterium in the present clas-
sification. Bifidobacteria are host specific and age specific. For example, in humans,
B. infantis and B. breve are predominant in infants whereas B. adolescentis and
B. longum are predominant in adults (Mitsuoka, 1982). It has been reported that
Bifidobacteria bind to host mucus glycoproteins and these vary from species to
species (Von Nicolai et al., 1981; He et al., 2001). Bifidobacteria adhere to mucus
and, in human studies, the number of Bifidobacteria in faeces and intestinal contents
reduces with increasing age of the host. Some strains also showed decreased adhe-
sion to mucus isolated from infants compared with that from adults (Ouwehand
et al., 1999). It has been reported that Bifidobacteria competitively exclude other
bacteria such as Proteus vulgarius and Klebsiella pneumoniae (Timoshko et al.,
1981). However, many factors have been shown to reduce the number of
Bifidobacteria in the GI tract of chickens including thermal kestoses (Patterson
et al., 1997). The mechanisms by which Bifidobacteria competitively exclude other
bacteria are beneficial to health and are considered to be: 1) inhibiting the growth of
pathogenic bacteria by producing bacteriocins; 2) lowering the gut pH by acetate
and lactate production; 3) colonization of the gastrointestinal tract; 4) production of
vitamins; and 5) stimulating the immune system (Deguchi et al., 1985; Fuller, 1989;
Mitsuoka, 1990; Ballongue, 1993). Lievin et al. (2000) described two Bifidobacteria
strains that expressed antagonistic activity against S. Typhimurium SL1344 in vitro.
The Bifidobacteria inhibited Salmonella cell entry and killed intracellular
Salmonella in Caco-2 cells and the effector was identified as a low molecular weight
lipophilic molecule. Additionally, in the axenic C3/He/Oujco mouse model, these

two Bifidobacteria strains colonized the intestinal tract and protected mice against a
lethal S. typhimurium oral dose.
In poultry, Bifidobacteria have been shown to be obligate inhabitants of the crop
and intestine of adult birds. In addition Bifidobacteria are found in high numbers in
the intestines. Interestingly, it has been shown that those inhabiting the intestine and
crop are specific to those tissues, respectively (Petr and Rada, 2001).
4. COMPETITIVE EXCLUSION
As early as the late nineteenth century the antagonistic interaction between some
bacterial strains was observed (Pasteur and Joubert, 1877; Metchnikoff, 1907).
However, the therapeutic use of bacterial cultures in human and veterinary medicine
is more recent for two main reasons. First, control of zoonotic pathogens by anti-
biotics is associated with increased antimicrobial resistance amongst many common
bacterial pathogens. Secondly, an increased awareness that antibiotic treatment
perturbs a native and therefore “protective” flora that may predispose to later infec-
tions. Thus, the use of bacteria for prophylaxis has become more commonplace
(Bengmark, 2000).
It has been recognized that the normal intestinal microflora of animals can be
divided into three categories. 1) The autochthonous microbiota – microbes that are
present at high population levels, usually throughout the life of an animal and found
in all members of a host population (e.g., Lactobacilli). 2) The normal microbiota –
populations of microbes frequently present in the digestive tract, but of variable
population size and absent from some populations (e.g., Escherichia coli). 3) True
pathogens (e.g., Salmonella enterica serotypes) – accidentally acquired and capable
of persisting in tissues and causing disease (Dubos et al., 1965).
The phenomenon of controlling or modifying the resident microflora is known
as “competitive exclusion” or the Nurmi concept (Pivnick and Nurmi, 1982).
Competitive exclusion is used either to protect young chicks by early establishment
of adult microflora or to compensate for the side-effects of antibiotic treatment in
older birds. However, little is known about the bacteria responsible for the barrier
effect or the mechanisms involved (Impey et al., 1982; Nuotio et al., 1992; Nuotio
and Mead, 1993).
Protection against colonization with bacterial pathogens depends upon oral
administration of viable non-pathogenic bacteria, especially anaerobes (Schneitz
and Mead, 2000). Indeed, Impey et al. (1982) described a 48-strain mixture of
species that comprised part of the mature flora of chickens that competitively
excluded Salmonella. Notable in that mixed population were 11 Lactobacillus spp.,
10 Clostridium spp., eight commensal E. coli, three Bacteroides spp., three
Streptococcus spp., two Eubacteria spp., two Bifidobacteria spp., various undefined
anaerobes and Bacillus coagulans.
With regard to the colonization of the chick gut by members of the

Enterobacteriaceae, undefined complex cultures were more effective than defined
treatments (Stavric et al., 1991). Also, the most effective exclusion agents were
derived directly from healthy adult chicken intestines (Cameron and Carter, 1992;
Spencer et al., 1998).
Competitive exclusion (CE) cultures may offer alternatives to antimicrobial
agents for disease prophylaxis in poultry (McReynolds et al., 2000). Following the
studies of Nurmi and Rantala (1973), it is widely recognized that the autochthonous
gut microbiota provides an effective barrier in poultry against colonization by human
food-borne pathogens such as Salmonella enterica serotypes Enteritidis and
Typhimurium.
It has been reported that chicks are immunologically naive (Jeurissen et al.,
1989) and prone to rapid and persistent colonization by both commensal and path-
ogenic bacteria in the first 3 to 4 weeks of life (Barrow et al., 1988). Recent studies
by Holt et al. (1999) indicated that the avian immune system may be compromised
by vaccination at 1 day old and that an optimum time for delivery of vaccines was
at about 4 weeks of age. Up to that age, birds remain vulnerable to infection
although maternally derived immunity may reduce colonization by pathogens, but
this is unlikely to be fully protective. Therefore, if vaccines alone cannot militate
against bacterial pathogens, the development of defined, reliable competitive exclu-
sion agents is essential. However, it is interesting to note that Weinack et al. (1984)
showed that the immune system played no role in the control of Salmonella in CE
treated birds and van der Wielen et al. (2000) showed that volatile fatty acid
concentrations were important in controlling microbial populations in the gut.
Additionally, it has been suggested that Bifidobacteria may increase antibody syn-
thesis through its mitogenic influence on B cells. Bifidobacteria induce spleen B cells
to be reactive to transforming growth factor beta 1 and interleukin-5, so resulting in
increased surface IgA expression (Ko et al., 1999). Bifidobacteria have also been
shown to have a protective effect against clostridia in quails (Butel et al., 1998).
Many of the commercially available agents are ill-defined mixtures of adult bird
gut content. However, recent studies have shown that defined single organism agents
can offer good protection to day-old chicks subsequently challenged with patho-
genic organisms. In one study, birds were dosed at 1 day old with Bacillus subtilis
spores and subsequently challenged with avian pathogenic E. coli O78:K80 24 h
later. In these studies, a significant reduction in E. coli O78:K80 colonization and
invasion was seen. Additionally, compared to a control group, the level of faecal
shedding of the pathogenic E. coli was reduced over a 35-day period (Stavric, 1992;
La Ragione et al., 2001).
The criteria for competitive exclusion agents include bile and acid stability, adhe-
sion to the intestinal mucosa, temporary colonization of the gastrointestinal tract,
production of antimicrobial components and safety in medical and veterinary use
(Wesney and Tannock, 1979; Saxellin et al., 1995). The most often used genera are
Lactobacilli and Bifidobacteria (Lee et al., 1999). It has also been suggested that
probiotic supplementation is of greatest benefit when birds are exposed to stressful
conditions (Jin et al., 1997). However, for organisms such as C. jejuni, occupation of
the same apparently unique niche by the competitive agent appears to be necessary
which is probably why competitive exclusion is not consistently successful. It has
been suggested that exclusion may only be achieved using other Campylobacter
strains (Wassenar et al., 1994b). If reduction of pathogenic Campylobacters in the
food chain is the aim, then non-pathogenic, genetically stable strains will be required.
5. REMOVAL OF ANTIBIOTICS FROM FOOD ANIMALS

Antibiotic growth promoters were introduced because of their association with the
benefits of decreased mortality, increased feed conversion efficiency and improved
welfare through improvements in litter quality. The risks associated with their use
appeared low as all antibiotics used as growth promoters are absorbed minimally
from the gut and do not have systemic action and, therefore, were considered to
present little or no risk of residues in meat. Recently, EU Agricultural Ministers
voted in favour of a ban on the use of four antibiotic growth promoters (zinc baci-
tracin, spiramycin, tylosin phosphate and virginiamycin) and these products were
banned in July 1999 (EC Council Regulation, 1999). As a consequence, only two
antibiotic growth promoters are currently commercially available: avilamycin and
bambermycin. However, they are closely related to antibiotics for human use and
their use is very limited. Additionally, studies by Elwinger et al. (1992) showed that
commercially available competitive exclusion agents are more effective than anti-
microbial growth promoters in controlling necrotic enteritis, at least.
Antibacterial substances have been used widely as growth promoters in animal
husbandry (Witte et al., 1999). In recent years, there has been growing concern over
the use of growth promoters in animal feed and the possible acquisition of resistance
by normal resident flora which may have a zoonotic potential. Different means of
interaction between microecological systems in different animal hosts and the envi-
ronment may occur during the transfer of resistant bacteria and their resistance
genes. Spread of resistance takes place in different ways with respect to clonal
spread of resistance strains by the spread of wide host range plasmids and trans-
locatable elements. Commensals in ecosystems have a special significance and
a pronounced capacity for acquisition and transfer of resistance genes as with
Enterococcus faecium and E. coli in the normal gut flora (Witte, 2000).
Certain antibiotics are used at subtherapeutic doses in poultry feed for growth
promotion and also at higher doses to treat clinical disease (Gustafson and Bowen,
1997). The removal of antibiotics from animal feed has led to a sharp increase
in infections caused by opportunistic pathogens. For example, the incidence
of necrotic enteritis which is caused by C. perfringens has been reported to have
increased sharply since the withdrawal of growth promoters. Of particular concern
is the increase in disease in poultry by potentially zoonotic pathogens such as

Salmonella enterica serotypes, E. coli and C. perfringens. Many of these diseases are
on the increase as primary predisposing infections are not susceptible to treatment.
For example, the incidence of avian colibacillosis is on the increase as primary
Mycoplasma infections leave birds predisposed to E. coli infections.
There is much information covering bacterial colonization in poultry although,
unlike in other species, such as pigs and sheep where specific host receptors for
bacterial colonization have been identified, no poultry-specific receptors have been
identified. Recently, some of the mechanisms of colonization such as flagella,
fimbriae and LPS of well studied bacterial pathogens (e.g., S. Enteritidis and E. coli)
have been elucidated. For other organisms such as Lactobacilli, Bifidobacteria,
Clostridium perfringens, Campylobacter and Spirochaetes, only limited information
exists and for these organisms the exact mechanisms of colonization and persistence
need to be elucidated. This is especially important as there is a desire to enhance the
stability of the native “protective” flora and eliminate zoonotic pathogens such as
Campylobacter jejuni. To this end, a greater understanding of the host–pathogen
relationship needs to be gained possibly through insight into the relationship
between the in vivo environment and in vivo bacterial responses. Apart from the
host–bacterium interactions, there is a wealth of data on the efficacy of competitive
exclusion agents reducing colonization by bacterial pathogens. However, there is
scant information on how these agents mediate their effect. Within this complex pic-
ture is bacterium–bacterium interactions about which little is known. It may be
assumed that bacteria do “talk” to each other as evidenced by the phenomenon of
quorum sensing. Is it fanciful to consider positive cross talk that enhances the “pro-
tective” flora and negative cross talk that eliminates the pathogens? Identifying
these putative messages, which may be small metabolites such as the homoserine
lactones, will be important in modulating the gut flora. Other mechanisms for mod-
ifying flora that could be developed include dietary management, addition of spe-
cific dietary supplements such as oligomannans, enhancing the secretory immune
responses by mucosal vaccines, passive immunization possibly by plant-derived
antibodies in feed, use of bacteriophage for selective lysis, antibacterial toxins and
novel antibiotics. The list of possibilities is long but the knowledge base limited.
There is much to do!
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13 Mucosal immunity and the bovine
entero-mammary link: evolutionary
established dialogue between antigen
and arms of immune system1
M. Niemialtowskia, A. Schollenberger a and W. Kluciński b

aImmunology Laboratory, Division of Virology, Mycology and Immunology,
Department of Preclinical Sciences, Warsaw Agricultural University, Grochowska
272, PL-03-849 Warsaw, Poland
bDepartment of Clinical Sciences, Faculty of Veterinary Medicine,
Warsaw Agricultural University (SGGW), Ciszewskiego 8, 02-786 Warsaw,

Poland
Immunology has all the beauty and fascination but also all the complexities
and uncertainties of research in biology (Zinkernagel, 2000).
Animals have the ability to develop an active response in their contact with the
variety of pathogens from their environment owing to their evolutionary adapted
immune system. The mammalian immune system consists of innate (non-specific)
and acquired (specific) humoral and cellular mechanisms of self defence which
were developed by the co-evolution between the organism and the environment.
A special role is played by the mucosal immune system (MIS) which, together
with the skin immune system (SIS), is critical for efficient protection against infec-
tious agents and also in developing a tolerance towards food antigens. Mucosal-
associated lymphoid tissues (MALT) form a network of functional integrity, which
is achieved by communication of cells via secreted cytokines/chemokines and by
trafficking of immune cells between different mucosae. Development of this part
of the immunity is also attributed to microflora colonizing mucosal surfaces. In
this review we present and discuss the mechanisms of immune protection in the
1Supported by the State Committee for Scientific Research (KBN, Grant No. 6 P06K 020 21) and by the Foundation for
Polish Sciences: (i) Professors of 2000 grants (No. 10/2000 for M. Niemialtowski), and (ii) IMMUNO Program (1999) –
both institutions in Warsaw, Poland.

294 M. Niemialtowski, A. Schollenberger and W. Kluciński
gastrointestinal tract and in the bovine mammary gland as an entero-mammary link,

which is extremely important for the newborn.
1. INTRODUCTION
Immunology, as an experimental and clinical science, developed at the end of the
eighteenth century and was directly associated with the work of Edward Jenner
(1749–1823), an English physician who, knowing nothing about the true nature of
smallpox, used the material from cowpox lesions as a “vaccine” to protect humans
against smallpox variolation. From the work of Louis Pasteur (1822–1895), followed
by others, the exogenous infectious nature of many human and animal diseases
became well established. Nonetheless, we still know little about the functioning of the
immune system from birth to maturity – from immune innocence to immune memory.
The new threats of the new transmissible agents prions have been described, and
evidence is growing that they may cross the interspecies barrier (reviewed in detail by
Prusiner, 1996; Johnson, 1998). Consumers fear that bovine spongiform encephalo-
pathy (BSE) agents may be present in beef and in bovine milk so that the food may
transmit the non-curable, deadly disease. There are also fears of “old” animal infectious
diseases such as foot-and-mouth disease (FMD) or the “new” diseases caused by HIV
in humans or SIV (simian immunodeficiency virus) in primates (Luciw, 1996).
Our understanding is based mostly on studies performed on laboratory animals,
which differ significantly from farm animals and also from humans. There is, how-
ever, a universal model (pattern) of cell-to-cell communication that can be achieved
by a variety of receptor–ligand interactions. These molecules, either expressed on
the cell surface or secreted from the cell, establish a functional network for homeo-
stasis within which integrity is achieved. The mucosal immune system, together
with the skin’s immune system, directly communicate with the outside environment
with its enormous number of foreign antigens, many of them being a pathological
threat for the host (VanCott et al., 2000). Local mechanisms of defence in the
gastrointestinal, respiratory and genitourinary tracts and also within the mammary
gland(s) are an integral part of the whole immune system. The immune system is
localized in every type of mucosa, and each is well adjusted to the function that it plays
(Savage, 1977; Kaganoff, 1996; Shanahan, 1997). In order to maintain homeostasis, a
balance must be kept between signals that activate necessary immune responses and
signals that may induce improper immune reactions leading to pathology, i.e.
autoimmune diseases (Abbas et al., 1994; Bell, 1998; Zinkernagel, 2000).
This short introduction is an effort to provoke questions about immune
tolerance/immune response towards commensal microflora. Ruminants, unlike other
mammals, depend entirely on microorganisms that enable them to utilize cellulose,
hemicellulose, lignin, gums, pectin and cellobiose from plants. Digestion of dietary
fibres takes place in the forestomach, mostly in the rumen, which is a huge fermen-
tative vat. A newborn does not have bacteria in its immature gastrointestinal (GI)
Mucosal immunity and entero-mammary link 295
tract and during the first few days of life when subsequent bacterial colonization
takes place, the GI tract also matures (Tizard, 2000). Thus, not only enzymatic/
digestive activity but also immune mechanisms need some time to be developed.
In horses and in pigs, degradation of plant fibres takes place in the colon where com-
mensal bacteria secrete cellulolytic enzymes and also synthesize vitamins. Intestinal
autochthonic microflora are very competitive so, in cooperation with local host
defences, create a hostile environment for many pathogenic microorganisms.
Therefore, favouring the growth of commensals by feeding animals with probioti-
cally formulated food is advantageous for the host.
A very special example of local immunity operates within the bovine mammary
gland (Salmon, 1999). Milk is a stable constituent of the human diet in most parts
of the world and the increasing rate of subclinical mastitis in highly genetically
selected dairy cattle presents a serious problem for consumer health. Inflammatory
responses within the mammary gland are also the cause of huge economical losses.
The immune response within the bovine mammary gland is subjected to enormous
physiological pressure if highly producing dairy cows have to give daily 20 litres of
milk. On the other hand the non-lactating period is characterized by massive apop-
tosis of secreting parenchymal cells. Immune cells located within mammary gland
are challenged by various modulatory factors in colostrums/milk during lactation
and by apoptotic signals during the dry period.
2. IMMUNOLOGICAL AND NON-IMMUNOLOGICAL PROTECTION

OF THE GASTROINTESTINAL TRACT
In mammals the surface area of all mucosal tissues exceeds the skin surface area by
more than 200 times. Mucosal-associated lymphoid tissues (MALT) are composed
of gut-associated lymphoid tissues (GALT), bronchus-associated lymphoid tissues
(BALT), nasal (nasopharynx)-associated lymphoid tissues (NALT) and lymphoid
tissues in the reproductive tract, urinary tract, mammary gland(s), lacrimal glands
and salivary glands. Immune cells are able to migrate within the tightly regulated
network of mucosal tissues, therefore local immune responses in one system can
result in stimulation of local immunity within the whole MALT (Stokes and Bourne,
1989; Herbert et al., 1995; Green et al., 1997; Holmgren and Rudin, 1999; Lee and
Mekalanos, 1999; McGhee et al., 1999).
The alimentary tract is the most important route for foreign antigen entry. It has
a unique property of simultaneous immune exclusion, immune elimination, devel-
opment of immune tolerance and certainty of mounting an efficient immune
response by associated lymphoid tissue. It requires perfect cooperation between
antigen presenting cells (APCs) and immune cells, and precise regulation through
the locally secreted cytokines/chemokines/hormones. Conditions within the alimen-
tary tract are challenging, yet healthy individuals survive, owing to the subtle inter-
play between various bioactive molecules and immune cells within the mucosa.
Non-specific, i.e. innate, defence mechanisms in the GI tract, include an acidous

gastric environment, mucosal protection, an epithelial barrier and peristalsis. An
important factor of host defence is autochthonic microflora in the forestomach of
ruminants and in the large intestine of monogastric animals. Intestinal peristalsis
mixes the food and promotes the broad contact of ingested nutrients with the
mucosa. In the colon, peristaltic movement prolongs such contact. Intestinal smooth
muscle contractions are under intrinsic (Meissner and Auerbach plexuses) and
extrinsic neural control. Primary and accessory cells involved in the immune and
nervous systems create a network through surface receptors of neurons and immune
cells, i.e. lymphocytes (Trautmann and Vivier, 2001). Khan et al. (2001) showed that
agrin (glycoprotein in neuromuscular junctions/synapses between neurons and
muscle cells) is also present in the immune system network and may participate
in rosette formation by APCs and T cells. Thus, agrin may be also critical for
presentation of foreign antigens to APCs which are located within the GI tract and
in regulation of the mucosal immune system. The GI tract mucosa in the small
intestine provides an absorptive function, extending into the lumen on the finger-like
projections of the innumerable villi. Their surface contains three major types of
epithelial cells: columnar absorptive cells that may also serve as APCs, mucin-
producing goblet cells and several endocrine cells. Within the crypts are undifferen-
tiated crypt cells, goblet cells and Paneth cells (Voynow and Rose, 1994; Ayabe
et al., 2000; Ganz, 2000; Hermann, 2000; Nordman, 2000).
In the colon, the mucosa does not absorb digested nutrients but retains water and
electrolytes from liquid content; it does not have villi and is almost flat, but its
epithelium does have columnar absorptive cells and goblet mucous cells. Within the
GI mucosa, different immune cells are distributed and communication via
cytokines, chemokines, chemotactic peptides, neuropeptides, leukotrienes, vasoac-
tive peptides and many more, allows not only the coordination of the absorptive and
digestive function but also the immune surveillance and protection of the host
(Voynow and Rose, 1994; Bagglioni and Loetscher, 2000). In reference to the GI
tract, antigen-specific immune cells (cytotoxic T lymphocytes, CTLs) have also
been identified between epithelial cells and lamina propria lymphocytes. These are
effector cells not only for their killing capacity but also for secreting many bioregu-
latory proteins/peptides (Garcia, 1999; Kerksiek and Pamer, 1999; Rothkötter et al.,
1999). It is worth stressing the extraordinary regenerative capacities of intestinal
epithelium. Cells proliferate within the crypt, then migrate out and upwards to the
tip of a villus, where they are shed into the lumen. It takes usually 96 to 144 h, so
renewal of the epithelium in the small intestine is possible within 4–6 days (Stokes
and Bourne, 1989).
The epithelial surface is covered with a layer of mucus that creates a barrier for
microorganisms and also facilitates the digestion and absorption of different nutrients
(Stokes and Bourne, 1989). The entrapment of some bacteria is due to their binding to
mucosal analogues of epithelial receptors. In this way bacteria may be easily expelled
from the GI tract. However, an important factor in their virulence is the production of
mucolytic enzymes that enable bacterial colonization of the intestine epithelium.
Mucus is also the best milieu for secretory IgA (sIgA), lysozyme, interferon (IFN) and
other humoral factors active in hosts’ protection (Shanahan, 1997).
Another important mechanism of intestinal defence, active mostly within the
duodenum and small intestine, is the bile acids (Stokes and Bourne, 1989). They
inactivate viruses having a lipid-containing envelope and also some enteropatho-
genic bacteria; however, enterococci and Bacteroides spp. can degrade bile salts.
Moreover, in some species (rats, rabbits and chicken) bile is very rich in IgA and
over 75% of IgA reaches the intestine by this route, whereas in ruminants, swine and
dogs less than 5% of IgA enters the bile.
Briefly, the GI tract immune system is a part of MALT, which consists of lym-
phoid nodules, mucosal lymphocytes, isolated lymphoid follicles and mesenteric
lymph nodes. The gut-associated lymphoid tissues (GALT) are the largest lymphoid
organ in the body and contain different effector mechanisms to fight potential
pathogens entering the host via food. Nodules of lymphoid tissue may lie within the
mucosa or span the mucosa and partially the submucosa. Peyer’s patches (PP) are
organized masses of lymphocytes and are macroscopically visible follicles in the
ileum. GALT may be divided into lymphoid compartments which communicate
within a functional network (reviewed by Cebra et al., 1999). In ruminants and in
pigs there are two types of PP in the small intestine. Jejunal PP are a secondary lym-
phoid tissue playing an important role in intestinal defence throughout life, whereas
ileocaecal PP are primary lymphoid tissues responsible for the development of
competent immune cells and they regress in animals over 6 months of age (Stokes
and Bourne, 1989; Tizard, 2000). The epithelium covering lymphoid follicles con-
tains columnar absorptive cells and epithelial microfold (M) cells which transcytose
macromolecules from the lumen to underlying lymphocytes, and thus are probably
the portal of entry for viruses and bacteria. In the fundus of mucosal crypts in the
intestine are Paneth cells which secrete lysozyme and peptides called cryptdins that
are related to α-defencins and are also toxic for microorganisms − lipopolysaccha-
rides (LPS), lipid A, lipoteichoic acid, Gram-positive and Gram-negative bacteria
may significantly elicit cryptdin production and secretion (Eisenhauer et al., 1992;
Pang et al., 1994; Ganz, 1999; Ayabe et al., 2000).
Throughout the intestines T cells (usually of CD8+ and largely γ /δ T cell recep-
tor (TCR) phenotype, however, TCR α/β T cells may be found in the lamina propria)
as intraepithelial lymphocytes (IELs) are spread within epithelium – these cells may
constitute up to 27% of the epithelial cell population (Emoto et al., 1996; Wyatt
et al., 1996, 1999; Marrack et al., 2000; Tizard, 2000). In ruminants as many as 90%
of IELs have γ /δ TCR and they are specialized for epithelial surveillance in the GI
tract (McGhee et al., 1999; Tizard, 2000). Their unique property is antigen recogni-
tion without previous processing. They also communicate with epithelial cells,
which do express major histocompatibility complex (MHC) class II molecules,
through E-cadherin and the novel integrin αEβ7 (CD103). However, IELs do not
respond by proliferation of conventional antigens; they have the ability to produce
cytokines and also to kill target cells just as natural killer (NK) cells do (Lanier,
2001). They are involved in regulation of IgA production by B cells; some are
suppressive while others express contrasuppressive activity.
Bovine lymphocytes are composed of three subsets: i) B lymphocytes bearing IgM
surface receptors and several non-immunoglobulin (non-Ig) molecules, ii) T lympho-
cytes with α/β TCR (TCR2), co-expressing either CD4 or CD8 molecules, thus repre-
senting bovine CD4 (BoCD4) (T helpers), and bovine CD8 (BoCD8) (T cytotoxic/
suppressors) subpopulations and iii) γ/δ TCR (TCR1) T cells (Tizard, 2000). The last
subset dominates in young animals; in calves 40–80% of peripheral blood lympho-
cytes and 35% of spleen lymphocytes represent the TCR1 phenotype. Professional
APCs, dendritic cells (DC), are also present within the intestinal epithelium. Epithelial
cells may express MHC class II molecules and may produce many cytokines that
regulate intestine inflammatory response and also interleukin-7 (IL-7), which is a
growth factor for γ/δ T cells.
B cells and plasma cells secrete sIgA which evolved to protect mucosal surfaces
(Shanahan, 1997). These cells are located in the submucosa especially in the crypt
region. Secretory IgA antibodies are responsible for immune exclusion, i.e. they
prevent adherence of microorganisms to the epithelium; however, they are not bac-
tericidal and activate complement only by an alternative pathway. They neutralize
viruses and some bacterial enzymes/toxins and also opsonize particles and may
operate in antibody-dependent cell-mediated cytotoxicity (ADCC). Recently,
Macpherson et al. (2000) showed that in C57 BL/6 (H–2b) mice maintained under
specific pathogen free (SPF) conditions, sIgA against commensals were produced in
a T cell independent manner. This took place within lymphoid follicles where the
B cell subpopulation, derived from B1 peritoneal cells, was induced to synthesize
IgA independently from helper T (Th) cells, thus lymphoid follicular organization
represents a premature (primitive, less sophisticated) form of specific humoral
response.
Another important component of local cellular defence are mast cells. Believed
to be the effector cells of anti-parasitic immunity and to play a key role during
immediate hypersensitivity reactions (Herbert et al., 1995; Miller, 1996; Smith and
Weis, 1996), these cells were also found to be of significance for local immune
response on mucosal surfaces. There are two subpopulations of mast cells:
mucosal mast cells (MMC), of a lymphoid appearance with sparse granules,
located within mucosal surface; their proliferation and differentiation is T cell
dependent and they are involved in anti-gastrointestinal nematode immune
responses as effector cells,
connective mast cells (CMC) with profuse cytoplasmic granules; T cell
independent, they are apparently associated with hypersensitivity reactions
and were found within fibrotic lesions within the gastrointestinal tract.
Although MMC need interleukin 3 as a growth factor, Miller (1996) described

the regulatory activity of these cells towards T lymphocytes, which makes MMC an
important link of a local immune network in the GI tract. During nematode invasion
MMC produce, as one of their effector molecules, specific serine protease and the
activity of these cells may be quantified by the level of chymases (chymotrypsin-
like proteases, MMCP-I and RMCP-II) released from their granules.
GALT should not be mobilized against food antigens mostly because immune
response to food may produce severe intestinal pathology to say nothing about seri-
ous reduction in food uptake, absorption and metabolic utilization. Therefore the
intestinal immune system has to discriminate between harmless food and potentially
harmful pathogens. It is well accepted now that every intake of food provides
enough intact proteins to easily stimulate immune responses if given by other routes.
Another challenge comes from the normal microflora in the intestines and/or in
forestomachs (rumen) in ruminants (Szynkiewicz, 1975; Lee, 1985; Rojas Vasquez,
1996; Cebra et al., 1999; Roos, 1999).
Improper immune responses to food antigens and the harmful effect on homeo-
stasis exerted by commensal GI bacteria may lead to serious pathological conditions
such as coeliac disease and inflammatory bowel disease in humans (Abbas et al.,
1994). To avoid unnecessary and dangerous specific immune response to food-
derived antigens, many inhibitory/suppressory mechanisms were developed that
lead to immune tolerance. Ingested proteins during short-term contact with GALT
are locally processed by antigen presenting cells (APCs) and presented in the con-
text of MHC II molecules (Picker, 1992). Depending on the nature of the antigen,
its quantity, the frequency of administration and the response of the host, T cell
energy, T cell deletion or antigen dose-related suppression of immune response is
developed. It is accompanied by production of inhibitory cytokines such as TGF-β
and by inhibiting co-stimulatory signals for CD4+ T lymphocytes. Intraepithelial
lymphocytes (IELs) of TcR γδ phenotype play a special role in establishing toler-
ance to food antigens. On the other hand it is obvious that the active immune
response may also develop, including local sIgA production and generation of spe-
cific and memory cellular defences. Moreover, owing to the trafficking of antigen-
primed immune cells, mechanisms operating in the GI tract may lead to generation
of a local immune response on other mucosal surfaces (i.e., in the respiratory tract)
as well as systemic immunity. Therefore this method of stimulation was chosen for
successful oral vaccination.
If dangerous pathogens target the epithelial cells they secrete chemotactic/acti-
vating molecules – chemokines/cytokines – that alert the surrounding immune and
inflammatory cells. Chemokines are small molecules produced almost instantly
under proinflammatory conditions and GI epithelial cells may secrete either type I
as IL-8, gro-α or ENA78 or type II chemokines as RANTES, MCP-1 and MIP-1α
(Agace et al., 1993). Certainly, immune cells are major producers of cytokines that
modulate and direct immune reactions within the GI tract. Major cytokines active
during inflammatory response are: TGF-β with IL-4 and IL-5, switching B cells in
lamina propria to IgA synthesis and IL-6, inducing IgA+B cells to differentiate into
sIgA-producing cells. One recently described reaction is the modulation (i.e. devel-
opment and activation) of IELs with IL-7 (Fujihashi et al., 1996). It is therefore
logical that epithelial cells and IELs of the mucosal surface regulate each other to
maintain an immunological homeostasis in the GI tract. Cytokines exert their
modulatory activity through autocrine, paracrine or endocrine pathways (Abbas
et al., 1994). They are responsible for the dialogue between immune cells. It may
result in establishing a humoral response, i.e. sIgA production or a cellular response,
i.e. generation of antigen-specific CTLs (Emoto et al., 1996). A well known mecha-
nism of cytokine cascade may be responsible for the clinical signs of endotoxaemia as
a result of LPS challenge (Abbas et al., 1994). Activation of macrophage MΦ takes
place in the presence of IL-1, tumour necrosis factor (TNF) and IFN-γ, whereas poly-
morphonuclear neutrophils (PMNs) are attracted by IL-8 (Pang et al., 1994).
Cytokines also can down-regulate cellular activities, such as TGF-β, which reduces
the lymphocyte proliferation rate. Within the Th cell population a cytokine network
functions, i.e. IL-4 and IL-10 inhibit the growth of Th1 cells and IFN-γ decreases the
proliferation of Th2 cells (Abbas et al., 1994; Tizard, 2000). Moreover, IL-1 has a direct
influence on intestinal glutamine utilization – the primary source of energy for epithelial
cells and also a substrate for DNA synthesis (Austgen et al., 1992). Changed gluta-
mine metabolism may result in altered permeability of the mucosa and induction
of bacterial translocation. Another cytokine, IL-6, together with IL-1 and IFN-γ, is
associated with inflammatory bowel disease in humans and it is suggested that its
role is to induce production of autoantibodies to epithelial antigens (Powrie, 1995;
Groux and Powrie, 1999).
Intestinal epithelial cells express very low levels of MHC class II proteins on
their surface and usually do not act as APCs. However, in contact with bacterial
endotoxins expression of these molecules is augmented which allows a response to
be mounted against epithelial cells, resulting in their damage. Enteropathogenic
bacteria have many virulence factors, such as adhesins, toxins/enzymes and trans-
missible resistance to chemotherapeutics. Amongst the exotoxins produced are:
i) enterotoxins, ii) cytotoxins, iii) toxins damaging the cytoskeleton, and iv) neuro-
toxins (Sears and Kaper, 1996). For example, the cytotoxic effect (CTE) of entero-
pathogenic E. coli in chinese hamster ovary (CHO) cells is shown in fig. 1
(Niemialtowski and Toka, 1996). These are bacterial exotoxins that act differently
in various systems so they are classified within more than one group. Enterotoxins
may stimulate secretory activity of intestinal epithelial cells, with virtually no dam-
age to cells, whereas cytotoxins may suppress FcR-dependent phagocytosis and
intracellular killing by phagocytic cells leading to their death (Niemialtowski et al.,
1993; Rappuoli et al., 1999).
Changes in organization of F-actin lead to cytoskeleton damage, but this does not
kill the cell. Cytotoxins, however, may inhibit migration of phagocytic cells and
Fig. 1. Cytotoxic effect of enteropathogenic

E. coli in chinese hamster ovary (CHO) cells
(B) and control untreated CHO cells (A).
Original magnification = 400 × (Olympus
BX60 microscope; own experiments).
diminish FcR-dependent phagocytic and bactericidal activity – they usually may

cause the death of cells (Niemialtowski et al., 1993; Rappuoli et al., 1999). The neu-
rotoxins are an important group produced by intestinal pathogens, which release
neurotransmitters within the intestinal wall and can modulate smooth muscle activity
(Sears and Kaper, 1996).
During each individual development, the pattern of immune response character-
izing mature T cells depends on interactions between genetic constitution and envi-
ronmental factors influencing maturation. Cytokine response of mononuclear blood
cells from newborns and the foetus indicates that differences occur when compar-
ing foetal cells with adult cells. Such immature responses are characterized by a pre-
ponderance of cytokines of Th2 type (Martinez et al., 1995). In mice, as in most
mammalian species there are three subsets of Th cells: Th1, which produce IFN-γ
(type 1 cytokines), Th2, which synthesize IL-4 (type 2 cytokines) and Th0 of indef-
inite cytokine pattern (Abbas et al., 1994; Tizard, 2000). One may suggest that a
strong Th1 response in utero and immediately after birth may result in serious harm
to the offspring or that trophoblasts are a source of a high level of Th1 inhibitors
such as IL-4, IL-10, progesteron, and prostaglandins. Functional immaturity of the
APC system may also be significant in favouring Th2 early postnatal responses.
Another factor, IL-12 pathway deficiency, can contribute in directing the cytokines
of newborns towards Th2 (Ridge et al., 1996). Animal experiments suggest strongly
that microorganisms are the primer for Th1 maturation and that removing such
stimuli (by creating a bacteria-free environment) locks the immune system into a
Th2 bias (Holt and Macaubas, 1997). The most important sources for these matura-
tion signals are commensals within the GI microflora (Sudo et al., 1997). Also
important seems to be the role of bacterial LPS in the maturation of APC precursors
and/or the upregulation of either bound or soluble CD14 by mononuclear cells,
at least in humans. However, many pathogens modulate the expression, secretion
and biological activity of host regulatory proteins, i.e. cytokines, MHC molecules
and adhesins, thus providing evidence for their delicate adaptive mechanisms of
escaping the immune recognition and response (Banks and Rouse, 1992;
Niemialtowski et al., 1997; Alcami and Koszinowski, 2000; Tortorella et al., 2000).
In bovines, the upregulation of IL-2Rα on B cells and MHC II expression on T and
B cells by bovine leukaemia virus (BLV) infection is well documented (Torre et al.,
1992; Stone et al., 1995).
Microbiota that colonize the intestinal ecosystem have evolved with their hosts
for over a 100 million years. More than 400 different species of bacteria, divided
into indigenous, transient and permanent inhabitants, and shed from other niches,
colonize the GI tract of the average animal species. This stable climax population is
associated with the intestinal epithelium and is required for IEL activation and NK
cell stimulation (Berg, 1996). The majority of them are anaerobic bacteria tightly
bound to the epithelial layer (reviewed in Cebra et al., 1999).
The GI tract of the newborn calf is colonized during the first hours of life by
Lactobacillus spp., E. coli and Enterococcus spp. and later by anaerobic bacteria of
rumen microflora from the forestomachs. As long as the calf is milk-fed, lactoferrin
and κ-casein exert their bactericidal activity against coliform bacteria and favour the
growth of Bifidobacterium spp. (reviewed by Schanbacher et al., 1997). Bovine milk
is also rich in proteins that may act as probiotics helping the GI microflora to
grow (Lee and Salminen, 1995; Dugas et al., 1999). Anaerobic conditions and very
low Eh (i.e. –280 mV or lower) support a friendly environment for Bacteroides spp.,
Ruminococcus spp., Selenomonas spp., Lampropedia spp., Oscillospira spp.,
Clostridium spp. and many more bacteria (over 30 species) that occur exclusively
within the rumen (Szynkiewicz, 1975; Holt et al., 1994). Maturation of the GI tract
together with enriching microflora with cellulolytic microorganisms enable growing
animals to utilize fibre from plants and become independent of their mothers’ milk.
The passive mucosal protection of the newborn animal until weaning is depend-
ent on the continuous supply of maternal antibodies and other milk proteins/
peptides (reviewed by Salmon, 1999). The lactogenic humoral immunity is linked
to the GI tract (mostly to the gut) and is called the entero-mammary link, because
of the translocation of IgA (in monogastric animals) and IgG1 (in polygastric ani-
mals) or the migration of primed lymphocytes to the mammary gland. Studies on
lymphocyte subsets within the mammary gland have sustained the view of a true
local immune response, depending on the udder stage development. Accordingly,
the increase of the lactogenic, i.e. maternal immunity focuses on: i) antigen priming
(inducing) sites; possibly the intestines and also the mammary gland, ii) increase of
cell trafficking from the gut into the mammary gland and iii) enhancement of Ig pro-
duction and secretion within the udder. A very special environment, rich in
cytokines and hormones is responsible for the IgM/IgA switch, the induction of
mucosal homing receptors on to lymphocytes/lymphoblasts, and the recruitment and
also the induction of mucosal vascular addressins (Brandtzaeg et al., 1999). Young
animals after weaning are able to mount an intestinal immune response and the
decreasing of the suckling period does not seem to be detrimental for its onset
(Dreau et al., 1994, 1995; Salmon, 1995; Butler, 1998).
Milk bioactive proteins such as insulin-like growth factors I and II not only are
important for GI physiology but also may modulate non-immune protection
(Grosvenor et al., 1992; Zinn, 1997). Another milk protein α-lactalbumin (α-LA)
(but not β-lactoglobulin, β-LG), that escapes degradation by digestive enzymes
within the GI tract, may alter the proliferation and/or maturation of intestinal epithe-
lial cells (as was shown in vitro with Caco-2 and HT-29 cell lines) (Hendriks, 1996;
Alston-Mills et al., 1997). Other factors are reviewed in detail by Schanbacher et al.
(1997) and by Weaver (1997).
3. IMMUNOBIOLOGY OF THE BOVINE MAMMARY GLAND IN THE

CONTEXT OF THE ENTERO-MAMMARY LINK
The mammary gland has evolved in all mammalian species to nourish neonatal off-
spring. Lactation is the final phase of the mammalian reproductive cycle and mater-
nal milk, especially in ruminants, is essential for the survival of newborns during the
neonatal period by providing not only nutrients but moreover colostral maternal
antibodies that protect the offspring from infections (passive immune protection).
Milk of various mammalian species contains a “cocktail” of bioactive and immuno-
regulatory substances, i.e. cytokines, digestive enzymes, hormones and hormonally
active peptides (Koldovsky and Goldman, 1999), as well as anti-infectious cells
(leukocytes/PMNs, MΦ, lymphocytes B and T), molecules (i.e. Ig, lysozyme, lacto-
ferrin, opsonins, glycoconjugates, oligosaccharides, lipids) and pathogenic contami-
nants (Weaver, 1997; Asai et al., 1998, 2000; Butler, 1999; Goldman and Ogra,
1999; van Kampen et al., 1999). Dairy cows were domesticated a long time ago and
through genetic selection their mammary gland yields far more milk than is needed
to nourish their offspring. Advanced milking technologies and other factors associ-
ated with intense dairy cow management have increased significantly the exposure
to many potential pathogens and also profoundly affect mammary gland immunity
which results in dramatically reduced resistance to mastitis (Hillerton et al., 1995).
The bovine mammary gland is most susceptible to invasion by different
pathogens, usually bacteria, during the period of transition from involution to
colostrogenesis and from lactation to the involution phase (Stokes and Bourne,
1989; Annemüller et al., 1999). Thus, during the dry period, mammary gland phys-
iology remains the fundamental factor for the production of high-quality milk
throughout the subsequent lactation. The early dry period and periparturient period
have been considered as extremely important for the control of bovine mastitis
(Hillerton, 1999). Since we cannot eliminate the selective pressure on dairy cattle to
increase production we should be aware that the only ways to combat mastitis are
genetic selection of cattle for mastitis resistance and rigorous hygiene procedures.
3.1. Development of mastitis

Mastitis is a very old problem and one of the most expensive diseases in farm ani-
mals (Schalm and Lasmanis, 1968; McDonald and Anderson, 1981). The infectious
agents gain entrance to the gland via the teat canal (McDonald, 1984). Bacteria that
cause mastitis are able to overcome the bactericidal barrier of the teat canal keratin
and also the physical barrier of the teat sphincter (Grindal et al., 1991). Moreover
they can escape bacteriostatic and bactericidal factors present in the milk and are not
sensitive to resident leukocytes. Many pathogens are able to adhere to epithelial
cells, some may have capsule and also produce exotoxins. Enterotoxins produced
by staphylococci, acting as superantigens, may modulate cytokine response, other
factors may protect microorganisms within phagocytic cells and thus establish intra-
cellular invasion (Almeida et al., 1999; Lee and Mekalanos, 1999).
As inflammation proceeds, with increased vascular permeability and subsequent
PMN infiltration of mammary parenchyma, the secretory activity decreases. The
duration and severity of the inflammatory response within the udder have a major
impact on the quality and quantity of produced milk. The most common pathogens that
are the cause of mastitis are staphylococci (S. aureus), streptococci (S. dysgalactiae,
S. agalactiae, S. uberis), E. coli, mycoplasmas (M. agalactiae) and, less frequently,
Corynebacterium bovis, Pseudomonas aeruginosa, Enterobacter aerogenes,
Klebsiella pneumoniae and Actinobacillus pyogenes (Holt et al., 1994; Quinn et al.,
1994; Cifrian et al., 1996; Calvinho and Oliver, 1998; Douglas et al., 2000).
Furthermore, any inflammatory process that is raised within the secretory parenchyma
causes damage to the parenchymal cells. Interestingly, Milner et al. (1995, 1996)
showed that changes in electrical conductivity of foremilk can be used for the detec-
tion of clinical mastitis caused by S. aureus and S. uberis before visible changes in
milk would occur. Routine application of this method requires automated sensors
and accurate computer software.
Bovine coliform mastitis is an example of compromised mammary gland defence,
thus an insufficient local immune response is a major factor in pathogenesis (Quinn
et al., 1994). The disease develops around the time of calving and is regarded as a
serious problem because of its severity and difficulty to control with antibiotics for-
mulated for intramammary administration. The reason why E. coli colonization is so
effective is the reduced antibacterial activity of PMNs migrating to the site of inflam-
mation. They indiscriminately ingest milk constituents, release their granule content and
lose the energy sources and the phagocytic activity for low opsonin concentration.
This is accompanied by elevated levels of glucocorticoids in the postpartum period

and lower chemotactic activity owing to copious milk production, so that intracellu-
lar killing of phagocytosed bacteria is severely reduced. Once the infection is estab-
lished, E. coli uses its own immunoregulatory strategy through endotoxin (endotoxic
shock) and exotoxin (enterotoxins and other factors) production.
In staphyloccocal mastitis, however, the pathogen exhibits different mechanisms
of infection (Quinn et al., 1994). S. aureus escapes mammary gland defence by
enterotoxins, proteases and selective adhesin production that usually leads to severe
immunosuppression. Properly activated PMNs can assist the host in clearing the
infection but, when dysfunctional, they become a reservoir of viable bacteria thus
protecting them from antibiotics. Day et al. (2001) showed by multilocus sequence
typing (MLST), a typing system based on DNA sequences of ~450 base pairs (bp)
from seven loci scattered throughout S. aureus chromosome, that a link exists
between virulence and ecological abundance of these strains. It was found that
pathogenic strains of S. aureus represent a restricted subset within the species and
are important for studying the mechanisms of mammary gland colonization, i.e. for
pathogenesis of staphylococcal mastitis.
It is well documented that PMNs within the mammary gland have much lower
phagocytic activity than PMNs from peripheral blood (Targowski and Kluciński,
1985; Targowski and Niemialtowski, 1986a,b, 1988). Since these cells migrate
to the udder in great numbers during the first days of lactation, this diminished
antibacterial activity may be identified as an immune paradox (Targowski and
Kluciński, 1985; Targowski and Niemialtowski, 1986a). It can also be accompanied
by a high level of immunoglobulins (IgG1 predominates in ruminants, whereas IgA
prevails in monogastric animals) that form immune complexes (Ic) and also by an
increased number of MΦ (Butler, 1998, 1999; Tizard, 2000).
3.2. Immune paradox

Targowski and Kluciński (1985) and Targowski and Niemialtowski (1986a) demon-
strated the presence of a certain factor/activity in the bovine colostrum, which was
lethal for phagocytic cells – their vitality was severely reduced when compared with
the bovine milk fraction(s) (P < 0.01). In a period from 3 days to 24 h before partu-
rition, PMN vitality dropped from 70 to 10%, respectively. Then, from parturition
up to day 21 of lactation, a systematic increase of viable cells up to 70% was
observed. When comparing colostral fractions, the cytotoxic factor was found in the
fat fraction. It was active against PMNs and lymphocytes isolated from bovine
peripheral blood. Simultaneously conducted studies showed that the reduced phago-
cytic activity of mammary gland PMNs and MΦ was also associated with saturation
of their FcR with Ic that together with non-limited ingestion of fat from the mam-
mary secretion, significantly diminished the ability to engulf and kill other antigens,
i.e. bacteria (P < 0.01). What is important is that α-LA and β-LG may also form Ic,
Fig. 2. Hallmark of rosette formation by

bovine milk leukocyte and sensitized sheep
red blood cells (SRBC). Original magnification =
400 × (Olympus BX60 microscope; own
experiments).
thus additionally abusing phagocytic cells (Kilshaw and Slade, 1981). In normal
milk the level of Ic is significantly lower.
As was shown by the rosette test with Ig coated sensitized sheep red blood cells
(SRBC), most of the colostral leukocytes have FcRs on their surface (Targowski and
Kluciński, 1985; Targowski and Niemialtowski, 1986a,b; Kluciński et al., 1990;
Worku et al., 1994). When isolated 3 days before parturition and consecutively
through the periparturient period up to day 21, the percentage of rosette formation
increased from 15 to 25% (fig. 2). Moreover, it was found that cytotoxic factor(s) is
(are) produced locally and amounts may differ as the viability of phagocytic cells
changes between udder quarters. All these findings may suggest that cytotoxic activ-
ity: i) is dependent on the local microenvironment, ii) is associated with the fatty
acids fraction, and iii) is yet to have its source and control of synthesis described.
Human female milk contains a cytostatic factor able to inhibit the proliferation of
mitogen-stimulated peripheral blood lymphocytes and that also has cytotoxic factor
activity. Colostrum-associated cytotoxicity was also found in women (Drew et al.,
1984). It seems that the role of these factors is to prevent excessive maternal
lymphocyte activity in the GI tract of newborns.
In human females and in cows, when colostral cytotoxic activity and phagocytic
cells are overloaded with fat droplets this significantly diminishes the bactericidal
activity of colostrum and phagocytic cells, and this should be considered the main
reason for an overwhelmed local immunity and recurrent bacterial infections (fig. 3).
Every immune response is orchestrated through a variety of cytokines (Butler, 1999;
Koldovsky and Goldman, 1999). This prompted the idea of prevention and treatment
of bovine mastitis with exogenous administration of colony stimulating factors
(CSFs) (G- and GM-CSF), interleukins (IL-1, IL-2), and IFN-γ. Their common
feature is to stimulate directly phagocytic cells and/or to inhibit production of other
cytokines such as TNF-α which may contribute in PMN and MΦ suppression, thus
increasing the severity of coliform mastitis (Sordillo et al., 1995).
Recently it was found that soluble Fas is present in human milk and it is assumed
that it is bound to FasL (also called CD95L or Apo-1L) on mammary cells. Thus,
Fas may modulate the apoptotic machinery (programmed cell death, PCD); an
Fig. 3. Phagocytosis by PMNs isolated from

bovine milk: (A) fat droplets (marked by
arrows), and (B) S. aureus (marked by arrows).
Original magnification = 400 × (Olympus
BX60 microscope; own experiments).
essential and complex regulated process for balancing cell numbers during animal
development and adult homeostasis (Khler et al., 1999; Porter, 1999; Srivastava
and Srivastava, 1999; Hunt and Evan, 2001). During the dry period almost no secre-
tion is produced and bovine mammary gland parenchyma is subdued in the invo-
lution process which is associated with apoptosis (Wilde et al., 1997). The
pro-apoptotic microenvironment can thus favour conditions facilitating the bacterial
invasion through the teat canal. Håkansson (1999) and Håkansson et al. (1995,
1999) described that the casein fraction of human milk contains multimeric α-LA
which induces apoptosis. In contrast, the monomeric form of α-LA, which is
present in the milk whey, does not induce apoptosis. These examples suggest that
elimination of secretory cells during mammary tissue involution is physiologically
regulated and is due by PCD.
Thus, mammary gland and GI tract immune mechanisms play an important role
in the entero-mammary link, a bridge between GI destruction (food and microbial
pathogens) and production of milk for the offspring.
4. CONCLUSIONS AND FUTURE PERSPECTIVES

This brief presentation of the MIS in the context of the entero-mammary link may
provide the reader with a basis for understanding more complex issues involving
immune defence against foreign antigens and the role of mucosal immunity. In the
past few years the danger of rapidly spreading animal infectious diseases such as
foot-and-mouth disease, and also the threat of new transmissible diseases (like spongi-
form encephalopathies) in humans and in animals, has been shown. There is also an
increasing risk of infection with antibiotic-resistant strains of bacteria (i.e. nosocomial
infections), which presents a real danger especially in the immunocompromised host.
Hazardous decisions on feeding of animals with prion-containing feed undoubtedly
caused BSE, and also set up the prospect of transmission of these agents to the human
population (infectious Creutzfeldt–Jakob disease/CJD/variant).
Viruses and bacteria evolve faster than the mammalian immune system may adapt
and moreover they develop very effective strategies of immune evasion. We are now
facing the real problem of an increasing number of antibiotic-resistant bacteria owing
to the many years of selective pressure applied by using antibiotics in farm animals
and overprescribing in human medicine. On the other hand many new perspectives
may arise from projects on safe, edible mucosal vaccines. Local immunity presents a
field of a great interest for immunologists (Sheoran et al., 1997). Studies on mecha-
nisms of apoptosis within the mammary gland and regulation of immune and neural
systems are in progress. Recently, there have been some interesting findings on the
role of agrin in establishing immunological synapses – the question on their role in
mucosal immunity will hopefuly be answered in the near future.
One has to remember that we are “in a race with the replicative capacity of micro-
organisms, immune responses must not be ‘too cold’ … neither ‘too hot’, … but ‘just
right’ – rapid, vigorous, properly modulated, and of the correct quality” (Germain,
2001). The many unresolved questions and doubts regarding the entero-mammary link
leave the door open to develop new theories and hypotheses. A better understanding
of the multifactorial regulatory network of the local immune system may help us
compete more successfully with faster contestants.
ACKNOWLEDGEMENT
We express our sincere appreciation to Professor Stefan G. Pierzynowski (Institute of Cell and Organism
Biology, Lund University, and Gramineer Int. AB, Lund, Sweden) for helpful scientific discussions and
fruitful long-term collaboration.
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14 Development of the immune response
in relation to bacterial disease in the
growing fish
A.E. Ellis
Marine Laboratory, Victoria Road, Aberdeen AB11 9DB, UK
Newly hatched fish do not have a functionally developed specific immune system and
protection against bacterial infection is provided by non-specific mechanisms and fac-
tors, some of which may be derived from maternal origins. There may be scope for
optimizing the transfer of these factors from mother to offspring or for improving the
ability of larvae to synthesize non-specific defence factors by manipulation of dietary
or environment parameters of the mother prior to ovulation. In the larvae, lympho-
cytes first appear in the thymus and T cells differentiate before B cells, which develop
first in the kidney. The gut becomes populated with T and B cells quite early in devel-
opment, with T cells being present in the mucosal epithelium and B cells in the lam-
ina propria. The ability to produce specific immune responses begins at the time of
first feeding in fish such as salmonids with large eggs and larvae, but in many marine
fish species, with small eggs and larvae, there is some time between the onset of feed-
ing and functional maturation of the specific immune response. This appears to be a
time when the fry are very susceptible to bacterial infection. Furthermore, there is evi-
dence that if exposure to some types of antigen occurs during this period, a state of
immunological suppression can be induced. Thus, there are important consequences
for the earliest time to vaccinate fish. The onset of maturation of the specific immune
response relates to the rate of general differentiation of the tissues and this is temper-
ature related in fish. For a given species there is a particular degree/days relationship
governing this differentiation. Thereafter, the magnitude of antibody responses
increases with increasing age of the fish until maximum levels are achieved. This may
take several months but is different for different species.

Immune response in growing fish 315
1. INTRODUCTION
Different fish species hatch at different stages of development but in most (if not all
species studied) the newly hatched fry do not have a functionally developed specific
immune system. Protection against diseases must, therefore, be based on non-
specific mechanisms, some of which may initially be derived from the mother.
Transfer of specific antibody to the egg from the mother does occur but in most
species this does not appear to be in sufficient amounts for protection. The lymphoid
system usually develops some time after hatching and takes further time to become
functionally mature. There is some evidence that antigenic exposure of very young
fry may result in immunological tolerance. Thus, knowledge of the functional devel-
opment of the immune system of fish is important for the strategic use of vaccines.
This chapter will review this knowledge, especially in relation to defences against
bacterial diseases.
2. MATERNAL INFLUENCES
Little is known about the influence of maternal investment in protection of eggs and
larvae of fish, especially with respect to the physiological status of the mother at the
time of egg development in the ovary. However, it is known that the ova of many
species of fish contain substances that are considered to play important roles in
defence against bacterial infections. These include C-reactive protein (CRP), lectins
and lysozyme (Ellis, 1999) which are present in fairly high concentration and pre-
sumably are selectively incorporated into the yolk. Lysozyme levels have been meas-
ured in the embryos and larvae of sea bass and these were elevated in embryos (48
and 72 h after fertilization) and larvae (24 h after hatching) originating from mothers
fed a diet enriched in vitamin C for 1 month prior to spawning (Cecchini et al., 2000).
Immunoglobulin (Ig) has been detected in the ova of fish but at very low con-
centration (3–12 μg/g egg weight, compared to about 12 mg/ml serum of adult fish;
Scapigliati et al., 1999) indicating that active secretion of Ig into fish ova is absent.
Attempts to protect larval Atlantic salmon against Enteric Redmouth by immuniz-
ing mothers prior to spawning were unsuccessful although some specific antibody
was detected in the eggs (Lillehaug et al., 1996).
It is interesting that newly hatched fry appear to be much more resistant to chal-
lenge by bacteria than older fry. Rainbow trout fry were completely resistant to
immersion challenge by Vibrio anguillarum at 2 and 4 weeks post-hatch while 20%
mortality was obtained in fry of 6 weeks post-hatch (0.2−0.3 g) and over (Tatner and
Horne, 1983). Similarly, when Atlantic salmon fry were challenged with Yersinia
ruckeri 2 weeks after hatching, 8% mortality occurred, rising to 60% mortality when
challenged 6 weeks post-hatch onwards (Lillehaug et al., 1996). This occurred in
offspring of both immunized and non-immunized mothers, suggesting that non-
specific factors of maternal origin were responsible and were depleted with time.
316 A.E. Ellis
The situation in mouth-brooding fish appears to be different. In these species,

relatively small numbers of eggs are produced and the larvae are carefully attended
by the parents. In the tilapia Oreochromis aureus, mothers that were vaccinated
against Ichthyophthirius multifiliis (a protozoan parasite) 4 weeks before spawning,
produced offspring that were highly protected against challenge following hatching
(10 days after spawning). It was further shown that this protection was not only pas-
sively transferred from the mother via antibodies in the eggs but also enhanced via
the mucus secretions in the mouth during mouth-brooding (Sin et al., 1994). In these
species it may be possible to effectively protect the fry against diseases before their
specific immune system is fully developed by vaccination of the mothers.
3. NON-SPECIFIC DEFENCE FACTORS OF ENDOGENOUS ORIGIN

IN FISH FRY
Little is known about the expression of non-specific defence factors in fish ontogeny
but recently the initiation of expression of pleurocidin (an antibacterial peptide) in
the fry of winter flounder has been demonstrated 13 days post-hatch (dph) (Douglas
et al., 2001). In the pre-larvae of tilapia, lysozyme has been detected immunohisto-
chemically in the epithelial cells of the primordial swimbladder and the hepatocytes
suggesting these organs produced lysozyme before the haemopoietic tissue devel-
oped in the kidney (Takemura, 1996).
4. DIFFERENTIATION OF THE PHAGOCYTIC SYSTEM

The development of this system in rainbow trout has been studied following
intraperitoneal (ip) injection of carbon particles (Tatner and Manning, 1985). The
major tissue sites containing phagocytic cells in trout of different ages are shown in
table 1. In the fry, phagocytic cells are mainly present in the integument, particularly
the gills. As the fish ages, the kidney and spleen become the major sites of phago-
cytic cells coinciding with the progressively developing lymphoid tissue in these
organs. In the carp, the use of monoclonal antibodies specific for monocytes/
macrophages, indicates that these cells differentiate very early, possibly in the yolk
sac, before other leucocytes and before the lymphoid organs, being detectable by
1 week post-fertilization (Romano et al., 1997).
Table 1. Development of the phagocytic system in tissues of the rainbow trout: days
after hatching
Age Gill Skin Gut Kidney Spleen
4 days ++ + + − −
18 days ++ − − ++ +
8 months − − − ++ ++
Data from Tatner and Manning, 1985.

5. DIFFERENTIATION OF LYMPHOID ORGANS

The larvae of different fish species hatch at different stages of development of their
various organ systems and lymphocytes differentiate within the lymphoid organs at
different times relative to hatching. For example, in Atlantic salmon, the thymus and
kidney are fully lymphoid at the time of hatching, while lymphocytes do not appear
in lymphoid organs of some other species, e.g. sea bream and plaice, until 6 weeks
after hatching. Nevertheless, the order in which lymphocytes first appear in the lym-
phoid organs is the same for all species studied. Lymphocytes first appear in the thy-
mus followed by the blood and pronephros and lastly, after some considerable delay,
in the spleen (table 2).
The origin of the thymic stem cells is not known, but most workers have identi-
fied haemopoietic blast cells in the kidney prior to differentiation of lymphocytes in
the thymus (Ellis, 1977; Razquin et al., 1990; Josefsson and Tatner, 1993) and it is
believed that the thymic rudiment is initially populated by stem cells from the yolk
sac or pronephros. Using in situ hybridization, genes associated with haemopoiesis
(e.g., GATA-1, c-myb) are first expressed in zebra fish in the yolk sac, 12 h post-
fertilization (hpf), and definitive haemopoiesis is established in the kidney by day 5
when Scl/tal-1 is first detected. Lymphocyte specific gene markers (e.g., Rag-1,
Rag-2) and the T cell specific lck gene have been found to be expressed in the zebra
fish thymus as early as 68 hpf (Trede and Zon, 1998).
Following proliferation of lymphocytes in the thymus, many lymphocytes can
be observed in the connective tissue separating the thymus from the pronephros,
Table 2. Appearance of lymphocytes in the developing lymphoid organs of fish: days prior to (−)
or post (+) hatching
Size when
lymphocytes
first appear in
Thymus Blood Pronephros Spleen thymus (mm) References
Plaice +28 NK +49 NK 8 Lele, 1933

Salmon −22 −14 −14 +42 NK Ellis, 1977
Rainbow +1–3 +5 +5 +14–25 17 Grace and
trout Manning, 1980;
Razquin et al., 1990
Carp +5 +7–8 +7–8 +8–9 4 Botham and
Manning, 1981
Sebasticus +21 NK +30 +44 NK Nakanishi, 1991
marmoratus*
Sea bream +47 +54 +54 >+77 6 Josefsson and
Tatner, 1993
Sea bass +21–27 NK +30 +44 NK Abelli et al., 1996;
Breuil et al., 1997
*Days post-birth.
NK, not known.
318 A.E. Ellis
leading to the suggestion that lymphocytes migrate from the thymus to populate the
pronephros (Tatner, 1985; Josefsson and Tatner, 1993; Abelli et al., 1996).
The thymus in fish first develops as separate buds situated dorsal to several gill
arches. These later coalesce into a single organ in each branchial cavity. The thymic
buds develop as collections of lymphocytes between the pharyngeal epithelial cells and
the epidermal basement membrane (Ellis, 1977). In young fish the fully differentiated
thymus is separated from the external environment only by a single layer of simple
epithelial cells. However, this still provides an effective barrier to entry of antigens into
the thymic tissue from the environment (Castillo et al., 1998). In older fish the epithe-
lium thickens and the organ progressively becomes encapsulated in connective tissue.
During the first few months there is intense mitotic activity of lymphocytes in the
thymus and then a decrease. Apoptosis of thymocytes has been reported, with an
onset in carp at about 4 weeks of age (Romano et al., 1999) and sea bass at 5 weeks
(Abelli et al., 1998). It is believed that many thymocytes migrate to peripheral
organs during the first 2–3 months post-hatching and signs of involution of the
thymus appear with the onset of sexual maturation. Histologically, lymphocytes
appear in the kidney and blood a few days after their appearance in the thymus while
splenic lymphocytes appear a considerable time later (table 2).
6. DEVELOPMENT OF T LYMPHOCYTES
At present monoclonal antibodies (Mabs) specific for mature T lymphocytes have
only been developed for sea bass (Scapigliati et al., 1995, 2000). Mabs have also
been produced which are specific for early thymocytes in the carp (Rombout et al.,
1997) or a carp mucosal T cell subpopulation (Rombout et al., 1998). These Mabs
provide evidence for initial differentiation of T lymphocytes within the thymus fol-
lowed by their migration to the gut mucosa, kidney and spleen. In sea bass, T cells
first appeared in the thymus at 30 dph, 3 days after the first appearance of lymphoid
cells, shortly after in the epithelium of the gut mucosa, and then in the kidney
(35 dph) and spleen (44 dph). Throughout development, T cells were very numer-
ous in the thymus and increased markedly in the intestinal mucosa from 44 dph
onward while they remained infrequent in the developing kidney and spleen (Abelli
et al., 1996; Picchietti et al., 1997). Using flow cytometry of larval homogenates,
cells with T cell determinants have been detected in sea bass as early as 12 dph,
which suggests they may originate in an extrathymic compartment as the thymus is
not lymphoid at this stage (dos Santos et al., 2000). Adult levels of T cells in sea
bass are attained by about 140 dph (dos Santos et al., 2000).
7. DEVELOPMENT OF B LYMPHOCYTES
A polyclonal antiserum to Atlantic salmon serum Ig, which stained all blood
lymphocytes of adult salmon was used to study the differentiation of surface
Ig positive (sIg+) cells in salmon fry (Ellis, 1977). Although small lymphocytes were
present in the thymus and kidney prior to hatching, sIg+ lymphocytes were not
detected in whole fry homogenates until 45 dph, coinciding with the time of first
feeding. By 48 dph, the majority of lymphocytes stained with this antiserum. It is
thus apparent, that while lymphocytes are present in the lymphoid organs of fish
from an early age they do not initially express the surface markers characteristic of
mature lymphocytes and a further period of time is necessary for functional differ-
entiation to mature.
Mabs that react with the sIg determinants expressed on B cells are available for
a number of species of fish (Scapigliati et al., 1999). Studies using these Mabs to
determine the appearance of B cells in the lymphoid tissues clearly demonstrate the
importance of the kidney in their development. In rainbow trout, B cells first appear
in the kidney at about 8 dph and in the spleen at about 30 dph (Razquin et al., 1990).
Using homogenates of embryos, B cells could first be detected as early as 8 days
prior to hatching in rainbow trout (Castillo et al., 1993). In sea bass, B cells develop
after T cells, first appearing in the kidney at about 38 dph (Breuil et al., 1997) and
in the spleen at about 50 dph (Picchietti et al., 1997). The proportion of lymphocytes
that are B cells in the different organs of adult sea bass is reached by about 140 dph
(dos Santos et al., 2000).
The proportion of sIg+ cells within the lymphoid organs in the developing
carp has shown them to first appear in the kidney about 2 weeks post-hatch and
their numbers increase with age to the adult levels (about 20%) at 30 weeks
(Koumans-van Diepen et al., 1994; Romano et al., 1997) (table 3). Plasma cells
were first detected in the kidney about 1 month after hatching. B cells first appeared
in the spleen at about 4 weeks, in the blood at 6 weeks and in the gut at about
11 weeks (Romano et al., 1997). The percentage of B cells in all these organs
continued to increase and did not reach adult levels until after 30 weeks of age. The
thymus never contained many sIg+ cells. These data indicate that the humoral
immune system in the carp begins to mature at about 2–4 weeks of age.
Table 3. First appearance (days after hatching) of lymphocytes expressing thymocyte (T)-specific
(T cells) or immunoglobulin (Ig)-specific (B cells) markers in lymphoid tissues of fish detected by
immunohistochemistry
Thymus Kidney Spleen
Mab reactivity T+ Ig+ T+ Ig+ Ig+ References
Carp 5 Absent 10 14 28 Secombes et al., 1983;

Romano et al., 1997
Sea bass 30 Absent 35 38 50 Abelli et al., 1996;
Picchietti et al., 1997;
Breuil et al., 1997
Rainbow trout 8 30 Razquin et al., 1990
320 A.E. Ellis
8. MUCOSA-ASSOCIATED LYMPHOID TISSUE (MALT)

The entire integument of fish is a mucous membrane that is in intimate contact with
a pathogen-rich aquatic environment. It is to be expected therefore that well devel-
oped immune responses will occur in the mucosal compartment but there is compa-
ratively little known. There is no well organized lymphoid tissue in the mucosae but
leucocytes are diffusely scattered throughout. These include lymphocytes, antibody-
secreting cells (ASCs), macrophages, granulocytes and eosinophilic granular cells
(EGCs, considered to be mast cell equivalents; Reite, 1998). EGCs form a dense
layer, the stratum granulosum, in the intestine of many fish species. In rainbow
trout, EGCs first appear about the time of first feeding (Bergeron and Woodward,
1982). Lymphocytes and ASCs are present in skin (Davidson et al., 1993a), gill
(Davidson et al., 1997; Lin et al., 1999) and gut (Davidson et al., 1993b; Lin et al.,
2000). In the intestine, ASCs and sIg+ (B) cells are present mainly in the lamina pro-
pria (Rombout et al., 1993; McMillan and Secombes, 1997). Most lymphocytes in
the gut mucosal epithelium of fish are sIg − (McMillan and Secombes, 1997). Such
sIg− lymphocytes are also present in the epithelium of skin and gills of carp and are
regarded as a distinct population of T lymphocytes in the mucosae, with some sim-
ilarities to CD8+ T cells of mammals (Rombout et al., 1998). Immuno-purification
of sIg− lymphocytes from the gut epithelium and blood of sea bass using a mono-
clonal antibody has been performed and these cells have been confirmed to be
T cells by their expression of the T cell receptor, TCR (Scapigliati et al., 2000).
While antibody-producing cells have been reported in the gill and gut, and antibody
is present in gill and skin mucus (Lumsden et al., 1995), Ig appears to be absent
from gut mucus, at least in salmonids (Hatten et al., 2001).
9. ONTOGENY OF IMMUNE RESPONSIVENESS

9.1. Antibody responses
Evidence from assays of Ig concentration in ova and larvae of fish indicates that
small amounts are present at the time of spawning and this decreases post-hatch
reaching a nadir at about 2 months post-hatch in rainbow trout (Castillo et al., 1993),
12 dph in tilapia (Takemura, 1993) and 5–15 dph in sea bass (Breuil et al., 1997).
Thereafter, Ig concentrations begin to increase, suggesting that the onset of endoge-
nous production occurs at the time of depletion of maternally derived Ig and yolk
and the start of feeding.
The timing of maturation of the immune system in fish is of importance in defin-
ing the earliest time at which different fish species can be vaccinated. As fish grow
more slowly at lower temperatures, development correlates better with size rather
than age. Accordingly the onset of immunomaturation has been claimed to correlate
better with the weight of the fish rather than time after hatch (Manning and Mughal,
1985). However, individual fish of the same hatch can grow at different rates but
this does not appear to affect the rate of development of T and B lymphocytes
(dos Santos et al., 2000). Thus, a developmental age expressed as degree/days may
be the best way of standardizing data for comparison purposes. As these parameters
(age in days, weight and rearing temperature) are infrequently reported in the liter-
ature, it is difficult to directly compare results from different studies. This is further
complicated because there is evidence that exposure to antigen at a very early age
of development may result in tolerance (see below).
In general the specific antibody titres induced by immunization increase with the
age of the fish. Sea bass fry are capable of specific antibody responses to immersion
delivery of Photobacterium damselae spp. piscicida vaccine when only 0.1 g but
higher numbers of antibody-producing cells were induced in 2 g fish and the response
was faster. These responses occurred mainly in the gill tissue (dos Santos et al.,
2001). The serum antibody titres in carp intramuscularly immunized with Vibrio
anguillarum when 85, 99 and 128 days old significantly increased with age (Joosten
et al., 1995). These results are in agreement with the finding of Koumans-van Diepen
et al. (1994) who suggested that the immune system of carp was completed between
3 and 8 months of age, based on the percentages of B cells and plasma cells found in
the lymphoid tissues (see table 4). In addition, van Loon et al. (1981) stated that the
adult level of serum Ig was attained when carp were 5–8 months of age.
Oral exposure of carp and gilthead sea bream (Sparus aurata) to V. anguillarum
antigen at an earlier age (58 dph, just prior to weaning from Artemia nauplii to
commercial diet; weight not reported) resulted in memory formation, as the anti-
body titres induced by an injection of the antigen 10 weeks after oral administration
were significantly higher than in the control group which had not received the oral
priming. However, when carp of only 15 and 29 dph were similarly exposed to
orally administered Vibrio antigens, the antibody titres induced by an injection route
10 weeks later were significantly lower than in the control group (Joosten et al.,
1995). This indicates that very young carp can develop a degree of immunological
tolerance to intestinal exposure to antigens which lasts for at least 10 weeks; an
important feature in devising protocols for oral priming of juvenile fish.
9.2. Ontogeny of memory versus tolerance

The age of onset of specific antibody production appears to depend upon the nature
of the antigen, possibly pertaining to whether it is T independent or T dependent, as
well as the route of exposure. As shown in table 4, rainbow trout fry, injected with
Aeromonas salmonicida antigens at 7 and 14 dph, were unresponsive; they showed
no antibody production, or tolerance on secondary immunization at a later stage
(Manning et al., 1982). On the other hand, injection of this antigen into rainbow
trout and carp at 3–4 weeks post-hatch, induced an antibody response and develop-
ment of memory (Manning et al., 1982; Manning and Mughal, 1985). Similarly,
with bath at this age, although primary antibody levels were low, the bath priming
improved the secondary response to subsequent injection immunization (Manning
and Mughal, 1985). In contrast, injection of human gamma globulin (HGG) or
Table 4. Development of immune responsiveness in fish
Rainbow trout Carp
Antibody response to Antibody response to
Age (weeks)
when first immunized, Vibrio (following Salmon
grafted or assayed A.s HGG Graft rejection A.s HGG Srbc oral priming)4 Graft rejection MLR6
1 −5 − (T)5 Incomplete7 −
2 −5 − (T)5 +7 S +(M)8,9
3 +5 − (T)5 +(M)9 −
4 +(M)1 +(M)1 −T1 −T2 S −
6 +
8 +5 +(M)1 +3 P
−, No response; +, positive response; M, memory induced; T, tolerance induced; S, P, suppressed or enhanced response to subsequent injection respectively;
MLR, mixed leucocyte reaction; HGG, human gamma globulin; Srbc, sheep erythrocytes; A.s, A. salmonicida.
Data from: 1Manning and Mughal (1985); 2Van Loon et al. (1981); 3Van Muiswinkel et al. (1985); 4Joosten et al. (1995); 5Manning et al. (1982); 6Ellis (1977);
7Tatner and Manning (1983); 8Botham and Manning (1981); 9Botham et al. (1980).
sheep red blood cells (Srbc) into trout and carp up to 4 weeks post-hatch resulted in
the induction of tolerance which persisted for up to 23 weeks (Van Loon et al., 1981;
Manning et al., 1982; Manning and Mughal, 1985; Van Muiswinkel et al., 1985).
A positive response and development of memory was not detected to these antigens
until the fish were 8 weeks of age.
This lack of tolerance induction to bacterial antigens by injection or immersion
exposure of young fish contrasts with the effect of oral exposure. As mentioned
above, oral priming of carp at 2 and 4 weeks post-hatch with V. anguillarum anti-
gens suppressed antibody production in response to injection of the antigen
10 weeks later in comparison to non-orally primed controls. However, oral priming
at 8 weeks of age resulted in enhanced responses to injected antigen 10 weeks later
compared with controls (Joosten et al., 1995).
Taken together, these data suggest that B cells and T suppressor functions in carp
mature at about 4 weeks of age but T helper functions and memory cells mature later
at about 8 weeks.
9.3. Ontogeny of immune protection

The earliest time to vaccinate fish is an important issue in aquaculture. Some studies
with salmonid species indicate that immersion vaccination with Vibrio anguillarum
vaccines is ineffective in fish below 1 g (Johnson et al., 1982a). Above this size,
duration of protection lengthened with age. Maximum duration of protection was
achieved in rainbow trout when they were immunized at about 4 g (Johnson
et al., 1982b). Other work indicates that rainbow trout fry could be effectively pro-
tected against vibriosis when immersion vaccinated at 0.5 g body weight (10 weeks
post-hatch) and challenged by ip injection 4 weeks later (Tatner and Horne, 1983).
These fish began feeding at 4 weeks post-hatch.
However, the situation in channel catfish may be quite different. Eyed ova were
immersed in a live attenuated Edwardsiella ictaluri vaccine 4 days prior to hatching.
When challenged by immersion 60 days post-vaccination significant protection was
observed compared to non-vaccinated controls (Shoemaker et al., 2002). However,
it is considered that channel catfish do not attain immuno-competence (antibody
production) until about 4 weeks post-hatch (Petrie-Hanson and Ainsworth, 1999).
The mechanism of the protection by in ovo vaccination is not understood and may
be to do with the vaccine being live and persisting until the immune system matures,
or stimulating the system in a different way to that of inactivated antigens. It may be
relevant that the mechanism of protection against enteric septicaemia in catfish has
been shown to be cell mediated rather than antibody mediated (Shoemaker and
Klesius, 1997; Shoemaker et al., 1997) and cell-mediated immunity (CMI) develops
slightly earlier than the antibody response (see below). Furthermore, it has been
demonstrated that live attenuated bacterial vaccines, such as the AroA Aeromonas
salmonicida, preferentially induce T cell responses relative to B cell responses in
rainbow trout (Marsden et al., 1996).
324 A.E. Ellis
9.4. Ontogeny of cell-mediated immunity

Development of the CMI response in young fish has been investigated using two
tests: the mixed leucocyte response (MLR) and the allograft rejection response
(table 4). In the Atlantic salmon the MLR develops at 45 dph, coincidentally with
first feeding (Ellis, 1977). Skin rejection responses in carp and rainbow trout fry are
developed by 16 dph (Botham and Manning, 1981) and 14 dph (Tatner and
Manning, 1983), respectively. In the trout, grafting at 5 dph resulted in incomplete
rejection by day 30 post-grafting but the experiment was not continued to establish
if allograft application at this age eventually results in rejection or tolerance.
The ontogeny of cell-mediated immune memory has been investigated following
the application of first set skin grafts to 26-day-old rainbow trout and 16-day-old carp
(Botham et al., 1980). In both cases memory developed. It is thus apparent that in trout
and carp the CMI system has fully matured by 2–4 weeks post-hatch and suggests that
this system, with the production of cytotoxic T-like cells, matures a little earlier than
the humoral immune response particularly to the T-dependent antigen, HGG.

Fish larvae appear to hatch when their specific immune responsiveness is still non-
functional and defence against bacterial infection is presumably provided by non-
specific factors derived from the yolk or endogenously produced. Little is known about
these factors or how they are affected by the health status of the mother. However, they
would be expected to play an important role in affecting “egg quality” and the chances
of survival of the larvae. Optimizing these factors with respect to resistance to impor-
tant diseases in aquaculture will be a useful challenge for future research.
As the yolk is utilized by the sac-fry, the lymphoid organs develop functional
maturity, which in some species, e.g. salmonids, coincides with the virtual depletion
of the yolk sac and the onset of first feeding. However, in some species, e.g. flatfish
and sea bass, where the eggs and larvae are very small, feeding on zooplankton
appears to precede the development of the lymphoid organs. For instance, Breuil
et al. (1997) reported that the onset of feeding on Artemia nauplii was at 7–10 dph
in their sea bass larvae, yet the thymus did not become lymphoid until 21 dph and
sIg+ cells began to increase after 30 dph, at about the time the fish were weaned on
to a commercial sea bass diet. These authors concluded that the fry are competent
for antibody responses when the weaning period is achieved at about 40 dph and
the fish were about 50 mg. Thus, in these species there appears to be a rather pro-
longed period from first feeding until the capacity for specific antibody responses.
During this time, defence against bacterial infection would appear to be entirely
dependent on non-specific mechanisms but it is also a period when sea bass fry
are highly sensitive to bacterial infection (Breuil and Haffner, 1989). Sea bass are
certainly capable of antibody responses to immersion immunization at 100 mg,
especially in the gill tissue (dos Santos et al., 2001), but the effect of earlier exposure
to antigen has not yet been reported. In larval carp, oral delivery of Vibrio antigens
bio-encapsulated in Artemia nauplii, before 58 dph resulted in immunosuppression
(Joosten et al., 1995). It will therefore be of great importance to define the timing of
vaccination of larvae of different fish species in order to achieve protection rather
than risk rendering the fish more susceptible to disease.
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15 Development of lactobacilli for mucosal
immunization
J.F.M.L. Seegers, C.E.G. Havenith, S.H.A. Kremer and P.H. Pouwels
Toegepast Natuurkundig Onderzoek (TNO) Prevention and Health,

Department of Infection and Immunology, Special Programme Infectious
Lactobacilli have a number of properties that render them highly suited as vehicles
for delivery to the mucosa of compounds that are of pharmaceutical interest. One
attractive property of many lactobacilli is that they survive passage through the
highly acidic stomach. As such, the desired compounds can be delivered in situ
without the risk of being degraded prior to reaching the actual target site. Indeed,
many strains of the genus Lactobacillus are capable of colonizing specific regions
of the body, e.g. the oral cavity and the gastrointestinal and urogenital tracts, where
they play an important role in maintaining a balanced ecosystem.
Recent years have seen an impressive growth in our understanding of the
molecular genetic properties of lactobacilli and how to exploit this knowledge for
the expression of foreign proteins. The immunomodulating capacity of lactobacilli
together with the possibility to target antigens to specific sites of the bacterium, offer
attractive opportunities for the treatment of infectious diseases through vaccination,
and of autoimmune diseases or other immune disorders by modulating the immune
response in a directed and predetermined way.
In this overview the present state of the art regarding Lactobacillus systems for
the high-level expression of foreign antigens and their delivery will be presented.
Some immunological properties of lactobacilli will be discussed and their potential
use as delivery vehicles for oral immunization purposes will be highlighted.
1. INTRODUCTION
Why mucosal immunization? First of all, delivery of vaccines to the mucosa (orally,
intranasally, vaginally) is simple and relatively inexpensive and does not require the

Lactobacilli for mucosal immunization 329
use of needles with all the risks of contamination involved. This is especially impor-
tant for large vaccination programmes in which large numbers of subjects are
involved. Moreover, for most pathogens mucosal sites are the porte d’entrée and
therefore the most likely sites for a vaccine to elicit the desired response. Parenteral
administration of antigens stimulates induction of systemic immune responses,
but is generally ineffective for induction of secretory immunoglobulin A (sIgA).
Vaccines administered via mucosal surfaces can elicit biologically active serum IgG
antibodies and cell mediated immune responses. Furthermore, they can elicit
mucosal sIgA and cell mediated immunity at mucosal surfaces to prevent pathogen
infiltration and inflammation (Levine and Dougan, 1998; van Ginkel et al., 2000).
Why lactobacilli? Several live microbial vaccine-vectors that are active at target
sites of mucosal immunization have been shown to be efficient delivery systems in
facilitating immune responses at mucosal and systemic sites concurrently (Walker,
1994). These vectors are usually derived from attenuated pathogenic microorgan-
isms such as Salmonella typhimurium, Listeria monocytogenes and Mycobacterium
bovis BCG. Several excellent recent reviews describe the use of these pathogens in
vaccination and their future development (Thole et al., 2000; Medina and Guzman,
2001; Mollenkopf et al., 2001). However, safety considerations, particularly the
immune status of the vaccine recipients in developing countries, make it doubtful
whether these vector candidates can be used on a large scale. Furthermore, these
organisms are highly immunogenic themselves, drawing unnecessary attention of
the immune system, and this might even hamper repetitive use of the carrier with
other antigens (Hone et al., 1991). Moreover, this system could be extremely useful
for the development of safe oral vaccines for infants of whom the immune system
has not been fully developed and for whom vaccines based on attenuated pathogens
could pose a serious health risk. Therefore, non-pathogenic, food grade or com-
mensal bacterial vectors have started to receive attention for their vaccine potential
(Pouwels et al., 1996; Slos et al., 1998; Shaw et al., 2000; Havenith et al., 2002).
These bacteria are in general amenable to genetic transformation, and the fact that
they can survive the passage through the stomach and can persist in the gastroin-
testinal tract makes them useful delivery vehicles of antigens to be presented to the
mucosal immune system.
A potential limitation of oral immunization is that the immunogenicity of anti-
gens administered in a soluble form is low. In fact, this administration route may
even induce tolerance. To circumvent this, antigens should be presented together
with an adjuvant, either chemically coupled to a carrier or in the form of a geneti-
cally engineered bacterium or virus. Proteins delivered in this way can induce a pro-
tective immune response in the host without the need for actual contact with the
pathogen itself.
The past decade has seen intensive research, focused on the use of lactic acid bac-
teria (LAB) as vaccine delivery vehicles. Overviews of earlier research in this field
have appeared in a number of articles (Wells et al., 1996; Pouwels et al., 1998;
330 J.F.M.L. Seegers et al.
Mercenier, 1999). In this chapter, an overview is given of the current state of the art
with respect to the development of lactic acid bacteria in general and Lactobacillus
spp. in particular as immune modulating entities with an emphasis on developments
over the past five years.
2. LAB AS LIVE MICROBIAL CARRIERS

Many LAB species are being used for the manufacturing of fermented food prod-
ucts, varying from wine and yoghurt to cheese and dried sausages. LAB species can
also be found as members of the natural microflora of living organisms and, as a
result, have long been the subject of scientific research. Over the past two decades
LAB have received enormous attention in international research, also as a result of
large financial funding from the EU. This has led to an explosive growth in our
knowledge on many aspects of these bacteria, such as carbohydrate metabolism,
genetics, stress regulation, and phage resistance. By now, the genomes of several
representatives of LAB have been determined, which will give a new stimulus to
research in this field.
The possibility to use LAB for vaccination purposes was seriously considered for
the first time about 10 years ago. Gerritse (1990) showed that killed lactobacilli that
carried the hapten trinitrophenyl chemically coupled to the cell surface, could be
used for the immunization of mice. Other groups exploited the use of Streptococcus
gordonii (Pozzi et al., 1992) or Lactococcus lactis (Wells et al., 1993) as expression
and delivery vehicles. A first joint effort was initiated through the EU Biotech 1
programme. This programme was organized as a single integrated project aimed
at the development of LAB for industrial application and combined the expertise of
36 participating laboratories. The project was divided into subgroups to facilitate a
targeted focus on key objectives. The key objective of one subgroup was LAB in
vaccines. This subgroup continued in the fourth framework programme under the
acronym LABVAC, comprising nine groups from all over Europe. Their work
resulted in the development of sophisticated cloning systems that allow expression
at the different cellular compartments and led to several interesting findings. It was
shown that the level of expression of the antigen is a crucial factor. This in itself is
not a surprise, but poses some limits to what can be administered. It was also shown
that the immune response could be enhanced when mice were immunized
intranasally with different expression strains of L. lactis which secreted IL-2 or
IL-6 in addition to the production of tetanus toxin fragment C (TTFC). In this
case, the anti-TTFC antibody titres increased more rapidly and were substantially
higher (Steidler et al., 1998). This group also showed that lactococci, expressing
and secreting IL-10, can be used for the treatment of colitis in mice, and this model
is currently being tested as a possible treatment for Crohn’s disease in humans
(Steidler et al., 2000). An overview of viral and bacterial antigens that have been
cloned in non-pathogenic Gram-positive bacteria is given in table 1.
Table 1. Antigens that have been cloned in non-pathogenic bacteria for vaccine development
Carrier strain Pathogen Antigen Reference
L. lactis Brucella abortus L7/L12 Ribeiro et al., 2002

L. lactis Helicobacter pylori Ure B Lee et al., 2001
L. lactis Rotavirus NSP4 Enouf et al., 2001
L. lactis Clostridium tetani TTFC Wells et al., 1993
Lb. fermentum HIV gp41 Turner and Giffard, 1999
Lb. fermentum Chlamydia psittaci OmpA Turner and Giffard, 1999
Lb. casei Bacillus anthracis PA Zegers et al., 1999
Lb. plantarum HIV Epitope gp41 Hols et al., 1997
Lb. casei Influenza virus Hackett epitope Pouwels et al., 1996
Lb. casei FMD virus fgmt VP1 Pouwels et al., 1996
S. gordonii Porphyromonas gingivalis FimA Sharma et al., 1999
S. gordonii Measles virus HA Maggi et al., 2000
S. gordonii Measles virus F Maggi et al., 2000
S. gordonii Escherichia coli LTB Ricci et al., 2000
S. gordonii HIV Epitope gp120 Pozzi et al., 1994
S. gordonii Bordetella pertussis Toxin S1 subunit Lee et al., 1999
S. gordonii Human papillomavirus E7 Pozzi et al., 1992
L., Lactococcus; Lb., Lactobacillus; S., Streptococcus.
Experiments performed by Drouault et al. (1999) with Lactococcus lactis express-

ing green fluorescent protein suggest that the antigens are being released as a result
of cell lysis either before or after uptake by macrophages and are thus exposed to
the immune system. This difference in immunogenicity could reflect a difference
in expression levels as a result of promoter activity or as a result of extracellular
proteolytic activity.
An ongoing topic of research is strain containment. It is desirable that recombinant
strains that have been used for immunization experiments are not able to survive
outside the treated subject in order to prevent the unwanted spread of these micro-
organisms in the environment.
3. SELECTION OF STRAINS
Several criteria can be defined for the choice of the proper strain. These might for
example be related to the capacity to stimulate or suppress an immune response, to
the efficacy with which such strains can express and secrete a foreign antigen, or to their
resistance to the hostile environment that is present in the gastrointestinal tract. There
is an increasing accumulation of evidence showing that LAB, notably lactobacilli, are
capable of influencing the expression of a number of cytokines, both in vivo and
in vitro. It has also been shown that different strains induce distinct mucosal cytokine
profiles and possess differential intrinsic adjuvanticity (Maassen et al., 2000), a pro-
perty that enables the investigator to aim at Th1- or Th2-dominated immune responses.
A strong Th2-dependent adjuvanticity of a Lactobacillus strain might be considered

as an advantageous property, if the strain is to be used for active vaccination.
However, for the deliberate induction of mucosal tolerance, e.g. in the case of expres-
sion of autoimmune antigens, strains with immunosuppressing properties might be
more suitable.
A number of Lactobacillus strains, in contrast to lactococci, can persist in the
gastrointestinal tract for an extended period of time and are found as part of the
normal microflora. Both in vitro and animal models have been used to select for
adherent Lactobacillus strains. In a study performed by the groups of Collins and
Marteau, the survival of four non-recombinant lactic acid bacterial strains that were
given orally to human volunteers has been analysed. The two major conclusions from
these studies are: i) there are considerable differences in the survival of different
strains, and ii) no strain colonized the gastrointestinal tract permanently (Dunne
et al., 1999; Vesa et al., 2000). Depending on the application, persistence of the strain
could be a major selection criterion.
Other selection criteria are the transformability of a selected strain and the level
at which it can express the antigen. Although a variety of protocols have been devel-
oped for the transformation of many Lactobacillus strains, some strains can still not
be transformed. In addition, the synthesis of antigens is not equally effective in all
Lactobacillus strains. An important observation has been that the efficiency with
which a promoter is recognized can differ greatly between strains. In studies per-
formed by McCracken and Timms (1999), mutations were introduced in a single
promoter, and the transcription levels were compared between two Lactobacillus
strains. In another study (McCracken et al., 2000), several promoters were isolated
and their activity was tested in three different Lactobacillus strains. These studies
have identified some attributes, such as a UP element and the TG DNA sequence
motif, that can play a role in the control of transcription in lactobaccilli. A selection
criterion therefore could be the level of expression from a certain promoter in a
specific strain. Another difference that can be observed between Lactobacillus
strains is codon usage (Pouwels and Leunissen, 1994). These differences are being
corroborated by the increasing availability of sequence data. The implications of this
have still to be assessed.
Some studies have been performed aimed at determining the fate of LAB after
oral or intranasal administration. The green fluorescent protein (GFP) and luciferase
genes have been used to determine survival and metabolic activity of L. lactis in
the gastrointestinal tract (Droualt et al., 1999). It was shown that the way in which
bacteria are administered had a dramatic impact. Lactococci that transit with the diet
appeared quite resistant to gastric acidity (90 to 98% survival), whereas only 10 to
30% of the bacteria survived in the duodenum. Viable cells were shown to be meta-
bolically active in each compartment of the digestive tract, while most dead cells
were subjected to rapid lysis. Furthermore, it was shown by in vitro studies that
trypsin strongly affected survival while pepsin, a stomach enzyme, had far less
influence on survival rates. These findings were corroborated by in vivo experiments

which indicated that viability of L. lactis was greatly reduced in the duodenum
where trypsin, secreted by the pancreas, is present. In another study with GFP-
expressing Lb. plantarum, ingestion of cells by bronchoalveolar macrophages
following intranasal administration could be shown (Geoffroy et al., 2000). As esti-
mated by flow cytometry, the proportion of macrophages having phagocytosed
lactobacilli could reach 10% of the total bronchoalveolar macrophage population.
4. LACTOBACILLUS AS DELIVERY VEHICLE FOR

ORAL IMMUNIZATION
As outlined above, lactobacilli are capable of persisting in the gastrointestinal tract
for an extended period of time and show a significant immunomodulatory capability.
One could argue that these criteria would favour lactobacilli over lactococci for
the development of live oral vaccines. As an added benefit many lactobacilli have
probiotic properties and can prevent adherence, establishment, and replication of
several enteric pathogens through different antimicrobial mechanisms such as
competition for nutrients, occupation of mucosal sites, and the production of anti-
microbial peptides. Apart from these intrinsic properties of lactobacilli, several other
criteria are important for the induction of an adequate immune response. These
involve the regime of immunization and the level of expression of the antigen as
well as the location of expression.
In the following section, probiotic properties, some immunogenetic traits of
lactobacilli, and the development of genetic tools will be discussed.
4.1. Probiotic properties

In recent studies sponsored by the EU, a consortium of laboratories have defined
and applied a number of criteria for the selection of probiotic strains (Dunne et al.,
1999). Some of these criteria that also apply to the selection of vaccine-carrier
strains, are acid resistance, bile tolerance and adherence to host epithelial tissues.
Many lactobacilli are acid resistant and bile salt resistant, which enables them to
survive the passage through the stomach and ileum, and to be maintained in large
numbers in the small intestine and upper part of the large intestine, despite the pres-
ence of considerable amounts of bile salts. The property of survival in the presence
of bile salts originates from the capacity of numerous lactobacilli to hydrolyse con-
jugated bile salts. Deconjugation not only renders bile salts less toxic for intestinal
bacteria, but also diminishes their concentration in the lumen, since deconjugated
bile salts can be resorbed in the duodenum into the blood, in contrast to conjugated
bile salts (Leer et al., 1993; de Roos and Katan, 2000).
Very little is known about the mechanisms that are used by lactobacilli to adhere
to the mucosa of the intestinal tract. This contrasts with the detailed knowledge that
has become available on the structure and properties of adhesins and the mode of
regulation of adhesin biogenesis of pathogenic bacteria such as Salmonella, Yersinia
and Listeria (Finlay and Cossart, 1997; Finlay and Falkow, 1997).
Persistence of lactobacilli in the gastrointestinal tract is a host-specific and
possibly also tissue-specific process that is mediated by a number of cell wall asso-
ciated components such as lipoteichoic acids (LTA), polysaccharides and proteins
(Tannock, 1999). A surface-associated protein has been identified that enhances the
adhesion of L. reuteri 104R to squamous epithelial cells and to mucus (Henriksson
and Conway, 1992). It was shown that mutants lacking this MapA protein had a
significantly reduced persistence in mice (Satoh, Leer, Conway and Pouwels,
unpublished observations). The fact that the strain still persisted for a period of more
than 3 to 4 days, suggests that other surface-associated bacterial factors are involved
in its adhesion to mucosal surfaces. Lactobacilli can also associate with gastroin-
testinal and urogenital cells and co-aggregate with microorganisms owing to the
presence of so-called surfactants at the bacterial surface (Reid et al., 2000). These
molecules, which are characterized by the ability to reduce liquid surface tension,
can inhibit the adhesion of enterococci and other uropathogens to solid surfaces
(Velraeds et al., 1996).
Yoghurt consumption has some well acclaimed benefits such as prevention,
reduction of duration or severity of infant diarrhoea and has even been linked to anti-
tumour effects. These benefits are thought to be largely a result of metabolic fermen-
tation products of the commonly used lactic acid bacterial strains Lb. bulgaricus and
Streptococcus thermophilus (Djouzi et al., 1997). Table 2 shows some health bene-
fits that have been attributed to a number of other Lactobacillus strains.
4.2. Intrinsic immunogenicity of lactobacilli

Immune responses against bacteria are usually and understandably directed against
cell surface associated moieties such as peptides, lipopolysaccharides (LPSs) and
carbohydrates. Since the composition of these elements differs strongly between
bacteria, they influence the response of the immune system in different ways. LPS,
for instance, is a strong immunogen, but is completely absent in Gram-positive
bacteria. It has been reported that lipoteichoic acid (LTA), a major constituent of the
bacterial cell wall of Gram-positive bacteria, is able to weakly stimulate cytokine
synthesis, and that this effect depends on the presence of D-alanine substituents
(Bhakdi et al., 1991; Tsutsui et al., 1991).
On the basis of specific antigenic determinants (mostly cell wall located sugar
moieties), lactobacilli have been classified into seven serological groups (Sharpe,
1981). More recent studies have shown that lactobacilli can stimulate or inhibit
the expression of several cytokines and can thus influence the immune response
(Yasui et al., 1999; Maassen et al., 2000). The intrinsic properties of lactobacilli to
modulate the immune system render them attractive for health applications, and in
Table 2. Reported activities of probiotic bacteria
Activity Mode of action* Species**
Intestinal health Balancing microflora Lb. acidophilus,

Lb. bulgaricus
Anti-cancer Enhancing the immune response, Lb. acidophilus, Lb. casei
binding/removal of carcinogens
Immune system modulation Production of immunopeptides Lb. reuteri, Lb. plantarum
Milk tolerance (lactose Lactose metabolism Lb. bulgaricus
intolerance/milk allergy)
Vaginal/urinary tract health Low pH, bacteriocin production, Lb. rhamnosus,
hydrogen peroxide production Lb. acidophilus,
Lb. fermentum
Stomach health Anti-Helicobacter pylori Lb. acidophilus
Hypertension Bioactive peptides resulting Lb. Helveticus, Lb. casei
from proteolysis
Cholesterol lowering ??? Lb. acidophilus
*Since in many cases the exact modes of action are unknown, only some likely possibilities are given.
All these activities and their possible modes of action are extensively reviewed in a recent issue
of the Journal of Nutrition that was in part dedicated to a symposium on probiotic bacteria, held
in Washington DC in April 1999 (J. Nutr. 130, 2000, 382S−416S).
**Most of the reported activities are not exclusive to the species that are mentioned. Other species
with these activities have been reported, but only the most important ones are listed.
particular for the in vivo production and delivery of biologically active molecules
(Geoffroy et al., 2000). Administration of lactobacilli can lead to activation of innate
immune effector functions, can affect cytokine expression in a specific or non-specific
manner, and can influence humoral responses.
With regard to the innate immune function, many studies have shown that differ-
ent strains of lactobacilli are able to activate macrophages and induce production of
TNF-α, IL-1, IL-6, IL-12, IL-18 and/or IFN-γ (reviewed by Maassen, 1999). Recent
studies indicate that in general Gram-positive bacteria induced more Th1-like
cytokines, e.g. IL-12, IL-18, and IFN-γ, than Th2-like IL-10 in human monocytes,
while Gram-negative bacteria induced more IL-10 than IL-12 (Mietinnen et al.,
1998; Haller et al., 2000; Hessle et al., 2000). The differential effects of various
bacteria, and especially of Lactobacillus strains, on the immune system are at least
partially due to differences in cell wall composition. Peptidoglycan, a major com-
ponent of the cell wall of Gram-positive bacteria, has been shown to induce the
production of IL-1, IL-6, and TNF-α by human blood cells (Schrijver et al., 1999).
However, it is still uncertain whether peptidoglycan is responsible for macrophage
activation (de Ambrosini et al., 1996). In addition, the cell wall components pepti-
doglycan and LTA, could not completely mimic the impressive IL-12 inducing
effect of intact Gram-positive bacteria (Hessle et al., 2000). It was suggested that yet
undefined component(s) of Gram-positive bacteria is (are) responsible for their
strong IL-12 inducing capacity. These data suggest that Gram-positive bacteria
could be especially suited as inducers of Th1-type responses. Recently, it has been
demonstrated that different types of Toll-like receptor (TLR) recognize different cell
wall components. This new class of receptors is indicated as a key initiator of innate
immunity by activating the NF-κB and AP1 pathway. The triggering of these recep-
tors leads to intracellular signalling and activation of a variety of inflammatory
mediators and cytokines. Different members of the TLR class are triggered by dif-
ferent bacterial components (Aderem and Ulevitch, 2000). TLRs have also been
identified on intestinal epithelial cells, and it has been speculated that they might
play a frontline role in monitoring lumenal bacteria (Cario et al., 2000). NF-κB has
been indicated as a central regulator of intestinal epithelial cell innate immune
responses induced by infection with enteroinvasive bacteria (Elewaut et al., 1999).
Recently it was shown that a non-pathogenic strain of Salmonella (commensal
bacterium) is able to abrogate the synthesis of inflammatory cytokines by gut
epithelial cells. The bacteria accomplish this by blocking degradation of IκB, an
inhibitor of NF-κB. It was speculated that this might represent one of the probiotic
working mechanisms of commensal bacteria in general (Neish et al., 2000).
4.2.1. Natural killer cells

Natural killer (NK) cells play an important role in innate immune resistance, partic-
ularly through the synthesis of the proinflammatory cytokine IFNγ. Besides
macrophages (vide infra), NK cells constitute the primary targets for bacterial stim-
ulation. NK cells do upregulate the IL-2Rα chain (CD25) and undergo proliferation
when stimulated by L. johnsonii. In the presence of bacterially primed macrophages,
expression of CD25 and/or secretion of IFNγ from purified NK cells was signi-
ficantly increased, indicating that full activation required both bacterial and cell
contact-based signals derived from accessory cells (Haller et al., 2000).
4.2.2. Immunoglobulins
The capacity of certain lactobacilli to enhance systemic and mucosal immunity in
general, has been shown both in animal model systems and in humans. There have
been several reports describing the effects of lactobacilli on upregulation of IgA pro-
duction, locally as well as systemically, and enhanced sIgA production and
increased numbers of IgA-producing plasma cells was observed (Maassen, 1999;
Perdigón et al., 1999; Erickson and Hubbard, 2000). For instance, oral administra-
tion of Lactobacillus GG (LGG) during acute rotavirus diarrhoea promotes recovery
via augmentation of the local immune defences. LGG enhanced non-specific humoral
responses during the acute phase of the infection, reflected in the IgG, IgA, and IgM
secreting cell numbers. Furthermore, a specific IgA response to rotavirus is endorsed,
which is possibly relevant in protection against reinfection (Kaila et al., 1992;
Isolauri et al., 1995). In addition, LGG has an immunostimulating effect on oral

rotavirus vaccination (DxRRV reassortant rotavirus vaccine), with higher serocon-
version (IgA and IgM).
A role for lactobacilli in treatment of allergic disorders was indicated by the
observation that oral feeding of killed Lb. casei (strain Shirota) was able to stimulate
the production of Th1 cytokines, resulting in repressed production of IgE antibodies
in an animal model. A potential role in attenuating autoimmune diseases came from
the observation that cofeeding of antigen with probiotic bacteria can suppress
both antibody and cellular immune responses. The tumour-suppressive activity of
lactobacilli seems to depend on the activation of macrophages producing IL-1 and
TNF-α (Maassen, 1999; Matsuzaki and Chin, 2000).
4.3. Molecular biological considerations

Over the past decade, considerable progress has been made in developing techniques
to genetically modify lactobacilli. As mentioned before, one of the great obstacles for
the genetic manipulation of lactobacilli is their often poor transformability. However,
for most strains, adequate transformation protocols have been established. To date
essentially all tools necessary for investigation of the properties of lactobacilli at the
molecular level and for their modification in a directed and predictable manner have
become available. Plasmid vectors have been constructed with which different
species of Lactobacillus can be genetically modified as well as vectors for general or
site-specific integration of vector material into the Lactobacillus chromosome.
Expression signals have been analysed and are currently being used in expression
vectors enabling the efficient and targeted expression of antigens or enzymes.
While site-specific chromosomal integration is possible for lactobacilli, its main
application has been the adaptation of an inducible system based on the autoregula-
tory properties of the bacteriocin nisin (Pavan et al., 2000). This system was derived
from L. lactis and extensive studies of its regulatory mechanisms have resulted in
the development of the nisin-controlled expression (NICE) system (Kleerebezem
et al., 1997). The original two-plasmid NICE system turned out to be poorly suited
to Lb. plantarum. In order to obtain a stable and reproducible nisin dose-dependent
synthesis of a reporter protein or a model antigen, the lactococcal nisRK regulatory
genes were integrated into the chromosome of Lb. plantarum. This gave satisfactory
results with regard to stability and regulation. This system turned out to be well
suited to determine a dose–response relationship between the amount of antigen
produced by the bacteria and the corresponding antibody titre. For vaccine develop-
ment using non-pathogenic Gram-positive microorganisms chromosomal integra-
tion is mainly being exploited for Streptococcus gordonii. This research has led to
some impressive results in eliciting immune responses to different antigens and
combating pathogens at vaginal and oral sites (Oggioni et al., 1999; Loeffler et al.,
2001; table 2).
Indeed, integration into the chromosome usually results in genetically fully stable
transformants. This method, however, results in multiple copies of the gene and
therefore generally gives rise to higher levels of expression, which has been shown
to be an important criterion in eliciting an appropriate immune response. At TNO
(the Dutch Organization for Applied Research), a series of expression vectors was
designed that allows direct cloning of antigens and expression of these antigens in
different cellular compartments. These vectors are organized in easily replaceable
cassettes, and their main differences are i) the promoter that is employed for the
expression of the antigen, ii) the presence or absence of a secretion signal, and iii) the
presence or absence of a cell wall anchor that, when present, results in surface expo-
sure of the gene product. The general structure of these vectors is depicted in fig. 1.
4.3.1. Promoter sequences

As for most prokaryotic promoters, the majority of Lactobacillus promoters show
typical and conserved –35 and –10 sequences. In some cases an additional TG motif
is found at position –16. The appearance of a TG motif often coincides with the
presence of a less conserved or even total absence of a –35 region. The TG motif,
which is not essential for promoter activity, might enhance a rate limiting reaction
such as docking of RNA polymerase near the –35 region (Pouwels and Chaillou,
2003). For the current series of vectors, two promoters were chosen. One promoter
(Pamy), driving the expression of the L. paracasei α-amylase gene, carries a cre
element to which the global repressor CcpA can bind in the presence of a rapidly
metabolizable energy source. Expression from Pamy can be repressed, e.g. by the
presence of glucose in the growth medium, and can be derepressed by replacing
glucose by other sugars such as mannitol or galactose. In other vectors, the promoter
of the Lb. casei lactose dehydrogenase gene (Pldh) is used. A new series of vectors
Fig. 1. General structure of the Lactobacillus

expression vectors that can be used for the
expression of antigens in a variety of
Lactobacillus species. mcs = multiple cloning
site, ss = secretion signal. All elements such as
promoter, selection marker, anchor sequences,
etc. are present in the vectors as cassettes that
can be easily exchanged between different
vectors. Upstream and downstream of the open
reading frame (orf) region a number of unique
restriction enzyme sites are present to allow
insertion or replacement of antigen encoding
sequences. For more details about
the structure and properties of these vectors,
see Pouwels, Leer and Boersma (1996) or
Pouwels et al. (2001).
is being developed for which a third promoter, the slpA promoter (PslpA) that drives
expression of the surface layer protein of Lb. acidophilus, is being employed (Boot
et al., 1996). Similar to the ldh promoter, expression from PslpA is constitutive, but
it has been shown to give higher levels of expression than the other two promoters.
Although protocols have been developed for the transformation of many
Lactobacillus spp., the transformation frequencies are still considerably lower than
those obtained for E. coli. Consequently, cloning of antigens is mostly carried out in
E. coli. As cloning of foreign genes in E. coli frequently results in structural insta-
bility with concomitant loss of parts of the plasmid vector (Pouwels and Leer, 1993),
a phenomenon which was also observed in the initial stages of the construction
of these vectors, a strategy was designed to circumvent instability in E. coli. The
observed instability seemed to be correlated with the activity on cloned fragments
of promoter sequences. For this reason, an 80-basepair cassette containing the
rho-independent terminator of the ldh gene, flanked by NotI sites, was inserted in all
vectors downstream from the promoter and the translation initiation region. This
resulted in complete structural stability of the hybrid Lactobacillus-E. coli vectors
in E. coli. Before the vectors are transferred to Lactobacillus, this terminator is
removed by NotI digestion and religation.
4.3.2. Secretion signals

In order to elicit an immune response, antigens have to be presented to the immune
system. Three possibilities exist for bacteria: intracellular, extracellular and surface-
bound expression. Extracellular and surface-bound expression require the presence of
an efficient secretion signal. The secretion signal that was used for the current series
of vectors normally drives the secretion of the α-amylase gene of L. amylovorus.
Another secretion signal that is currently under investigation, is that of the highly
expressed surface layer protein slpA (Boot et al., 1993).
4.3.3. Anchor sequences

Surface exposure of the antigen is obtained by adding a peptide anchor sequence
to the protein in addition to the secretion signal. Five different anchors can be
distinguished, tentatively named A1 to A5 (for reviews, see Leenhouts et al., 1999;
Navarre and Schneewind, 1999). The A1 anchor comprises a transmembrane span-
ning domain (TMD) that links the antigen to the cytoplasmic membrane. This
method, however, requires the addition of at least 100 amino acids between the
TMD and the protein of interest in order to span the cell wall so that the antigen
becomes exposed at the cell surface. Its efficacy has not yet been tested in intact
cells. The A2 anchor binds the antigen covalently to the lipid bilayer through N-acyl
diglyceride modification of a cysteine residue, located immediately C-terminal to
the signal sequence cleavage site (Pugsley, 1993). The A3 anchor is responsible for
covalent binding to the cell wall through a sorting reaction that is characterized by
a LPXTG box, a hydrophobic region and a charged tail (Navarre and Schneewind,
1999). This region is preceded by a 50 to 125 amino acid residues long region to
span the cell wall, and has a high percentage of proline/glycine and/or threonine/
serine residues. Binding of the immunoglobulin binding protein A and of numerous
other Gram-positive bacterial surface proteins, occurs through a type A3 anchor.
A fourth type of anchor (A4) has a modular design and is applied by nearly all bac-
terial cell wall hydrolases. The cell wall binding domain often comprises repeated
amino acid sequences and the nature of binding to the cell wall is as yet unknown,
but is most likely of a non-covalent nature. Surface layer proteins (S-proteins) of
bacilli and lactobacilli are anchored with the outer part of the cell wall by hydrogen
bonds and/or ionic interactions through a region (A5) that is highly positively
charged and contains a large number of tyrosine residues. The tyrosine residues may
be involved in binding of the S-proteins to saccharide moieties in the cell wall.
S-proteins of bacteria belonging to the acidophilus group A, such as Lb. acidophilus,
Lb. crispatus and Lb. helveticus contain a highly conserved C-terminal region
(one-third of the protein) that interacts with the cell wall, while the anchoring
region in bacilli and L. brevis is found near the N-terminal end (Jarosch et al., 2000;
Smit et al., 2001).
The anchor sequence that is used in the vectors that are presented in fig. 1 is
derived from the L. paracasei protease PrtP and belongs to the A3 family of anchors.
It has successfully been employed for surface exposure of β-glucuronidase of
E. coli, rotavirus structural proteins VP7 and VP8 and Urease A and B of H. pylori
(Pouwels et al., 1998), and tetanus toxin fragment C (TTFC) (Maassen, 1999).
Other anchor types that are reported to have been successfully employed in lacto-
bacilli are an A4 type anchor from the AcmA protein of L. lactis for surface exposure
of β-lactamase, and an A5 type anchor of the surface layer protein SlpA of L. brevis
(Leenhouts et al., 1999).
4.4. Future development of vectors

Results from several groups indicate a strong correlation between the level of expres-
sion and the immune response that is elicited. Therefore, high-level expression of the
antigens seems to be a prerequisite. This can be obtained by choosing strong pro-
moters. It has been shown, however, that a strong promoter in one Lactobacillus
strain is not necessarily a strong promoter in another strain (McCracken and Timms,
1999). This necessitates the evaluation of the used promoter in the chosen strain and,
when necessary, either optimizing the promoter or selection of another promoter that
does display strong expression levels for the desired strain.
Another variable is the immunogenicity of the chosen antigen itself. Some
proteins hardly elicit an immune response while other proteins, such as TTFC,
are potent stimulants of the immune system. In this respect, translational coupling
of a low immunogenic substrate such as glutathione S-transferase with TTFC has been
successfully employed to elicit an immune response against this substrate (Khan et al.,
1994b). The immune response could further be enhanced by coupling TTFC with mul-
tiple tandem copies of a short peptide of this protein (Khan et al., 1994a).
A necessary further development is the construction of food grade vectors. The
current vectors carry antibiotic resistance genes as selection markers and these will
have to be replaced by adequate food grade selection markers to allow their use in
humans without risking the inadvertent release of the antibiotic resistance trait into
the environment. Several metabolic genes such as the lacF gene, which is essential
for lactose utilization, have been employed as food grade selection markers
(MacCormick et al., 1995; Platteeuw et al., 1996). This requires inactivation of
the concomitant gene on the chromosome and limits its use to that specific strain.
A wider application can be obtained with bacteriocin resistance (immunity) markers.
An example of this is the L. johnsonii LafI, which confers immunity to lactacin F
(Allison and Klaenhammer, 1996). Since lactacin F does not inhibit growth of all
lactobacilli, additional resistance markers against different bacteriocins are required.
5. LACTOBACILLI AS VACCINE CARRIERS: A CASE STUDY

In the previous subsection, we mentioned that one of the factors that can influence the
immune response is the immunization regime. In our group, oral and nasal immu-
nizations of mice have been performed with vaccines constructed through an original
system for heterologous gene expression in Lactobacillus in which the 50 kDa TTFC
is expressed as either an intracellular or a surface-exposed protein (Shaw et al., 2000).
Following nasal delivery of recombinant Lb. plantarum expressing TTFC intra-
cellularly, antigen-specific antibody secreting cells (ASC; IgA as well as IgG) could
be detected in the cervical lymph nodes, indicating that local mucosal activation has
occurred. These observations are underlined by the results from an analysis of frag-
ment cultures of nasal-associated lymphoid tissues (NALT), in which TT-specific
IgA could be detected. In addition, activation of the local gut mucosa occurred
following oral delivery of recombinant Lactobacillus, as indicated by the presence
of TT-specific ASC in mesenteric lymph nodes (MLN) and Peyer’s patches (PP),
and by the presence of TT-specific IgA in fragment cultures of the duodenum.
Substantial levels of TTFC-specific immunoglobulin G (IgG) in serum were induced
after nasal delivery of live recombinant lactobacilli, confirming the efficiency of the
nasal route for immunization. Although less efficient, oral application also induced
significant levels of TTFC-specific IgG in serum (fig. 2).
These results led us to the hypothesis that nasal delivery could facilitate the
induction of gastrointestinal tract responses by subsequent oral deliveries. Indeed,
it was found that induction of systemic, and more importantly of gastrointestinal
tract responses are augmented in mice that received recombinant lactobacilli via the
intranasal route before intragastric deliveries were applied (fig. 3). Moreover, the
Fig. 2. TTFC-specific IgG following immunization of groups of mice with live recombinant lactobacilli,
measured by ELISA. (A)16 C57BL/6 mice were immunized with three doses of 5×109 Lb. plantarum
pLP503-TTFC intranasally in 20 μl of phosphate buffered saline (PBS) ( ) or orally in 200 μl of NaHCO3 (▲)
•
on days 1–3. Identical booster immunizations were administered on days 28–30. (B) Three C57BL/6 mice
were immunized with 5×109 Lb. plantarum pLP503-TTFC intranasally in 20 μl of PBS on day 1 and day 28
(■) or three doses of Lb. plantarum pLP401-TTFC intranasally in 20 μl of PBS on days 1–3 followed by a
booster with either 5×109 Lb. plantarum pLP503-TTFC on days 28–30 (▲), or Lb. plantarum pLP401-TTFC
on days 28–30 and 49–51 (◆), intranasally in 20 μl of PBS. This figure is adapted from Shaw et al. (2000).
results gave further proof of the compartmentalization of the immune system, and
indicated that responses found at the gastrointestinal level were not due to accidental
intranasal cross-contamination.
The goal for successful mucosal vaccination will be to optimize priming of both
systemic and mucosal immune compartments. Our data indicate that systemic and
local activation of the mucosal sites occurs, in which both sites, nasal and intestinal,
can be targeted. Intranasal application leads to better induction at the nasal mucosal
site, whereas oral application leads to better induction of local responses at the intes-
tinal site. Intranasal priming, in addition, can enhance the local responses at the
intestinal sites following oral booster applications. This indicates that recombinant
lactobacilli interact with the mucosa and opens up considerable potential for the
generation of more potent mucosally administered vaccines.
Fig. 3. Measurements of TT-specific IgA antibodies in fragment cultures of the NALT (I), and duodenum
and ileum (II) of animals immunized intranasally (in) and/or intragastrically (o) with live recombinant
Lb. plantarum expressing TTFC. Each animal was given one prime and two boosters each 2 weeks apart.
Group A was primed intranasally followed by two intranasal boosters, group B primed intranasally followed
by two intragastric boosters and group C primed intragastrically, followed by two intragastric boosters.
The absorbance (OD405nm) values of each individual animal sample are given.
In a comparative study, Lb. plantarum 256 turned out to be more effective in

eliciting an immune response than Lb. casei 393 (Shaw et al., 2000). These strains were
initially selected for study because of their different periods of persistence in the GI
tract. It is interesting to speculate that differences in persistence could explain the
differences found in immunogenicity. Moreover, the levels of TTFC expressed intra-
cellularly were higher in Lb. casei than in Lb. plantarum. However, sustainable levels
of non-degraded TTFC, interaction of the bacteria with M cells, dendritic cells and
macrophages, and intrinsic adjuvanticity will each have its influence. These character-
istics may help to increase the immunogenicity of the antigen delivery vehicle. In addi-
tion, the influence of immunization schemes, with variations in time between priming
and boost, and combinations of application routes, seem to have their influence on the
efficiency of the induction of responses. Furthermore, delivery of TTFC expressed as
an intracellular antigen turned out to be more effective than cell-surface expression
under the conditions tested (fig. 2). All these factors together have to be taken into con-
sideration in order to optimize strain and expression system combinations for a rational
augmentation of the efficiency of lactobacilli as vaccine delivery vehicles.
6. PASSIVE VACCINATION
Following infection with a pathogen, the immune system will need several days to
mount a full blown attack against this pathogen. It was discovered over 100 years
ago by Adolph von Behring that rabbits and guinea pigs could be protected from
diphtheria or tetanus toxin by administering serum of previously immunized
animals (Behring and Kitasato, 1890). For this work he was awarded the first Nobel
Prize for medicine in 1901. It was discovered later that this passive immunization
depends on the presence of antibodies in the serum, specific for these toxins.
The introduction of monoclonal antibody (MAb) technology in the 1970s has led to
a renewed interest in this immunization strategy. Its potential is now well recognized
and 80 monoclonal antibodies are in clinical development, not just for infectious
diseases but also for therapy in chronic immune-mediated diseases. A major prob-
lem in the oral application of antibodies is their instability in the digestive tract.
Yet, most pathogens enter the host exactly via this route which therefore remains the
preferred anatomical site for delivery. The use of lactobacilli for delivery of anti-
bodies appears an excellent solution to this dilemma. While bacteria cannot produce
the full structure of the different disulphide-linked immunoglobulin chains that
together make up a normal antibody, there is a way around this. By combining the
antigen-specific portions VH and VL from an original MAb, an artificial single-chain
protein is constructed that retains the original antigen specificity, but is sufficiently
simple to be produced by bacteria. Several groups have demonstrated the feasibility
of this approach by producing such so-called single-chain Fv’s (scFv) in E. coli.
ScFv’s are now widely studied for therapeutic use, for example in cancer treatment.
An additional advantage of scFv’s is the relative ease with which the genes encoding
Fig. 4. Expression of anti-Shiga toxin scFv, coupled to an E-tag for detection purposes, in Lb. casei.
(A) Western analysis. Lane 1: total cell free extract of Lb. casei, expressing cell wall anchored scFv;
lane 2: total cell free extract of Lb. casei, expressing cell wall anchored GusA; lane 3: concentrated
extracellular proteins of Lb. casei, secreting GusA; lane 4: total cell free extract of Lb. casei, secreting scFv;
lane 5: concentrated extracellular proteins of Lb. casei, secreting scFv. (B) Fluorescence activated cell
sorter (FACS) analysis showing Lb. casei with cell wall bound scFv. Bound anti-E-tag antibody was
detected with optimally diluted fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody. 10 000
bacteria were analysed for each experiment.
them can be manipulated, for example to achieve higher specificity, or to fuse them
with other bioactive molecules. In addition, by directing scFv’s to a bacterial sur-
face, a bacterium can be made to adhere to predetermined structures.
To test the feasibility of using lactobacilli for the production of scFv’s to combat
infections we have cloned a scFv that was derived from a hybridoma cell line that
expresses the anti-Shiga toxin monoclonal antibody 13C4 in E. coli and subse-
quently in Lactobacillus expression vectors. Both secreted and cell wall anchored
scFv’s could be detected using either western analysis or a fluorescence activated
cell sorter (fig. 4). Multiple bands in the sample of cell wall anchored fraction are a
result from cell wall fragments that remain attached to the protein, that is composed
of the scFv and a cell wall anchoring domain. The cell free extract of the cells that
are secreting the scFv shows two bands. The upper band represents precursor scFv,
while the lower band represents processed scFv from which the signal sequence has
already been cleaved off. In ELISA assays Shiga toxin binding activity could be
shown for this scFv. Tests are currently being performed to determine the in vivo
neutralizing capacity of the Lactobacillus generated anti-Shiga toxin scFv.
The past decade has witnessed a considerable increase in our knowledge of
the molecular genetics of lactobacilli, a biotechnologically very important group
of microorganisms. The structure of regulatory elements such as promoters has been
elucidated and the mode of regulation of gene expression has been investigated in
detail. Tools have been developed for the directed integration of foreign DNA into
the chromosome of various Lactobacillus species and plasmid-derived vectors have
been constructed with which foreign DNA sequences can be efficiently expressed,
and their products be targeted to the cytoplasm, the cell surface or the medium.
Yet, much needs to be learnt on how cellular processes interact, how signals are
transmitted and how the cell responds to such signals.
Lactobacilli are natural commensals of the gastrointestinal and urogenital tracts.
We have, however, little understanding of the mechanisms through which lacto-
bacilli colonize the mucosa. To be “on speaking terms” with its host, e.g. to avoid
an immune response against the bacterium, lactobacilli have to closely interact with
the mucosal cells and emit signals to the host. How they do that is as yet unknown.
To get a better understanding of these mechanisms, two adherence factors have been
studied in some detail. The main conclusions from this research are that coloniza-
tion most likely is a multifactorial mechanism in which several adhesion factors
participate. The structure and mode of interaction with the target receptors on the
mucosa of these adherence factors can greatly differ, making a multidisciplinary
approach necessary if we are to fully understand how the interaction of a bacterium
and a host cell takes place. Recent developments in genomics, proteomics and
DNA chip technology are expected to result in dramatic changes in knowledge
acquisition in these areas in the near future. The nucleotide sequences of the genome
of L. acidophilus, Lb. plantarum and L. sakei have recently been determined. Once
the complete genome sequences are available, we will be in a position to determine
the function of all individual genes and to exploit that information for a detailed
analysis of the regulatory mechanisms. These technological developments, which
mark a revolution in biology, will have a great impact on industrial applications of
lactobacilli, not only for their traditional market, the food industry, but also for
applications in public and animal health.
The developments in genetics and the demonstration of immunogenicity follow-
ing delivery of recombinant lactobacilli by the oral route in animal models will
endorse the development of safe Lactobacillus-based oral vaccines. Accelerating the
kinetics of the response, and increasing the specific immune responses at systemic
and mucosal levels, by defining optimal host–vector combinations, will provide a
sound basis to analyse protective efficacy. Application of this technology to other
pathogens, which manifest their pathology through the mucosal surfaces, has to be
assessed. Further improvements of the mucosal immuno-adjuvanticity of lactobacilli
and understanding of the basic mechanisms behind this, will also be a prerequisite to
obtaining optimal protective efficacy. Recent discoveries in the involvement of regu-
latory mechanisms through NF-κB in distinguishing between self and non-self in the
microflora could provide clues for a further development of mucosal vaccines, using
non-pathogenic microorganisms.
Application of the Lactobacillus delivery systems and combination of these sys-
tems with existing or newly developed mucosal vaccine systems should lead to more
knowledge of the mucosal immune system and finally to safe vaccines for both the
human as well as animal population.
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16 The influence of the lactic acid bacteria
and other resident microflora on the
immune system of the growing animal1
G. Perdigón a,b, R. Fuller c and M. Medina a

a
Research Centre for Lactobacilli (CERELA), Chacabuco 145, 4000 Tucumán,
Argentina
b
Immunology Department Faculty of Biochemistry, National Tucumán University,
Argentina
c
59 Ryeish Green, Three Mile Cross, Reading RG7 1ES, UK
The intestinal flora exerts a strong effect on gut-associated lymphoid tissue (GALT)
activation and development of the regulatory mechanisms involved in the mainte-
nance of the healthy state at the intestinal level. GALT is under constant exposure
to environmental antigens, and the digestive flora is the main antigenic stimulus.
Bacterial numbers and composition vary considerably along the gastrointestinal
tract, constituting a complex ecosystem which depends on the physiology of the
host and on the interaction between bacteria and eukaryotic cells. The gut microflora
is an important protective barrier against intestinal infections by bacteria, fungi and
viruses. This knowledge is the basis for the use of live microbial food supplements
(probiotics) as a method for repairing microbial deficiencies and enhancing resist-
ance to disease. Lactic acid bacteria (LAB) have been used for many centuries in the
preparation and processing of food. These microorganisms are the main candidate
to be used as probiotics. Currently, lactobacilli are also known for their health-
stimulating activities by inducing an effect on the immune system. The knowledge of
the role played on the immune system and on the functioning of the systemic immune
response as a whole, is essential for the successful development of probiotics for use
in animals and humans. The natural route for the majority of pathogens to enter
the body is via the mucosa of the gastrointestinal–respiratory and urogenital tracts.
1
This research was supported by Grants from CIUNT 26D/127 and PICT/97 No. 05-02312.

352 G. Perdigón, R. Fuller and M. Medina
Most of the information about the mechanisms on the pathway of internalization

refers to the interaction between host and pathogen resulting in disease; however,
these interactions do not always result in disease, depending on many factors such
as the immune state of the host, and the virulence and pathogenicity of the microbes.
There are few reports about the interaction of non-pathogen bacteria such as LAB
with the intestine and the mechanisms involved in the adjuvant capacity on the gut
mucosal immune activation. There is no doubt of the immunomodulatory effect
played by some probiotics administered by the oral route. However, how these
microorganisms interact with the intestinal epithelium and with the immune cells
associated to modulation of the immune response is still poorly understood. Here,
we review the role of the normal microflora in the maintenance of adequate immune
stimulation and we also analyse the different immunomodulating activities of LAB
which would be related to the mechanisms of interaction or to the pathway of inter-
nalization by these microorganisms with the intestine. The intensity of such inter-
action induces the intercellular signals between epithelial and immune cells
associated with the gut to stimulate the immune system. Both parameters are essen-
tial for the induction and/or downregulation of the immune response.
1. ACQUISITION AND DEVELOPMENT OF GUT FLORA

The origin and development of the microflora colonizing the intestinal tract varies
between animal species, but for all animals there is a very large number of different
microorganisms resident in the lower gut; this may comprise over 400 different
types of microorganisms, including numerous variants of each microbial species
present. This complex population is acquired shortly after birth and forms a symbi-
otic association with the host contributing to its nutrition and immune states.
The newborn animal (or newly hatched bird) is sterile and rapidly acquires its
flora from the mother, other adults and the general environment. From the vast array
of different organisms which the young animal will ingest, it must select those that
are required to form its characteristic microflora. Various host and flora-derived
factors allow the “right” organisms to colonize the gut. By far the most important
source of gut microorganisms is the mother, because a) she has intimate contact with
the infant and b) she provides microorganisms that are components of the flora
characteristic for that species of animal.
In the case of the newborn human infant, the high level of hygiene practised in
hospitals and homes of developed countries aims to prevent faecal contamination of
the baby. However, in spite of the precautions taken, transfer of microorganisms
from the mother to the infant does occur (Tannock et al., 1990) but, no doubt, there
will be a delay in the development of the normal flora resulting from this restricted
access to the mother’s microorganisms. In a sense we are being too hygienic, but we
have to strike a balance between preventing contamination by pathogens and allow-
ing the transfer of desirable bacteria which constitute the indigenous microflora.
Microflora and the immune system 353
The development of probiotics containing defined microbial species has been influ-
enced by this consideration.
So there is a progression from a sterile gut to one in which a large number of dif-
ferent microbial species become established and influence the host animal. The speed
and composition of this change will depend on the species of animal and the nature of
the environment. Animals will vary in the structure of the gut and the food which they
eat. The ruminant animal is a unique example which has a large sac (the rumen) at the
anterior end of the gastrointestinal tract. This contains a very complex population of
microorganisms with the strictly anaerobic species being dominant. This arrangement
allows for the digestion of fibre which forms a large part of the ruminant’s diet.
The more normal organization of the gut in mammals is to have the major micro-
bial presence in the lower gut (caecum and colon). For monogastric herbivores the
digestion of fibre occurs in the lower gut and supplies significant amounts of nutri-
ents. For omnivores, such as the pig and humans, the digestion of fibre is less impor-
tant and the volatile fatty acids (VFAs) produced in the lower gut form only a small
part (about 7%) of the energy requirements of the host animal. Graminivorous birds,
such as the chicken have two caeca attached to the rectum. These organs contain a
large number of microorganisms but no fibre digestion occurs. Even grazing birds,
such as the goose, are unable to metabolize cellulose and they depend on the cell
contents of the grass for nutrients.
Humans have a low stomach pH (like carnivores) which tends to limit the
survival and growth of bacteria in the stomach and upper small intestine. Humans
are also unique in that a large proportion of their food is heated or processed in some
way that reduces the number of viable bacteria ingested. Despite the inimical
conditions in the stomach, some organisms, with special attributes, can exist there.
For example, Helicobacter pylori can survive in the mucous layer of the stomach
antrum. Although this organism can be the cause of gastric or duodenal ulcers, it is
often present in the gut of healthy individuals (Lee et al., 1993). This is an excellent
illustration of the delicate balance that exists between the host animal and its gut
microflora, where, according to the prevailing conditions, an organism can be a
harmless commensal or a dangerous pathogen.
The total number of microorganisms in the healthy stomach rarely exceeds about
104 CFU/g. But, in achlorhydric patients this will increase, showing the importance of
pH in flora control in this region of the gut. In the duodenum the pH rises and becomes
slightly alkaline, but the rapid transit time of food in the small intestine prevents any
large increases in bacterial numbers. It is not until the food enters the ileum (lower third
of the small intestine) that numbers begin to increase. This may be due to an increase
occurring in this region or to backflow from the colon. Lactobacilli and enterococci
which formed the dominant flora in the stomach and small intestine, are joined in the
lower gut by enterobacteriaceae and obligate anaerobes. The flora in this region
exploits the availability of the time allowed for growth by the stasis which occurs in this
part of the gut. The breakdown of polysaccharides by one section of the flora releases
simple carbohydrates which can be used by other members of the colonic flora. There
is a nutritional interdependence of the various components of the gut flora, creating an
important balance which must be maintained to ensure the optimal health of the host.
The bacteria in the gut are not uniformly distributed through it. They colonize the
whole of the lumen by occupying numerous habitats. These include:
1. Bacteria living in the crypts at the base of the villi.
2. Bacteria attached to the cell membrane of the epithelial cells. A good example
of this is the Lactobacillus flora of the chicken crop which adheres to the
squamous epithelial cells (Barrow, 1992).
3. Bacteria entrapped in the mucous of the gut.
4. Bacteria floating in the lumen contents.
5. Bacteria attached to food particles. This is an important habitat for rumen
organisms that digest cellulose (Latham et al., 1978). The intimate association
of the bacteria and the food particle ensures a high concentration of the enzyme
on the substrate surface.
Association of bacteria with the epithelial surface is an important ecological
niche. By immobilizing itself on the gut wall, the microorganism can resist being
removed by the peristaltic flow of the intestinal contents. These attached bacteria are
some of the most important organisms inhabiting the intestinal tract; they are in a
position where they can benefit from nutrients and oxygen concentrations not avail-
able to those organisms that colonize sites remote from the gut wall. They are also
able to exert their influence on the host animal either adversely by the production of
toxins or positively by modulation of the immune response (see later).
Attachment is also a very effective colonization factor. The epithelial cells with
attached bacteria are continuously being sloughed off and will inoculate the incom-
ing food ensuring that the attaching bacteria remain as major components of the gut
microflora. The attachment of non-pathogenic indigenous bacteria, such as lacto-
bacilli, may protect the animal host against infection by not allowing the pathogens
to attach and colonize the gut. This is a feature which has been made use of in the
selection of organisms for inclusion in probiotic products. There is in vitro evidence
that lactobacilli can compete with enteric pathogens for adhesion receptors on
the surface of gut epithelial cells (Bernet et al., 1994). This ability to compete for
adhesion receptors and the potential for immunomodulation are the most important
features for determining the benefits derived from probiotic microorganisms.
1.1. Factors affecting the gut flora

The gut flora that develops in the adult animal is a complex mixture of organisms
which are interacting with each other and which are themselves influencing the host
animal in adverse or beneficial ways. If the animal is to remain healthy, it must keep
this interaction under control; the balance of the gut flora must be maintained.
However, there are several factors which can affect this balance.
1.1.1. Change in the diet

The effect of diet on the microflora is best assessed by studying the changes in meta-
bolic activity (Mallett and Rowland, 1988). Changes in enzyme activity and end
product concentration have been related to differences in polysaccharide, carbo-
hydrate and protein content of the diet.
The change from milk to solid foods that occurs when mammals are weaned can
have marked effects on flora especially when, as is the case with most farm animals and
many human infants, the weaning is done at an earlier age than would occur naturally.
1.1.2. Stress
The word stress can cover a multitude of conditions such as overcrowded transport
to the slaughter house or other locations or withdrawal of food, and these have all
been related to changes in the gut microflora (Tannock, 1983). These claims should
be regarded with caution because it is often not possible to separate stress from other
environmental factors such as diet. Often the stress causes a reduction in the
numbers of lactobacilli with a corresponding increase in the numbers of entero-
bacteriaceae. It is tempting to conclude that the one is responsible for the other. The
reasons for the effect of stress on gut flora are not well defined, but it is suggested
that it may be related to the autonomic nervous control of the gut musculature
modifying transit times, or the change in hormone balance which could affect the
gut mucous living.
1.1.3. Medication
Oral administration of antibodies can often result in a condition known as
pseudomembranous colitis caused by Clostridium difficile (normally present in the
gut in small numbers). Although Clostridium difficile is pathogenic for human adults
it can occur in large numbers in the gut of infants without causing adverse effects
(Corthier, 1997). The gut of human infants may also contain large numbers of
Staphylococcus aureus without showing symptoms of disease (Kay et al., 1990).
Even though these microorganisms are producing the relevant toxins, they are
without effect because the toxin receptors are absent from the gut wall. This again
illustrates the complicated relationship between the host and its gut flora. The
mere presence of an organism in the gut does not induce a predictable response in
the host.
1.1.4. Environment
As mentioned earlier, the use of antiseptics and a high level of hygiene can delay the
transfer of microorganisms from the mother to the infant. A difference in the species
of Bifidobacterium found in baby’s faeces can be detected between hospitals and
even between wards of the same hospital (Lundequist et al., 1985). The age of the
infant at weaning will also be important. Premature removal of pigs and calves from
the dam is a common practice on farms which will seriously affect the development
of the gut flora. The effect of environment on gut flora is seen in an extreme form
in the case of the incubator reared chicken. There the chick hatches into a clean envi-
ronment totally divorced from any contact with the mother hen. On hatching, the
chick acquires organisms from the incubator and is deficient in those bacteria that
give protection against intestinal infections. It was found that when newly hatched
chicks were dosed with a faecal suspension from a healthy adult chicken, the resist-
ance to infection found in farmyard reared chickens was restored (Nurmi and
Rantala, 1973). Although effective, the administration of faeces to day old chicks is
undesirable and much effort has gone into identifying the organisms responsible for
this so called “competitive exclusion” phenomenon (Barrow, 1992). Other prepara-
tions based mainly on lactic acid bacteria (LAB) (probiotics) have also been used to
repair the deficiencies in the flora created by incubator rearing techniques.
1.2. The probiotic concept

The very large and complex population of microorganisms that develops in the
gastrointestinal tract is an important part of the overall metabolic activity of the host
animal. In addition to the nutritional aspects of the association, the gut microflora is
an important protective barrier against intestinal infection by bacteria, fungi and
viruses. But, as mentioned earlier, the gut flora is subject to several factors that tend
to change it adversely and it is important, if optimal health is to be enjoyed, that
these changes be reversed.
This forms the basis of the move towards the use of live microbial food supple-
ments as a method for repairing the microbial deficiencies and enhancing resistance
to disease: the so-called probiotic effect. The precise mechanisms of this important
new approach are still not clearly understood, but the gut flora presents a formidable
antigenic challenge to the host animal’s immune system and it seems likely that
immunomodulation will be a significant aspect of the way in which probiotics
manifest their beneficial effects.
The remainder of this chapter is devoted to a critical review of the published
work on the immune potential of LAB which are the most common microorganisms
used in probiotic preparations.
2. HOST RESPONSE TO THE GUT MICROFLORA. DEVELOPMENT

AND MAINTENANCE OF THE MUCOSAL IMMUNE SYSTEM
Discussions of the host–microbe interaction usually concern the behaviour of
pathogens or descriptions of the effects of particular organisms rather than attempt-
ing to define a more general host–microbe relationship. Commensal microbes also
interact with the host but without harmful effect. Certain members of the intestinal
microflora are always present in high numbers throughout life and may confer
benefits on the host (autochthonous); others are incidental and generally innocuous
inhabitants (normal flora) that can colonize the gut from the environment, and
others are transient. The presence of pathogens in the gut does not always result in
disease. The harmful effect will depend on whether the pathogen is present in a high
number, its virulence and the immune state of the host (Casadevall and Pirofski,
2000). Beneficial associations between bacteria and eukaryotic cells have been
studied by many researchers in recent years, and they have discovered a whole
spectrum of interactions, ranging from obligatory symbiosis to loose associations.
The benefits derived from these interactions include nutritional exchange and
protection from infection. This has been described as important adaptations to
environmental gradients (Polz et al., 1994), as well as less integrated associations
for bacteria involved in cellulose degradation in the bovine rumen.
The understanding of the diversity of ecological niches and impact of bacteria–
eukaryote cell interaction is in the early stages. It has been known for decades that
gut commensal microbes or indigenous bacteria colonizing the neonatal mammal
can influence intestinal immune function. The effect is related to the activation and
development of the systemic immune system, especially to the increase of circulat-
ing specific and “natural” antimicrobial antibodies (Tlaskavlová-Hogenová et al.,
1983, 1994; Shroff et al., 1995). It was also demonstrated that microbes or micro-
bial products participate in the granulocyte lineage formation in the bone marrow
(Tada et al., 1996). Immune development does not occur in the intestine of germ-
free animals. Stimulation of the intestinal immune system development by the intes-
tinal microflora and host tolerance of the microflora indicate a complex symbiotic
mechanism, which is only beginning to be explored at the mechanistic level.
The following generalizations are the result of numerous studies comparing
germ-free and conventional animals, and observations with germ-free animals
colonized with specific microbes. It has been established that: a) conventional
animals develop “natural” antibody-secreting cells in the spleen and the peripheral
lymph nodes (PLN) against autoantigens and bacterial antigens (especially
lipopolysaccharides, LPS), whereas in germ-free neonates the development of
antibody-secreting cells is greatly delayed (Tlaskalová-Hogenová and Stepánková,
1980; Tlaskalová-Hogenová, 1997); b) there is a profound hypotrophy of lymphoid
tissues and lymphoid cells reactive to mitogenic stimuli (Moreau et al., 1978).
Bacterial antigens such as LPS may regulate B cell development in neonates
(Monroe et al., 1993). There is a delay in the competence of neonatal B cells, but
neonatal and adult B cells are equally responsive to mitogenic stimulation with LPS.
Studies with germ-free animals have demonstrated alterations of immune parame-
ters which include under-developed Peyer’s patches and mesenteric lymphoid nodes
(MLN) and smaller but more numerous goblet cells; macrophages are present, but their
activities (intracellular killing of bacteria) are diminished (Wells and Balish, 1980;
Rodming et al., 1982; Sotoh, 1988). Intestinal lymphocyte population and antibody
profiles are altered in germ-free animals. The intraepithelial lymphocytes (IEL) sub-
population is also altered; in conventional animals the number of antigen T cell recep-
tor (TCR) αβ+ is approximately the same as or greater than the number of γδ+,
depending on the mouse strain, whereas in germ-free mice the population of γδ+ IEL is
present in higher numbers than that of αβ+ IEL (Kaeaguchi et al., 1993).
When germ-free animals are associated with bacteria, the gut morphology and
the immune system develop quickly. Peyer’s patches develop antibodies, particu-
larly those specific for intestinal bacteria. After conventionalization, the IEL popu-
lation is predominantly αβ+ IEL, and the γδ T population acquires the ability to
regulate oral tolerance (Carter and Pollard, 1971; Kaeaguchi et al., 1993; Ke et al.,
1997). The role of individual bacteria or groups of bacteria in stimulating immunity has
been studied using germ-free rodents; it was demonstrated that some bacterial species
induce a host immune response, whereas others are poorly or non-immunogenic (Berg
and Savage, 1975; Shroff et al., 1995; Umesaki et al., 1996). However, it is difficult
to predict which indigenous bacterial species will be immunogenic. Mice and other
mammals normally harbour an extensive bacterial flora, not only in the large intes-
tine, but also in the small intestine, and it is very difficult to determine the exact role
of specific populations in a gut microflora that includes facultative anaerobes and
obligate anaerobes such as lactobacilli, enterococci and enterobacteria. The small
and large gut are first colonized by lactobacilli, then enterococci followed by bac-
teroides in the large intestine. The enterobacteria are a minor component of the com-
plete gut flora. Certain populations of enterobacteria and enterococci decrease after
having reached a maximum level. The presently available immunological evidence
shows that the gut mucosal immune response to some luminal microbes does not
prevent their continued successful colonization (Van der Waaij et al., 1994; Friman
et al., 1996). A high proportion of the normal bacteria of the gut are found to be
coated with “natural” IgA secreted into the gut lumen. It is not easy to determine the
conditions for immunogenicity with allochthonous or autochthonous strains. It was
also suggested that an autochthonous, non-immunogenic organism can induce anti-
body responses if encountered outside its normal habitat. The question of why some
non-pathogenic bacteria induce immunological development, while others are appar-
ently ignored by the host, remains unanswered. Although an individual autochtho-
nous species may not induce a response from the host, the same strain in germ-free
animals can induce an immune response. Certain bacterial species corresponding to
the normal microflora, individually stimulate rapid development of the intestinal
immune response. Understanding the difference between homologous bacteria which
are able or not able to influence the immune system in the same way as heterologous
bacteria, will be crucial for the characterization of immunogenic strains in probiotic
preparations.
The indigenous microflora is occasionally implicated in disease, for example
ulcerative colitis and Crohn’s disease (Sartor, 1995), although the exact mechanisms
responsible for disease induction remain unknown. It is also uncertain if bacteria

initiate the disease process, or if bacterial-induced inflammation is a secondary con-
sequence of intestinal immune dysfunction. These observations should be considered
before recommendations are made for ingestion of high concentrations of live organ-
isms. Some of the microbial components that are highly effective inducers of
inflammatory reaction, can take part in the induction of autoimmune mechanisms
(Kotzin et al., 1993). These microorganisms are capable of stimulating T or B cells
and inducing pathological symptoms, among them bacterial translocation by epithe-
lial modification of tight junctions. They exhibit high antibody titres for many species
of intestinal bacteria.
In conventional animals, the incomplete knowledge of the complex microflora of
the intestinal tract makes it difficult to relate immune response to specific micro-
organisms. Novel advances in molecular technologies are increasingly being used
for the identification and analysis of the intestinal ecosystem and yield a better
understanding of the interaction between host and microbes in the intestinal tract
(Vaughan et al., 1999, 2000). Populations of intestinal bacteria have been mapped
by molecular techniques and shown to be constant over time (Tannock, 1999). There
is a continuous natural process of selection exerted perhaps by the immune system
and matched by selective adaptation of specific strains of microorganisms. Recently,
the influence of the major histocompatibility complex (MHC) on the composition of
the gastrointestinal flora was also described, indicating that monozygotic twins that
have identical MHC also have identical faecal floras (Toivanen et al., 2001). These
studies highlight the intimate relationship between microflora and immune system.
3. BACTERIAL ADHESION TO GUT MUCOSAL SURFACE

The mucosal colonization by bacteria is preceded by attachment to epithelial cells
or to the mucin coating these mucosal cells. Cellular and molecular studies have
revealed that many bacteria express adhesins on their surfaces, which bind to recep-
tors on the surface of mucosal cells. This specific binding allows the bacteria to
attach firmly to particular sites on the mucosal surfaces and resist dislocation by
forces that act on these surfaces. Adhesion of a bacterium leading to mucosal colo-
nization determines its site and density. Other post-adhesion events enable the bac-
teria to establish themselves successfully on the mucosal surfaces; even pathogenic
bacteria to initiate infection need to upregulate virulence factors and to induce phys-
iological changes on the mucosal surface, such as proliferation of epithelial cells,
increased mucus secretion and induction of pro- and anti-inflammatory mediators,
by mucosal and submucosal cells (Ofek and Beachey, 1980; Sharon, 1996). The fac-
tors that determine the stability of the normal microflora are numerous.
Microorganisms on mucosal surfaces such as the gut are separated from cells of the
mucosal immune system by epithelial barriers. To enter the external environment it
is necessary for the epithelial tissue to transport the antigen across these barriers
without compromising the integrity and protective functions of the epithelium. Most
of the knowledge on bacterial adhesion to mucosal cells is based on studies of adhe-
sion of pathogens, which includes interaction of adhesin–receptor (lectin, carbo-
hydrate recognition) protein–protein recognition, and interaction between hydrophobic
moieties of protein and lipids, on the host cells with the bacterial surface (Silvester
et al., 1996; Sharon and Lis, 1997). On the other hand, mucosal cells are covered
by a layer of mucus composed of a heterogeneous mixture of proteins with various
degrees of glycosylation. This layer includes several different glycoproteins includ-
ing mucins, collagens, elastin, fibronectin, laminin and proteoglycans; fibronectin is
an important receptor for bacterial pathogens (Hasty et al., 1994). Many mucosal
colonizers express adhesins that specifically recognize one or more of those con-
stituents. These findings have stimulated the development of anti-adhesin drugs for
the prevention and therapy of microbial infections in humans.
The mechanism of adhesion of non-pathogenic bacteria to mucosal surfaces is
not well known. The polysaccharides or the lipopolysaccharides of Gram-negative
bacteria can interact with lectins on macrophages and initiate an immune response
(Jacques, 1996) when the antigen crosses the epithelial barrier. Bacteria possess a
repertoire of strategies for triggering the immune response; however, often, mucosal
responses are marked by the development of a tolerogenic response, for example by
responses elicited by a common mucosal antigen associated with the resident micro-
bial flora or with food proteins. The oral tolerance phenomenon has been most
extensively studied for soluble antigens in various animal models. The ability to
mount both an immunologic and a mucosal tolerogenic response posed the question
of how the mucosal immune system knows which type of response is most appro-
priate in any given circumstance. To answer this question, it was suggested that the
tolerogenic response is a failure of cellular signalling elicited by the antigen admin-
istered. Conversely, the immunogenic response is the result of cellular signalling
elicited if the antigen is accompanied by a mucosal adjuvant or is by itself a mucosal
adjuvant. Both immunogenic and tolerogenic mucosal responses are characterized
by the generation of immune effector cells. Thus, the immunity is always generated
but, the magnitude of the response is reduced. Bacteria and all antigens derived from
them have the ability to induce an immune response owing to the intimate interac-
tion occurring at the mucosal surfaces. Microbial signals are seen by the gut
immune system as being immunogenic, but we cannot ignore the tolerance to com-
mensal bacteria, which may maintain a level of suppressor mechanisms. It was
demonstrated that oral tolerance against enteric microorganisms is broken in the
event of an inflammatory mucosal response, where both humoral and cellular immu-
nity can be generated against intestinal bacterial populations (Duchman et al.,
1995). Many reports have shown the complexity of the immune mechanisms that
operate to maintain oral tolerance (Friedman and Weiner, 1994; MacDonald, 1995;
Gaborious-Routhian and Moreau, 1996). This becomes even more complex if we
consider the presence of the normal microflora and the live microorganisms that
enter the gut with the consumption of food (Chin et al., 2000). This implies that sev-
eral mechanisms may be operative, and the possibility of inducing an immunological
response to foreign bacteria is strong. It will depend on the strength of interaction
with the epithelial cells of the gut and also on the interaction with immune cells
associated to the lamina propria of the intestine. This aspect will be discussed later.
3.1. Bacterial interaction with gut epithelium and associated immune cells
Foreign antigens and bacteria (pathogens or non-pathogens) on the gastrointestinal
and all mucosal surfaces are separated from cells of the mucosal immune system by
an epithelial barrier. Thus, in order for the antigens from the external environment
to induce an effect on the mucosal immune system, it is necessary for the epithelial
cells to transport the antigen across this barrier, without compromising the integrity
and protective functions of the epithelium. However, this pathway of internalization
of antigens carries the risk that pathogens may exploit the mechanisms to cross
epithelial barriers and invade the body. Antigen sampling strategies at diverse
mucosal sites differ dramatically, because they are adapted to the cellular organiza-
tion of the local epithelial barrier (stratified or simple).
The gastrointestinal tract is lined with a single layer of epithelial cells; in this
simple epithelium, the intercellular spaces are sealed by tight junctions and special-
ized epithelial M cells which deliver samples of foreign material from the lumen to
organized lymphoid tissues within the mucosa. Tight junctions are generally effec-
tive in excluding peptides and macromolecules with antigenic potential (Madara
et al., 1990; Matter and Mellman, 1994).
The uptake of particulate antigens or bacteria across the intestinal epithelium can
occur only by active transepithelial vesicular transport, and this is restricted by mul-
tiple mechanisms including local secretions containing mucins and secretory IgA
antibodies. The epithelial cells have rigid microvilli and a glycocalyx (thick layer of
glycoproteins). The glycocalyx of enterocytes is an effective diffusion barrier that
contains negatively charged mucin-like molecules (Maury et al., 1995; Neutra et al.,
1996b). It prevents the uptake of bacteria (pathogens or not) by enterocytes, and also
is impermeable to most macromolecular aggregates, particles and viruses
(Amerongen et al., 1994).
There is considerable evidence that the pathways of antigen internalization can
be: a) through the enterocytes that transport small amounts of intact proteins and
peptides across the epithelium (Neutra and Kraehenbuhl, 1993), although the induc-
tion of immune response or immune tolerance is still controversial, and b) through
the Peyer’s patches that are the inductor sites of mucosal immunity. In these mucosal
lymphoid follicles the epithelium is characterized by special epithelial cells called
follicle-associated epithelium (FAE) cells and M cells. Both FAE and M cells allow
access of macromolecules, particles and bacteria from the surface, and promote their
uptake by transepithelial transport (Neutra et al., 1996a). The M cell apical surface
differs from the intestinal absorptive cells in that they lack the highly organized
brush border with microvilli typical of enterocytes, and also the uniform thick
glycocalyx seen on enterocytes. Thus, the hydrolytic enzymes present in the glyco-
calyx are often reduced or absent on M cells. M cells are the major pathway for
endocytosed material and for induction of a mucosal immune response. From the
characteristic of M cells, the microorganisms with a hydrophobic surface can inter-
act selectively with them. The M cells are the gateways to immune inductive sites
where the bacteria are destroyed as well as processing and presenting as antigen,
which will induce immunoglobulin A (IgA) specific antibodies. Thus IgA plays an
important role as a second line of defence, eliminating invasive bacteria and so pre-
venting disease (Kerr, 2000). Summarizing, the antigen uptake can be through the
epithelial cell and more specifically by M or FAE cells from Peyer’s patches.
However, even when epithelial M cells of the intestine are continuously exposed to
the lumen of the gut and are relatively accessible to attachment and invasion of
pathogens, the occurrence of mucosal disease may be reduced by the close interac-
tions of the FAE cells with professional antigen processing and presenting cells of
mucosal lymphoid tissues immediately under the epithelium. Nevertheless, the
transport of pathogens by M cells may result in initiation of mucosal and/or sys-
temic infections. A wide range of pathogenic Gram-negative bacteria, such as Vibrio
cholerae, Salmonella typhi, Salmonella typhimurium, Shigella and Yersinia can
selectively bind M cells and initiate a variety of molecular mechanisms including
recognition (lectin–carbohydrate interaction) followed by more intimate associa-
tions that conclude with the recruitment of bacteria to the interaction site. They can
also react with the immune cells associated with Peyer’s patches, and initiate an
immune response or systemic invasion, depending on the ability of M cells and
associated phagocytic cells to digest and inactivate microorganisms. When patho-
genic microorganisms bind to the host epithelial cells, they activate signal transduc-
tion with membrane activation; such signalling molecules may directly or indirectly
upregulate different adhesion molecules (addressins) on mucosal small venules, to
facilitate polynuclear and mononuclear cell extravasation. Those immune cells enable
some enteropathogens such as Salmonella typhimurium to disseminate into deep
tissue by apoptosis induction of infected macrophages (Van der Velden et al., 2000).
It is well known that the ability of a pathogen to initiate an infection is dependent on
factors such as dose and virulence. Some pathogens can be efficiently transported by
M cells, but they are not equipped to survive in the mucosa or spread systematically,
as is the case for V. cholerae or S. flexneri. S. flexneri is unable to invade the apical
surfaces but it infects by inducing release of chemotactic signals which attract inflam-
matory cells causing breakdown of normal epithelial function (Perdomo et al., 1995).
The commensal microflora plays a crucial role in the development of organized
mucosal lymphoid tissues. This microflora is able to influence the differentiation
programme of mucosal epithelial cells, thereby creating favourable niches while
shaping the host’s adaptative mucosal system (Bry et al., 1996). The microorganisms
also modulate the migration of professional antigen presenting cells and

lymphocytes into simple or stratified epithelia (Eckmann et al., 1995).
Most of the information about the mechanisms or pathways of internalization
refers to pathogens and in particular the Gram-negative bacteria. There are few reports
on Gram-positive pathogens and even fewer for Gram-positive non-pathogenic bacte-
ria, e.g. lactic acid bacteria. Enterococcus faecalis, a Gram-positive, facultative anaer-
obic bacterium that belongs to the normal flora of the intestinal tract, has increasingly
gained attention as a pathogen, especially those strains resistant to all antimicrobial
agents commonly used for therapy. The mechanisms involved in extraintestinal
dissemination of enterococci are related to an aggregation substance protein involved
in virulence. It is expressed on the surface of E. faecalis and facilitates bacterial adher-
ence and internalization by epithelial cells from the colon and duodenum, but not by
cells derived from the ileum (Sartingen et al., 2000; Wells et al., 2000). Enterococcal
translocation through the epithelial cells was not observed, but these studies indicate
that for some Gram-positive bacteria the route of internalization is not via M cells, but
via epithelial cells allowing the establishment of infection by these microorganisms.
The normal microflora by itself exerts a barrier effect against pathogens. It was
demonstrated that some microorganisms from the flora, e.g. Bifidobacterium can
help in the maintenance of barrier function by developing antimicrobial activity
against pathogens (Liévin et al., 2000). The improvement of the intestinal microflora
and the immune response increase the host’s resistance to intestinal infections.
It was shown that non-pathogenic, segmented filamentous bacteria (anaerobic Gram-
positive microorganisms) colonize the ileum of many young animals by attaching
to intestinal epithelial cells. These bacteria strongly stimulate the mucosal immune
system and induce intestinal epithelial cells to express major histocompatibility
complex class II molecules. These segmented filamentous bacteria can be phago-
cytosed into the ileal epithelial cells and intracellularly processed by lysosomal
heterophagy, and this phagocytosis could be the first triggering step for the immuno-
logical response induced (Yamauchi and Snel, 2000).
The knowledge that microorganisms from the normal microflora can stimulate the
immune system and preserve the barrier functions, led to the use of non-pathogenic
microorganisms for the prevention and treatment of intestinal infections. In this
sense, Saccharomyces boulardii (non-pathogenic yeast) was used in the treatment of
infectious diarrhoea, because it preserves an effective barrier and can modulate the
signal transduction pathway induced by enteropathogenic E. coli (Czerucka et al.,
2000; Qamar et al., 2001). S. boulardii exerts a protective effect on epithelial cells
after adhesion of E. coli by modulating the signalling induced by bacterial infection
and provides protection against intestinal lesions caused by the pathogen.
Lactic acid bacteria are non-pathogenic, Gram-positive bacteria frequently used as
probiotics. They are associated with fermented products and, if consumed in the daily
diet, LAB can improve the intestinal microflora and the immune state. There is no
doubt about the beneficial effect of LAB on the mucosal immune system (see later).
However, the way in which these bacteria influence the immune system, how they
interact with the gut and how they are internalized to make contact with the immune
cells associated with the mucosa, are information that is essential for understanding
the behaviour of the different LAB on the mucosal immunostimulation.
In our laboratory, we have studied the pathway of internalization of different LAB,
e.g. Lactobacillus casei CRL 431, L. acidophilus CRL 724, Lactobacillus plantarum
CRL 936, Lactobacillus delbrueckii spp. bulgaricus CRL 423 and Streptococcus
thermophilus CRL 412. These LAB were selected on the basis of previous studies or
their effect on the mucosal immune system, after oral administration, where we
demonstrated that the LAB induced different immune responses, e.g. non-specific
(inflammatory), specific or both (Vintiñi et al., 2000). These results led us to analyse
these different responses, by studying the mechanisms of gut interaction, internaliza-
tion and contact with the immune cells associated with the mucosa. Our aim was to
understand how the LAB induce immunostimulation and therefore to know 1) why
some of them enhance the inflammatory immune response and others the specific
response and 2) why not all LAB, as Gram-positive microorganisms carrying the basic
structure in their cell wall (muramyldipeptide), induce the same immunostimulation.
We performed animal studies of immunofluorescence and transmission electron
microscopy (TEM). For the first assay, the LAB were labelled with fluorescein iso-
thiocyanate (FITC) (Perdigón et al., 2000). We looked for fluorescent bacteria in histo-
logical slices from the small intestine, Peyer’s patches and the large intestine. We
demonstrated that all LAB were present in the Peyer’s patches, all except L. delbrueckii
spp. bulgaricus and L. acidophilus were present in the villi of the small intestine, and only
L. acidophilus and L. plantarum were present in the large intestine (figs 1 a,b and 2 a,b).
These observations led us to outline what was the pathway of internalization to Peyer’s
patches, M cells or FAE cells. To investigate this, we performed TEM studies.
Unlabelled bacteria were administered by intubation and we analysed by histological
slices of Peyer’s patches and epithelial cells from the small and large intestine, the
Fig. 1. (a) Histological slice of Peyer’s patch tissue from control animal that received unlabelled lactic acid
bacteria. Magnification × 40. (b) Peyer’s patch from mice that received labelled L. plantarum (108 cells) by
oral intubation. Numerous FITC-labelled bacteria are observed in Peyer’s patch. Magnification × 40.
Fig. 2. (a) Histological slice of large intestine tissue from control animals treated with unlabelled bacteria.
Magnification × 40. (b) Large intestine from mice that were given labelled L. plantarum. Fluorescent
bacteria can be seen. Magnification × 40.
lysosomal activation induced by antigen interaction as an indirect measure of cell acti-

vation by LAB. We found that L. casei and L. plantarum interact with M cells and FAE
cells. L. acidophilus, L. delbrueckii spp. bulgaricus and S. thermophilus were internal-
ized to Peyer’s patches only by FAE cells and no activity in M cells was observed.
L. acidophilus and L. delbrueckii spp. bulgaricus also induced a marked lysosomal acti-
vation in the epithelial cells of the small intestine and L. acidophilus and L. plantarum
interacted with the epithelial cells of the large intestine (figs 3 a,b and 4 a,b).
Fig. 3. (a) Transmission electron micrograph of murine Peyer’s patch containing M and FAE cells from the
control animal. Magnification × 7800. (b) Electron micrograph of lymphoid cell associated with M cell from
mice following oral inoculation of L. plantarum. Intense lysosomal activity in lymphoid cell and FAE cell are
observed. Magnification × 7800.
Fig. 4. (a) Micrograph of control mouse epithelial cell from large intestine. Magnification × 7800.
(b) Micrograph of epithelial cell from large intestine after oral administration of L. plantarum. Lysosomal and
reticule ergoplasmic activity is observed. Magnification × 7800.
Taking into account the above results, we proposed for the LAB studied a model
of interaction with the gut as shown in fig. 5. These findings confirm that Gram-
positive non-pathogenic bacteria such as LAB can stimulate the immune system by
different pathways of antigen uptake with M or FAE cells from Peyer’s patches or
epithelial cells. These observations are important because they explain the different
immunostimulations observed. The pathway of antigen internalization in the mucosal
Fig. 5. Scheme of pathways of internalization of different lactic acid bacteria. Oral administration of LAB
could modulate different immune responses depending on the route of entry.
immune system is important in the generation of a variety of distinct responses in the

host, so, if the route of entry is by M cells, a specific immune response with antibody
production and increase in the number of IgA secreting cells will be induced. If the
internalization is by FAE or epithelial cells, an inflammatory response would be
elicited due to the transported antigen interacting with dendritic cells or macrophages
associated with the lamina propria of the intestine with the consequent cellular
migration, release of cytokines and T cell stimulation (Sozzani et al., 1995).
There is information that the intestinal flora is the driving force of an excessive
T cell response (Th1) inducing a slight inflammatory state. If stimulation is by
exogenous antigens, this Th1 response can lead to undesirable effects such as colonic
inflammation by the cytokines that the Th1 cells release (Strober et al., 2000).
Although we demonstrated the different ways of gut internalization by LAB
which influence the mucosal immune response, other questions should be asked. For
example, if the LAB are internalized giving a short residence in the gut, how can we
explain the effect of probiotic microorganisms as a repair of a deficient microflora,
or in their metabolic activity by beneficial enzyme production? Does it justify the
long period of administration that is required to produce the desired effect? If this is
the case, it is important to adjust the dose to avoid oral tolerance. Similarly, the use
of a heterologous strain as a probiotic should be restricted to those that give the
effect that we want to obtain. Finally, how important is the use of homologous
strains? We think that this last aspect must be considered carefully for animal
probiotics because of the diversity of the intestinal ecosystem and microflora. For
humans, the situation is different because not all the strains used in commercial
products are isolated from humans. Thus, the importance of host specificity in the
choice of probiotic strains (human and animal) should be reassessed.
4. INFLUENCE OF LAB ON THE INTESTINAL IMMUNE RESPONSE

In recent years, the food industry has indicated the importance of the consumption of
functional food as dietary supplements to promote health. Such dietary supplements
are called probiotics, and the development of probiotics is based on the knowledge
that the gut microflora is involved in resistance to disease. A probiotic is defined as
a “live microbial feed supplement which beneficially affects the host animal by
improving its intestinal microbial balance” (Fuller, 1989). Probiotic administration
causes changes in the composition and/or activity of the gut microflora and, although
the word probiotics originally referred to feed for farm animals, the concept has more
recently been applied to humans. The major consumption by humans is through dairy
fermented products containing species of lactobacilli and bifidobacteria. Several
studies have demonstrated that some lactic acid bacteria that can inhabit the lumen
of gastrointestinal tract are beneficial to animal and human health (Holzapfel
et al., 1998). It was demonstrated that fermented milk can improve digestion and
assimilation of lactose in patients suffering from lactose maldigestion. It also
prevents diarrhoea and constipation and decreases the serum cholesterol level
(Gilliland and Walker, 1990; Sanders, 1993; Saavedra et al., 1994).
Most of the protection attributed to probiotics may result from colonization
resistance, but need not necessarily be related to the establishment of the microbial
species administered in the gut. Inhibition of colonization by other strains may be
by competition for nutrients or attachment sites, change in pH, and production of
bacteriocins or other antimicrobial substances (Rolfe, 1996). In addition to prevent-
ing colonization resistance by pathogens, the probiotics contribute to the enhance-
ment of the immune response (systemic and mucosal) and can reinforce the
epithelial barrier and prevent bacterial translocation. Numerous studies were con-
ducted to demonstrate that probiotic strains can prevent or decrease the number of
pathogens (bacteria or virus) occurring in human or animal intestinal infections
(Muralidhara et al., 1977; Kaila et al., 1995; Kimura et al., 1997).
Probiotics have also been reported to stimulate non-specific immunity including
increased macrophage activation, natural killer (NK) cell numbers and inflamma-
tory release of cytokines (Sato et al., 1988; Schiffrin et al., 1995; Haller et al., 2000;
Maasen et al., 2000). Acquired or specific immune response is also enhanced
including activation of lymphocytes with antibody production and anti-tumour
activity (De Simone et al., 1993; Link-Amster et al., 1994; Kato, 2000). A primary
mechanism by which probiotics mediate immune improvement is through an adju-
vant effect. This adjuvant effect is induced by whole bacteria as well as by products
of their cell wall. Probiotics have been used in immunosuppressed hosts (Perdigón
and Oliver, 2000). Most information is derived from experiments with animals and
it may not always be possible to extrapolate to humans.
With regard to the immunomodulatory effect of LAB on the mucosal immune
system, since they are usually ingested as part of the normal daily diet, it is impor-
tant to know their influence on the immune cells associated with the gut. In this
respect there are many reports analysing their behaviour. Although most studies were
performed using experimental models (Yasui et al., 1992; Famularo et al., 1997) they
are a useful basis for the design of human experiments (Perdigón et al., 2001).
In our laboratory we evaluated the mucosal adjuvant capacity of different LAB
orally administered with particular reference to L. casei CRL 431 (Perdigón et al.,
1991, 1993, 1995; Perdigón and Pesce de Ruiz Holgado, 2000; Alvarez et al., 1998).
We determined that our viable strain of L. casei was able to 1) protect against a
Sal. typhimurium experimental infection in the mouse, 2) enhance the secretory IgA
(S-IgA) specific for the pathogen Sal. typhimurium, 3) increase the number of IgA+ cells
associated with the lamina propria of the small intestine, 4) protect against Salmonella
up to 5 days post-treatment and 5) have a boosting effect on day 15 post-priming and
to increase the protective effect against a new challenge with the pathogen (table 1).
We also determined the effect of the oral administration of L. casei, L. acidophilus,
L. rhamnosus, L. delbrueckii spp. bulgaricus, L. plantarum, S. thermophilus and
Lactococcus lactis on bronchus immunity, by analysing the IgA secreting cells
Table 1. Effect of Lactobacillus casei protection against Salmonella typhimurium and

gut immunity
Test group Control group

(treated with L. casei) (untreated with L. casei)
Assays 2 × 109 cells/day/mouse treated with NFM 10%
Protection against Sal. typhimurium. ++ −

Optimal dose of 2 days
Levels of S-IgA anti-Salmonella.
OD = 493 nm 2.5 ± 0.03 1 ± 0.05
IgA+ secreting cells/10 villi 100 ± 10 85 ± 5
Duration of preventive effect after 5 days −
priming optimal dose
Effect of boosting on day 15 ++ −
post-priming in prevention of
Sal. typhimurium infection
Lactobacillus casei was administered at an optimal dose of 2 days, previously determined. Values
are the mean of n = 5 ± SD. Results expressed as + or − represent positive or negative protection
against Sal. typhimurium challenge.
NFM, non-fat milk; OD, optical density.
associated with bronchus. We showed that, with the exception of L. acidophilus, the
LAB were able to increase the IgA-producing cells at bronchus level (Perdigón et al.,
1999a). These results were very interesting because the demonstration that oral admin-
istration of LAB induced immunity at bronchus level means that those bacteria
increased the IgA cycle by cellular migration of IgA B cells. This led us to study the
number of CD4+ T cells on the lamina propria of the small intestine after LAB feeding.
We saw that only L. casei and L. plantarum induced CD4+ T cell migration from
Peyer’s patches and they increased the number of these T cells on the lamina
propria of the small intestine (Vintiñi et al., 2000).
The interaction with M cells of Peyer’s patches can induce cellular migration.
Considering that the LAB produced cellular migration and a strong immuno-
stimulation, we analysed whether or not the LAB are presenting and processing as
antigen, by determining antibodies specific against their epitopes (a-LAB). We
found antibodies against L. casei, L. plantarum, S. thermophilus and L. rhamnosus
(Perdigón et al., 1999b) (table 2).
We demonstrated that not all the LAB are able to stimulate the mucosal immune
system in the same way, and that the immunostimulatory capacity was not related to
the genus or species, but it was strain specific. The different immunomodulating
activities of LAB would be related to the mechanisms of interaction of these
microorganisms with the intestine, and with the intensity of such interaction that ini-
tiates the intercellular signals of transductions to stimulate the immune system. Our
research has yielded a sounder knowledge of interactions of the existing probiotic
LAB, however, it may also be necessary to search for new probiotic organisms with
better probiotic activity and improved immunomodulating powers.
Table 2. Gut immunostimulation by lactic acid bacteria
Increase of Increase of Increase of IgA antibody

Microorganisms IgA B cells in IgA+ cells in CD4+ T cells a-LAB
assayed LP intestine bronchus in LP intestine response
L. casei ++ ++ + +
L. acidophilus + − − −
L. rhamnosus + + − +
L. plantarum + + + +
L. delbr. spp. bulgaricus ++ ++ − −
S. thermophilus ++ + − +
Lac. lactis + + − −
Lactic acid bacteria were orally administered to mice. Animals were sacrificed and small intestines
were removed for histological techniques. B and T cells were determined by fluorescence test.
IgA antibodies anti-LAB were measured in the intestinal fluid by ELISA test. Results expressed
as + or – represent enhancement of cells or antibody detection.
LP, lamina propria.
5. CONCLUSIONS
Progress in the knowledge of the intestinal microflora and their interaction with host
cells is essential for the use of viable microorganisms to improve animal and human
health. Since the human digestive tract is different in anatomy and physiology to
those of animals, the research in that field will require improved understanding of
host intestinal physiology and its relationship with intestinal microbes. The identi-
fication in the host cell surface of receptors able to bind microbes (indigenous or
exogenous non-pathogenic) will allow selection of desirable microorganisms for
probiotic use. A better understanding in the conditions necessary for adhesion of
bacteria to mucin or the host cell may help to establish a reliable test for the selec-
tion of strains. The value of single- or multi-strain probiotic preparations should also
be assessed. The correct characterization and identification by molecular biology of
the strains isolated from intestinal microflora from each ecological niche is neces-
sary for the complete understanding of the significance of the gut flora. The studies
on immune effects of non-pathogenic microbial strains should be performed in
in vivo animal models with a complete gut flora (conventional). Although the use
of mono-associated animals has provided a reasonable understanding of the role of
microflora on the immune system, direct transpose of these findings to explain what
is happening in the conventional animal, is not correct. Similarly, while some
in vitro tests could be useful for the understanding of the probiotic effect, they
should not be extrapolated directly to the in vivo situation.
It is also necessary to determine the importance of the viable probiotic strains,
because we cannot ignore several studies that suggest that the use of cell wall or lysate
preparations is also active. The modifications induced in the intestinal micro-
flora after long-term microbial administration of probiotics should be evaluated.
The mechanisms of action and potential side-effects of each selected strain must be
carefully studied to avoid compromising the important role of the intestinal
microflora as a barrier to intestinal infections.
ACKNOWLEDGEMENTS
The authors wish to thank Dr Marta Medici for typing the manuscript, and also the co-worker from the
Immunology Laboratory at CERELA and Tucumán University.
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17 Prospects of fish probiotics
L. Grama and E. Ringøb

a
Danish Institute for Fisheries Research, Department of Seafood Research,
Søltofts Plads, c/o Technical University of Denmark bldg. 221, DK-2800 Kgs.
Lyngby, Denmark
b
Section of Arctic Veterinary Medicine, Department of Food Safety and Infection
The chapter provides a “state of the art” for the use of probiotics (live microbial cul-
tures that improve the health of the host) in aquaculture. In fish farming, probiotic
cultures are not exclusively aimed at the gastrointestinal tract but may just as well
act via skin or gills. Some cultures can therefore be added directly to tank or pond
water. Current strategies for selecting potential fish or crustacean probiotic cultures
are outlined, including in vitro antagonism, survival, adhesion to mucus and aggre-
gating abilities. However, there is a lack of scientific evidence comparing in vitro
activities with in vivo effects. A multitude of studies have, based on in vitro tests,
reported on the isolation of what are believed to be potential fish or crustacean
probiotic cultures. The chapter emphasizes the need for in vivo testing in challenge
model or field trials as a prerequisite before the term probiotic can be used about a
culture. Many in vivo trials are hampered by too few replicates and/or poor statisti-
cal analyses and the need for improvement in this area is discussed. An important
area of future research will be to gain insight into mechanisms, stability, resistance
and specificity of fish probiotics, and knowledge of bacterial physiology and molecu-
lar approaches is needed. Despite the shortcomings outlined, several trials do docu-
ment that, in some systems, live microbial cultures can indeed be used to reduce
disease and/or decrease mortality. Probiotics should especially be investigated in the
rearing of fin fish larvae or shellfish where vaccination is difficult or not possible.
1. INTRODUCTION
World fish production, including wild catches, has increased steadily during the past
several decades (FAO, 1998), however, catches from the wild populations have

380 L. Gram and E. Ringø
Fig. 1. World global fish

production – catch and
aquaculture (FAO, 1998).
remained static at approximately 90 million metric tonnes (mt) per year since 1990.
This is currently believed to be the maximum sustainable yield from this resource.
In contrast, the contribution from reared fish is increasing, and currently approxi-
mately 30 million mt per year are produced by mari- and aquaculture (fig. 1). In
1996, aquaculture provided 20% of global fisheries production (and 29% of food
fish) (FAO, 1998). This makes aquaculture the fastest growing protein producing
sector in the world. This development is desired as one source of food to the grow-
ing world population. The aquaculture industry has also become a major source of
income for several countries, particularly in Southeast Asia, such as Thailand, where
the culture of crustaceans is an expanding industry (fig. 2).
Fig. 2. Cultured and wild-captured

shrimp harvests in Thailand
(Dierberg and Kiattisimkul, 1996;
cited from FAO/NACA, 1995).
Prospects of fish probiotics 381
Disease is one of the major constraints on the aquaculture sector, as fish in inten-
sive rearing experience stress and rapid spread of infections. Thus, the sustainable
control of fish diseases has become one of the most important prerequisites for the
future development of aquaculture. In the early days of commercial large-scale
aquaculture, treatment with antibiotics was the method of choice for controlling fish
disease. The widespread use of high amounts of antibiotics has caused much
concern because of the risk of developing antibiotic resistance (Sørum, 1999).
Antibiotic resistance may be detrimental to commercial fish production as the fish
pathogen is no longer sensitive (Alderman and Hastings, 1998). Thus, prophylactic
use of antibiotics in a shrimp (Penaeus monodon) hatchery led to antibiotic resistant
Vibrio harvyei that was responsible for mass mortalities and could not be
controlled by addition even of very high levels of antibiotics (e.g., 1000 μg/ml of
chloramphenicol, erythromycin and nifurpirinol) to which the organism is normally
sensitive (Karunasagar et al., 1994).
Although the resistance of fish pathogens has immediate serious effects for the
aquaculture sector even worse scenarios can be envisaged if such resistance spreads
to human pathogens (Levy, 1998). Several studies have shown that the microflora in
aquaculture environments is more resistant to antibiotics than microorganisms in
environments not exposed to antibiotics (Austin and Al-Zahrani, 1988; Spanggaard
et al., 1993; DePaola et al., 1995; Schmidt et al., 2000). Thus, during treatment with
oxytetracycline, approximately 80% of the intestinal Gram-negative bacteria from
channel catfish (Ictalurus punctatus) were resistant to the compound (DePaola et al.,
1995). Despite reports on occurrence of antibiotic resistance in fish farms, there is
currently no information available about the potential transfer to human pathogenic
bacteria (MAFF, 1998) and the risk may be smaller than assumed. More recently, the
US Food and Drug Administration (FDA) conducted an investigation of the preva-
lence of Salmonella in imported foods and the level of antibiotic resistance (Zhao,
2001). Of 187 Salmonella strains investigated, 15 were resistant to one or several
antibiotics, and of these strains, 10 were isolated from imported seafoods, particu-
larly from Southeast Asian countries. Whilst this could be caused by an uncontrolled
use of antibiotics in clinics or agriculture eventually leaking to the aquatic environ-
ment, it could also be linked to improper use of antibiotics in aquaculture.
No statistics are available on the worldwide use of antibiotics in aquaculture.
In a study of 11 000 aquaculture farms in Southeast Asia in 1995, it was found that
only 5% of inland carp farms used antibiotics (FAO/NACA/WHO, 1999) and,
similarly, few of the coastal shrimp farmers used antibiotics. In contrast, in intensive
shrimp farming, there was a higher frequency of antimicrobial use with oxytetra-
cycline and oxolinic acid being the main compounds.
During the past 10 years, significant advances have been made in several more
environmentally compatible disease control measures. Of major importance is the
development of fish vaccines that now enable the control of a range of bacterial fish
pathogens in several fish species. Thus, the use of antibiotics in Norway has
decreased from 48 570 to 591 kg active substance per year from 1987 to 1999 –
whilst the production of Atlantic salmon has increased from 60 000 to 464 000 mt
per year during the same period (Grave et al., 1990, 1996; Alderman and Hastings,
1998; Norm-Vet, 1999). This significant reduction in the use of antibiotics is
primarily due to the introduction and use of efficient vaccines. However, vaccination
of fish smaller than 35 g is not recommended. Also, during hatching and in the
larval stage, vaccination may not be possible due to the slow development of the fish
immune system and the small size of the fish. Owing to the lack of acquired immu-
nity in crustaceans and molluscs, conventional vaccination cannot be used as a
disease control strategy for such organisms.
One alternative means of disease prevention that has attracted attention during
recent years is the addition or inclusion of assumed beneficial bacteria to rearing
water or feed, the so-called probiotics (Gatesoupe, 1999; Gomez-Gil et al., 2000;
Verschuere et al., 2000a). This chapter reviews some of the findings in terms of
(in vitro) strategies for selecting probiotics and their use in vivo. Whilst the overall
conclusion of the chapter is that there is indeed reason to follow this path, the reader
should be alerted that one common feature of probiotic research whether in warm-
and cold-blooded animals is the huge variability of experimental results, particularly
in in vivo studies (Berg, 1998). As will become evident, no studies so far have shown
which strategy is optimal for selection of probiotic cultures, and a better under-
standing of the fish indigenous flora and of the basic ecological principles control-
ling its development must be built. Further, results from in vitro antagonism tests
must be compared with in vivo experiments. To understand the areas in which the
probiotic concept is viable, we must understand the mechanisms by which such
treatments work (Atlas, 1999). This will require detailed knowledge of:
the pathogen, its virulence, its proliferation and invasion sites;
the host, its immune defence, and its natural microflora;
the surrounding environment, including nutrients, microorganisms, etc.;
the probiont, its functional features, its mechanisms of action, and its effect
on the general microflora, etc.
A range of microorganisms has either been suggested or evaluated as fish
probiotics. These include lactic acid bacteria (LAB) (Gatesoupe, 1994; Gildberg
et al., 1995, 1997; Gildberg and Mikkelsen, 1998; Robertson et al., 2000), Bacillus
species (Moriarty, 1998), Pseudomonas species (Bly et al., 1997; Gram et al., 1999;
Spanggaard et al., 2001), Vibrio species (Austin et al., 1995) and other Gram-
negative bacteria (Nogami et al., 1997).
As well as adding a beneficial microorganism to the system, attempts have also
been made to increase the disease resistance in fish by other means than probiotics.
Several compounds, such as glucans and vitamins, can have an immunomodulating
effect (Raa, 1996; Sakai, 1999), and it has been suggested that certain polysaccha-
ride compounds can be used as prebiotics, i.e. host-indigestible compounds that can
be metabolized by “beneficial” microorganisms colonizing the digestive tract
(Roberfroid, 1993, 1998, 2001). However, to date no information is available on

prebiotics and their effect on intestinal fish microflora and fish welfare. Other
disease control attempts include the use of “natural compounds” as agents against
pathogens: e.g. the reduction of Vibrio alginolyticus infection in juvenile rockfish
(Sebastes schlegeli) fed an aloe-containing diet (Kim et al., 1999).
2. PROBIOTICS: DEFINITION OF TERMINOLOGY

When initially introduced in the 1970s, the term “probiotic” described microbial
feed supplements (Berg, 1998) and stems from combining the Latin word pro (for)
with the Greek word bios (for life) (Zivkovic, 1999). Fuller (1989) further detailed
this as “live microbial feed supplements which beneficially affect the host animal by
improving its intestinal microbial balance”. Since then, many variations to the
definition(s) have been proposed (table 1). As discussed below, the term “microbial
balance” is ill-defined and “The Lactic Acid Bacteria Industrial Platform” at their
workshop in 1995 provided a more concise definition of the term as “oral probiotics
are live microorganisms which upon ingestion in certain numbers, exert health
benefits beyond inherent basic nutrition” (Guarner and Schaafsma, 1998). In this
chapter, we define a probiotic as a live microbial preparation that when added to the
fish, crustaceans or molluscs (larvae, fry, young or adult animals) has a beneficial
effect on the health of the host.
Some points should be made with respect to the definitions proposed.
The term probiotic originates from warm-blooded animals where the importance
of the gastrointestinal tract for onset of diseases and of the mucosal immune system
is clearly recognized. In contrast, in fish (and other cold-blooded animals), areas such
as skin and gills are probably as important sites for proliferation of pathogens and
initiation sites for the infection. Therefore, in the context of fish and aquatic animals,
a probiotic is a live microbial supplement added to the host environment or feed.
By expanding the area of application, the term probiotic parallels the “biocontrol”
term used for microbial cultures applied to prevent plant (rhizosphere) disease.
Several definitions imply that the probiotic “beneficially affects the microbial
balance”. The term “microbial balance” is not well defined and whilst it must be
assumed that this refers to a change in the host microbial community to diminish
numbers and/or metabolism of pathogenic organisms, it may be difficult and time
consuming to measure. There are examples in the literature where addition of a
probiotic culture (Bacillus) to pond water decreased the numbers of potentially
pathogenic bacteria (luminescent vibrios) in penaeid culture and subsequently
increased survival of the animals (Moriarty, 1998). Furthermore, Skjermo et al.
(1997) showed that so-called “microbial maturation” of water may increase the
survival of Atlantic halibut (Hippoglossus hippoglossus). This process involves
changing the microflora of pond water from opportunistic, substrate-(nutrient)-
requiring microorganisms (r-strategists) to non-opportunistic so-called K-strategists
Table 1. Definitions of probiotics by various authors
Definition Reference Comments
Microbial compounds which promote body functions and beneficial microorganisms Vergin, 1954 Products – not live culture?
Microbially produced “factors” which promote growth of other organisms Lilly and Stillwell, 1965 Growth promotion
Animal feed supplements – organisms and substances that have a beneficial effect Parker, 1974 Only feed what is “microbial
on the host animal by contributing to its intestinal microbial balance balance”
Live microbial feed supplements which beneficially affect the host animal by Fuller, 1989 Only feed what is “microbial
improving its intestinal microbial balance balance”
A microbial dietary adjuvant that beneficially affects the host physiology by Naidu et al., 1999 Only feed what is “microbial
modulating mucosal and systemic immunity, as well as improving nutritional balance”
and microbial balance in the intestinal tract
Live microorganisms supplemented in food or feed which give beneficial effects Gildberg et al., 1997 Only feed what is “microbial
on the intestinal microbial balance balance”
A live microbial supplement which beneficially affects the host animal by Gram et al., 1999 What is “microbial balance”?
improving its microbial balance
Is based on elimination of harmful microflora from the animal’s digestive tract Bogut et al., 1998 Elimination? Only dietary tract
Mono or mixed cultures of live microorganisms which when applied to animal Havenaar et al., 1992 What are “properties of
or man, beneficially affect the host by improving the properties of the Conway, 1996 indigenous microflora”?
indigenous microflora Holzapfel et al., 1998
Viable microorganisms (bacteria or yeasts) that exhibit a beneficial effect on the Salminen et al., 1998
health of the host when they are ingested
Beneficial bacteria which may override pathogens by producing inhibitory Riquelme et al., 2000 What is “override”?
substances, or by preventing pathogenic colonization of the host
Beneficial bacteria that displace pathogens by competitive processes or by release Moriarty, 1997
of growth inhibitors
Live intestinal bacteria that are added to promote the viability of the host, but the Skjermo and Vadstein, 1999 How is “colonization” regulated?
term is also proper for bacteria able to regulate colonization of the outer surfaces
Live microbial adjunct which has a beneficial effect on the host by modifying the Verschuere et al., 2000a
host-associated or ambient microbial community, by ensuring improved use of the
feed or enhancing its nutritional value, by enhancing the host response towards
disease, or by improving the quality of its ambient environment
Oral probiotics are living microorganisms, which upon ingestion in certain Guarner and Schaafsma,
numbers, exert health benefits beyond inherent basic nutrition 1998
Live microbial cultures added to feed or environment (water) to increase viability This chapter
(survival) of the host
with high substrate (nutrient) affinity (Salvesen et al., 1999). Despite these studies
that show that alterations in microbial composition may affect fish survival, this may
not be a prerequisite for a successful probiotic culture. Therefore, we propose that
the effect of a probiotic be measured by its ability to decrease frequency of disease
and/or increase survival from lethal diseases.
Live microbial cultures may also be used in aquaculture for other purposes such
as feed or water quality improvement that indirectly will benefit the health/survival
of the aquatic organism (Moriarty, 1997). If a clear effect on, e.g. health condition
(disease survival) can be documented, we suggest that such preparations be denoted
probiotics. Otherwise, they should be classified as “feed”, “water treatment” etc.
(Gatesoupe, 1999). Examples of this include the nitrification (e.g. the conversion of
− −
toxic NH3 and NO2 to NO3 ) of water by various bacterial species such as
Nitrosomonas and Nitrobacter (Hagopian and Riley, 1998), and the enhancement of
fish growth by microorganisms that has been observed in several studies (Byun
et al., 1997; Bogut et al., 1998; McCausland et al., 1999; Thompson et al., 1999).
In an attempt to clarify some of the inconsistencies, we define a probiotic as
a live microbial preparation that when added to the fish, crustacean or mollusc
(larvae, fry, young or adult animals) has a beneficial effect on the health of the host.
Most commonly a beneficial effect (which it can be argued is somewhat imprecise)
refers to reduced mortality (or increased survival). We do not use “probiotic” to
describe microorganisms that improve water quality, e.g. by denitrifying, or
microorganisms that serve as food supply. Whilst most studies have used bacterial
cultures as potential probiotics, yeast (Scholz et al., 1999) has also been suggested
as a disease control measure. The addition of microalgae to the rearing waters of
larvae presents an intermediate. Whilst this use of “green water” improves the
growth rate of several larvae, and thereby classifies the algae as feed, it also
improves survival and thereby qualifies as a probiotic (Alves et al., 1999; Planas and
Cunha, 1999). It has also been suggested that the addition of viruses specific for the
fish/larval pathogen could be used to prevent disease (Park et al., 2000).
3. MICROFLORA OF FISH
Whilst no clear strategy exists for the isolation and selection of fish probiotic cul-
tures, most studies have based their work on isolates from the natural microflora of
fish or fish larvae. This is due to the assumption that pathogen-antagonizing bacte-
ria from the fish or fish larvae are better adapted for this niche than cultures derived
from terrestrial or other sources. Fish and other aquatic organisms harbour a micro-
bial flora on the surfaces of skin, gills and in the intestinal tract, either in the lumen
or associated to mucus or epithelial cells. Fish live in extreme environments, from
the cold Arctic and Antarctic oceans to tropical freshwater lakes. Despite these dif-
ferences in environmental conditions, certain general patterns emerge in terms of
composition of the culturable microflora (Cahill, 1990; Hansen and Olafsen, 1999;
Ringø and Birkbeck, 1999; Gram and Huss, 2000; table 2).
Table 2. Bacterial genera associated with various raw finfish and crustaceans, and their percentage of the total microflora (ICMSF, 2004)
% composition
Acinetobacter –
Gram-positive
Other or not
Bacillus spp.
Moraxella
Vibrionaceae
Cytophaga
Gram-negative
Coryneforms
cocci
Flavobacterium –
Pseudomonas
Other
Fish type Reference
Marine fish, temperate

North Sea fish (1932) 5 1 56 11 5 — 23 — — Shewan, 1971
North Sea fish (1960) 16 2 23 27 10 18 4 — — Shewan, 1971
North Sea fish (1970) 22 1 41 10 21 — 1 — — Shewan, 1971
Haddock (North Atlantic) 26 2 45 15 — 4 4 2 — Laycock and Regier, 1970
Flatfish (Japan) 21 29 22 13 — 13 2 — — Simidu et al., 1969
“Pescada” (Brazil) 32 — 35 5 18 4 4 — 3 Watanabe, 1965
Shrimp (North Pacific) 10 — 47 22 — 3 7 4 8 Harrison and Lee, 1969
Scampi (UK) 3 — 11 2 — 81 — — 3 Walker et al., 1970
Marine fish, tropical
Mullet (Australia) 18 — 9 8 — 12 51 2 — Gillespie and Macrae, 1975
Prawn (India) 11 10 23 6 — 13 6 — — Surendran et al., 1985
Sardine (India) 20 28 30 4 — — 7 — 11 Surendran et al., 1989
Shrimp (Texas Gulf) 22 2 14 9 1 40 11 — 2 Vanderzant et al., 1970
Shrimp (Texas, pond) 2 — 15 25 12 43 3 0.5 1 Vanderzant et al., 1971
Freshwater fish, temperate
Pike (Spain) 10 — 15 — 55 10 — 5 5 González et al., 1999
Brown trout (Spain) — 6 — — 40 — 15 — 34 González et al., 1999
Trout (Spain, reared) 11 — 7 — 26 5 45 — 6 González et al., 1999
Trout (Denmark, reared) 19 3 50 — 18 6 4 — — Spanggaard et al., 2001
Freshwater fish, tropical
Nile perch (Kenya) 6 2 43 — 9 5 30 5 — Gram et al., 1990
Catfish (India) — — 10 — — — 50 40 — Venkataranan and Sreenivasan, 1953
Carp (India) — — 20 11 30 — 39 — — Venkataranan and Sreenivasan, 1953
It should be borne in mind that whilst the majority of the bacteria in the
gastrointestinal tract and on fish surfaces are probably culturable (Spanggaard et al.,
2000a), some studies using molecular techniques clearly demonstrate large propor-
tions of unculturable bacteria associated with the gastrointestinal tract and fish
surfaces (Bernadsky and Rosenberg, 1992; Huber et al., 2004). Thus, this area of
microbial ecology would benefit from the use of molecular techniques such as dena-
turing gradient gel electrophoresis and in situ rRNA hybridization methods for the
evaluation of microflora composition.
3.1. Fish eggs and larvae

The interior of the fish egg is assumed to be sterile, but the outer surface of Atlantic
halibut, Atlantic cod, turbot and Atlantic salmon eggs is typically occupied by non-
fermentative Gram-negative bacteria such as Cytophaga, Flavobacterium and
Pseudomonas (Bell, 1966; Cahill, 1990; Bergh, 1995; Hansen and Olafsen, 1999).
Upon hatching, the yolk-sac larvae start to drink (Magnor-Jensen and Adoff, 1987;
Reitan et al., 1998), and, as a natural consequence, the intestinal tract of non-
feeding larvae becomes colonized by the same genera present in the rearing water
(Strøm and Ringø, 1993; Bergh et al., 1994). Furthermore, some studies (Ringø
et al., 1996; Makridis et al., 2000b) have shown that the gut microflora of early
developing turbot larvae fed rotifers reflect the water microflora by adding Vibrio
pelagius (Ringø et al., 1996) or unidentified Gram-negative bacteria (Makridis et al.,
2000b) to the water. Upon feeding, a dramatic shift occurs in the intestinal
microflora which becomes dominated by fermentative Gram-negative rods such as
Vibrio and Aeromonas (Munro et al., 1993, 1995; Bergh et al., 1994; Bergh, 1995;
Blanch et al., 1997). In agreement with these results, Gatesoupe (1990) reported that
the microflora on both rotifers and turbot larvae feeding on the rotifers were domi-
nated by Vibrio spp. The occurrence of Vibrio species as the dominant gut micro-
organisms has also been reported in white shrimp (Litopenaeus vannamei) larvae in
a recent study by Vandenberghe et al. (1999). The exact role of different bacterial
groups in larval disease or disease prevention is not known, however, it has been
suggested that some members of the natural flora are part of the non-specific dis-
ease defence of the larvae (Bergh et al., 1994; Makridis et al., 2000b). Accordingly,
some studies have shown that bacteria isolated from larvae may be inhibitory – in
laboratory based in vitro assays – against potential fish pathogenic organisms
(Bergh, 1995). However, in vitro studies may not reflect the actual role of each
organism in its ecological niche as will become evident from studies that have
compared in vitro and in vivo antagonism. Even though few studies have identified
specific microbial larval pathogens, there seems no doubt that they are the cause of
the significant mortalities often experienced in larval rearing. Thus, ultraviolet (UV)
treatment of rotifers used for feeding turbot larvae, reduced bacterial numbers
by more than 90% and caused a significant increase in survival of the larvae as
compared to larvae fed non-UV-treated rotifers (Munro et al., 1999), and Munro
et al. (1995) obtained survival of up to 100% of larvae in bacteria-free larval rearing.
Also, surface disinfection of eggs results in a much higher survival of the hatched
larvae than when non-disinfected eggs are used (Vadstein et al., 1993).
Readers with special interest in bacterial species colonizing the gastrointestinal
tract of larvae and fry are referred to the recent reviews of Hansen and Olafsen
(1999) and Ringø and Birkbeck (1999).
3.2. Skin flora

Despite large variations in the environment of fish (cold marine fjords, tropical
freshwater ponds) and the variation this imposes on the microbiology of cold-
blooded animals, several groups of microorganisms are typical of fish skin. In
general, the microflora of fish skin has been found to reflect the microflora in the
surrounding water (Horsley, 1973; Cahill, 1990). Thus, fish caught in temperate
waters harbour microflora dominated by Gram-negative bacteria belonging to
Pseudomonas, Acinetobacter, Vibrionaceae (including Photobacterium spp.),
Flavobacterium and Moraxella, with Gram-positive bacteria represented by species
such as Kurthia, Streptococcus, Micrococcus and the coryneforms. In warmer
waters, the percentage of Gram-positive bacteria tends to be higher, and Bacillus
and Micrococcus are more often isolated. Also, Enterobacteriaceae, which are
isolated from temperate water fish, typically occur in higher numbers in tropical
fish species (Gram and Huss, 2000).
3.3. Gill flora

In his early study, Horsley (1973) demonstrated that the major microbial groups of the
gill microflora of Atlantic salmon were similar to those present in the water, which
supports the hypothesis that the external fish flora is a reflection of the environment.
In contrast, Mudarris and Austin (1988) demonstrated in a study with turbot that bac-
teria isolated from the surface of gills were quite distinct from the species isolated
from the surrounding water. Also, Austin (1982, 1983) demonstrated that the surface
microflora isolated from turbot did not closely reflect the type of bacteria found in
either the seawater that supplied the tanks, or the water in which the fish lived. By
using scanning electron microscopy of gill preparations, Mudarris and Austin (1988)
found that only protected areas on the gill, such as clefts between adjacent secondary
lamellae were colonized by bacteria-like objects. The gill flora is somewhat similar to
the skin microflora and is typically dominated by non-fermentative Gram-negative
rods (Gennari and Tomaselli, 1988; Mudarris and Austin, 1988; Cahill, 1990) and
Vibrionaceae (Mudarris and Austin, 1988; Cahill, 1990). Thus, Spanggaard et al.
(2001) found that 43% of the culturable flora were non-fermentative Gram-negative
rods whilst 35% belonged to Vibrionaceae and Enterobacteriaceae (table 3).
Table 3. Composition of the culturable microbial flora (1018 strains) of rainbow trout (isolated from 49 fish) and the number of strains capable of inhibiting
Vibrio anguillarum in an agar-well diffusion assay (modified from Spanggaard et al., 2001; Huber et al., 2004)
Skin flora Gill flora Gut flora
No. of No. of No. of No. of No. of No. of

Group/species isolates % antagonists isolates % antagonists isolates % antagonists
Pseudomonas 27 19 1 99 24 24 23 5 3
Acinetobacter /Moraxella 73 50 1 133 32 4 32 7 0
Vibrionaceae 4 3 0 60 15 4 100 22 0
Enterobacteriaceae 0 0 0 46 11 1 167 36 3
Other Gram-negatives1 26 18 0 50 12 0 78 17 0
Gram-positives2 15 10 1 23 6 0 38 8 3
Yeasts 0 0 0 1 <1 0 23 5 0
Total 145 100 3 412 100 33 461 100 9

1
Include Shewanella, Flavobacterium, Plesiomonas, Xanthomonas.
2
Include Streptococcus, Staphylococcus, Carnobacterium.
3.4. Gut flora

The fish gut microflora is markedly different from the environment and is typically
dominated by a few genera. However, there is a large variation in numbers of micro-
organisms associated with epithelial mucosa and in the lumen of individual fish
(Ringø and Olsen, 1999; Huber et al., 2004). Faecal bacterial flora (Sugita et al.,
1988, 1990) and the gut microflora of one fish species may vary between different
geographical regions. Furthermore, it is well known that dietary components affect
the gut microflora (Sugita et al., 1988; Strøm and Olafsen, 1990; Ringø, 1993;
Ringø and Strøm, 1994; Ringø and Olsen, 1999) and there may also be large day-
to-day fluctuations in both numbers and composition (Sugita et al., 1987, 1990). The
change in microflora from the outer surfaces is probably due to both a selective pres-
sure from the gut acid and bile, and just as much to the nutrient-rich, low-oxygen
environment selecting for fermentative Gram-negative rods. Thus, Vibrionaceae and
Enterobacteriaceae are typical of the fish gut where they may occur in high num-
bers (106–108 CFU/g) (Austin and Al-Zahrani, 1988; Cahill, 1990; Spanggaard
et al., 2001; Huber et al., 2004) although the overall number of bacteria in fish gut
may vary from 103 to 108 CFU/g (Huber et al., 2004). Most of these bacteria seem
to reside in the lumen with only a minor proportion of the bacterial cells present
along the gut wall (Austin and Al-Zahrani, 1988; Huber et al., 2004). In Arctic charr,
scanning and transmission electron microscopy has shown that bacterial cells are
closely associated with epithelial cells of pyloric caeca, midgut and hindgut
(Lødemel et al., 2001; Ringø et al., 2001).
In most studies on intestinal microflora of fish, only aerobic or facultative
anaerobic bacteria have been isolated (Hansen and Olafsen, 1999; Ringø and
Birkbeck, 1999). However, some investigations revealed the occurrence of signifi-
cant numbers of strict anaerobic bacteria in fish gut (Trust and Sparrow, 1974; Trust,
1975; Trust et al., 1979; Sakata et al., 1981; Huber et al., 2004). Huber et al. (2004)
reported that an unculturable bacterium with a gene-sequence clustering amongst
anaerobic bacteria accounted for 99% of the gut microflora of rainbow trout, but
then only in a few fish. Gram-positive bacteria such as LAB are also commonly
isolated from fish gut (Ringø and Gatesoupe, 1998), and Ringø et al. (2000) reported
that for Atlantic salmon sampled in Northern Norway approximately 30% of a
gut flora of 10 3 CFU/g were LAB, in particular carnobacteria (Carnobacterium
piscicola).
4. SELECTION STRATEGIES FOR PROBIOTIC CULTURES

Studies on the use of probiotics in warm-blooded animals and, as outlined above, in
fish have often been non-conclusive or even contradictory (Conway, 1996).
A variety of traits have been considered important, including the production of
anti-pathogen substances and the ability to survive and colonize the host. Amongst
the parameters listed for selecting a probiotic strain are (Conway, 1996):
specified target
host origin
survival in vivo/in situ
colonization potential
non-pathogenic/safe
biological activity against target
demonstrable efficacy
stability/robustness.
At present little is known about the in vivo mechanisms that are operational when
probiotic culture is used with success, and a scientific rationale for the selection
of the best species or strains for use as probiotics is not possible without more
information on the mechanisms by which probiotics exert their beneficial effect
in vivo (Berg, 1998). For instance, Moriarty (1998) found that a preparation of
Bacillus spp. was effective in reducing shrimp disease but also that a constant
supply was required, indicating that colonization did not take place and was not a
prerequisite. Ringø and co-authors (Ringø, personal communication, 2001) recently
demonstrated by transmission electron microscopy (TEM) that a pathogenic Vibrio
(V. anguillarum) attached to the gut epithelial cells of the midgut and hindgut
of common wolffish (Anarhichas lupus L.) fry, whereas a potential probiotic
LAB strain, a Carnobacterium divergens originally isolated from wolffish fry,
colonized enterocytes of the pyloric caeca and foregut. Such studies by using
TEM and scanning electron microscopy (SEM) emphasize the need to understand
the pathogen proliferation and infection sites, so that the probiotic culture can be
targeted to these sites.
4.1. In vitro antagonism

Most – if not all – studies initiate the search for probiotic microorganisms by evalu-
ating the in vitro inhibitory activity of a collection of microorganisms against the
target pathogen, although it has not been shown that inhibitory substances produced
in vitro are actually effective in vivo (Atlas, 1999).
Agar-based diffusion assays exist in several forms, where the target organism is
incorporated into an agar layer and exposed to the test organism as either a live cul-
ture (Gram, 1993; Bly et al., 1997) or the inactivated test organism (Lemos et al.,
1985; Dopazo et al., 1988; Westerdahl et al., 1991; Bergh, 1995; Sugita et al.,
1996a,b, 1997a; Jöborn et al., 1997). Also, the two organisms may be cross-streaked
against each other (Smith and Davey, 1993).
The test organism may be cultured in liquid media and the filter-sterilized culture
supernatant (or extracts thereof) tested for its inhibitory activity against the pathogen.
This is typically done following the growth of the pathogen by absorbance meas-
urement (Smith and Davey, 1993; Jöborn et al., 1997; Gram et al., 1999; Ringø
et al., 2000). Some studies have also used co-culture in broth systems to evaluate the
inhibitory properties of an organism (Gram et al., 1999, 2001; Spanggaard et al.,
2001), however, such studies require that a specific, quantitative method exists for
enumeration of the pathogenic target organism.
Using such approaches, a range of bacteria with inhibitory properties against fish
pathogenic bacteria have been identified. Typically, only a few per cent of the cul-
turable flora from fish can inhibit fish pathogenic bacteria in vitro (Sugita et al.,
1996a,b; Riquelme et al., 1997; Spanggaard et al., 2001) but some studies have
reported that almost 1/3 of the microflora were inhibitory to target organisms
(Westerdahl et al., 1991; Burgess et al., 1999). A high proportion (20%) of bacteria
from intertidal seaweeds was also inhibitory to other bacteria (Lemos et al., 1985).
Media composition strongly influences the results of such screenings, as the
inhibitory activity of the test strains is medium dependent. One classical example is
the antimicrobial properties of fluorescent pseudomonads in which iron limitation
causes production of iron chelating siderophores which deprive the competing
pathogen of iron. Under iron-surplus conditions, no inhibition is detected, whereas
under iron-limited conditions, a pronounced inhibition is seen (Gram, 1993; Smith
and Davey, 1993). Bearing the influence of media and culture conditions in mind,
it is perhaps not surprising that no clear pattern emerges in terms of which bacterial
species harbour anti-pathogen strains. Some studies have identified Vibrio (and
Aeromonas spp.) as the most inhibitory (measured in in vitro assays) as compared
to other culturable bacteria isolated (Westerdahl et al., 1991; Bergh et al., 1995),
whereas others have identified Pseudomonas and Alteromonas as potent inhibitors
(Lemos et al., 1985; Tanasomwang et al., 1998; Spanggaard et al., 2001). Also,
a large proportion of LAB is inhibitory to fish pathogens (Ringø and Gatesoupe,
1998; Ringø et al., 2000). In the studies by Sugita et al. (1996a,b, 1997b) almost
2000 strains were isolated from the intestinal tract of various fish species. No
specific bacterial group emerged as more inhibitory than others.
4.2. Adherence to mucus and survival of intestinal conditions

Probiotic bacteria must persist – either transiently or permanently – in the environ-
ment in which they are to act. Thus, they must be able to adhere to the fish surfaces.
Several authors pursuing fish probiotics have used the ability of an organism to
adhere to intestine or skin mucus as a selection criterion (Olsson et al., 1992; Jöborn
et al., 1997; Nikoskelainen et al., 2001a). Whilst the organisms do adhere, it should
be noted that fish mucus does not, in general, cause an increase in numbers of
adhered bacteria, as adhesion to neutral surfaces, e.g. polystyrene, may be as high
or better (Nikoskelainen et al., 2001a).
Also, if a probiotic microorganism is to act in the intestinal system, it must be
able to resist compounds that may act as antimicrobials. Thus, Nikoskelainen et al.
(2001a) found that a range of LAB that were tested for potential as probiotics
resisted 10% fish bile for 11/2 hours.
4.3. Colonization and persistence

“Colonization potential” has been suggested as one of several selection criteria for
probionts (Conway, 1996). In this chapter, colonization is defined not only as the
ability of an organism to proliferate for some time in a niche to which it is added
(in high numbers), but also as the ability to establish itself more permanently and
remain after exogenous supply has stopped. Innumerable studies of the mammalian
tract have found that unless the existing microflora is eliminated, e.g. by antibiotic
treatment, it is virtually impossible for an exogenous microorganism to become
established. This ability of the existing microbiota in a niche to resist invaders has
been called the colonization resistance (van der Waaj et al., 1971). As described
below, this phenomenon appears important in aquatic organisms as well.
Robertson et al. (2000) found 106–107 CFU of Carnobacterium spp. per gram in
the gut, when trout were fed a diet containing approximately 107 per gram of the
organism, indicating that no significant growth took place. However, when the fish
reverted to the control diet without Carnobacterium, the level dropped to less than
1 per gram in 6 days indicating that no colonization had taken place at all and that
the supplement was unable to compete with the natural flora. Similarly, Strøm and
Ringø (1993) added 10 5 CFU/ml of an Atlantic cod-derived Lactobacillus plantarum
to cod fry. This resulted in an immediate dominance of this organism, however, after
9 days the flora of the Lactobacillus treated fry was similar to that of non-treated fry.
Colonization by added microflora may be possible if very young, and pre-
sumably not completely colonized animals are exposed to an exogenous source
of microorganisms. Electron microscopy revealed extensive colonization of the cae-
cal mucosal epithelium of 3-day-old chicks that were fed a mixture of 29 different
microorganisms on the day of hatch (Droleskey et al., 1995). This so-called com-
petitive exclusion treatment increased resistance to colonization with Salmonella
and reduced Salmonella contamination of floor pen litter (Corrier et al., 1998).
4.4. Aggregation with pathogens

Some potential probiotic bacteria may have the ability to aggregate the pathogenic
organisms, and it has been suggested that this may prevent the adhesion of the
pathogen to the mucosa. This has been studied in bacteria relevant to mammalian
systems where Spencer and Chesson (1994) isolated five strains of lactobacilli able
to aggregate Escherichia coli 0129-K88+ within a sample of 43 lactobacilli strains.
Later, Kmet and Lucchini (1999) isolated 20 strains of lactobacilli and demonstrated
aggregation activity between six homofermentative autoaggregative lactobacilli and
three strains of pathogenic E. coli with F4, F5 and F6 fimbriae. As no information
is available about aggregation of probiotics with pathogens in fish, this topic might
be of some interest in future studies on fish. One could argue that if such aggrega-
tion occurs, it may also prevent the probiont from reaching attachment sites and
thereby reduce the potential beneficial effect.
4.5. Site specificity in vivo

A probiotic microorganism may exert an “overall” effect on the environment,
e.g. producing a diffusible antibacterial compound that affects the pathogenic organ-
ism. However, it is possible that the effect of a probiotic microorganism has to be
exerted against the pathogen in very specific spatial areas. Therefore, the probiotic
microorganism must be targeted to the niches where the pathogenic organism
resides, proliferates or invades. This will require better knowledge of fish pathogens
than currently available. As mentioned, Ringø and co-workers (unpublished data)
observed using TEM and SEM that a pathogenic Vibrio adhered to another part of
the intestinal tract than a potential probiotic Carnobacterium. Such observations
could point to selection of probiotic strains from the same species as the pathogen,
assuming that they will compete more efficiently for the same nutrients and space
as the pathogen.
5. USE OF MICROBIAL CULTURES FOR PREVENTION OF DISEASE

IN AQUACULTURE
The number of studies testing probiotics in aquaculture is rapidly increasing. Whilst
initial studies were carried out with grown fish, the focus has shifted to fish larvae,
crustaceans and molluscs where vaccines are unlikely to be available within the near
future. This section gives an overview of studies that have involved in vivo evaluation
of probiotics as disease preventive measure, and major results are summarized in
tables 4, 5 and 6. In vivo trials have typically evaluated a probiotic effect by compar-
ing the accumulated mortality of control (infected) tanks or ponds to that of treated
(infected) tanks or ponds. Unfortunately, the statistical treatment of such
survival/mortality data is often lacking or is based on simple comparisons of mean
values, e.g. using analysis of variance (Gram et al., 1999) or student’s t-test (Queiroz
and Boyd, 1998). It would be statistically more appropriate to use other means of
analysis commonly used to analyse survival data. The standard analysis for analysing
survival data, when the exact survival times are observed, is the proportional hazard
model (Cox, 1972; Cox and Oakes, 1984). When the survival times are only observed
at certain points in time, this model reduces to a so-called generalized linear model
with a clog-log link (Fahrmeir and Tutz, 1994; Spanggaard et al., 2001).
As mentioned, in vivo infection trials may be difficult to standardize and, even
with tight control of fish, water temperature, oxygen concentration, etc., large vari-
ations, e.g. in accumulated mortality may be seen between tanks. Thus, in a set of
trials testing the probiotic bacterium P. fluorescens strain AH2 (Gram et al., 1999),
Table 4. Effect of addition of probiotic microorganisms on fish and crustacean larval survival
Presumed probiont Pathogen Host organism Effect on survival Reference
Carnobacterium divergens Vibrio anguillarum cod fry no effect Gildberg and

Mikkelsen, 1998
Carnobacterium divergens Vibrio anguillarum cod fry increase accumulated survival from 40 to 60% Gildberg et al., 1997
after 3 weeks
Carnobacterium divergens Aeromonas Atlantic salmon fry decrease accumulated survival from 50 to Gildberg et al., 1995
salmonicida 30% after 4 weeks
Lactic acid bacterium Vibrio P turbot larvae increase accumulated survival from 19 to 28% Gatesoupe, 1994
(expt 1) after 48 hours or from 19 to 23%
(expt 2) and from 8 to 50%
(expt 3) after 72 hours
Vibrio pelagius not known turbot larvae increase accumulated survival from 6 to 9% on Ringø and Vadstein, 1998
day 12 and from 0 to 3% on day 16 after
hatching
Vibrio mediterranei Q40 not known turbot larvae increase accumulated survival (5 days post Huys et al., 2001
hatching) in five separate experiments; e.g. 14
to 55% in trial 1 or 75 to 81% in trial 4
Aeromonas media Vibrio tubiashii oyster larvae increase survival after 6 days from 4 to 100% Gibson et al., 1998
Pseudomonas and unknown Vibrio anguillarum scallop larvae increase survival from 5 to 60% after 14 days Riquelme et al., 1997
strain like
Pseudomonas and Vibrio field trial? not known scallop larvae same survival after 48 h as antibiotic treated Riquelme et al., 2001
tanks
Thalassobacter utilis field trial not known crab larvae increase survival from 16 to 26% (see table 7) Nogami and Maeda, 1992;
(Vibrio spp.) Nogami et al., 1997
Table 5. Effect of addition of probiotic microorganisms on fish survival
Presumed probiont Pathogen Host organism Effect on survival Reference
Bacteriophage1 Pseudomonas ayu increase survival from 35 to 75% Park et al., 2000
plecoglossicida
Tetraselmis suecica several Gram-negatives salmon increase survival from 0–15 to 20–100% Austin et al., 1992
Bacillus spp. field trial channel catfish increase survival from 56 to 80% Queiroz and Boyd, 1998
not known
Carnobacterium spp. Vibrio anguillarum salmon no effect on survival Robertson et al., 2000
Vibrio ordalii increase survival from 23 to 74%
Yersinia ruckeri increase survival from 42 to 71%
Aeromonas salmonicida increase survival from 0 to 20%
Aeromonas salmonicida trout increase survival from 32 to 74%
Vibrio alginolyticus Aeromonas salmonicida salmon increase survival from 0 to 82% Austin et al., 1995
Vibrio anguillarum increase survival from 10 to 26%
Vibrio ordalii increase survival from 0 to 26%
Yersinia ruckerii no effect on survival
Pseudomonas fluorescens Aeromonas salmonicida salmon increase healthy fish from 25–70 to 90–100% Smith and Davey, 1993
Pseudomonas fluorescens Vibrio anguillarum trout increase survival from 50 to 70% Gram et al., 1999
Pseudomonas spp. Vibrio anguillarum trout increase survival (some strains) Spanggaard et al., 2001
no effect (some strains)
Pseudomonas fluorescens Aeromonas salmonicida salmon no effect on survival Gram et al., 2001
1
It is debatable whether or not a bacteriophage qualifies as a probiont, “a live microbial...”, because of the inert nature of viruses.
Table 6. Effect of addition of probiotic microorganisms on crustacean (shrimp) survival
Presumed Host
probiont Pathogen organism Effect on survival Reference
Saccharomyces not known shrimp increase survival Scholz et al., 1999

cerevisiae from 60 to 80%
Phaffia not known shrimp increase survival Scholz et al., 1999
rhodozyma from 60 to 80%
Bacillus spp. field trial shrimp increase survival Moriarty, 1998
luminescent
vibrios
Bacillus S11 Vibrio harveyi tiger shrimp increase survival Rengpipat et al., 2000
from 36 to 54%
Bacillus S11 field trial tiger shrimp increase survival Rengpipat et al., 1998
Vibrio harveyi from 16 to 33%
increase survival
from 30 to 100%
rainbow trout were immersion infected with V. anguillarum and the fish divided into
16 tanks each with 30 fish (Slierendrecht and Gram, 2001, unpublished data). Eight
tanks were treated with AH2 by adding the probiont to the water on a daily basis.
The accumulated mortality in control tanks varied from 64 to 89% whereas the
accumulated mortality in treated tanks varied from 28 to 63% with an outlier of 84%
(fig. 3). The importance of knowing the inherent variation in a particular infection
trial system and of using an appropriate number of replicates cannot be under-
estimated. With a large variation, a too low number of replicates may mask
Fig. 3. Accumulated mortality of rainbow trout following immersion infection with Vibrio anguillarum.
Approximately 480 fish were infected; half of which were also submerged in Pseudomonas fluorescens strain
AH2 and subsequently treated with AH2 on a daily basis. Fish were divided into 16 tanks: eight served as
controls and eight were treated with AH2 (Slierendrecht and Gram, 2001; unpublished data).
Fig. 4. Accumulated mortality of Atlantic salmon following co-habitant infection with Aeromonas
salmonicida. Six tanks were infected with furunculosis and three tanks were treated with Pseudomonas
fluorescens AH2 three times per day (see also Gram et al., 2001).
a significant effect of a treatment. Conversely, single determinations may, by chance,

be different and appear to show an effect, yet may be only reflecting the normal
window of variation in the experiment.
By contrast, if the variation is smaller, a lower number of replicates is required.
This is depicted in fig. 4 where the accumulated mortality of Atlantic salmon
co-habitant infected with furunculosis, varies from 66 to 76% in the control and
from 66 to 78% in the tanks treated with AH2 (Gram et al., 2001).
5.1. Disease prevention in Artemia

Despite many attempts, it has not been possible to develop commercial, non-live feed
for fish larvae. Thus in a very sensitive stage of the life cycle, fish larvae depend on
live feed, typically small shrimp, Artemia, and rotifers. As these cultures also expe-
rience microbial disease, probiotic disease prevention has been studied for the live
feed. Rico-Mora et al. (1998) showed that the number of the pathogenic Vibrio
alginolyticus in a diatom culture could be reduced by the addition of an Aeromonas
species. Later, Verschuere et al. (2000b) tested nine bacterial strains isolated from
well-performing Artemia cultures for their in vivo effect on Artemia survival follow-
ing Vibrio proteolyticus infection. All Artemia in infected cultures to which no other
bacterial strains were added, died within 2 days. Five of the nine bacterial strains had
a probiotic effect as their addition resulted in survival (90%) comparable to the non-
infected Artemia. These data were derived 48 h following infection. Two strains were
also tested over a 4 day period, and one strain (belonging to the Vibrionaceae) also
increased survival over this longer period. This strain had the highest colonization
potential (measured as CFU/Artemia) and caused a slower growth of the pathogen
V. proteolyticus, resulting in a lower cell density of V. proteolyticus in the culture

water. Interestingly, this Vibrio strain was also tested as a food source for rotifers
where it caused a significant reduction in growth rate of the rotifer (Rombaut et al.,
1999). Addition of filter-sterilized supernatant from the probiotic Vibrio strain did not
increase survival of Artemia and none of the nine strains showed in vitro activity
against the pathogen Vibrio alginolyticus when tested by Dopazo’s double layer tech-
nique. The addition of potential probiotic microbial cultures to Artemia or rotifer
cultures has also been studied as a way of introducing the probionts to the larval
rearing systems (Gatesoupe, 1994). Introducing a LAB culture to rotifers (adding
outgrown culture to the rotifer medium) increased the counts of LAB in the larvae a
100-fold from 10 2 to 10 4 per larvae (Gatesoupe, 1994).
5.2. Fish and crustacean larvae

For a range of fish species, in particular marine species, the main obstacle for cultiva-
tion is a very high mortality in the larval stage. In these marine larvae, the pathogenic
organisms have not been unequivocally identified but trials have shown that culturing
turbot larvae under bacteria free conditions or at very low bacterial densities enhances
survival dramatically (Munro et al., 1993, 1995), thus indicating the importance of
bacteria for disease development. Also, exposure to V. anguillarum increases mortal-
ity of turbot larvae (Munro et al., 1995). The live food (rotifers, Artemia) can be a
source of pathogenic bacteria (Makridis et al., 2000a). These bacteria may be removed
by disinfection regimes, however, re-colonization with opportunistic, potentially patho-
genic bacteria may occur rapidly (Skjermo and Vadstein, 1999). Therefore, it has been
suggested that the live food, following disinfection, may be exposed to bacteria bene-
ficial to larvae which are then allowed to colonize the rotifers or Artemia and
subsequently transferred to the fish larvae (Makridis et al., 2000a).
Gatesoupe (1994) added LAB originally isolated from the non-dominant flora of
rotifers (Brachionus plicatilis) to rotifers which were used as feed for turbot larvae
that subsequently were infected with a Vibrio in three separate experiments. In one
of the experiments, the author reported a marginal effect on survival, whereas in the
two other trials addition of LAB increased larval survival from 13 to 28% and from
8 to 50%, respectively. Unfortunately, the study does not give mortality kinetics but
only survival percentages at 48 and 72 h after infection. LAB addition did not alter
the number of vibrios in the tank water. In a recent study, Huys et al. (2001)
searched for beneficial bacterial strains for turbot larviculture and found increased
survival of larvae by addition of a Vibrio mediterranei Q40 originally isolated from
sea bream larvae and an unknown organism isolated from turbot larvae at a concen-
tration of 10 5 bacteria per ml water.
Several studies from Tromsø, North Norway, have focused on the possible use of
LAB, in particular carnobacteria, as oral probiotics for marine fish larvae and fry.
The accumulated mortality of Atlantic salmon fry fed a diet with C. divergens and
challenged with Aeromonas salmonicida reached 65% in 3 weeks whereas the con-
trol group was at approximately 40% after the same time period (Gildberg et al.,
1995). The LAB used in this study showed no in vitro antagonism, neither as sterile
filtered supernatant nor in co-inoculation trials where equivalent numbers were used
as inoculum. In two studies, the feed for Atlantic cod larvae was supplemented
either with freeze dried C. divergens-like (Gildberg et al., 1997) or with a live
C. divergens-like culture (Gildberg and Mikkelsen, 1998). Upon challenge with
V. anguillarum, the mortality was lower in the group fed LAB-containing feed than
in the group fed the same diet with no supplementation of LAB (Gildberg et al.,
1997). However, a control group fed a third (non-LAB containing) diet, had the
lowest accumulated mortality of all. Following infection with V. anguillarum,
all fish reached 80% accumulated mortality after 3 weeks. In in vitro co-culture,
V. anguillarum was not inhibited by C. divergens. However, the cultures were inocu-
lated at similar cell densities (10 6 CFU/ml), so no inhibition is to be expected.
Bacillus strains have been used successfully as probiotics in shrimp culturing
(Moriarty, 1998) and some data exist on their potential use in larval rearing. Addition of
a Bacillus spp. combined with a slight decrease in salinity, resulted in excellent survival
(80 –100%) of larvae of common snook in commercial systems (Kennedy et al., 1998).
In two studies, Nogami and Maeda (1992) and Nogami et al. (1997) added 10 5–
6
10 CFU/ml of bacterial culture isolated from shrimp pond to seawater used for crab
(Portunus trituberculatus) culture. The strain was a Gram-negative, non-fermentative,
motile rod identified as Thalassobacter utilis (Maeda and Liao, 1992; Nogami et al.,
1997). The culture, which was added once every 7 days, was selected based on its
ability to improve survival in in vivo infection trials. The organism also inhibited
growth of V. anguillarum, in vitro. By adding the culture, a decline in concentration of
Vibrio spp. in the seawater occurred and survival was significantly improved (table 7).
It should be emphasized that the addition of five other microbial cultures (e.g.
a Bacillus subtilis) accelerated mortality of the larvae (Nogami and Maeda, 1992).
Gatesoupe (1997) tested a siderophore-producing vibrio as probiotic by feeding
infected turbot larvae with rotifers enriched by the vibrio. This improved survival
when measured 48 h after infection. However, after 10 days, no difference was seen
in survival. In contrast, Ringø and Vadstein (1998) found that addition of Vibrio
pelagius to early developing turbot (Scophthalmus maximus) larvae had no short-
term effect but caused a slight increase in survival after 12–16 days. V. pelagius was
taken up by the larvae if added to the tank water at the day of hatching (Ringø et al.,
1996). It is, however, not known whether the bacterium had colonized the gut and
would persist if the larvae were removed from the Vibrio-containing tank water.
5.3. Scallop and oyster larvae

The bivalve molluscs (e.g. oysters) are of increasing interest in the aquaculture
sector. These invertebrate organisms do not possess acquired immunity (Roch, 1999)
Table 7. Survival and production of a swimming crab (Protunus trituberculatus) in five

consecutive years when comparing untreated systems to systems treated with the bacterium
Thalassobacter utilis. The bacterium was added at a density of 10 5 –10 6 per ml once every
6 to 8 days (modified from Nogami et al., 1997)
Avg. survival Final No. of

Addition of No. of Larval no. rate (%) production production
Year biocontrol experiments at start to 1st crab stage (ind/m3) failures
1989 − 10 46 960 000 22.0 5158 0

+ 4 20 300 000 30.4 7703 0
1990 − 9 42 930 000 6.8 1617 4

+ 7 30 570 000 26.7 5838 0
1991 − 7 34 150 000 10.4 2543 4

+ 7 34 790 000 27.9 6938 0
1992 − 8 35 710 000 17.8 3963 3

+ 9 37 410 000 28.8 5994 1
1993 − 8 33 200 000 21.6 4474 1

+ 6 26 610 000 27.7 6150 0
Total − 42 192 950 000 15.7 3605 12

+ 33 149 680 000 28.2 6397 1
and disease prevention by vaccination is therefore difficult. The animals do possess

an innate immunity and are capable of specific humoral and cellular defence reac-
tions (Roch, 1999).
Riquelme et al. (1997) investigated 506 bacterial strains for their potential
probiotic effect in Chilean scallop (Argopecten purpuratus Lamarck 1819) larval
culture. Initially, both a Pseudomonas isolate showing in vitro activity against a
V. anguillarum-related strain and an unidentified isolate with no in vitro inhibitory
activity were found to improve survival from 5% in the non-probiotic treated to
60% in the probiotic treated over a 14 day period. Screening the 506 bacterial
isolates, the authors found that only 2.2% of them were able to inhibit growth of a
V. anguillarum related bacterium associated with mortality of scallop larvae.
However, several strains showing in vitro activity increased mortality of scallop
larvae. Thus, this study demonstrates the importance of in vivo testing, as strains
with in vitro effect may be dangerous to the animals – and strains with no in vitro
effect may have probiotic effects in vivo.
In a recent study, Riquelme et al. (2001) reported that growth and survival in field
trials with scallop larvae treated with pathogen-antagonizing bacteria at 10 3 CFU/ml
were the same as when the larvae were treated with antibiotics. The antagonizing
bacteria were added to the water at the initiation of the experiment and again after
48 h. Controls with no treatment were not included as the commercial producer
experienced rapid mortality when no treatment was used. Earlier, Riquelme et al.
(2000) studied the uptake of pathogen-inhibiting bacterial cultures in Chilean scallop
larvae, and found that an Arthrobacter was ingested in significant numbers. This can be
a way of continuously adding the probiotic culture to the scallop larvae. The
Arthrobacter strain was not tested in in vivo infection trials.
Bacterial probiotics have also been tested in the culturing of Pacific oyster
(Crassostrea gigas) larvae. Gibson et al. (1998) added an Aeromonas media strain, iso-
lated from Koi carp (Cyprinus carpio) from the Hawksbury River (Gibson, personal
communication, 2001), to waters of oyster larvae which had been challenged with
V. tubiashii. The challenge caused an increase in numbers of the pathogen and a com-
plete kill of the oyster population in 5 days but addition of the A. media strain together
with the V. tubiashii, reduced numbers of the pathogen and resulted in complete sur-
vival of the population. These results are in accordance with earlier results demonstrat-
ing that additions of both algae (McCausland et al., 1999) and bacteria (Douillet and
Langdon, 1994) have been found to improve growth of Pacific oyster larvae.
5.4. Grown fish

Prevention of bacterial disease in grown fish is somewhat easier than in larvae and
juveniles. For a range of bacterial pathogen–host combinations, good vaccines have
been developed, and their optimization and use will be facilitated as further under-
standing of the pathogen virulence factors and of the host immune system emerges.
Some of the studies described below should therefore rather be regarded as trials of
the probiotic concept than as suggestions for actual use of probiotics. Several Gram-
positive and Gram-negative bacteria have been tested and experiments cover both
additions to the rearing water and feed supplementation.
Austin et al. (1992) found that feeding Atlantic salmon with a diet containing
1% of the alga Tetraselmis suecica resulted in significant improvement of survival rate
when the fish were subsequently challenged with a range of Gram-negative pathogens
such as A. salmonicida, A. hydrophila, V. anguillarum, V. ordalii, Y. ruckerii and
S. liquefaciens. The algae produced an antibacterial compound as tested by agar-
diffusion assays and the protective effect displayed a dose–response relationship with
improvement in survivals seen with 0.25 to 1% in the feed.
Incorporation of a killed (by exposure to air and lyophilization) preparation of
Clostridium butyricum into feed of rainbow trout reduced susceptibility to Vibrio
infections (Sakai et al., 1995). Whilst this does not concur with the definition in this
chapter of probiotics being a “live microbial supplement” it contributes to the under-
standing of the potential applications of probiotics.
In order to improve water quality of channel catfish ponds, a commercial bacte-
rial inoculum (BioStart®) based on Bacillus spp. was added (at 10 3–10 4 CFU/ml) to
pond water three times per week for 5 months (Queiroz and Boyd, 1998). This did
not change water quality parameters but resulted in significant improvement of
survival from 56 to 80%. Although the fish were smaller in the treated ponds, over-
all production was 30% higher here.
A Carnobacterium inhibens strain, originally isolated from Atlantic salmon
(Jöborn et al., 1999) which has a strong in vitro antagonism against two fish pathogens
(V. anguillarum and A. salmonicida) (Jöborn et al., 1997), was incorporated into fish
feed at levels of 1 × 10 6–5 × 107 CFU/g and fed to Atlantic salmon and trout (15–20 g
sizes) for 7–28 days. Subsequent co-habitant challenge with several Gram-negative
bacteria, showed higher percentage survival in groups fed 14 days or more with the
probiont (Robertson et al., 2000). The study – like most studies – unfortunately has
not recorded the kinetics and replicates. Also, variances are not presented. It is not pos-
sible, as is the case with most probiotic studies, to evaluate if differences are statisti-
cally significant. The effect of a Lactobacillus rhamnosus strain on mortality of
furunculosis co-habitant infected rainbow trout was studied (Nikoskelainen et al.,
2001b). The lactobacilli (LAB) were incorporated in the fish feed and a level of
109 LAB per gram feed reduced accumulated mortality from 50 to 20%. However,
incorporation of 10 12 LAB resulted in an accumulated mortality of 45%. The infection
trial was only done with one tank per treatment and thus, the variation of survival/
mortality curves is not known. Single determinations may be far from sufficient,
bearing in mind the variation in mortality that sometimes is seen (fig. 3).
Bathing of salmon for 10 min in suspensions containing Vibrio alginolyticus
originally isolated from a shrimp hatchery in Ecuador, resulted in establishment of
the culture as it was isolated 21 days after the immersion (Austin et al., 1995).
Improved survival of the fish following infection with A. salmonicida was noted.
Some effect on survival from Vibrio infections was also seen, whereas the probiotic
treatment had no effect on infection with Yersinia ruckerii.
Smith and Davey (1993) reported that bathing of Atlantic salmon pre smolts in
5 × 105 CFU/ml fluorescent pseudomonad suspension, reduced subsequent disease
from stress-inducible furunculosis. Thus, between 0 and 10% of pseudomonad
treated fish became infected whereas 30–72% of the non-treated fish were infected.
In contrast to this, Gram et al. (2001) did not find improved survival in Atlantic
salmon co-habitants infected with furunculosis and treated with Pseudomonas
fluorescens strain AH2. Levels of AH2 varied from 10 to 10 5 CFU/ml. In other
experiments, this strain caused a reduction in accumulated mortality of rainbow
trout bath infected with V. anguillarum (Gram et al., 1999; Spanggaard et al., 2001).
When rainbow trout were infected with V. anguillarum, the organism proliferated to
high numbers on the skin surface before significant growth was detected on the gills
and in the intestinal tract. The beneficial action of pseudomonads added to the rear-
ing water could be explained by their direct action on the outer skin surface of the
fish where V. anguillarum proliferated (Spanggaard et al., 2000b). Studies have not
been performed determining the proliferation and infection sites of A. salmonicida
in co-habitant infected salmon, but if this occurs in areas (e.g. the gut) where the
pseudomonads are not favoured, this could explain the different outcomes.
Whilst most studies have aimed the search for probiotic candidates at the bacterial
population, it has been demonstrated that pathogen-specific bacteriophages can inhibit
the pathogen in vitro and reduce subsequent fish mortality in vivo (Park et al., 2000).
Phage treatment of ayu (Plecoglossus altivelis) infected with Pseudomonas
pleccoglossicida resulted in reduction of accumulated mortality from 60–70% to
20–25% in 10 g fish and from 73–80% to 0–13% in 2.4 g fish (Park et al., 2000).
5.5. Shrimps
Shrimp and other crustaceans are important aquaculture animals. The immune
defence system of shrimp is poorly developed and, in spite of a primitive immune
system relying on, e.g. phagocytosis and lysis activity of the haemocytes (cited from
Scholz et al., 1999), vaccines are not likely to be successful. Therefore, several stud-
ies have been dedicated to alternative disease control measures in crustacean culture.
Scholz et al. (1999) reported that addition of yeasts (Saccharomyces cerevisia
and Phaffia rhodozyme) to shrimp feed improved survival from 60 to 80% over a
7 week period. Interestingly, phenoloxydase activity, which is an indicator of bacte-
rial clearance ability, was lowest in the animals treated with the Phaffia. In contrast,
phenoloxydase and other immunity indicators in tiger shrimp (Penaeus monodon)
showed an increase due to inclusion of a Bacillus strain S11 in the feed. This addi-
tion also caused an increase in survival as compared to a control feed when shrimp
were infected with Vibrio haryvei (Rengpipat et al., 1998, 2000). The supplement of
Bacillus S11 into the feed also increased survival in unchallenged shrimp
(Rengpipat et al., 1998) where average survival after 100 days was 33% in the
Bacillus treated tanks as opposed to 16% in the control systems (fig. 5).
Fig. 5. Survival of shrimp (Penaus monodon) fed a diet with () and without (■) a Bacillus S11 diet.
Feed contained 1010 CFU per gram of S11 and the shrimp were fed three times per day (modified from
Rengpipat et al., 1998).
Bacillus S11 was also used to suppress levels of luminescent vibrios in shrimp
ponds. Vibrio levels reached almost 107 CFU/ml in untreated ponds but remained
at 10 2–10 4 CFU/ml in ponds treated with Bacillus S11 (Rengpipat et al., 1998).
Moriarty (1998) similarly found that a Bacillus-based probiotic addition lowered
the level of luminescent vibrios. In untreated ponds, the level varied from 44 to
281 CFU/ml, whereas the level in treated ponds varied from 0 to 75 CFU/ml.
6. USE OF MICROBIAL CULTURES AS FEED

Microorganisms are an important part of the diet of some fish food organisms and
fish (Moriarty, 1997; Thompson et al., 1999). Therefore, trials have been carried out
to evaluate the growth rate of both fish larvae and rotifers following addition of
microorganisms or microbial products to rearing water. Results from such studies
vary and are typically based on “trial and error”, and with few attempts to under-
stand the microbial ecology and mechanisms of the system.
6.1. Microorganisms as feed for larval feed

Douillet (2000a,b) found that addition of bacteria significantly enhanced the growth
rate of rotifers (Brachionus plicatilis) grown in synxenic culture. A range of Gram-
positive and Gram-negative bacterial cultures were tested, both laboratory isolates
and commercial preparations. None of the commercial preparations showed consis-
tent positive effects, but particularly a laboratory isolate of Alteromonas spp. repeat-
edly increased growth rate. Rombaut et al. (1999) and Gatesoupe (1991) also found
that a range of bacterial isolates had positive effects on growth rates of rotifers.
Makridis et al. (2000a) did not evaluate growth rates, but showed that both
rotifers (B. plicatilis) and Artemia franciscana grazed on bacteria added to culture
water, and that a significant proportion of their bacterial flora consisted of the intro-
duced bacteria. This “bioencapsulation” may be a way to introduce probiotic bacte-
rial cultures to fish larvae.
6.2. Microorganisms as larval feed

Addition of high numbers (10 7 CFU/ml) of a bacterial culture (CA2) to Pacific
oyster larvae (Crassostrea gigas Thunberg) caused a depression of both survival and
growth, whereas addition of lower bacterial numbers (10 4–10 6 CFU/ml) increased
growth rate of the larvae (Douillet and Langdon, 1994). Also, the addition of various
algal preparations enhanced growth of oyster larvae (McCausland et al., 1999).
McIntosh et al. (2000) added BioStart HB-1 or HB-2 with different Bacillus species to
tank water of juvenile saltwater shrimp (Litopenaeus vannamei), and saw no effect on
weight gain, survival or water quality parameters. Unfortunately, no bacteriological
data are included and the concentration and persistence of the added bacteria cannot
be evaluated. Similarly, no weight gain was found for sea bass when placed in recircu-
lating water with a bacterial cell density of 10 4 to 10 5 CFU/ml (Leonard et al., 2000).
6.3. Bacteria as fish feed

Several reports indicate that addition of bacteria or microalgae to fish feed can cause
an increase in weight gain (McCausland et al., 1999). Ramesh et al. (1999) added
plant carbohydrates to fingerlings of carp (Cyprinus carpio) and rohu (Labeo rohita).
This caused a doubling of bacterial numbers in the water and significant biofilm
growth on the carbohydrate particles. The increased weight gain of the carp being
20–50% higher than the control, was attributed to this increase in microbial biomass.
Also, the addition of Enterococcus faecium (strain M74) to fish feed at a level of
5 × 10 5 CFU/g caused an increase in weight gain of carp (Cyprinus carpio) fry
(Bogut et al., 1998). Incorporation of a Lactobacillus spp. in flounder (Paralichthys
olivaceus) feed at a level of 5 × 10 3 CFU/g caused an increase in the intestinal counts
of Lactobacillus spp. and an increase in weight gain of the fishes (Byun et al., 1997).
McCausland et al. (1999) found that the growth of juvenile Pacific oysters
(Crassostrea gigas) increased significantly if fed a diet containing live microalgae
but the addition had no effect on water quality parameters and disease survival.
7. USE OF MICROBIAL CULTURES AS WATER TREATMENT

A range of commercial bacterial products are used to control water quality in aqua-
culture (Moriarty, 1997). This is due to the ability of the bacteria to participate in the
turnover of organic nutrients in the ponds. However, there are few scientifically docu-
mented cases in which bacteria have assisted in bioaugmentation, with the notable
exception of manipulating the NH3/NO2/NO3 balance (Verschuere et al., 2000a) in
which nitrifying bacteria are used to remove toxic NH3 (and NO2).
Fish expel nitrogen waste as NH3 or NH4+ resulting in rapid build up of ammonia
compounds which are highly toxic to fish (Hagopian and Riley, 1998). Nitrate, in con-
trast, is significantly less toxic being tolerated in concentrations of several thousand mg
per litre. Several bacteria, e.g. Nitrosomonas, convert ammonia to nitrite and other bac-
teria, e.g. Nitrobacter, further mineralize nitrite to nitrate. Nitrifying bacteria excrete
polymers (Hagopian and Riley, 1998), allowing them to associate with surfaces and
form biofilms. Recirculating systems must employ biofilters to remove ammonia, and
Skjølstrup et al. (1998) demonstrated a 50% reduction in both ammonia and nitrite in
an experimental fluidized biofilter in a rainbow trout recirculating unit.
8. PREBIOTICS
The addition of high doses of probiotic strains to established microbial communi-
ties of fish can provoke a temporary change in the composition of the microbial
community as described above. However, within a few days after administration had
stopped, the added strains disappeared from the system (Jöborn et al., 1997; Ringø
and Gatesoupe, 1998). Prebiotics are a way to increase the population level of
already colonized beneficial bacteria with antagonistic ability (Roberfroid, 1993,
1998, 2001). Prebiotics have been defined with reference to the intestinal tract. They
are “a non-digestible food ingredient beneficially affecting the host by selectively
stimulating the growth and/or activity of one or a limited number of bacteria in the
colon”. In mammalian systems focus has been on the non-digestible oligosaccha-
rides, in particular inulin-type fructans. Inulins occur naturally in several vegetables
and greens, such as garlic, chicory, asparagus and grass (Roberfroid, 1993; Van Loo
et al., 1995). Several LAB are able to ferment inulin and other fructans – thus
Lactobacillus plantarum strains isolated from Thai fermented fish products ferment
inulin (Paludan-Müller et al., 1999). Some of the Carnobacterium strains isolated
from fish intestinal tract are also capable of fermenting inulin (Ringø et al., 1998;
Ringø and Olsen, 1999). It is also known that dietary inulin resulted in damage to
intestinal enterocytes of the salmonid fish Arctic charr (Salvelinus alpinus L.)
(Olsen et al., 2001), and that dietary inulin alters the adherent gut microbiota of
Arctic charr (Ringø, Myklebust, Mayhew and Olsen, unpublished results). However,
the effects of dietary inulin on fish welfare are not yet known.
Another aspect related to fish health is modulation of immune response and
barrier function by dietary means (Sakai, 1999). It is well known from endothermic
animals that any method leading to an improved feed intake in the first days after
weaning, could reduce inflammatory symptoms, and improve both intestinal mor-
phology and resistance against pathogens (Bosi, 2000). However, knowledge about
dietary tools to modulate the immune response and barrier function in the fish is
incompletely known, and this is a topic for further study. Even at a very young stage
some immunostimulation may occur, as Vadstein et al. (1993) showed that feeding
halibut larvae with alginate rich in mannuronic acid polymer enhanced viability
during 4 weeks from 10 to 55%. Such effects may be explained by increase in, e.g.,
complement activation as was found for sea bass (Dicentrarchus labrax) which were
fed a glucan and ascorbic acid containing diet (Bagni et al., 2000).
Even though several studies have shown that the probiotic concept has potential in
the aquaculture sector, much work is still needed. Some of the most promising data
stem from field trials where addition of probiotics to the water on a routine basis
increased survival of fish or crustaceans (Nogami et al., 1997; Moriarty, 1998;
Queiroz and Boyd, 1998; Rengpipat et al., 1998). Several aspects, however, need to
be dealt with, in order to develop this concept further. Owing to the relatively high
numbers of bacteria that must be added, fish probiotics currently should be developed
for hatcheries, larval rearing units and recirculating systems.
It is crucial that the mechanisms involved in the in vivo probiotic effect be deter-
mined (Berg, 1998; Atlas, 1999). Some go as far as stating that “without specific
cause and effect relationships that can be substantiated scientifically, the use of
probiotics remains controversial and should not be endorsed by the scientific com-
munity” (Atlas, 1999). Even with a slightly less rigorous attitude (e.g. acknowledg-
ing the data from several field trials), understanding mechanisms is a requirement
for any long-term commercial use as this is needed to determine any possible side
effects on the environment, e.g. will the addition of probiotics alter the microbial
community on a permanent scale and will this subsequently affect turnover of
organic and inorganic compounds in the particular environment? Thus, the anti-
microbial effect of some Bacillus and Pseudomonas species is caused by production
of antibiotics (Marahiel et al., 1993; Duffy and Défago, 1999; Msadek, 1999) and
this is obviously not a viable path in an attempt to find non-antibiotic substitutes for
disease control. An understanding of the in vivo mechanism(s) would also allow for
a much more efficient and intelligent selection of potential probionts. At present, not
a single study has seriously compared in vitro and in vivo antagonism. Therefore,
it is not known if the screening of thousands of isolates for antagonistic activity in
in vitro assay has any importance for their in vivo effect. Determining mechanisms
of activity is not an easy task, however, some options exist. Comparing phenotypic
characteristics and disease suppressing abilities (against phytopathogenic fungi) of
fluorescent pseudomonads has shown that for some strains, production of cyanide is
important (Ellis et al., 2000). Mutant strains, e.g. constructed by random transposon
mutagenesis, could allow for identification of clones with no disease preventive
effect. Subsequent cloning and sequencing of the genes affected by the knockout
could help clarify mechanisms. It has been hypothesized that iron chelation is
important for the antagonism of pseudomonads in the rhizosphere and this hypoth-
esis has been tested by comparing the in vivo disease suppressing effects of a wild
type strain and siderophore negative mutants (Buysens et al., 1996).
A particular aspect concerns the testing of probiotic cultures. The use of field
trials under real conditions is obviously the ultimate test. However, an intermediate
step in terms of infection model systems using live hosts, is often needed. Owing to
the very high inherent (biological) variation in such systems, the model infection
studies should be carried out with a sufficient number of replicates to allow for
proper statistical treatment. As discussed in Section 5, analyses normally used to
describe and compare survival data must be used. Even with more appropriate
statistical analysis, the development of the probiotic principle would benefit greatly
from more stable infection models.
It must also be recognized that a particular probiont which may work in one sys-
tem (Gram et al., 1999; Spanggaard et al., 2001) may be completely ineffective in
another host–pathogen system (Gram et al., 2001). Therefore, more detailed knowl-
edge of the pathogenic agents, their virulence factors and their interaction with the
host would be of great importance.
Different approaches have been used for introducing the probiont to the system.
The organism may be live or in a freeze dried state. It can be added directly to the
water or incorporated in the feed; either pelleted or live feed. Nothing is known
about how each of these treatments affects the viability of the organisms or the
probiotic effect. Knowledge of proliferation and invasion sites of the pathogen
would assist in determining whether a water borne or food borne vehicle is the most
appropriate. Such understanding is required for further technological developments.
Several studies have found that a single treatment with probiotic culture is not
enough and that the organism(s) must be added on a more continuous basis (Nogami
et al., 1997; Moriarty, 1998; Gram et al., 1999), however, the robustness of the
systems, e.g. required concentration of probiont, required frequency of addition,
effects of changing temperature, has not been documented.
Finally, legal matters must be resolved. Is probiotic treatment classified as a
medical issue (treating animals) or an environmental issue (treating water) and in
either case, who is responsible for control? Also, no cost–benefit analysis has yet
been carried out. Whilst the application of probiotic technology is likely to increase
costs per se, it must be emphasized that if used succesfully (e.g. table 7), there may
be tremendous benefits due to a more stable and therefore higher production. Also,
as some uses of antibiotics may be prohibited, use of probiotics may gain wider
interest.
In conclusion, there are several promising developments for fish probiotics,
however, this will certainly not become the cure for all maladies.
ACKNOWLEDGEMENTS
Critical reviewing and valuable comments from Dr Harry Birkbeck, University of Glasgow, Dr Jorunn
Skjermo, SINTEF, and Mette Hjelm, Danish Institute for Fisheries Research are appreciated.
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18 Antimicrobial activity of lactic acid
bacteria isolated from aquatic
animals and the use of lactic acid
bacteria in aquaculture1
E. Ringøa, U. Schillingerb and W. Holzapfelb

aSection of Arctic Veterinary Medicine, Department of Food Safety and Infection
bInstitute of Hygiene and Toxicology BFE, D−76131 Karlsruhe, Germany
This review presents updated knowledge on the antagonistic ability of lactic acid
bacteria (LAB) isolated from aquatic animals. Numerous strains of LAB including
the genera Lactobacillus, Carnobacterium, Aerococcus-like, Lactococcus and
Streptococcus, are capable of producing antibacterial substances against different
potential fish-pathogenic bacteria such as Aeromonas salmonicida subsp. salmonicida,
Aeromonas hydrophila, Aeromonas caviae, Flavobacterium psychrophilium,
Photobacterium damselae subsp. piscicida, Vibrio salmonicida, Vibrio anguillarum,
Vibrio splendidus and other Vibrio species, Carnobacterium piscicola, Streptococcus
milleri, several strains of Listeria including Listeria monocytogenes, several strains
of Enterobacteriaceae and Clostridium tyrobytyricum. In addition, many LAB have
the ability to inhibit growth of closely related bacteria including strains of carnobac-
teria, lactobacilli, lactococci, leuconostoc and pediococci. This review will focus on
the antagonistic ability of LAB, the effect of LAB administration on intestinal micro-
biota, in vivo challenge tests and the use of commercial preparations of live LAB in
aquaculture.
1. INTRODUCTION
The aquaculture industry is plagued by many disease problems. Presently, the pre-
vention and control of these diseases have concentrated on good husbandry practices
1Financial support from the Norwegian Research Council (Grant No. 122851/122) is gratefully acknowledged.

Lactic acid bacteria in aquaculture 419
and the use of vaccines and antibiotics. However, treatment or feeding with anti-
biotics may cause the development of resistant bacteria (Aoki et al., 1985; Amàbile-
Cuevas et al., 1995; Towner, 1995). An alternative method of prevention is the use
of probiotics (non-pathogenic bacteria) able to inhibit colonization of and to exert
inhibitory effects against undesired microorganisms, and that support the natural
host microbial defence mechanisms. Six recent reviews have hinted at the impor-
tance of the inhibition of pathogens by antibacterial substances produced by the fish
mucosal microbiota (Ringø and Gatesoupe, 1998; Gatesoupe, 1999; Hansen and
Olafsen, 1999; Ringø and Birkbeck, 1999; Gomez-Gill et al., 2000; Verschuere
et al., 2000; Gram and Ringø, 2005, Chapter 17 in this book). The presence of a non-
pathogenic microbiota with antibacterial ability on mucosal surface is of importance
for protection as skin, lateral line, gills and the gastrointestinal tract (GIT) or a com-
bination of these organs, are suggested to be infection routes of pathogenic bacteria.
From their studies, Tatner et al. (1984), Muroga and De La Cruz (1987), Kanno
et al. (1989, 1990), Magarinos et al. (1995), and Svendsen and Bøgwald (1997) con-
cluded that skin is involved as an infection route, while other investigations (Evelyn,
1984; Hjeltnes et al., 1987; Baudin Laurencin and Germon, 1987; Svendsen et al.,
1999) suggest that gills served as an essential route of penetration. In contrast to
these results, Chart and Munn (1980), Campbell and Buswell (1983), Horne and
Baxendale (1983), Muroga et al. (1987), Chair et al. (1994), Olsson (1995), Grisez
et al. (1996), Olsson et al. (1996), Romalde et al. (1996), Jöborn et al. (1997), and
Lødemel et al. (2001) suggest that the alimentary tract is involved in Vibrio and
Aeromonas infections. Olsson et al. (1992) put forward the hypothesis that the GIT
is a site of colonization of Vibrio anguillarum as the pathogen could utilize diluted
turbot intestinal mucus as the sole nutrient source. In a later study, Garcia et al.
(1997) concluded that Atlantic salmon (Salmo salar L.) intestinal mucus is an excel-
lent growth medium of V. anguillarum. This information reveals an important aspect
of the pathogenesis of this bacterial species.
Numerous autogenic factors generated by some members of the GIT microbiota
are believed to influence the capacity of other microorganisms to associate with
epithelial surfaces. The role of the indigenous microbiota in infection control with
regard to exclusion or reduction of pathogen adhesion has been extensively studied
in humans and other endothermic animals (Fuller, 1997; Naidu et al., 1999), and the
antimicrobial compounds known to be produced in the GIT and to be inhibitory to
microbial cells are lactic acid, short-chain fatty acids (SCFA), H2S and macro-
molecular substances such as bacteriocins. These proteinaceous antimicrobials are
ribosomally produced by certain bacterial strains, and may kill or inhibit sensitive
strains of closely related taxonomic groups. During the past two decades in vivo
experiments have clearly demonstrated inhibitory effects of bacteria associated with
the mucosal surfaces of fish and aquatic organisms against both bacterial pathogens
and closely related species (for review see Gatesoupe, 1999; Gomez-Gill et al.,
2000; Verschuere et al., 2000). Considering the gut microbial ecology, it seems
420 E. Ringø, U. Schillinger and W. Holzapfel
important to take into account the antibacterial effect of the microbiota associated
with the gills, skin and digestive tract, and to enhance the level of beneficial bacte-
ria by antibacterial effects against undesirable bacteria.
Many research laboratories have attempted to improve and modify the microbial
balance by probiotics in the fish digestive tract and larval aquatic organisms, and a
growing number of scientific papers are dealing with this strategy for microbial con-
trol (for review, see Ringø and Gatesoupe, 1998; Gatesoupe, 1999; Ringø and
Birkbeck, 1999; Skjermo and Vadstein, 1999; Gomez-Gill et al., 2000; Verschuere
et al., 2000; Gram and Ringø, 2005, Chapter 17 in this book). In the review by Ringø
and Gatesoupe (1998) devoted to LAB in teleost fish, some information was pre-
sented on antagonistic activity of LAB, a more specific overview is, however,
needed on antagonistic activity, the use of LAB in aquaculture, and the effects of
LAB administration on intestinal microbiota, survival, growth and competition/
interactions in vivo.
The term LAB is used to characterize a broad group of Gram-positive, usually non-
motile, non-sporulating bacteria, which are generally catalase-negative, usually lack
cytochromes, utilize carbohydrates fermentatively and produce lactic acid as a major
or sole product of sugar fermentation. Members of this group are widespread in nature
and comprise both rod-shaped (Carnobacterium, Lactobacillus, Weissella) and coccus
forms (Aerococcus, Enterococcus, Lactococcus, Leuconostoc, Pediococcus and
Streptococcus) as single cells, pairs or chains. Their distribution is typically associated
with habitats containing high concentrations of soluble carbohydrate, protein break-
down products and vitamins. In endothermic animals, lactobacilli are normal inhabi-
tants of the intestinal tract (for review see Juven et al., 1991; Naidu et al., 1999), whilst
the same organisms are seldom isolated from the alimentary tract of fish where
carnobacteria seem to be more common but not numerically dominant (Ringø and
Gatesoupe, 1998). However, during recent years, some attempts have been made to
increase the level of the lactobacilli and carnobacteria GIT population by nutritional
factors such as: 1) chromic oxide (Ringø, 1993b,c), 2) dietary polyunsaturated fatty
acids (Ringø et al., 1998), 3) level of dietary lipid (Ringø and Olsen, 1999), 4) dietary
lipid source (Ringø et al., 2002a) and 5) dietary inulin (Ringø et al., unpublished
results). Furthermore, Ringø and Gatesoupe (1998) discussed that the population
levels of adherent lactobacilli and carnobacteria in the digestive tract are affected by
environmental factors such as handling stress, hierarchy formation and salinity. These
factors described above must certainly be taken into consideration when discussing
antagonism of carnobacteria and other LAB isolated from aquatic animals. Today, it
is well documented in several investigations that LAB are a part of the native micro-
biota of aquatic animals from hatching and onwards (table 1a). Furthermore, several
studies during the past decade have reported on the isolation of LAB from cold-
smoked fish and fermented fish (table 1b).
In their recent review on probiotic bacteria as biological control agents in aqua-
culture, Verschuere et al. (2000) discussed that, as LAB normally account only for
Table 1a. Lactic acid bacteria isolated from aquatic animals
Lactic acid bacteria isolated from
Whole
intestinal Small Large
Source tract Stomach intestine intestine Faeces Gills References
Lactobacillus spp.
Arctic charr A + Ringø, 1993a
A + + Ringø, 1993b
A + + Ringø, 1993c
A + Ringø, 1994
A + + + + Ringø et al., 1998
A + + + Ringø and Strøm, 1994
Atlantic cod + Strøm and Olafsen, 1990
L + Strøm and Ringø, 1993
Atlantic salmon + Westerdahl et al., 1998
A + + Ringø et al., 2000
A + + + Ringø and Bendiksen (upd)
Brown trouta Gonzáles et al., 2000
Herring + Kraus, 1961
+ Olsen et al., 1994
Various fish + Kvasnikov et al., 1977
Lb. plantarum-like
Arctic charr A + + + + Ringø et al., 1998
Saithe + Schrøder et al., 1980
Carnobacterium spp.
Arctic charr A + + + + Ringø et al., 1998
A + Ringø and Olsen, 1999
Rainbow trout + Wallbanks et al., 1990
+ + Jöborn et al., 1997
J intestinal content Spangaard et al., 2000
C. divergens-like Ringø et al., 1997
Arctic charr A + Ringø and Olsen, 1999
A + + Strøm, 1988
Atlantic cod J/A + + Strøm, 1988
Atlantic salmon A + + Gonzáles et al., 2000
Brown trouta
Saithe A + + Strøm, 1988
Wolffish F + Ringø et al., 2001a
C. funditum-like
Arctic charr A + + Ringø et al., 2002a
C. inhibens
Atlantic salmon + Jöborn et al., 1999
C. mobile-like
Arctic charr A + Ringø and Olsen, 1999
C. piscicola-like Ringø et al., 1998
Arctic charr A + + + +
A + Ringø and Olsen, 1999
Atlantic cod A + + Strøm and Ringø (upd)
A + Ringø (upd)
Atlantic salmon A + + Ringø et al., 2000
A + Ringø and Holzapfel, 2000
J + Ringø (upd)
J Strøm and Ringø (upd)
+ + + Gonzáles et al., 2000
Continued
Table 1a. Lactic acid bacteria isolated from aquatic animals—cont’d
Lactic acid bacteria isolated from
Whole
intestinal Small Large
Source tract Stomach intestine intestine Faeces Gills References
Brown trouta
Fisha + Stoffels et al., 1992a
Rainbow trout + Starliper et al., 1992
Saithe A + + Strøm and Ringø (upd)
Turbot J + Ringø (upd)
Aerococcus spp.
Atlantic salmon + Westerdahl et al., 1998
Enterococcus spp.
Common carp + Cai et al., 1999
Enterococcus
faecium
Lactococcus spp.
Lac. garvieae
Lac. lactis
Rotiferb Harzevili et al., 1998
Leuconostoc spp.
Arctic charr A + Ringø and Strøm, 1994
Leu. mesenteroides-like
Arctic charr A + + + Ringø et al., 1998
Pediococcus acidilactici
Streptococcus spp.
Arctic charr A + + Ringø, 1993b
A + + Ringø, 1993c
A + Ringø, 1994
A + + Ringø and Strøm, 1994
A + + + Ringø et al., 1998
A + + Ringø and Olsen, 1999
Atlantic salmon A + + Ringø et al., 2000
Carp + Sugita et al., 1985
Eel, European + Esteve and Garay, 1991
Eel, Japanese intestinal content Sugita et al., 1996
Goldfish + Sugita et al., 1988
Rainbow trout intestinal content Sugita et al., 1996
Various salmonids + + + Trust and Sparrow, 1974
Turbot J + + + Ringø (upd)
Yellowtail + Takemaru and Kusuda,
1988a,b
Vagococcus spp.
aNo further information was given.
bIsolated from whole animal.
A, adult; F, fry; J, juvenile; L, larvae; upd, unpublished data.
Table 1b Lactic acid bacteria isolated from cold-smoked and fermented fish
Sources Lactic acid bacteria References
Carnobacteria
vacuum-packed cold-smoked salmon C. piscicola Leroi et al., 1998
spoiled cold-smoked salmon Carnobacterium spp. Hansen and Huss, 1998
Lactobacilli
chilled, stretch-wrap-packed channel Lb. plantarum Fricourt et al., 1994
catfish
spoiled cold-smoked salmon Lb. curvatus, Hansen and Huss, 1998
Lb. sakei
Lb. plantarum
fermented fish Lb. pentosus, Tanasupawat et al., 1998
low-salt fermented fish products Lb. plantarum
spoiled, vacuum-packaged, Lb. pentosus, Paludan-Müller et al., 1999
cold-smoked rainbow trout Lb. plantarum
Lb. plantarum Lyhs et al., 1999
Jeot-gal, a Korean fermented fish Lb. brevis Lee et al., 2000
food fermented fish Lb. acidipiscis spp. nov Tanasupawat et al., 2000
Lactococci
low-salt fermented fish products Lac. lactis subsp. lactis Paludan-Müller et al., 1999
Jeot-gal, a Korean fermented fish food Lac. lactis Lee et al., 2000
Leuconostoc
spoiled cold-smoked salmon Leuconostoc spp. Hansen and Huss, 1998
low-salt fermented fish products Leu. citreum Paludan-Müller et al., 1999
spoiled, vacuum-packaged, Leu. citreum Lyhs et al., 1999
cold-smoked rainbow trout Leu. mesenteroides
subsp. mesenteroides
Pediococci
low-salt fermented fish products P. pentosaceus Paludan-Müller et al., 1999
a marginal part of the intestinal microbiota of fish, it can be questioned if bacte-

riocins produced by LAB can effectively contribute to the health status of aquatic
animals. However, the main reason for not isolating LAB from aquatic animals
might be that these LAB from fish are generally slow-growing microorganisms, and
Ringø and Gatesoupe (1998) recommended an incubation time of up to 4 weeks at
low temperature (4 and 12°C). In addition, as for the human GIT, it can also be
expected that a number of LAB representatives are not yet culturable by existing
methods.
The ability of LAB to produce antibacterial substances has historically long been
used to preserve foods. Since the days of Metchnikoff, efforts have been made to
improve the normal microbiota of the intestine of endothermic animals by LAB
(Tannock, 1995, 1999; Fuller, 1997; Naidu et al., 1999; Sanders, 1999). During the
past two decades, numerous experiments have evaluated the ability of LAB (lacto-
bacilli, carnobacteria, enterococci, lactococci and streptococci) isolated from sev-
eral fish species and live food (Brachionus plicatilis) to inhibit growth of obligate
pathogenic bacteria, and antagonism seems to be common among LAB. On the
basis of these results, it is suggested that LAB along with other bacteria that belong
to the indigenous microbiota of aquatic animals are an important part of the defence
mechanism against fish pathogens. In addition to the antagonistic microorganisms
colonizing the mucus surface as part of the natural microbial defence mechanisms,
it has been shown that the surface mucus also plays a role in the prevention of
colonization by parasites, bacteria and fungi. Readers with special interest in the
antiviral activity of gastrointestinal mucus and antibacterial activity of epidermal
mucus of fish are referred to the papers of Harrell et al. (1976), Austin and McIntosh
(1988), Fouz et al. (1990), Takashashi et al. (1992), Magarinos et al. (1995), Cain
et al. (1996), Lemaitre et al. (1996), Romalde et al. (1996), Cole et al. (1997),
Robinette et al. (1998) and Ebran et al. (1999, 2000). Moreover, it is generally
accepted that in adult fish local mucosal and secretory immunity is important in pro-
tection against bacterial infections (Trust, 1986; Hart et al., 1987, 1988).
A review of the literature shows that there are several papers that report the anti-
bacterial abilities of LAB isolated from fish and other aquatic animals, their poten-
tial to adhere to the GIT and how LAB administration affects the GIT microbiota.
The purpose of this article is to review the current knowledge on this subject and to
present data from challenge tests in vivo, where antagonistic LAB are included in
the feed and to summarize information on purification and characterization of the
antibacterial compounds responsible for the inhibitory effect.
2. DEFINITION OF ANTIMICROBIAL COMPOUNDS

Antimicrobial substances of eukaryotes and prokaryotes are produced and function
in entirely different modes and settings (Nissen-Meyer and Nes, 1997). However,
apparently diverse biological systems often have many elements in common at a
molecular level.
2.1. Bacteriocins
Many bacteria produce proteinaceous agents that inhibit or kill closely related
species or even different strains of the same species. These agents are called bacte-
riocins to distinguish them from antibiotics, which generally have a wider spectrum
of activity (Madigan et al., 1997) and a different mode of action. Bacteriocins are
peptides or protein complexes with bactericidal activity that are ribosomally syn-
thesized in contrast to antibiotics that are synthesized by different mechanisms. The
structural gene for the bacteriocin and the genes encoding the proteins involved with
processing of the bacteriocin are often carried out by plasmids or transposons.

A general feature of most bacteriocins of LAB is the presence of an own dedicated
immunity protein whose gene is located physically next to the bacteriocin’s struc-
tural gene. Bacteriocins are named in accordance with the species of organism that
produce them. According to the definition of Tagg et al. (1976), the general criteria
characterizing these molecules are a) a narrow activity spectrum, b) the proteina-
ceous nature of the molecule, c) a bactericidal mode of action, and d) receptors on
the cell surface necessary for attachment. Nevertheless, the inhibitory spectrum of
many bacteriocins produced by LAB includes various more distantly related Gram-
positive bacteria such as strains of Listeria monocytogenes, Bacillus cereus and
Staphylococcus aureus (Klaenhammer, 1988; Stevens et al., 1991; Vandenbergh,
1993; Ouwehand, 1998).
2.2. Antibiotics
Antibiotics are chemical substances produced by certain microorganisms that in small
quantities inhibit or kill other microorganisms (Madigan et al., 1997). They constitute
a special class of chemotherapeutic agents, distinguished from growth factor ana-
logues because they are natural products often resulting from secondary microbial
metabolism. They are an important class of pharmaceutical substances produced by
large-scale industrial processes. For instance, many bacilli produce antibiotics, and
production seems to be related to the sporulation process (Madigan et al., 1997).
Furthermore, it is important to remember that antagonism may be mediated not
only by antibiotics, but also by many other inhibitory substances such as organic
acids, hydrogen peroxide (reviewed by Lindgren and Dobrogosz, 1990; Ringø and
Gatesoupe, 1998) and siderophores (Gram and Melichiorsen, 1996).
3. DETERMINATION OF GROWTH INHIBITION

It is generally believed that the intestinal mucosal barrier, including the epithelial
cells, tight junctions controlling paracellular pathways, and a superficial mucous
layer form an effective physical barrier which separates the individual from the
complex microbial populations that constitute the normal intestinal microbiota
(Mims et al., 1995). The factors considered important in mammals in influencing
the gut microflora, such as gastric acid, bile salts, and proteolytic enzymes, will be
present at various stages of development of fish but little is known on their role in
the natural defence system of fish against invading pathogens.
The presence of bacteria associated with mucus capable of inhibiting the growth
of pathogenic bacteria may probably constitute a barrier against colonization and
proliferation by pathogens. Several techniques have been proposed as in vitro antag-
onism tests such as: 1) the disc diffusion method, 2) the diametric-streak technique,
3) the double-layer method, 4) liquid growth medium and 5) the microtitre plate assay.
As the double-layer method and the microtitre plate assay are most frequently used
in the determination of antagonism of LAB, a brief description is presented of these
two techniques.
3.1. Double-layer method

Inhibition of a given microorganism can easily be made visible by using a double-
layer method as described by Dopazo et al. (1988), and during the past decade this
method or its modifications have been used in numerous investigations (Westerdahl
et al., 1991, 1998; Bergh, 1995; Sugita et al., 1996, 1997, 1998; Jöborn et al., 1999;
Ringø, 1999a). Briefly, the method of Dopazo et al. (1988) is as follows: cultures
of the producer strains are spotted on the surface of appropriate agar plates. After
incubation at 20°C for 4 days, the producer cells are killed with chloroform vapour
(15 min) and an overlay containing, e.g. the pathogenic strain is poured on the plate.
After a diffusion period of 15–30 min at 20°C, plates are incubated for 2−4 days at
the optimum temperature for each species. After incubation, a clear zone of inhibi-
tion around the growth of the producer strain indicates antibacterial activity. Readers
with special interest in the modified methods are referred to the investigations
presented above.
3.2. Microtitre plate assay

An alternative to the double-layer method is to pre-culture the test bacteria in a
flask, withdraw samples, centrifuge the cells, filter-sterilize the supernatant and test
possible inhibition of the extracellular extracts on the growth of, e.g. a fish pathogen.
It is recommended to add 50 μl of the sterile supernatant to 150 μl fresh medium in
microtitre wells and inoculate 10 μl of a dilution of the fish pathogen at CFU values
of approximately 106 per ml. The use of 50 μl of the sterile supernatant to 150 μl
fresh medium is recommended as some LAB are strong acid producers, and as 100 μl
of the sterile supernatant to 100 μl fresh medium may give false positive results.
Alternatively, the pH of the culture supernatant may be adjusted to a neutral pH
before using in the microplate assay. In controls, the indicator strain (e.g. the fish
pathogen) is inoculated in 200 μl fresh sterile medium. Growth is estimated as opti-
cal density at 600 nm (OD600) using a microplate reader. To obtain correct results,
triplicates must be used, and e.g strong inhibition is only obtained when complete
inhibition is recovered in all triplicates. As the microtitre plate assay seems to be
more convenient and not as time consuming as the double-layer method several
recent studies have used the microtitre plate assay (Stoffels et al., 1992a,b; Byun
et al., 1997; Jöborn et al., 1997, 1999; Gildberg and Mikkelsen, 1998; Gram et al.,
1999; Ringø et al., 2000; Ringø and Holzapfel, 2000). Moreover, this assay can be
used to quantify the bacteriocin activity by using two-fold dilutions of the super-
natant (Holo et al., 1991).
4. ANTAGONISM OF LACTIC ACID BACTERIA FROM

AQUATIC ANIMALS
The antimicrobial effect of LAB has been the basis of food preservation by fermen-
tation throughout the history of mankind, and the ability of LAB to produce
proteinaceous antagonistic substances as one of the mechanisms of antimicrobial activ-
ity is well documented (for review see Fernandes et al., 1987, 1988; Klaenhammer,
1988; Piard and Desmazeaud, 1992; Parente and Ricciardi, 1994, 1999; Paik and Oh,
1996; Ennahar et al., 2000). Since the late 1920s and early 1930s, when the discovery
of nisin initiated the investigation of proteinaceous antimicrobial compounds from
LAB, a large number of chemically diverse bacteriocins have been identified and
characterized, particularly in recent years (for review see Piard and Desmazeaud, 1992;
Nettles and Barefoot, 1993; Paik and Oh, 1996; Parente and Ricciardi, 1999; Ennahar
et al., 2000).
In addition to bacteriocins, LAB can produce other compounds that inhibit
growth of microorganisms, e.g. H2O2 and organic acids such as lactic acid con-
comitantly lowering pH. According to Piard and Desmazeaud (1992), bacteriocins
of most LAB are active against the lactic acid flora itself. On the other hand, most
studies on antagonistic activity of LAB isolated from fish have concentrated on
the capability of LAB to inhibit undesirable microorganisms (table 2). In fish
studies carried out on antagonistic effects of LAB, the Gram-negative species Vibrio
anguillarum and Aeromonas salmonicida were most frequently used, as these fish
pathogens have caused the heaviest economical losses in the Atlantic salmon (Salmo
salar L.) and turbot (Scophthalmus maximus L.) industry in western Europe.
Currently, the prophylactic and therapeutic control of, e.g. vibriosis, comprises the
feeding of antibiotics, but an alternative treatment could be the administration of
probiotic bacteria that exert inhibitory effects against V. anguillarum, and support
the natural host microbial defence mechanisms. In addition to the fish pathogens as
target organisms, L. monocytogenes is also frequently used.
4.1. Lactobacillus
During the past two decades, five investigations reported on the antibacterial effects
of lactobacilli isolated from the digestive tract of fish. These involved Lactobacillus
plantarum UTC 101-11 isolated from saithe (Gadus virens) (Schrøder et al., 1980),
Lb. plantarum BF001 isolated from chilled stretch-wrap-packaged channel catfish
(Ictalurus punctatus) (Fricourt et al., 1994), Lactobacillus sp. DS-12 isolated from
flounder (Paralichthys olivaceus) (Byun et al., 1997), Lactobacillus-like bacteria
isolated from Atlantic salmon (Salmo salar) (Westerdahl et al., 1998) and two
Lactobacillus isolates (8M851 and 8M852) isolated from Atlantic salmon (Ringø,
unpublished data) (table 2).
In their early study, Schrøder et al. (1980) demonstrated that Lb. plantarum pro-
duced inhibitors against a Vibrio sp. isolated from the gut of saithe, when this strain was
Table 2. Antagonistic activities of lactic acid bacteria isolated from aquatic animals and fish products against different bacteria
Bacterial genera Isolated from Inhibitory effect against References
Lactobacillus-like Atlantic salmon GIT Vibrio anguillarum HI 11360 Westerdahl et al., 1998
Lb. plantarum UTC101 Saithe GIT Vibrio sp. Schrøder et al., 1980
Lb. plantarum BF001 Channel catfish Lactobacillus; Lactococcus; Listeria; Micrococcus; Leuconostoc; Fricourt et al., 1994
Pediococcus; Staphylococcus; Streptococcus; Salmonella;
Pseudomonas
Lactobacillus sp. DS-12 Flounder GIT Aer. hydrophila; Edwardsiella tarda; Pasteurella piscida; Byun et al., 1997
V. anguillarum
Lactobacillus sp. 8M851/8M852 Atlantic salmon Aer. salmonicida AL 2020; V. anguillarum Ringø (unpublished
V. salmonicida data)
Carnobacterium sp. strain K1 Atlantic salmon GIT V. anguillarum HI 11360; Aer. salmonicida ATCC 14174 Olsson, 1995; Jöborn
et al., 1997, 1998
Carnobacterium sp. strain K1 Atlantic salmon GIT Aer. hydrophila; Aer. salmonicida; Flavobacterium psychrophilum Robertson et al., 2000
Atlantic salmon Photobacterium damselae subsp. piscicida; Streptococcus milleri
V. anguillarum; V. ordalii
Carnobacterium spp. Atlantic salmon LI Aer. salmonicida; V. salmonicida Ringø et al., 2001b
C. inhibens Atlantic salmon LI V. anguillarum; Aer. salmonicida Jöborn et al., 1999
C. piscicola Atlantic salmon GIT V. salmonicida Strøm, 1988
C. piscicola UI49 Fisha a 6 strains of carnobacteria; 19 strains of lactobacilli Stoffels et al., 1992a
15 strains of lactococci; 3 strains of pediococci
19 unidentified lactic acid isolates from fish
C. piscicola UI49 Fisha a Lactobacillus lactis LMG 2115 Stoffels et al., 1992b
C. piscicola Trout GIT 8 strains of Listeria including L. monocytogenes; Pilet et al., 1995
2 strains of C. divergens; 1 strain of C. piscicola; 2 strains of lactobacilli;
1 strain of leuconostoc; 1 strain of pediococci; 1 strain of enterococci;
Clostridium tyrobutyricum
C. piscicola V1 Trout GIT L. monocytogenes Duffes et al., 1999
C. piscicola 9F 251 Arctic charr F Aer. salmonicida AL 2020; V. anguillarum; V. salmonicida Ringø (1999a;
C. piscicola CCUG34645 unpublished data)
C. piscicola Fisha L. monocytogenes Duffes et al., 2000
C. piscicola Arctic charr F Aer. salmonicida
C. piscicola Arctic charr G Aer. salmonicida; V. anguillarum; V. salmonicida Ringø, and Holzapfel, 2000
C. piscicola 203 Arctic charr LI Aer. salmonicida AL 2020; Vibrio sp. LFI 5000; Vibrio splendidus Ringø, (unpublished data)
V. anguillarum; V. salmonicida; C. piscicola CCUG34645
C. piscicola 204 Arctic charr LI Aer. salmonicida AL 2020; Vibrio sp. LFI 5000; V. splendidus Ringø, (unpublished data)
V. anguillarum; V. salmonicida
Carnobacterium mobile Arctic charr LI Aer. salmonicida AL 2020; Vibrio sp. LFI 5000 Ringø, 1999a
C. divergens Lab 01 Atlantic salmon GIT Vibrio spp.; V. anguillarum; V. salmonicida; Proteus vulgaris Strøm, 1988
C. divergens-like Atlantic salmon, GIT Vibrio spp.; V. anguillarum; V. salmonicida; Pro. vulgaris Strøm, 1988
saithe, Atlantic cod
C. divergens V41 Trout GIT L. monocytogenes** Duffes et al., 1999
C. divergens Lab 01 Atlantic salmon GIT Aer. salmonicida AL 2020; V. salmonicida Ringø (1999a;
unpublished data)
C. divergens WF2201 Wolffish GIT Aer. salmonicida AL 2020; V. anguillarum; V. salmonicida Ringø (unpublished data)
C. divergens 6251 T Arctic charr SI Aer. salmonicida; V. anguillarum; V. viscosus LFI 5000 Ringø et al., 2002b
C. funditum Arctic charr LI Aer. salmonicida; V. anguillarum; V. salmonicida Ringø et al., 2002a
Carnobacterium-like Atlantic salmon GIT V. anguillarum HI 11360 Westerdahl et al., 1998
Aerococcus-like (isolate K1) Atlantic salmon GIT V. anguillarum HI 11360; Aer. salmonicida; Aer. hydrophila Westerdahl et al., 1998
Lactococcus lactis Brachionus plicatilis V. anguillarum Q19 Harzevili et al., 1998
Streptococcus spp. Japanese eel, IT Aer. caviae ATCC 15468; Aer. eucrenophilia ATCC 23309 Sugita et al., 1996
rainbow trout Aer. jandaei ATCC 49568; Aer. sobria ATCC 43979,
Plesiomonas shigelloides ATCC 14029
aNo further information was given.

* Chilled processed channel catfish; ** vacuum-packed cold smoked.
GIT, gastrointestinal tract; S, stomach; SI, small intestine; LI, large intestine; IT, intestinal contents; F, faeces; G, gills.
grown in the presence of a culture filtrate of Bacillus thuringiensis. However, no

inhibitory effect was observed without the culture filtrate, showing the limitation of
such in vitro antagonism tests. Fricourt et al. (1994) demonstrated that Lb. plantarum
BF001 produced an antimicrobial substance active against selected strains from the
genera Lactobacillus, Lactococcus, Listeria, Micrococcus, Leuconostoc, Pediococcus,
Staphylococcus, Streptococcus, Salmonella and Pseudomonas. The antimicrobial
effect did not result from acidic fermentation products or hydrogen peroxide.
Culture extracts showed a bactericidal mode of action, displayed optimal activity at
pH 3.5, and retained full activity after 30 min at 100°C (pH 3.5). The molecular
weight of the substance was estimated by ultrafiltration to be less than 10 000
daltons. Byun et al. (1997) investigated the antagonistic activity of a Lactobacillus
tentatively identified as Lactobacillus sp. DS-12 against the fish pathogens
Aer. hydrophila, Edwardsiella tarda, Pasteurella piscicida, Pseudomonas
fluorescens, Ps. anguilliseptica, Enterococcus faecalis and V. anguillarum. When the
target organisms were transversely streaked against the lactobacilli grown overnight
in MRS agar, the highest antibacterial activity was found against E. tarda and
V. anguillarum, moderate activity against Aer. hydrophila and P. piscicida, but no
activity against the Pseudomonas and Ent. faecalis strain. However, when the paper
disc assay with neutralized supernatant was used, no antagonistic activities were
observed against any of the target organisms. On the basis of their results, the
authors assumed that the antagonistic activity was not due to bacteriocin production,
but to the organic acids produced. In a recent study, Westerdahl et al. (1998) using
a double-layer method demonstrated antagonistic activity for strains K2 and K7,
characterized as Lactobacillus-like and isolated from Atlantic salmon, against
V. anguillarum HI 11360.
Ringø (unpublished data) tested the antagonistic activity of two Lactobacillus
isolates from stomach of Atlantic salmon, by the microtitre plate assay. The two
strains were slow growing, and both inhibited growth of Aer. salmonicida subsp.
salmonicida, V. anguillarum, V. salmonicida and a pathogenic Carnobacterium
piscicola strain. The antagonistic activity was considered to be not due to acid
production, which was low in both exponential and stationary growth phases.
4.2. Carnobacterium
The genus Carnobacterium was described by Collins et al. (1987) as atypical non-
aciduric lactobacilli unable to grow on acetate agar at pH 5.6. During the past two
decades, a number of authors have reported antibacterial activities of carnobacteria
isolated from the GIT of fish (Strøm, 1988; Stoffels et al., 1992a,b; Olsson, 1995;
Pilet et al., 1995; Jöborn et al., 1997, 1999; Duffes et al., 1999, 2000; Ringø, 1999a;
Ringø et al., 2000, 2001a,b; Robertson et al., 2000; Ringø, unpublished data) and
vacuum-packed cold-smoked salmon (Duffes et al., 2000; Schillinger, unpublished
data) (table 2). In most of these studies, the fish pathogenic strains of Vibrio and
Aeromonas and the pathogen L. monocytogenes have been the most abundantly used
target organisms.
Strøm (1988) was first to report antagonism of intestinal carnobacteria against fish
pathogens. In this study, she proved that the Atlantic salmon bacterium Lb. plantarum
Lab01, later reclassified as Carnobacterium divergens Lab01 (Ringø et al., 2001a),
and eight other strains isolated from the GIT of adult Atlantic salmon, wild-captured
saithe and wild-captured Atlantic cod (Gadus morhua L.) reared in seawater and
characterized as C. divergens-like, inhibited growth of Vibrio spp., V. anguillarum,
V. salmonicida and Proteus vulgaris in in vitro culture experiments. Furthermore, she
reported that two strains of C. piscicola isolated from Atlantic salmon parr fed
a commercial dry pellet, possessed antagonistic activity to V. salmonicida. Later,
Stoffels et al. (1992a) investigated the properties of a bacteriocin-producing
C. piscicola isolated from fish against six strains of carnobacteria, 37 strains of lacto-
bacilli, 21 strains of lactococci, 12 strains of pediococci and 19 unidentified LAB
isolates from fish. Of these 95 strains tested, the six strains of carnobacteria, 19 lacto-
bacilli, 15 lactococci, three pediococci and all 19 unidentified LAB isolates from fish
were sensitive to carnocin UI49.
In two recent studies, Olsson (1995) and Jöborn et al. (1997) demonstrated that
Carnobacterium K1, later identified as Carnobacterium inhibens (Jöborn et al.,
1999), isolated from the gastrointestinal tract of Atlantic salmon, produced non-
characterized inhibitory substances against the two common fish pathogens
V. anguillarum and Aer. salmonicida. The antagonistic activity was demonstrated
in vitro both in mucus and faecal extracts and in laboratory media. Furthermore, the
strain was able to adhere to intestinal mucus, and to survive and proliferate in the gas-
trointestinal tract of fish in vivo, indicating a probiotic potential (Jöborn et al., 1997).
Pilet et al. (1995) showed activity of two bacteriocins produced by C. piscicola
and C. divergens isolated from fish which were active against eight strains of
Listeria including five strains of L. monocytogenes. However, the bacteriocins
piscicocin V1 and divercin V41 differed in their spectra of activity against other
LAB as piscicocin VI inhibited growth of two strains of C. divergens, one strain of
C. piscicola, two strains of lactobacilli, one strain of Leuconostoc and one strain of
pediococci, while divercin V41 only inhibited growth of one strain of C. divergens
and two strains of C. piscicola (table 2). MRS broth was chosen for production stud-
ies because its higher carbohydrate level provided better growth and better bacterio-
cin production than Elliker broth. The inhibitory substances were produced in the
early exponential growth phase and maximum production of the bacteriocins
occurred at the beginning of the stationary growth phase and was also higher when
the producer strain was grown at 20°C than at 30°C (Pilet et al., 1995). In contrast
with piscicocin V1, maximum activity of carnocin UI49 was observed at 34°C with
completely abolished activity at 15°C (Stoffels et al., 1992a).
In view of the finding by Ringø et al. (1998) that dietary fatty acids affect the
population level of LAB associated with the GIT, these intestinal LAB were
screened for the presence of antibacterial activity (Ringø, unpublished data). Of the
153 strains of C. piscicola examined, 121 strains were able to inhibit growth of the
pathogen Aer. salmonicida subsp. salmonicida in an agar diffusion assay, indicating
a high frequency of antagonists. However, it was interesting to note that the ability
of C. piscicola to inhibit Aer. salmonicida subsp. salmonicida strain LFI 4038 was
highest (19 out of 19) in isolates isolated from fish fed 4% dietary α-linolenic acid
(18:3 n-3) and highly unsaturated fatty acids (HUFA) (25 out of 27), compared to
fish fed linoleic acid (18:2 n-6) where only 11 out of 21 inhibited the pathogen.
On the basis of these results it is recommended that greater attention should be given
to the subject of how to increase the level of intestinal carnobacteria with inhibitory
effect against fish pathogens by dietary manipulation. The results obtained from fish
fed dietary 18:3 (n-3) may lead to the conclusion that it is desirable to increase level
of dietary 18:3 (n-3) in commercial diets in order to obtain a higher population level
of intestinal strains of C. piscicola able to inhibit growth of Aer. salmonicida.
However, in this respect it is worthwhile to note that feeding the charr high levels
(>15%) of dietary 18:3 (n-3) increased accumulation of lipid droplets in the entero-
cytes and cell damage which may increase the risk of microbial infections (Olsen
et al., 1999, 2000).
Under conditions of artificial rearing of teleost fish, it is not uncommon that fish
may be subjected to situations of excessive stress. It is well known that teleosts placed
under stress show a set of physiological reactions, and stress (excessive handling or
crowding) is assumed to be one of several factors contributing to the increased
susceptibility to infectious diseases in the fish farming industry (Wedemeyer, 1970;
Snieszko, 1974; Mazeaud et al., 1977; Peters et al., 1988; Espelid, 1991). The possible
involvement of the GIT as a possible route of infections has mostly been overlooked,
although several investigations have suggested the GIT as an infection route of
pathogens (Chart and Munn, 1980; Campbell and Buswell, 1983; Horne and Baxendale,
1983; Muroga et al., 1987; Chair et al., 1994; Olsson, 1995; Grisez et al., 1996; Olsson
et al., 1996; Romalde et al., 1996; Jöborn et al., 1997; Lødemel et al., 2001). It is well
known that chronic stress alters the intestinal microbiota of endothermic animals
(Tannock and Savage, 1974; Tannock, 1983). The same seems to apply to fish (Lesel
and Sechet, 1982; Ringø et al., 1997) although reports are scarce. However, in a
recent work, it was demonstrated that population levels of C. piscicola-like isolates
in the GIT of Atlantic salmon reared in seawater were affected neither by starvation
nor by daily handling stress (Ringø et al., 2000). Out of the 196 C. piscicola isolates,
139 showed the ability to inhibit growth of Aer. salmonicida subsp. salmonicida LFI
4038 (Ringø et al., 2000), while Vibrio viscocus LFI 5000, the causative agent of
winter ulcers, was only inhibited by 21 C. piscicola-like isolates using the double-
layer method (Ringø, unpublished data). As the frequency of C. piscicola-like isolates
able to inhibit Aer. salmonicida subsp. salmonicida LFI 4038 (Ringø et al., 2000) and
V. viscocus LFI 5000 (Ringø, unpublished data) decreased during the experiment
(11 days of regular handling stress), it was suggested that the antagonistic strains are
more loosely associated to the epithelial mucosa of the GIT than other C. piscicola-
like strains. This controversial hypothesis implies that the loss of these beneficial
C. piscicola-like strains may lead to colonization of pathogens if present and if the
stress occurs over several days. This hypothesis is controversial and calls for further
investigations.
In a recent study, Ringø and Holzapfel (2000) evaluated the ability of 26 isolates
of carnobacteria from gills of Atlantic salmon to inhibit growth of Aer. salmonicida,
V. anguillarum and V. salmonicida using the microtitre plate method. Nine strains
characterized as C. piscicola by 16S rDNA and amplified fragment length poly-
morphism (AFLP™), produced an antagonistic compound active against all three
fish pathogens. The nature of the inhibitory compound was not further investigated.
As the gills may be an infection route of pathogenic bacteria, these results illustrate
that carnobacteria associated with the gills may play a role in the disease defence of
fish through contact with infected fish or contaminated water (Evelyn, 1984; Baudin
Laurencin and Germon, 1987; Hjeltnes et al., 1987; Svendsen et al., 1999).
Duffes et al. (2000) recently showed that C. piscicola SF668 isolated from
Norwegian cold-smoked salmon was able to produce bacteriocins with a strong
antilisterial activity and to inhibit L. monocytogenes growth in a simulated cold-
smoked fish system at 4°C.
4.3. Aerococcus
Antagonistic activity of five Aerococcus-like isolates from the GIT of Atlantic
salmon against fish pathogens has been reported in one recent study (Westerdahl
et al., 1998). One of these isolates (strain K1) inhibited growth of V. anguillarum HI
11360, Aer. salmonicida and Aer. hydrophila in liquid media (table 2). The authors
present further information on growth inhibition of V. anguillarum HI 11360 in
liquid media supplemented with 50% sterile supernatant from strain K1. However,
as growth of V. anguillarum HI 11360 depended on the pH of the media, it may be
concluded that the inhibitory effect not only was the result of antagonist production
but may also be related to acid production by strain K1.
4.4. Lactococcus
Lactococcus strains are rarely isolated from aquatic animals. Yet in a recent
study, Harzevili et al. (1998) isolated a Lactococcus lactis strain AR21 from the
rotifer Brachionus plicatilis (Müller) which exhibited an inhibitory effect against
V. anguillarum Q19 (table 2).
4.5. Streptococcus
During the past decade, several authors have reported on strains of the genus
Streptococcus as indigenous to the intestine of fish (for review see Ringø and
Gatesoupe, 1998), but less research has been done on their antibacterial activity.
In a recent study, Sugita and co-authors (1996) isolated 12 strains of Streptococcus spp.
from intestinal contents of Japanese eel (Anguilla japonica) (11 isolates) and rainbow
trout (Oncorhynchus mykiss) (one isolate). Of these 12 strains, three showed antag-
onistic activity against Aer. caviae, two against Aer. eucrenophilia, one against
Aer. jandaei and five against Aer. sobria, while Plesiomonas shigelloides was sensitive
to two Streptococcus spp. (table 2).
4.6. Aggregation with pathogens

Aggregation may be an interesting feature of strains for probiotics, as the aggregation
by LAB may expel pathogens not adhering to the mucus or surface of enterocytes
from the gut. Spencer and Chesson (1994) reported that five strains out of 43 lacto-
bacilli were able to aggregate E. coli 0129-K88+. Similarily, Kmet and Lucchini
(1999) isolated 20 Lactobacillus strains from the oesophagus and vagina of farrowing
sows, and aggregation activity was observed between six homofermentative auto-
aggregative strains and three strains of pathogenic E. coli with F4, F5 and F6 fimbriae.
At present, no information on this topic is available on probiotic candidates isolated
from fish, and this topic deserves further study.
4.7. Purification and characterization of inhibitory substances from LAB

Information on purification and characterization of bacteriocins produced by LAB
associated with food systems is provided in numerous reports (for review see Piard and
Desmazeaud, 1992; Nettles and Barefoot, 1993; Paik and Oh, 1996; Parente and
Ricciardi, 1999; Ennahar et al., 2000). Yet, there is a paucity of such information on
bacteriocins of LAB from aquatic animals. Knowledge on the identity of inhibitory
substances produced by intestinal LAB is crucial for assessing the potential of different
isolates and their significance as antagonists in the digestive tract of fish. Information
on bacteriocin purification and characterization is only available for Carnobacterium
strains from aquatic animals; these results are summarized in table 3. Still, knowledge
of bacteriocins produced by this relatively new group of bacteria is limited.
Stoffels et al. (1992a,b, 1993) purified a bacteriocin designated carnocin UI49
from C. piscicola UI49 using a purification protocol including XAD chromatogra-
phy and cation exchange chromatography. It is a small peptide molecule containing
lanthionine and therefore belongs to the class of bacteriocins termed lantibiotics.
It was produced during the mid-exponential phase of growth at temperatures between
15 and 34°C. As known for other bacteriocins, its bactericidal mode of action results
in the lysis of sensitive cells of closely related LAB.
The bacteriocins named piscicocin V1 and divercin V41 were primarily charac-
terized in a study of Pilet et al. (1995). They were shown to be heat resistant for up
to 30 min at 100°C, but the activity was completely lost after autoclaving for 15 min
Table 3. Purification and characterization of bacteriocins from carnobacteria isolated from fish
Number
Heat of Amino
Producer stability pH Sensitivity to Purification Molecular amino acid
Bacteriocin strain Medium at 100°C stability proteases scheme mass (Da) acids sequence References
Carnocin C. piscicola MRS Partial 2–10 Trypsin: + ASP, desalt on 4635 35–37 ND Stoffels et al., 1992a
UI49 UI49 inactivation α-Chymotrypsin:+ CFC, CEC, XAD Stoffels et al., 1993
after 60 min Pepsin: ± chromatography,
CEC (large scale)
Piscicocin C. piscicola MRS No inactiva- ND Trypsin: + ASP, desalt with 4416 (V1a) 44 piscicolin Pilet et al., 1995
V1 V1 tion after Proteinase K:+ Sep-Pack, HPLC 4526 (V1b) 43 126*
30 min Pronase E: + carnobac- Bhugaloo-Vial
teriocin et al., 1996
BM1*
Divercin C. divergens MRS No inactiva- ND Trypsin: + ASP, desalt with 4509 43 similar to Pilet et al., 1995
V41 V41 tion after Proteinase K:+ Sep-Pack, HPLC enterocin Metivier et al., 1998
30 min Pronase E: + A and
pediocin
PA1
*The amino acid sequences are identical with those of piscicolin 126 from C. piscicola JG126 isolated from ham (Jack et al., 1996) and
carnobacteriocin BM1 from C. piscicola LV17B from meat (Quadri et al., 1994), respectively.
ASP, ammonium sulphate precipitation; CFC, gel filtration chromatography; CEC, cation exchange chromatography; XAD, adsorption media, polyaromatic;
HPLC, high-performance liquid chromatography.
ND, no data available.
at 120°C. They exhibited an antilisterial activity and were sensitive to various pro-
teolytic enzymes (pronase E, proteinase K and trypsin) suggesting their proteina-
ceous nature. A later study (Bhugaloo-Vial et al., 1996) revealed that the
antibacterial activity of piscicocin V1 was due to two different bacteriocins (piscico-
cin V1a and piscicocin V1b) showing identical inhibitory spectra. Both substances
were purified to homogeneity and were found to be non-lantibiotic small heat-
stable bacteriocins with molecular masses of 4416 dalton and 4526 dalton. The
purification and determination of the amino acid sequence of divercin V41 revealed
that this bacteriocin shows a high homology with pediocin PA-1 and enterocin A
produced by strains of Pediococcus and Enterococcus (Metivier et al., 1998).
Jöborn (1998) described inhibitory substances greater than 1000 dalton from LAB
strain K3 isolated from Atlantic salmon and probably a representative of C. piscicola
(Westerdahl et al., 1998). By contrast, other strains of LAB including Lactobacillus-
like, Aerococcus-like and Carnobacterium-like isolates produced inhibitory
substances less than 1000 dalton when tested in an in vitro assay (Jöborn, 1998).
5. USE OF LACTIC ACID BACTERIA IN AQUACULTURE

Specific bacterial pathogens can be an important cause of mortalities in fish hatch-
eries, as intensive husbandry practices often result in breakdown of the natural host
barriers. Research laboratories and commercial hatcheries have attempted to over-
come this problem by disinfection of water supplies and food, stimulation of host
resistance, and the prophylactic or therapeutic use of antibiotics. However, the indis-
criminate use of antibiotics in disease control in many sections of the aquaculture
industry has led to selective pressure of antibiotic resistance in bacteria, a property
which may be readily transferred to other bacteria (Aoki et al., 1985; Amàbile-
Cuevas et al., 1995; Towner, 1995; Sørum, 1999). An alternative approach by which
opportunistic infections of fish pathogens may be reduced is by manipulation of the
gut flora either by adding antagonistic bacteria to the diet or by dietary manipulation,
in order to increase the proportion of health-promoting bacteria in the gut microflora.
An advantage of these methods is that they can be implemented during the early
stages of development when vaccination by injection is impractical. In this regard,
the stability of the antagonistic feature is a very important trait of probiotic LAB.
According to Olsson (1995) several turbot (Scophthalmus maximus L.) and Atlantic
salmon LAB isolates lost their capacity to inhibit growth of V. anguillarum after
being subcultured a limited number of times and stored at −70°C. Similar observa-
tions were made by Westerdahl et al. (1998) who described that antagonistic activity
of several fish intestinal bacteria was rapidly lost after storage and subculturing.
5.1. Effects of LAB administration on intestinal microbiota

Several studies on endothermic animals have demonstrated that administration of
LAB affects intestinal microbiota (for reviews see Gorbach, 1990; Juven et al., 1991;
Chateau et al., 1993; Naidu et al., 1999). However, a number of factors determine the
type and extent of colonization and continued presence of bacteria within the digestive
tract. These factors have been extensively reviewed by Savage (1983) and include:
1) gastric acidity (Gilliland, 1979), 2) bile salts (Floch et al., 1972), 3) peristalsis,
4) digestive enzymes (Marmur, 1961), 5) immune response, and 6) indigenous
(autochthonous) microorganisms and their antibacterial compounds. Attempts to control
the intestinal microbiota by LAB administration in aquatic animals are rather scarce.
It is well known that LAB under normal circumstances are not numerically dom-
inant in the digestive tract of fish (Ringø and Gatesoupe, 1998). In order to increase
the proportion of LAB, some investigations have attempted to increase their popu-
lation level by dietary factors such as: 1) chromic oxide (Ringø, 1993b,c), 2) differ-
ent oils (Ringø et al., 1998, 2002a), 3) high and low dietary lipids (Ringø and Olsen,
1999), and 4) inulin (Ringø et al., unpublished results). Another important criterion
for the use of LAB in commercial aquaculture is the colonization potential of LAB
in the fish gut, as Vibrionaceae may also persist for days or weeks in fish (Austin
et al., 1995; Munro et al., 1995; Ringø and Vadstein, 1998). Some recent studies
have demonstrated that carnobacteria strains are able to survive for several days in
the intestine of larval and juvenile fish (Strøm and Ringø, 1993; Jöborn et al., 1997;
Gildberg and Mikkelsen, 1998; Ringø, 1999b). Three of these studies (Jöborn et al.,
1997; Gildberg and Mikkelsen, 1998; Ringø, 1999b) have suggested that there is
apparently no host specificity with regard to colonization of the fish gut with
carnobacteria, in contrast to endothermic animals where adhesion of LAB appears
to be complicated by host specificity (Lin and Savage, 1984; Fuller, 1986; Conway,
1989). However, the colonization site in the fish gut is also an important criterion.
In a recent study, Gildberg and Mikkelsen (1998) administered two C. divergens
strains originally isolated from the intestine of mature Atlantic cod and Atlantic
salmon, to Atlantic cod juveniles via the food. When the Atlantic cod isolate was
used, the authors only detected LAB in pyloric caeca, while the concentration of the
bacteria was approximately ten-fold higher in the pyloric caeca than in the intestine
when the salmon isolate was used.
Transient bacteria may also be efficient if the cells are introduced at high dose.
Moreover, as LAB may exert antibacterial effects against undesirable microbes,
some investigators have attempted to increase the proportion of LAB associated
with the fish digestive tract. In a study with 4-day-old Atlantic cod larvae, Strøm and
Ringø (1993) used an antagonistic LAB strain which, when added to the rearing
water, favourably influenced the intestinal microbiota of the larvae by increasing the
proportion of LAB from approximately 5 to 70% and by a subsequent decrease in
the proportion of the bacteria genera Pseudomonas, Cytophaga/Flexibacter and
Aeromonas (table 4). These results indicate that the LAB were able to colonize and
may comprise a major part of the autochthonous microbiota in the gut of the larvae.
A similar increase in intestinal LAB was also found in Atlantic cod fry fed a diet
containing C. divergens (Gildberg et al., 1997) (table 4). In a study with Atlantic
Table 4. Effect of LAB administration on intestinal microbiota
Bacterial genera isolated and proportion of microflora population

LAB
Fish species used Before administration (control) After administration After challenge References
Atlantic cod – C. divergens Pseudomonas 42.5; Cytophaga/Flexibacter 42.5 C. divergens 70; Pseudomonas 20 * Strøm and Ringø,
larvae Aeromonas 10; C. divergens 5 1993
Atlantic cod – C. divergens No information was given No information was given C. divergens 75 Gildberg et al.,
fry Pseudomonas-like 25 1997
Atlantic C. divergens Pseudomonas, Enterobacteriaceae C. divergens 100 Aer. salmonicida 90 Gildberg et al.,
salmon – fry Gram-positive cocci C. divergens 10 1995
Turbot – larvae C. divergens C. divergens n.d C. divergens (8 × 103) * Ringø, 1999b
Floundera Lactobacillus sp. Enterobacteriaceae 4.3 (5/5); G(+) Enterobacteriaceae * Byun et al., 1997
4.6 (5/5) 4.8 (5/5); G(+) 4.3 (5/5)
DS-12 Yeast 4.6 (5/5); haemolytic bacteria 5.8 (2/5) Lactobacillus sp. DS-12 7.0 (3/5)
Mucoid colony form 4.8 (1/5); aerobes Clostridium 4.3 (1/5); yeast 4.3 (1/5)
8.5 (5/5)
Anaerobes 7.6 (5/5) Haemolytic bacteria 5.1 (1/5)
Aerobes 7.3 (5/5); anaerobes 6.6 (5/5)
Carpb Ent. faecium Enterobacteriaceae 6.2; E. coli 4.2 Enterobacteriaceae 6.2; E. coli n.d * Bogut et al., 1998
Ent. faecalis 3.3; Staph. aureus 3.7 Ent. faecalis 3.5; Staph. aureus 4.0
Bacillus spp. 7.0; Clostridium spp. 2.9 Bacillus spp. 7.0; Clostridium spp. 2.7
Sheat fishc Ent. faecium Escherichia coli 3.1; Enterobacteriaceae 3.0 Escherichia coli 1.1; * Bogut et al., 2000
Enterobacteriaceae 1.9
Staph. aureus 4.7 Staph. aureus 1.4
Bacillus 6.0; Clostridium 2.1 Bacillus 5.6; Clostridium n.d
a Data are presented as log 10 and frequency is shown in parentheses.

b Data are presented as log 10 after 4 weeks of feeding.
c Data are presented as log 10 after 58 days of feeding.
n.d, not detected; *, challenge test not done.

salmon fry, Gildberg et al. (1995) demonstrated that administration of LAB reported
as Lb. plantarum, but later reclassified as C. divergens (Ringø et al., 2001a) increased
the proportion of adherent LAB to intestinal wall from nil to 100% (table 4).
Recently, Byun et al. (1997) evaluated the effect of LAB (Lactobacillus sp. DS-12)
administration via the feed on the intestinal microbiota of flounder (Paralichthys
olivaceus) after 1 month of feeding (table 4). Lactobacillus sp. DS-12 was not
detected in the intestine of the control group, but 107/g LAB were found in the GIT
when the fish were fed a LAB supplemented feed.
In a recent study, Bogut et al. (2000) evaluated the effect of Ent. faecium on the
intestinal microbiota of Sheat fish (Silurus glanis). In this study, the fish were exposed
to Ent. faecium by including the bacteria in the diet. After approximately 2 months of
feeding, some interesting differences in the intestinal microbiota were observed
between the two rearing groups. Ent. faecium administration decreased the population
level of Staph. aureus, E. coli and other bacteria of the family Enterobacteriaceae, and
resulted in complete elimination of Clostridium spp. (table 4).
Only one investigation has evaluated the influence of a commercial LAB prepa-
ration on the allochthonous intestinal microbiota. Supplementation of 1 gram of Ent.
faecium M74 per 100 kg feed influenced the intestinal microbiota of 0 + Israeli carp
(Cyprinus carpio) to some extent (Bogut et al., 1998). While Escherichia coli disap-
peared from the intestinal microbiota of the fish after 14 days and onwards by feed-
ing the probiotic preparation (table 4), the population levels of Enterobacteriaceae,
Ent. faecalis, Staph. aureus, Bacillus spp. and Clostridium spp. were not reduced as
a result of including Ent. faecium into the diet (Bogut et al., 1998). The authors
suggested a high adhesive ability in the epithelium of carp digestive tract for Ent.
faecium. However, as they isolated the allochthonous intestinal microbiota, convinc-
ing experimental evidence was not provided.
When dealing with the potential of probiotics (for example LAB) in aquaculture
the fundamental question arises whether it is possible to colonize and maintain the
probiotic bacteria within the digestive tract. This is particularly important when
long-term exposure may be required for the probiotic effect. In this respect, electron
microscope investigations are a useful tool (Ringø et al., 2003). Furthermore, read-
ers with special interest in prospects of fish probiotics, are referred to the recent
review on this topic by Gram and Ringø (2005, Chapter 17 in this book).
During the past decade some reports have been published on the nutritional con-
tribution of LAB to the production rate of rotifer Brachionus plicatilis (Gatesoupe
et al., 1989; Gatesoupe, 1990, 1991a), while the control of the microbiota of rotifer
cultures has received less attention.
5.2. Effects of LAB administration on survival and growth of fish

Some research has been conducted on the effect of LAB administration on survival
(Garcia de la Banda et al., 1992; Hamácková et al., 1992; Gildberg et al., 1995;
Ringø, 1999b; Ottesen and Olafsen, 2000) and growth of fish (Hamácková et al.,
1992; Noh et al., 1994; Gildberg et al., 1995; Byun et al., 1997; Bogut et al., 1998)
(table 5). In their study with turbot larvae, Garcia de la Banda et al. (1992) claimed
that administration of commercial preparations of live LAB (Lactococcus lactis and
Lb. bulgaricus) via enrichment of rotifer and Artemia increased survival of the
larvae 17 days after hatching. Accelerated growth of Sheat fish after Ent. faecium
M-74 administration was also reported by Hamácková et al. (1992). Ottesen and
Olafsen (2000) working with Atlantic halibut (Hippoglossus hippoglossus L.) larvae
demonstrated that Lb. plantarum (originally isolated from Atlantic cod) administra-
tion to the culture water improved larval survival. At 12 days post-hatching (the first
critical stage of initial feeding), larval survival was approximately 96% compared
to 81.5% survival in the control group (unsupplemented with bacteria). This posi-
tive trend in the LAB rearing group was also observed after 32 days, as survival
was significantly higher in the larval group incubated with Lb. plantarum (68.4%)
compared to the control group (58.2%). Contrary to these results, Ringø (1999b) did
not observe any positive effect on survival of turbot larvae exposed to C. divergens
(originally isolated from Atlantic salmon) compared to larvae not exposed to bacteria
(control).
Commercial preparations of Ent. faecium improved the growth and feed efficiency
of Israeli carp (Noh et al., 1994; Bogut et al., 1998) and Sheat fish (Hamácková et al.,
1992). Gildberg et al. (1995) included a C. divergens strain (originally isolated from
Atlantic salmon) to the diet, but the authors did not observe increased growth of
Atlantic salmon fry as a result of LAB administration. Byun et al. (1997) checked the
feeding effects of Lactobacillus sp. DS-12 on body weight of flounder (Paralichthys
olivaceus) in two feeding trials. In the first trial, a significant effect of LAB adminis-
tration was observed. However, the second experiment showed no significant differ-
ences between the groups although there was a tendency of greater increase in body
weight as a result of LAB administration.
No increase in the growth rate of the rotifers was observed after addition of Lac.
lactis AR21 through the diet under optimal conditions (Harzevili et al., 1998). Under
a suboptimal feeding regime where the food was reduced to 45%, Lac. lactis coun-
teracted the growth inhibition of rotifers due to V. anguillarum in two of the three
experiments performed. However, the authors recovered neither Lac. lactis nor
V. anguillarum from the rotifer after 24 h.
5.3. Challenges in vivo

The major factors involved in the biocontrol of bacterial pathogens in the gastro-
intestinal tract are primarily those regulating the composition, functions and inter-
actions of indigenous microbial populations with the animal tissues. This concept is
supported by repeated observations that strains of transient enteropathogens can col-
onize intestinal habitats of endothermic animals. The fact that fish contain intestinal
Table 5. Effect of LAB administration on survival and growth
LAB isolate used Host Way of administration Effect Reference
Survival
Lac. lactis and Turbot larvae Enrichment of rotifer Increased survival of larvae Garcia de la Banda et al.,1992
Lb. bulgaricus and Artemia 17 days after hatching
Ent. faecium M-74 Sheat fish fry Addition to the diet Increased survival Hamácková et al., 1992
Lb. plantarum a* Atlantic halibut larvae Addition to the culture water Increased survival of larvae Ottesen and Olafsen, 2000
2 weeks after hatching
C. divergensb Turbot larvae Addition to the culture water No significant effect on larval Ringø, 1999b
survival
Growth
Str. thermophilus, Lb. helveticus Turbot larvae Enrichment of rotifer Enhanced growth Gatesoupe, 1991a
or Lb. plantarum
Ent. faecium M-74 Sheat fish fry Addition to the diet Enhanced growth Hamácková et al., 1992
Ent. faecium Israeli carp adult Addition to the diet Enhanced growth Noh et al., 1994
Ent. faecium Israeli carp juvenile Addition to the diet Enhanced growth and feed Bogut et al., 1998
conversion
Ent. faecium Sheat fish juvenile Addition to the diet Enhanced growth Bogut et al., 2000
C. divergensb Atlantic salmon fry Addition to the diet No significant effect on growth Gildberg et al., 1995
Lactobacillus sp. DS-12 Flounder juvenile Addition to the diet Varied results Byun et al., 1997
aIsolated from Atlantic cod by Strøm, 1988.

bIsolated from Atlantic salmon by Strøm, 1988.
* Reclassified from Lactobacillus plantarum to Carnobacterium divergens by Ringø et al., 2001a.
microbiota with antagonistic effects against fish pathogens has prompted investiga-
tors to conduct challenge experiments with LAB during the past decade (Gatesoupe,
1994; Gildberg et al., 1995, 1997; Gildberg and Mikkelsen, 1998; Harzevili et al.,
1998). However, in these studies some conflicting results on the mortality were
reported when the control group was compared with probiotic treatment (table 6).
Gatesoupe (1994) suggested that in vivo experiments with turbot larvae using
rotifers grown on LAB strains (resembling those of Lb. plantarum or
Carnobacterium sp.) improved the disease resistance in challenge tests with patho-
genic vibrio (V. splendidus strain VS11). However, the results reported in this study
were registered after 48 and 72 h, beyond which the mortality pattern was not dis-
cussed. In three papers, Gildberg and Mikkelsen (1998) and Gildberg et al. (1995,
1997) have used two LAB strains originally isolated from Atlantic salmon and
Atlantic cod by Strøm (1988). These two isolates were recently identified by 16S
rDNA and AFLPTM fingerprinting as C. divergens (Ringø et al., 2001a). In challenge
trials with co-habitants with Aer. salmonicida, Gildberg et al. (1995) in contrast to
expectations, registered the highest mortality of Atlantic salmon fry with fish given
the diet containing C. divergens, originally isolated from Atlantic salmon intestine.
In their study with Atlantic cod fry, Gildberg and Mikkelsen (1998) observed the
same cumulative mortality independent of whether the C. divergens isolates supple-
mented to the commercial feed were originally isolated from the digestive tract of
Atlantic cod or Atlantic salmon, when the fish were bath exposed to V. anguillarum.
On the other hand, an improved disease resistance of Atlantic cod fry was observed
by supplementing a commercial dry feed with a strain of C. divergens originally
isolated from the cod (Gildberg et al., 1997). The explanation for these conflicting
results has not been elucidated. Gildberg and Mikkelsen (1998) put forward a
hypothesis that bacteriocin production can be inducible and may not occur if the
bacteria are not frequently challenged with inhibitors as previously demonstrated by
Schrøder et al. (1980). Furthermore, a recent study by Nikoskelainen et al. (2001)
used the human probiotic Lb. rhamnosus in a challenge test with Aer. salmonicida
with promising results (table 6). These results should stimulate fish microbiologists
to use human probiotic LAB in future studies.
If the intestine is involved in infection, the fundamental question arises of
whether there are differences between the posterior part of the intestine and the
hindgut region of the intestine. It is well established that the intestine in an imma-
ture or inflammatory state has an enhanced capacity to absorb intact macromole-
cules (for review see Olsen and Ringø, 1997). Furthermore, some studies report
endocytosis of bacteria by enterocytes in the epithelial border of hindgut of herring
(Clupea harengus) larvae (Hansen et al., 1992; Hansen and Olafsen, 1999), herring
and Atlantic cod larvae (Olafsen and Hansen, 1992) and 36-day-old juvenile turbot
(Grisez et al., 1996). It is generally accepted that mature and non-inflammatory
intestines of adult salmonids are not permeable to microparticulates, in contrast to
the mammalian GIT where M cells are active in phagocytosis. However, a recent
Table 6. Challenge tests of fish with LAB
Effect in
Way of challenge Suggested mode
LAB isolate used Host Pathogen administration test of action Reference
Carnobacterium spp.a Turbot larvae V. splendidus Enrichment of rotifers + Antagonism and/or Gatesoupe, 1994
improved nutritional
value of the rotifers
C. divergens b Atlantic salmon fry Aer. salmonicida Addition to the diet – Not specified Gildberg et al., 1995
C. divergens c Atlantic cod juveniles V. anguillarum Addition to the diet + Antagonism Gildberg et al., 1997
C. divergens b Atlantic cod fry V. anguillarum Addition to the diet +e Gildberg and Mikkelsen, 1998
C. divergens c Atlantic cod fry V. anguillarum Addition to the diet – Gildberg and Mikkelsen, 1998
Lb. rhamnosus d Rainbow trout Aer. salmonicida Addition to the diet + Nikoskelainen et al., 2001
+, Improved disease resistance; –, no significant effect.

a Isolated from rotifer.
b Isolated from intestine of Atlantic salmon (Strøm, 1988).
c Isolated from intestine of Atlantic cod (Strøm, 1988).
d A probiotic for human use.
e 12 days after infection significant reduced cumulative mortality was recorded in fish given feed supplemented with C. divergens isolated from Atlantic salmon,
but no effect was detected 4 weeks after infection.

Fig. 1. Endocytosis of bacteria demonstrated in enterocyte in the hindgut region (a) (arrowhead) and
bacteria between the microvilli (arrows) (Ringø et al., 2002a), and pyloric caeca (b) (arrow) of Arctic charr
(Ringø, unpublished data). Bar = 1.0 μm.
study demonstrated endocytosis of bacteria by enterocytes in the epithelial border of

hindgut of adult salmonid fish (fig. 1a), as well as in the posterior part of the intes-
tine (pyloric caeca) (fig. 1b) (Ringø et al., 2001b). These results are in accordance
with observations made by Vigneulle and Laurencin (1991) and Tamura et al. (1993)
who measured phagocytosis of fixed V. anguillarum in the posterior intestine of
rainbow trout (Oncorhynchus mykiss), sea bass (Dicentrarchus labrax), turbot
(Scophthalmus maximus) and eel (Anguilla anguilla).
The observations of Vigneulle and Laurencin (1991), Tamura et al. (1993) and
Ringø et al. (2001b, 2002a, 2003) indicate that the intestine is involved in bacterial
translocation. Yet no clear evidence is available on possible differences between dif-
ferent parts of the intestine with regard to bacterial infection.
It is well known that rotifers are often suspected of being a vector for bacterial
infections to the predatory organisms (Muroga et al., 1987; Perez-Benavente and
Gatesoupe, 1988; Tanasomwang and Muroga, 1988; Nicolas et al., 1989). It is there-
fore surprising that studies dealing with the proliferation of larval pathogens in
rotifer cultures are so scarce (Gatesoupe, 1991a; Harzevili et al., 1998). Gatesoupe
(1991a) reported that the proliferation of Aer. salmonicida that accidentally
appeared in the experimental rotifer culture was inhibited by treatment with
Lb. plantarum. Harzevili et al. (1998) reported that administration of the probiotic
strain Lac. lactis AR21 under a suboptimal feeding regime counteracted the growth
inhibition of the rotifers owing to V. anguillarum.
6. CONCLUSIONS
The intestine and its associated microbiota can be considered as a complex ecosystem.
Interactions and competition within the resident population, as well as dietary inputs,
environmental conditions, and possibly the host immune system, influence the com-
position of the enteric community and its ability to inhibit pathogens. On the basis of
the results presented in this paper, it is suggested that LAB with antagonistic activity
against fish pathogens are potential candidate probiotics in future aquaculture, but
further studies on how LAB can improve the disease resistance in challenge tests with
pathogenic bacteria and the influence of LAB on the GIT microbiota are necessary.
Future studies on the effect of LAB administration on gut microbiota of fish should
include molecular approaches to analyse bacterial communities as described by
Raskin et al. (1997), Wallner et al. (1997) and Hugenholtz et al. (1998).
As most probiotic studies on fish have not seen any clear effect in disease studies, we
suggest a new strategy. We must think as the military and use military strategy. If we
are going to defeat our enemies, the pathogens, we have to be at the same place and
time as them. For example, if probiotic bacteria mostly colonize the pyloric caeca,
the probionts will have no effect if the pathogen mostly colonizes the mid or hindgut
regions and translocates in these regions. In this respect, electron microscopical stud-
ies may be a useful tool in evaluating which part of the gastrointestinal tract the pro-
bionts colonize and where the main translocation of the pathogen occurs.
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19 Enhancement of the efficacy of
probiotic microorganisms in nutrition
and prevention of diseases of the
young animal
A. Bomba, R. Nemcová and D. Mudroňová
University of Veterinary Medicine, Komenského 73, 041 81 Košice, Slovak

Republic
Probiotics may represent an effective alternative to the use of synthetic substances in

nutrition and medicine. The data concerning the efficacy of probiotics are often
contradictory. It is therefore important to search for ways to improve the efficacy of
probiotic microorganisms. In order to improve the selection and enhance the efficacy
of probiotics, additional knowledge is required on the mode(s) of action. The efficacy
of probiotics may be potentiated by several methods: the selection of more efficient
strains; genetic modification; combination of several strains; and the combination of
probiotics and synergistically acting components. Combination with synergistically
acting components of natural origin seems to be one of the best ways of potentiating
the efficacy of probiotics and is widely used in practice. By this method, more effec-
tive probiotic preparations (potentiated probiotics) may be developed.
1. INTRODUCTION
Probiotics are biopreparations containing living cells or metabolites of stabilized
autochthonous microorganisms that optimize the colonization and composition of
gut microflora in both animals and humans, and have a stimulatory effect on diges-
tive processes and the immunity of the host (Fuller, 1992). From the viewpoint of
the practical use of probiotics, it is of particular importance that probiotics have both
local and general biomedical effects, an inhibitory effect against pathogens, an opti-
mizing effect on digestive processes, an immunostimulative effect, and possibly
even anti-tumour and cholesterol reducing activities.

Enhancement of efficacy of probiotics 455
Probiotics are being widely used in the food industry, agriculture, and human and
veterinary medicine (O’Brien et al., 1999; Shortt, 1999). Their applications include
uses in farm animal nutrition, for feed preservation, for improving the conversion of
feed nutrients, and for improving production (Nousiainen and Setälä, 1993). They are
also applied for modulating the functional development of the digestive tract of young
animals (Wallace and Newbold, 1992), and in the prevention and therapy of diseases
in humans and farm animals (Watkins et al., 1982; Bomba et al., 1997). Lactobacillus
bulgaricus, L. acidophilus, L. casei, L. helveticus, L. lactis, L. salivarius, L. plantarum,
Streptococcus thermophilus, Enterococcus faecium, Enterococcus faecalis,
Bifidobacterium spp., and particular strains of E. coli are most frequently used for
probiotic purposes. All the above-mentioned microorganisms, except L. bulgaricus
and Streptococcus thermophilus, which are starter cultures of yoghurt, form natural
components of the gut microflora (Fuller, 1989).
Despite extensive knowledge obtained in recent years, the mode of action of pro-
biotics has not been fully elucidated yet. The mode of inhibitory action of probiotics
against pathogens may be mediated by competition for receptors on the gut mucosa,
competition for nutrients (Freter, 1992), the production of antibacterial substances
(Piard and Desmazeaud, 1991), and the stimulation of the immune system (Perdigón
and Alvarez, 1992). Probiotics influence digestive processes by the improvement of
the microbial population beneficial for the macroorganism by enhancing its enzyme
activity, and by improving digestibility and feed utilization (Burgstaller et al., 1984).
The optimization of digestion can be exhibited by growth, stimulation effect, and
greater weight increase. The anti-tumour activity of probiotics may be realized in
three ways: a) the inhibition of tumour cells, b) the suppression of bacteria produc-
ing beta-glucosidase, beta-glucuronidase, and azoreductase, which catalyse the con-
version of procarcinogens to proximal carcinogens, and c) by the destruction of
carcinogens such as nitrosamines, and by the suppression of nitroreductase activity
which is involved in their synthesis. Probiotics may influence the blood cholesterol
level by the inhibition of cholesterol synthesis, or decrease its level directly by
assimilation (Zacconi et al., 1992).
2. METHODS OF POTENTIATING THE EFFICACY OF PROBIOTICS

Probiotics as natural bioregulators help to maintain the balance of the digestive tract
ecosystem by a variety of mechanisms and prevent the colonization of the digestive
tract by pathogenic bacteria (Ávila et al., 1995). The data concerning the efficacy of
probiotics in practice are often contradictory (Simmering and Blaut, 2001). In the
application to pigs of probiotics based on lactobacilli, many authors have recorded
a growth and stimulation effect (Pollmann et al., 1980; Nousiainen and Setälä,
1993). Some authors, on the other hand, did not observe growth improvement with
the administration of probiotics (Hines and Koch, 1971; Kornegay and Thomas,
1973). The data concerning the efficacy of probiotics in the prevention of diarrhoeic
456 A. Bomba, R. Nemcová and D. Mudroňová
diseases in young animals are contradictory too. The preventive effect of lactobacilli
and bifidobacteria against diarrhoea in pigs was confirmed by Maeng et al. (1989),
Depta et al. (1998), and Bomba et al. (1998). Some authors, however, have not
confirmed the preventive effect of probiotics against diarrhoeic diseases of piglets
(Wu et al., 1987; De Cupere et al., 1992; Bekaert et al., 1996).
The variation in efficacy of probiotics under different conditions may be attrib-
utable to the probiotic preparation itself or may be caused by external conditions.
The most frequent reasons and factors that may lead to variability in the efficacy of
probiotics are the following: low survival rate and instability of the strain, the use of
a non-specific strain relative to the host, low dose and frequency of administration
of the probiotics, interactions with some medicines, the health and nutritional status
of the animal, stress, genetic differences among animals, age and type of animal.
Research experience points to the fact that probiotics are most effective in animals
in the period of microflora development or in cases when physiological stability is
impaired (Stavric and Kornegay, 1995). A production strain of a probiotic should be
able to tolerate the conditions of the digestive tract, and, preferably, to adhere in
high numbers to the digestive tract mucosa; it should maintain high viability in
processing, lyophilization, and storage, and should re-vitalize rapidly in the diges-
tive tract; it should be able to produce inhibitory substances against pathogens and
stimulate the immune system (Chesson, 1993). The production strain must be non-
pathogenic. Some of the above-mentioned criteria for the selection of microorgan-
isms for probiotic purposes can be tested in vitro, but most of them must be verified
in vivo. Some properties of microorganisms observed under laboratory conditions
have not been confirmed in animal trials (Chateau et al., 1993; Bomba et al., 1996).
Despite increasing knowledge gained, the mode of action of probiotics has not been
fully explained yet. In order to enhance the efficacy of probiotics, it is necessary to
obtain additional important knowledge on the mechanisms mediating their effect in the
digestive tract (Stavric and Korgenay, 1995). The antibacterial effect of each probiotic
microorganism or its beneficial effect on the host may be mediated by one or a number
of mechanisms that may be expressed to different degrees. This is the starting point for
potentiating the efficacy of probiotics that may be realized either by intensifying one of
the functions, or by extending the range of functions of the probiotic organism.
The efficacy of probiotics may be enhanced by the following methods:
the selection of more efficient strains of a particular microorganism/species
genetic modifications,
the combination of a number of strains of one or more species,
the combination of probiotics and synergistically acting components.
More effective microbial strains can be selected under field conditions. This
procedure is, however, very time consuming, costly, and impracticable. The number
of microorganisms tested under conditions of practice may be reduced by using
laboratory tests (Fuller, 1989). This method uses natural properties of the strains.
Gene manipulations can be another way of enhancing the efficacy of probiotics.
Using techniques of gene manipulations would make it possible to connect the
ability of microorganisms to survive in the digestive tract with the ability to produce
metabolites that are carriers of probiotic effects (Fuller, 1989).
The advantage of probiotics based on a number of strains consists in their
efficacy under a range of conditions and in a variety of animal types. These
multiple-strain preparations can exhibit different characteristics (health, productive)
typical of these bacteria and, thus, offer a wider spectrum of biomedical effects.
Bacteria of the genus Bacillus in multiple-strain preparations may have an effect
as potential growth stimulators for Streptococcus and Lactobacillus species
(Pollmann et al., 1980). Although Bacillus spp. do not adhere to the intestinal
microvilli, these bacteria do grow in the mucous biofilm over the intestinal villi and
render the mucus more suitable as a nutrient source for other probiotic bacteria
(Porubcan, 1990).
The combination of probiotics with synergistically acting components of natural
origin seems to be the best way of enhancing the efficacy of probiotic preparations
from the practical point of view. It seems that in order to potentiate the effect of
probiotics, a number of suitable components may be used such as oligosaccharides,
phytocomponents, nutrients and growth factors, proteins, polyunsaturated fatty
acids, organic acids and bacterial metabolites (Pollmann et al., 1980; Gibson and
Roberfroid, 1995; Yadava et al., 1995). The enhancement of the efficacy of probi-
otics by their combination with synergistically acting components is frequently used
in practice.
3. SYNBIOTICS
Future challenges include the incorporation of one or more probiotics together or in
combination with suitable prebiotic substrates to enhance the efficacy of the prepa-
rations for clinical use (Salminen et al., 1998). A prebiotic is a non-digestible food
ingredient that beneficially affects the host by selectively stimulating the growth
and/or activity of one or a limited group of bacteria in the colon. In order for a food
ingredient to be classified as prebiotic, it must 1) be neither hydrolysed nor absorbed
in the upper part of the gastrointestinal tract, 2) be a selective substrate for one or a
limited number of beneficial bacteria commensal with the colon, which are stimu-
lated to grow and/or are metabolically activated, 3) consequently, be able to alter the
colonic flora in favour of a healthier composition, and 4) induce luminal or systemic
effects that are beneficial to the host’s health (Gibson and Roberfroid, 1995).
Some oligosaccharides comply with all criteria for prebiotics. Oligosaccharides
of various origin are found as natural components in many common foods including
fruit, vegetables, milk and honey. Oligosaccharides can be obtained by: a) extraction
from plant sources, b) controlled enzymatic hydrolysis of polysaccharides, and c) enzy-
matic synthesis. A wide variety of oligosaccharides (fructo-oligosaccharides, gluco-
oligosaccharides and galacto-oligosaccharides) is commercially available as feed additives.
The supplementation of feed with oligosaccharides improves feed conversion,
increases weight gains, and beneficially affects the health of animals (Bastien, 1990).
The mode of action of oligosaccharides may be explained as follows:

1. Partial or full resistance to the effects of the host’s digestive enzymes. Not
absorbed or metabolized in the upper part of the digestive tract, and therefore,
able to reach the colon, where they may react with microflora and enterocytes.
2. Used as specific growth substrates by beneficial bacteria (lactobacilli,
bifidobacteria, streptococci) and not by pathogens or potential pathogens
(coliform bacteria, salmonellae, clostridia). Induce the enzyme systems of
useful bacteria that break them down into organic acids and gases. In this way,
good conditions are created for the colonization of the digestive tract by useful
bacteria, while the growth of undesirable microflora is suppressed, which may
reduce the occurrence of diarrhoea (Modler et al., 1990). Higher weight gains
in animals may also be explained by the induction of the enzyme activity of gut
bacteria, which may be caused by feeding a limited amount of oligosaccharides.
3. Binding to the surface of bacterial cells thereby inhibiting the adhesion of
various bacterial pathogens to epithelial cells (Aniansson et al., 1990) or
binding to protein receptors on the surface of immunocompetent cells of the
intestinal mucosa activating the immune response. It is also thought that
oligosaccharides, acting as a soluble fibre, may decrease bacterial translocation
and thus promote the preservation of systemic immunity.
A way of potentiating the efficacy of probiotic preparations may be the combi-
nation of both probiotics and prebiotics to synbiotics. These may be defined as a
mixture of probiotics and prebiotics that beneficially affects the host by improving
the survival and implantation of live microbial dietary supplements in the gastro-
intestinal tract, by activating the metabolism of one or a limited number of health-
promoting bacteria and/or by selectively stimulating their growth improving the
host’s welfare (Gibson and Roberfroid, 1995). Nemcová et al. (1999) confirmed
the synergistic effect of Lactobacillus paracasei and fructo-oligosaccharide combi-
nation on faecal microflora of weaned pigs. This effect was demonstrated by
increased total anaerobes, aerobes, lactobacilli, and bifidobacteria counts, as well as
by decreased clostridia, Enterobacteriaceae, and E. coli counts. Kumprecht and
Zobač (1998) showed that biological preparations containing stabilized living
microorganisms, mainly lactic acid bacteria (LAB) as well as extracts of the cellular
wall of Saccharomyces cerevisiae (mannan-oligosaccharides) could have positive
effects on pig growth and feed conversion in addition to a positive effect on health.
The combination of probiotics and non-digestible carbohydrates may be a way of
stabilizing and/or potentiating the effect of probiotics.
4. POTENTIATED PROBIOTICS
Synbiotics appear to be preparations whose potentiated protective and stimulative
effects occur only in the colon. Taking into account the pathogenesis of diarrhoeic
diseases in young animals there is a need for protecting the digestive tract mucosa
Table 1. The localization of protective and stimulative effects in the digestive tract
Preparation Small intestine Colon
Probiotic + +
Prebiotic − +
Synbiotic + ++
Potentiated probiotic ++ ++
+, protective and stimulative effect.

+ +, potentiated protective and stimulative effect.
−, no effect.
throughout its length, i.e. also in the small intestine so that the adhesion of patho-
genic microorganisms can be prevented. In various disorders of the gastrointestinal
tract, there occur conditions for the translocation of conditioned pathogens or
pathogens from the colon into the front part of the digestive tract. Potentiated
probiotics are defined as biopreparations containing production strains of micro-
organisms and synergistically acting components of natural origin which amplify
their probiotic effect on both the small intestine and the colon, and their beneficial
effect on the host by intensifying a mechanism or by extending the range of their
probiotic action. Potentiated probiotics must comply with the criteria as follows:
a) they must be more effective than their components separately, b) their potentiated
protective and stimulative effects must be expressed in all parts of the digestive tract
(table 1).
On the basis of the above-mentioned criteria, a synbiotic could be regarded as a
potentiated probiotic, provided a component potentiating the probiotic effect on the
small intestine is added. From this it also follows that potentiated probiotics will
probably be multicomponent preparations.
5. COMBINATION OF PROBIOTIC MICROORGANISMS WITH

NON-SPECIFIC SUBSTRATES
Prebiotics are specific substrates selectively fermented in the colon. It has been
demonstrated that to enhance the efficacy of probiotics, non-specific substrates can
be used as well.
Hawley et al. (1969) suggested that large quantities of lactose are necessary for
lactobacilli to become established in the gut. Pollmann et al. (1980) studied the
effect of Lactobacillus acidophilus on starter pigs fed a diet supplemented with
lactose. Pigs receiving lactose in combination with the Lactobacillus inoculum had
the highest Lactobacillus counts and the best average daily gain in comparison with
other groups. The effect of caecal flora, cultured in lactose-based broths, against
Salmonella was enhanced by adding lactose to chick diets. The reduction in caecal
colonization was accompanied by an increase in the concentration of volatile fatty
acids and a decrease in the caecal pH (Corrier et al., 1990).
The combination of peptides and lactobacilli reduced mortality following diar-

rhoea, halved the incidence of digestive disorders and improved animal growth
significantly. While peptides or LAB alone improved animal productivity, their
combination resulted in a synergy of action (Lyons, 1987). Mordenti (1986) found
that the growth promoting effect which he obtained by feeding Enterococcus
faecium to pigs could be improved synergistically by the addition of whey peptides.
The use of whey and bovine blood plasma as non-specific substrates could be an
alternative processing route for probiotic production and/or for potentiating their
effect. Whey, an abundant by-product of the dairy industry, contains lactose, soluble
proteins, lipids, vitamins and mineral salts. It is used directly in animal feed
mixtures (Burnell et al., 1988). Supplementation of whey in a diet for turkey poults
enhanced the effect of Lactobacillus reuteri by increasing body weight gain and
resistance to salmonellae (Edens et al., 1991).
Bury et al. (1998) reported the use of whey protein concentrate as a nutrient
supplement for LAB. The addition of this protein significantly increased both the
cell numbers and lactic acid production by lactobacilli and streptococci. The whey
proteins, α-lactalbumin and β-lactoglobulin, were found to be excellent growth
promoters of bifidobacteria (Petschow and Talbott, 1990). The proteolytic enzymes
of the lactic acid bacteria are of great importance in the generation of free amino
acid and vitamins in fermented whey (Law and Haandrikman, 1997). More recently,
it has been recognized that several of the whey proteins confer antibacterial and
immuno-associated protection to the neonate against disease and that these and
other whey proteins also have putative biological effects when ingested, including
an anti-cancer action (McIntosh et al., 1998). Romond et al. (1998) suggested that
consumption of cell-free whey from a selected strain of bifidobacteria is capable of
modifying the ecosystem of the human intestine.
The effect of a preparation containing nutrients and growth factors which stimu-
late rumen bacteria was investigated in suckling lambs. The preparation contained
dried skim milk, glucose, ascorbic acid, nutritious broth, liver hydrolysate, and
enzymatic casein hydrolysate. A higher molar proportion of propionate and valerate
and a higher alpha-amylase activity in rumen fluid were observed in the lambs of
the experimental groups. The counts of streptococci and lactobacilli in the rumen
content and of those adhering to the rumen epithelium were significantly higher in
the experimental animals, too. Scanning electron microscopy showed longer and
more differentiated rumen papillae in an experimental lamb at the age of 4 weeks in
comparison with lambs of the control group (Bomba and Žitňan, 1993).
Bomba et al. (1999) investigated the influence of the preventive administration
of Lactobacillus casei subsp. casei and maltodextrin KMS X-70 on Escherichia coli
08:K88 adhesion in the gastrointestinal tract of conventional and gnotobiotic
piglets. L. casei alone had almost no inhibitory effect on the adherence of E. coli to
the jejunal mucosa of gnotobiotic and conventional piglets while the lactobacilli
administered together with maltodextrin decreased the jejunal mucosa E. coli counts
Fig. 1. The numbers of E. coli 08:K88

adhered to the jejunal mucosa in 7-day-old
gnotobiotic pigs after administration of
Lactobacillus casei and maltodextrin
KMS X-70.
(■) Group E: E. coli 08:K88.
( ) Group L: Lactobacillus casei + E. coli
08:K88.
( ) Group M: Lactobacillus casei + maltodex-
trin KMS X-70 + E. coli
08:K88.
of gnotobiotic piglets by 1 logarithm (4.95 log/cm2) in comparison with the control

group (5.96 log 10/cm2) (fig. 1). L. casei administered in combination with malto-
dextrin decreased the number of E. coli colonizing the jejunum of conventional piglets
by more than two and a half logarithms (4.75 log 10/cm2, P < 0.05) in comparison with
the control (7.42 log 10/cm2, fig. 2). The pH values of jejunum contents were lowest
in the group of gnotobiotic and conventional piglets administered with L. casei and
maltodextrin KMS X-70.
6. PROBIOTICS AND METABOLIC PRODUCTS

OF MICROORGANISMS
Lactic acid bacteria – among them many probiotics – have been found to produce
antimicrobial substances (Ouwehand et al., 1999). They include toxic metabolites of
oxygen, the lactoperoxidase-thiocyanate system, organic acids, the effect of pH, and
bacteriocins (Nemcová, 1997).
The ability to generate organic acids, particularly lactic and acetic acids, presents
one of the mechanisms by which lactobacilli perform their inhibitory effect upon
pathogens (Piard and Desmazeaud, 1991). Organic acids together with probiotics
Fig. 2. The numbers of E. coli 08:K88

adhered to the jejunal mucosa in 7-day-old
conventional pigs after administration of
Lactobacillus casei and maltodextrin
KMS X-70.
(■) Group E: E. coli 08:K88.
( ) Group L: Lactobacillus casei + E. coli
08:K88.
( ) Group M: Lactobacillus casei + maltodex-
trin KMS X-70 + E. coli 08:K88.
and specific carbohydrates (yeast cell walls) are often suggested as alternatives to
the use of antibiotic growth promoters (Jensen, 1998). A few studies have attempted
to correlate changes in the gastrointestinal ecosystem in response to diet acidifica-
tion. Sutton et al. (1991) reported that fumaric acid decreased E. coli numbers in the
stomach. Gedek et al. (1992) observed a decrease of several enteric bacteria including
E. coli in the gastrointestinal tract of weaned piglets fed a diet supplemented with
fumaric acid. Bolduan et al. (1998) and Kirchgessner et al. (1992) found that formic
acid decreased the population of coliform bacteria in the gastrointestinal tract of
weaned piglets. Propionic acid reduced the population of coliform bacteria in the
stomach (Bolduan et al., 1988) and in the small intestine (Cole et al., 1968).
Thomlinson and Lawrence (1981) found that the addition of 1% lactic acid to the
drinking water of creep-fed piglets significantly decreased the gastric pH, delayed
the multiplication of enterotoxigenic E. coli and lowered the mortality rate.
An alternative to the use of organic acids in combination with probiotics in pig
diets is the use of fermented feed. Under certain conditions probiotic strains may be
used as the sole fermenting agent in milk. However, in many cases use of a support
culture is preferable. The combination of the probiotic culture and the support
culture enhanced the acidification rate (Saxelin et al., 1999). Fermented liquid feed
is characterized by high LAB and yeast counts, and a high concentration of lactic
acid. Fermented milks are claimed to contain a number of biologically active
components. These include bacteria used for fermentation, their metabolic products,
and components derived from milk. Milk contains a large variety of proteins and
peptides. These bioactive peptide properties include antimicrobial, anti-cancer,
antihypertensive, immunomodulatory, and mineral carriers (Meisel, 1997). Feeding
milk fermented with Lactobacillus casei and Lactobacillus acidophilus to mice
resulted in an increased resistance to Salmonella typhimurium. Perdigón et al.
(1991) and Nader de Macías et al. (1993) described the immunostimulant effect of
fermented milk (lymphokines and macrophages) responsible for the elimination of
the pathogens from the liver and spleen in E. coli and Listeria challenged mice. LAB
used in fermented milk alter the immunogenic properties of milk proteins (Perdigón
et al., 1986). The administration of milk fermented with Lactobacillus acidophilus
LA-2 caused a remarkable decrease (71.9% on the average) in faecal mutagenicity
and increased Lactobacillus spp. and Bifidobacterium spp. populations in the human
intestine (Hosoda et al., 1996).
Several investigations have shown fermented liquid feed to improve growth
performance in pigs and to establish a prophylactic barrier against gastrointestinal
disorders. Another way of administering organic acids with probiotics would be to
use water as a vehicle. It appears to have given more consistent advantages than diet
acidification. Because the volume of water intake is about 2.5-fold higher than feed
intake, and especially because newly weaned piglets take in high amounts of water,
water acidification provides a greater scope for delivering higher and more regular
doses of acids to piglets (Jensen, 1988).
The in vitro bactericidal activity of certain fatty acids has been known for a long
time (Kanai and Kondo, 1979), and may be of importance in vivo in preventing,
eliminating, and, in some cases, treating infections. The number of LAB associated
with the epithelial mucosa and from faeces was highest in the digestive tract of fish
that were fed diets with added 7% linolic acid (18:3 a-3) or 4% of a polyunsaturated
fatty acids mix. It is suggested that dietary fatty acids affect the attachment sites for
the intestinal microbiota, possibly by modifying the fatty acid composition of the
intestinal wall (Ringø and Gatesoupe, 1998).
Bacteriocins, which are proteinaceous compounds produced by some strains of
LAB, inhibit the growth of Gram-positive bacteria including food spoilage organ-
isms and pathogens and are expected to be used increasingly as natural food preser-
vatives (Dykes, 1995). Several methods for the control of diarrhoeagenic E. coli
have been described (Giese, 1994). The use of biopreservatives, including bacteri-
ocins, has been proposed for the control of many food-borne pathogens. Nisin,
a bacteriocin from Lactococcus lactis subsp. lactis, which acts exclusively against
selected Gram-positive bacteria, has served as a model for the application of other
bacteriocins as food biopreservatives. Colicins are the classical bacteriocins
produced by E. coli, which specifically inhibit E. coli and closely related strains.
There is potential for using colicins in foods and agriculture to inhibit sensitive
diarrhoeagenic E. coli strains (Murinda et al., 1996). Colicin-sensitive cells have
colicin-specific receptors located in the outer membrane of their cell envelope and
are killed by colicins that attach to these receptors. It is suggested that combining
probiotic microorganisms with bacteriocins could improve their positive effect
on the host.
7. COMBINATION OF PROBIOTIC MICROORGANISMS

AND PLANT EXTRACTS
Mitchell and Kenworthy (1976) considered that LAB might prevent coliform
diarrhoea by interaction with the enterotoxins. Such interaction might be indirect by
influencing E. coli populations or metabolism or might be more direct by neutralizing
the enterotoxin itself. Eleven species of lactic bacteria have been investigated for the
ability to neutralize cell-free enterotoxins, in a bio-assay. Action against the
E. coli enterotoxin was found in two species, L. bulgaricus and L. faecalis. Part of
the activity in the former species was in the cell-free fraction. Further investigation
of the cell-free anti-enterotoxic action from L. bulgaricus showed that it had a low
molecular weight, probably less than 103, was not very stable, and was independent
of the anti-E. coli activity. Broths containing L. bulgaricus fermented to produce high
levels of anti-enterotoxin were beneficial when added to diets for early weaned pigs.
It was inferred that this effect was likely to be caused by the anti-enterotoxic action.
Plant extracts seem to have a similar ability, which might be used in potentiating
the neutralization effect of lactobacilli against enterotoxin-producing E. coli.
A crude alcohol extract of Coleus froskohlii Briq. roots showed a marked inhibitory
action against an E. coli toxin-induced secretory response at a dose of 300 mg/loop
in ileal loops of rabbits and guinea pigs. Coleonol A and B obtained by column chro-
matography of a benzene fraction of the alcohol extract of Coleus froskohlii roots
exhibited antisecretory (antidiarrhoeal) action at a dose of 1 mg/loop. Coleonol A
showed nearly 79% inhibition against heat-labile (LT) toxin and 67% against heat-
stable (ST) toxin-induced secretions. Coleonol B exhibited less activity against LT
(66.2%) but slightly higher activity against ST (68.3%) enterotoxins as compared
with the widely used antidiarrhoeal (antisecretory) drug Loperamide (an opiate ago-
nist) which showed 77 and 65% inhibition against LT and ST toxins, respectively,
at a similar dose (Yadava et al., 1995).
It is interesting to consider potentiating the efficacy of probiotics in the control
of the post-weaning diarrhoea syndrome of piglets. Weaned piglets usually show a
malabsorption syndrome known as non-infectious diarrhoea which is characterized
by increased excretion of fatty acids and carbohydrates in the faeces, watery stools,
and degenerative changes in villi of the small intestine (Kyriakis, 1989). In the
majority of these cases, opportunistic pathogens, particularly enterotoxigenic
Escherichia coli (ETEC) strains and rotaviruses, take advantage of the presence of
this diarrhoea and cause the post-weaning diarrhoea syndrome (PWDS). The essen-
tial oils derived from the plant Origanum were proved to have in vitro antimicrobial
action against various bacteria, including E. coli (Sivropoulou et al., 1996). Kyriakis
et al. (1998) studied the possible effect of two different dosages in feed applications
of 5% of etherous oils of flower and leaf of Origanum on the control of post-
weaning colibacillosis in piglets. The medication with the Origanum essential oils
seemed to be effective in the control of PWDS, resulting in a mild atypical illness
in the animals combined with very good growth performance. The unique conclu-
sion of this study is the discovery of the possibility of PWDS control without the use
of “classical” antibacterials. Within the first days after weaning, the E. coli popula-
tion in weaned pigs increases and the lactobacilli population in the digestive tract
decreases. That is why the application of probiotics in combination with plant
components exhibiting antimicrobial effects may be useful.
Babic et al. (1994) demonstrated that purified ethanolic extracts of carrots had an
antimicrobial effect against a range of food-borne microorganisms: Leuconostoc
mesenteroides, Listeria monocytogenes, Staphylococcus aureus, Pseudomonas
fluorescens, Candida albicans and Escherichia coli. The antimicrobial activity was
not linked to phenolic compounds, but was presumably due to apolar components.
Free dodecanoic acid and methyl esters of dodecanoic and pentadecanoic acids were
identified in the purified active extracts of carrots and could be responsible for the
antimicrobial activity. An extract from the cortex of the African Okoubaka tree has
been successfully used for the treatment of enterotoxin-induced diarrhoea in horses.
It has been shown that some phytochemical components stimulate the production
of lactic acid by lactobacilli. Nakashima and Yoshikai (1980) have shown that
phytins, including phytic acid (a naturally occurring compound formed during the
maturation of seeds and cereal grains) stimulated the growth of LAB in a skim milk
medium as measured by the number of live bacteria and the amount of acid
produced. Nakashima (1997) reported that the stimulating effect of the phytin prepa-
ration on acid production by Lactobacillus casei is not attributable to phytin, but is
exerted mainly by the Mn in the preparation, while the presence of other inorganic
materials also augments the effect to some extent. Similar results were reported for
green laver and sea lettuce.
8. PROBIOTICS AND TRACE ELEMENTS

Some microbes have the ability to bind metal ions present in the external environ-
ment at the cell surface or to accumulate them in the cell. These properties have been
exploited also in probiotic preparations.
Selenium: yeasts and lactobacilli (Lactobacillus delbrueckii subsp. bulgaricus,
L. casei subsp. casei, L. plantarum) (Calomme et al., 1995; Ingledew, 1999) are able
to concentrate selenium from their growth media into their cells in high concentra-
tions and both produce an organic form of Se from inorganic Se. Yeasts produce
above all selenomethionine, as well as a wide variety of Se compounds, many of
which are unidentified (Wolffram, 1999). Lactobacilli contain a mixture of Se in
various chemical forms too, but their main product is selenocysteine (Calomme
et al., 1995). Recently, Se-enriched yeasts and lactobacilli have been commercial-
ized as a Se supplement, which is much more bio-available for useful metabolism
and storage than an inorganic salt (Calomme et al., 1995; Ingledew, 1999).
Chromium: trivalent chromium is the active constituent of the glucose tolerance
factor (GTF), which is a cofactor needed to potentiate insulin. Yeast is able to pro-
duce GTF and thus to serve as a feed supplement of the bioactive and non-toxic
form of chromium (Ingledew, 1999).
Iron: the position of iron in bacterial metabolism is specific. Iron is an essential
nutrient for all microbes, except the genus Lactobacillus, which uses manganese and
cobalt instead. On the other hand, iron can react with reduced forms of oxygen,
leading to the production of free radicals responsible for the peroxidative alteration
of cell membranes, and therefore iron homeostasis has to be strictly controlled.
Bifidobacteria are capable of accumulating large amounts of Fe2+ from a medium
and they contain an intracellular ferro-oxidase, which in the presence of O2
catalyses the oxidation of Fe2+ to insoluble and less available Fe3+. Lactobacillus
delbrueckii spp. bulgaricus and L. acidophilus also have the ability to oxidize Fe2+
and to bind Fe(OH)3 to their cell surfaces (Kot et al., 1997). The binding of iron by
bifidobacteria and lactobacilli, used extensively as probiotics, can serve to reduce
the availability of iron to pathogenic microorganisms.
Manganese: certain species of lactobacilli (e.g. L. plantarum, L. casei subsp.
casei) are able to concentrate high levels of manganese, which supports their
pseudocatalase and superoxide dismutase activity. These strains are more oxygen-
and hydrogen peroxide-radical tolerant. The addition of Mn to the growth medium
increases the viable count of L. casei spp. casei and L. plantarum compared to
unsupplemented cultures (Calomme et al., 1995).
Zinc: zinc is important for both the stability and action of alcohol dehydrogenase
as well as for many other microbial Zn-metallo-enzymes. However, zinc’s antimi-
crobial effects are well known. Zinc has inhibited the growth of Escherichia coli,
Streptococcus faecalis, some species of soil bacteria, and fungi (Collins and Stotzky,
1989). This inhibiting effect of zinc has been used successfully in the treatment of
E. coli diarrhoea in post-weaning piglets (Holm and Poulsen, 1996).
9. COMBINATION OF PROBIOTIC MICROORGANISMS

AND ANTIBIOTICS
Probiotics are often considered as “natural” substitutes for feed antibiotics. On the
basis of the results of some studies, it may be feasible to combine the probiotic and
antibiotic treatments to obtain an additive advantage (Stavric and Kornegay, 1995).
It has been suggested that the natural microflora resist the invasion of both harmful
and probiotic bacteria. Therefore, the probiotic bacteria may be established more
easily in the digestive tract of animals if the natural flora is weakened by the use of
an antibacterial feed additive (Nousiainen and Setälä, 1993). Pollmann et al. (1980)
found an additive effect in the combination of lincomycin and Lactobacillus spp.
The disruption of the ecological balance of the gastrointestinal tract by antibiotics
applied per os and by other immunosuppressive drugs enables bacteria to over-
multiply, and facilitates the translocation of these bacteria from the gastrointestinal
tract into other organs. Prophylactic use of lactobacilli-containing preparations may
protect against the side effects of antibiotics. The principal risk of the therapeutic use
of antibiotics consists in bacterial translocation with potential subsequent septicaemia
caused by an increase in the number of resistant species and a decrease in the number
of more sensitive bacteria. Each antibiotic is associated with an increase in the
number of specific microorganisms (Lambert-Zechovsky et al., 1984). The application
of L. casei, L. acidophilus and L. bulgaricus in conjunction with antibiotics prevented
both the increase in the number of ampicillin-resistant bacteria and their translocation
into the liver. Wasson et al. (2000) suggested that oral administration of a
Lactobacillus-containing product is ineffective in preventing clinical disease in guinea
pigs administered clindamycin phosphate. The gastrointestinal tract microflora greatly
influences the numbers and distribution of lymphoid cells from lymphatic tissue
associated with the digestive tract. The impairment of this microflora interferes with
the regulation of the gut mucosa’s immune response (Porter and Allen, 1989).
Lactobacilli, when applied with antibiotics, may eliminate the impairment of the gut
microecosystem by maintaining the equilibrium between microbial types in the gut,
thus preventing the impairment of the mucosa’s immune response.
10. COMBINATION OF PROBIOTICS AND VACCINES

Methner et al. (1999) tested the combination of competitive exclusion (CE) and
vaccination against Salmonella infection in chickens of different ages. The results of
their model study demonstrated principally that a combination of CE and immunization
against Salmonella infection results in a degree of protection considerably beyond that
afforded by the use of one of the two methods. The administration of the live Salmonella
vaccine strain prior to or simultaneously with the CE culture revealed the best protec-
tive effect, because these combinations ensure an adequate persistence of the vaccine
strain as a prerequisite for the expression of inhibitory effects in very young chicks and
the development of strong immune protection in older birds. The combination of
Lactobacillus acidophilus-, Enterococcus faecium-, and S. typhimurium-specific anti-
bodies when administered via spray to broiler chicks at 1 day of age, and administered
orally for the first 3 days after placement, significantly reduced S. typhimurium colo-
nization of the caecum and colon in market-aged broilers (Promsopone et al., 1998).

If probiotics are to represent a real and effective alternative to antibiotics and
chemotherapeutics, it is absolutely necessary to ensure their consistently high effi-
cacy. To ensure the high efficacy of probiotic preparations requires a complex solu-
tion aimed at the product and its mode of application.
Regarding the product itself, future research should be aimed at the selection of
strains with strong probiotic effects that will comply with the main criteria of selection.
It will be important to search for ways to potentiate the efficacy of probiotic micro-
organisms in all parts of the digestive tract. In addition to prebiotics which potentiate
the effect of probiotics in the colon, there should be components that, in combination
with probiotic preparations, will ensure their high efficacy in the small intestine also.
With regard to the form of application, research and development should be
aimed at methods that will ensure the maximum efficacy of probiotics at the time of
their consumption. Current knowledge confirms that probiotic preparations as well
as fermented products are most effective in a fresh state when given together with a
particular medium or substrate. On the basis of current knowledge it may be
expected that the developments in the field of biotechnological research will result
in a very simple “fermentor” which will enable customers to prepare probiotic
preparations and fermented products directly.
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20 Strategies for the prevention of E. coli
infection in the young animal1
E. Van Driessche and S. Beeckmans
Laboratory of Protein Chemistry, Institute of Molecular Biology and

Biotechnology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium
Attachment of disease-causing E. coli to the tissues of the host is an initial but essen-
tial first step in pathogenesis. Consequently, any strategy that prevents attachment
will interfere with the development of disease.
Diarrhoea is a common result of the colonization of the small intestine by entero-
toxigenic E. coli (ETEC). In this chapter, several strategies that can prevent colo-
nization of the gut by these microorganisms will be discussed and compared. We
will first describe how E. coli, by virtue of fimbriae,2 attach to the intestinal wall,
and which molecules are involved, both at the bacterial surface and on the intestinal
epithelium. Then, attention will be paid to several approaches to prevent attachment
of enterotoxigenic E. coli to the small intestinal mucosa, i.e. passive and active
vaccination, the use of receptor analogues, modification of intestinal receptors, and
finally the potential of probiotics will be described.
1. INTRODUCTION
Escherichia coli is a well-known member of the complex colonic microflora of
mammals and birds. However, some E. coli strains have the possibility to colonize
other tissues as well, and this causes severe and often life-threatening diseases such
1The Belgian “Ministerie van Landbouw en Middenstand”, VEOS n.v. (Zwevezele, Belgium), the Flemish FWO (Fonds
voor Wetenschappelijk Onderzoek), VLIR (Vlaamse Interuniversitaire Raad), and DGIS (Directoraat-Generaal voor
Internationale Samenwerking) are gratefully acknowledged for financially supporting the authors’ research related
to the topic covered by this chapter.
2In this chapter, the F-terminology has been followed to designate fimbriae: F1, F2, F3, F4, F5 and F6 correspond
respectively with type-1, CFA/I, CFA/II, K88, K99 and 987P.

Prevention of E. coli infection 473
as diarrhoea. The relation between diarrhoea and E. coli was noticed soon after the
first description of this bacterium by Theodore Escherich in 1885. If not treated
properly and adequately after the onset of the first symptoms, diarrhoea is a devas-
tating disease.
In western countries, E. coli infections in humans and livestock are generally
treated with antibiotics. The occurrence of resistance and even multiresistance
towards the available antibiotics is a very serious problem we will have to face in
the future. In the tropics, diarrhoea, especially in children, remains life threatening
and is a major cause of death, often because adequate treatment is lacking. All over
the world, colibacillosis causes important economic losses because of death of
animals, veterinary costs, reduced performance, etc. Modern husbandry practices
create optimal conditions for the fast spreading of any infectious agent and
especially newborn animals and animals at the age of weaning are very sensitive to
infection by bacteria and viruses. The overuse of antibiotics during past decades has
resulted in the selection of multiresistant E. coli that can no longer be tackled with
the available antibiotics. Moreover, recent changes in the attitude of the consumer
necessitate husbandry practices that avoid, or at least strongly reduce the use of
antibiotics. For these reasons, alternatives for the treatment of colibacillosis have
become a priority in research efforts.
The understanding of the principles that govern the attachment of E. coli to host
tissues opens new avenues to treat diseases caused by these microorganisms. In this
chapter, we will restrict ourselves to enterotoxigenic E. coli that cause diarrhoea by
colonizing the small intestine, and that produce heat-stable (ST) and/or heat-labile
(LT) enterotoxins (Gyles, 1992; Spangler, 1992) that will cause massive water and
electrolyte losses, resulting in watery diarrhoea. As well as enterotoxigenic E. coli
(ETEC), other types such as enteroinvasive (EIEC), enteropathogenic (EPEC),
enterohaemorrhagic (EHEC), enteroaggregative (EAEC) and uropathogenic
(UPEC) strains are also known as important virulent agents (Nataro and Kaper,
1998; Nagy and Fekete, 1999).
In view of the importance of enterotoxigenic E. coli in provoking disease in
humans and animals, including important livestock species, ETEC have been investi-
gated very intensively for more than two decades, and hundreds of research papers
have been published on this topic. Consequently, it cannot be expected that this vast
literature can be completely covered in this chapter. Rather we will focus on some
general principles of biosynthesis, purification and application of fimbrial lectins of
ETEC for the elaboration of protective vaccines and on possible attachment–inhibition
strategies that have been developed based on the understanding of the mechanisms
ETEC use to colonize the small intestine. Although ETEC are important causative
agents of diarrhoea in humans and several animals, emphasis will be put on calves and
pigs. When appropriate, reference will also be made to ETEC from human origin,
especially when strategies to prevent attachment of these micoorganisms to the intes-
tinal mucosa are concerned.
474 E. Van Driessche and S. Beeckmans
2. E. COLI SURFACE LECTINS AS VIRULENCE FACTORS

Since the beginning of the 20th century it has been known that some E. coli strains
are able to agglutinate human and/or animal erythrocytes. In the mid 1950s, Collier
and De Miranda (1955) showed that this agglutination could be specifically inhib-
ited by D-mannose and some mannose derivatives. In 1957, Duguid and Gillies
(1957) observed that the mannose-sensitive agglutination is correlated to the pres-
ence of long proteinaceous filaments at the surface of the bacteria, and that these
E. coli can also attach in a mannose-sensitive way to enterocytes. Ofek and
co-workers (1977, 1978) showed that destruction of oligosaccharides at the surface
of enterocytes, or incubation of these cells with the mannose-specific lectin ConA,
prevents binding of the E. coli that provoke mannose-specific haemagglutination.
Moreover, these researchers showed that yeast mannan is a strong inhibitor of
attachment, and that yeast cells are agglutinated by E. coli that display mannose-
sensitive haemagglutination. From the observations mentioned, it was hypothesized
that the E. coli under investigation express at their surface filamentous mannose-
specific lectins (fimbriae) that recognize complementary receptors at the surface of
erythrocytes or enterocytes. Definitive proof that fimbriae are lectins responsible
for haemagglutination and attachment was provided by Salit and Gotschlich (1977),
who were able to isolate these structures in a highly purified form, and could show
them to cause mannose-inhibitable haemagglutination as well as binding to epithelial
cells in a mannose-inhibitable fashion. Finally Krogfelt and co-workers (1990)
succeeded in identifying the protein molecule within these fimbriae that is respon-
sible for the mannose-specific adhesion (i.e. the adhesin or lectin subunit, see below).
Apart from mannose-sensitive haemagglutination, some E. coli strains were
shown to provoke mannose-resistant haemagglutination, which is also associated
with fimbriae. The former fimbriae are referred to as F1 fimbriae or common
fimbriae because they are expressed by both commensal and pathologic E. coli,
while the latter are called host-specific, are produced by pathogenic E. coli and are
responsible for host and tissue specificity. In table 1, some important properties of
both types of fimbriae are compared.
Although haemagglutination is the most straightforward and easiest test for the
detection of surface lectins on E. coli, some lectins may not be detected by this
method. For example for F6, F18 and CS31A lectins, no erythrocytes are known to
be agglutinated by E. coli expressing these antigens. Also, haemagglutination does
not allow us to discriminate between fimbrial and non-fimbrial lectins. This differ-
ence can, however, be readily made by electron microscopy after negative staining
of the E. coli cells with uranyl acetate (see Van Driessche et al., 1995, for a review).
Electron microscopy can also be used for the unequivocal identification of surface
adhesins when monospecific antisera against known and highly purified adhesins
are available. However, if specific antibodies are available, a simple slide aggluti-
nation test is easier to perform when a newly isolated E. coli strain is assayed for the
Table 1. Characteristics of fimbriae of enterotoxigenic E. coli (ETEC) strains that colonize livestock, and that are assembled through the chaperone/usher pathway
Alternative Chaperone/ Major subunit Gene Erythrocytes Inhibiting

Fimbriae name Natural host Morphology usher MM (Dalton) localization agglutinated MS/MR sugars
FGS chaperone
F1 Type-1 Common fimbriae Rigid, FimC/FimD FimA Chromosome Guinea pig MS Mannose,
(not host-specific) ø 7 nm 17 000 mannosides
F4* K88* Pig Flexible, FaeE/FaeD FaeG Plasmid Guinea pig, MR Galactosides
ø 2.1 nm 23 000–27 000 chicken
F5 K99 Pig, calf, lamb Flexible, FanE/FanD FanC Plasmid Horse, sheep MR Sialic acid
ø 5 nm 18 500
F6 987P Pig Rigid, FasB/FasD FasA Plasmid None MR Not known
ø 7 nm 20 000
F17** various Calf, lamb, goat Flexible F17-D/F17-C F17-A Chromosome Bovine MR GlcNAc
17 500–20 000
F18*** F107 Weaned pig Rather rigid, FedC/FedB FedA Plasmid None MR Not known
ø 3–4 nm 15 000
F41 Pig, calf, lamb Flexible, Fim41a Chromosome Horse, sheep, MR GalNAc
ø 3.2 nm 29 500 guinea pig,
human
FGL chaperone
CS31A Calf, lamb, goat Flexible, ClpE/ClpD ClpG Plasmid Not known MR Not known
ø 2 nm 30 000
MM, molecular mass; MS, mannose sensitive; MR, mannose resistant.

*Different variants are designated K88ab, K88ac, and K88ad.
**F17 refers to a family of highly related GlcNAc-specific fimbriae, including F17a (from bovine ETEC), F17b (isolated from septicaemic and diarrhoeic calves
and lambs), F17c (previously called 20K fimbriae, causing bovine septicaemic diarrhoea, or G-fimbriae, initially isolated from human UPEC), and F17d
(previously called F111 and isolated from calf ETEC) (see Lintermans et al., 1988a,b; Bertels et al., 1989; El Mazouari et al., 1994; Saarela et al., 1995, 1996;
Bertin et al., 1996; Martin et al., 1997; Cid et al., 1999).
***Different variants are designated F18ab and F18ac.
expression of known surface lectins. When a new E. coli strain is to be investigated,

several techniques can thus be used for the detection and identification of surface
lectins, i.e. haemagglutination, attachment to isolated villi, isolated enterocytes or
cell lines such as Caco-2, or attachment to glycoproteins covalently immobilized on
a solid support (Van Driessche et al., 1995). Attachment studies on villi or entero-
cytes require fixation, a process that might affect the lectin receptors and prevent
them from interacting with lectins. An easy to use in vitro attachment system using
Eupergit-C beads, which can be easily derivatized with glycoproteins (including
solubilized brush border membranes or mucus) has been developed by Van
Driessche et al. (1988). These beads can be stored for years at 4°C and allow the
investigation of attachment as well as attachment-inhibition. For example, Eupergit-
C-glycoprotein beads were successfully used to investigate the expression by E. coli
strains of F17 fimbriae for which initially no erythrocytes were known that were
recognized by these fimbriae (Lintermans et al., 1991). Similarly, with Eupergit-
glycoprotein beads we were able to demonstrate the carbohydrate-binding hetero-
geneity of F17 fimbriae expressed by different E. coli strains (Van Driessche et al.,
1988). Today, multiplex polymerase chain reaction (PCR) has developed into a fast
and reliable technique for diagnosis and characterization of fimbrial lectin genes, as
well as for the genes encoding LT and ST toxins (see Osek, 2001, as an example).
Not only is the expression of lectins at their surface important for E. coli to be
able to attach to host tissues, but also lectins have been shown to be involved in
attachment of Gonococcus species, Salmonella species, Yersinia species, Proteus
mirabilis, Bordetella pertussis, Klebsiella pneumoniae, and others (see Soto and
Hultgren, 1999, for a review). Moreover, there is a close correlation between the
expression of surface lectins, the in vitro binding properties to cells, membranes,
and glycoconjugates, and in vivo infectivity of the strains expressing surface lectins.
Obviously, E. coli expressing surface structures that allow them to colonize host tis-
sues have quite some advantages over those that do not express adhesion molecules.
Indeed, attached E. coli are able to withstand the mechanical cleansing mechanisms
in, for example, the intestine or the urinary tract, they have considerable growth
advantage, and they display increased resistance to deleterious agents.
Whether an E. coli strain will be able to colonize a host tissue is dependent not
only on the expression of surface adhesins, but also on the presence of tissue recep-
tors that can be recognized by the lectins. This statement is best exemplified by the
susceptibility of piglets to infection by E. coli expressing F4 fimbriae. Investigations
of Sellwood in 1980 have shown that sensitive animals possess F4 receptors, while
resistant animals are lacking them. Several E. coli adhesin receptors have been
isolated and characterized, and were shown to be glycoproteins and/or glycolipids
present in the enterocyte membrane and/or mucus covering the intestinal epithelium
(Dean and Isaacson, 1985; Teneberg et al., 1993; Erickson et al., 1994; Khan and
Schifferli, 1994; Khan et al., 1996; Billey et al., 1998; Francis et al., 1998, 1999;
Al-Majali et al., 2000; Fang et al., 2000; Jin and Zhao, 2000; Jin et al., 2000a;
Mouricout and Védrine, 2000; Van den Broeck et al., 2000; Van Driessche et al.,
2000). Age-dependent variation of the oligosaccharide composition and structure
can explain why susceptibility often depends on the age of the animals. Similarly,
tissue-specific glycosylation explains the tissue tropism displayed by pathogens.
In the case of F6 fimbriae, Dean et al. (1989) demonstrated that the age-dependent
sensitivity of piglets to colonization by E. coli F6 is correlated to the presence of F6
receptors in the mucus layer. Indeed, both neonates (sensitive) and older piglets (not
sensitive) express F6 receptors at the enterocyte surface. However, mucus receptors in
older piglets proved to be low molecular weight glycoproteins, trace amounts of which
are only detectable in newborn animals. These results show that resistance to E. coli
F6 resides in the presence of attachment blockers secreted in the small intestinal
mucus layer in older piglets. Besides glycoproteins, Khan et al. (1996) showed that the
brush border membranes also contain two glycolipids that act as F6 fimbrial receptors.
Age-dependent expression of F5 fimbrial receptors has been investigated by Yuyama
et al. (1993). These investigators found a good correlation between the postnatal
changes of the F5 glycolipid receptor and the susceptibility to E. coli F5 infection.
3. STRUCTURAL FEATURES OF ENTEROTOXIGENIC

E. COLI FIMBRIAL LECTINS
By now, the structure of several fimbriae has been unravelled. Upon isolation (see
below), SDS-PAGE analysis of purified fimbriae generally shows only one band.
Initially, it was thought that fimbriae are very simple oligomeric structures, built
up of a single subunit. However, genetic analysis of several gene clusters encod-
ing fimbriae shows that several genes are involved, and their presence is necessary
in order to get the expression of functionally active fimbriae, i.e. able to bind car-
bohydrates. Whether or not the encoding genes will eventually be expressed may
depend on different physical factors such as temperature, pH, composition of the
medium, etc. (De Graaf, 1988, 1990). This finding also implies that, in vivo,
E. coli do not necessarily express the same repertoire of fimbriae as in in vitro
conditions.
It was shown that fimbriae biosynthesis requires a complex and meticulously
regulated interplay of several genes and the polypeptides they encode (De Graaf,
1988, 1990; Lintermans et al., 1988a,b, 1991, 1995; Moon, 1990; Krogfelt, 1991;
Mouricout, 1991, 1997; Mouricout et al., 1995; Pohl et al., 1992, 1995; Imberechts
et al., 1992, 1997a; Hultgren et al., 1993; Van Driessche and Beeckmans, 1993; Khan
and Schifferli, 1994; Van Driessche et al., 1995, 2000; Gaastra and Svennerholm,
1996; Mol and Oudega, 1996; Smyth et al., 1996; Nataro and Kaper, 1998; Nagy
and Fekete, 1999; Soto and Hultgren, 1999; Sauer et al., 2000; Van den Broeck
et al., 2000). From the information available today it becomes obvious that at least
the following polypeptides are required. 1) A large outer membrane “pore” protein,
called the “usher”, which is implicated in the translocation of fimbrial subunits
Fig. 1. Genes involved in fimbriae biosynthesis form a cluster, which is generally composed of regulatory
genes (stippled), and genes encoding a variety of polypeptide chains. In this figure, the final polypeptides
are coloured in the same way as the genes encoding them. The number of genes in the cluster, as well as
their order and length, is different for different ETEC strains.
The ultimate fimbrial structure is composed of a large number of identical major subunits (white ovals),
minor subunits (lightly hatched spheres), and special minor subunits recognizing carbohydrates (the lectin
subunits, heavily hatched). The fimbrial structure is attached to the bacterial surface through a large, pore-
forming outer membrane (OM) usher protein (vertically striped cylinder). Another essential component of the
system is the boomerang-shaped chaperone (grey).
During biosynthesis of the above-mentioned series of polypeptides in the bacterial cytoplasm, the emerging
chains cross the cytoplasmic membrane (CM) through the general transport apparatus, See. Within the periplasmic
space, each of them is captured by a single chaperone molecule. This results in fimbriae subunits reaching an
assembly-competent state, also preventing them from premature aggregation. The chaperone–subunit complexes
travel towards an assembly site comprising an usher protein. Once arrived there, the chaperone–subunit complex
falls apart, the subunit being transported through the pore and being incorporated in the growing fimbrial structure,
and the chaperone being recycled. Minor subunits are required to initiate fimbriae formation, they also control the
length of the fimbrial structure, and they are implicated in growth termination. Usually, carbohydrate recognition
is due to the presence of another class of minor subunits (the lectin subunits). Lectin subunits often are located at
the tip of the fimbrial structure, in other ETEC strains they occur at regular distances along the whole fimbriae.
Occassionally, however (as is the case in K88 fimbriae), all major subunits display lectin activity.
Subunits that are not successfully trapped by chaperone molecules misfold and aggregate in the periplasm.
These non-functional entities activate the Cpx system that is composed of a CpxA kinase embedded in the
cytoplasmic membrane and a CpxR response regulator. The latter responds to the trigger from the misfolded
chains in several ways, one of which is the induction of expression of periplasmic proteases such as DegP,
which will degrade the misfolded subunits.
The fimbrial biosynthetic pathway is indicated with dotted arrows. The activation pathway involing Cpx
and the degradative pathway are respectively shown wth dashed and full line arrows.
across the outer membrane, and also serves as a mould upon which fimbriae
polymerization occurs. 2) A multifunctional periplasmic protein involved in stabi-
lizing non-polymerized fimbrial subunits and in transporting subunits from the inner
to the outer membrane. This protein obviously acts as a chaperone (see e.g. Edwards
et al., 1996; Mol et al., 1996a,b; Soto and Hultgren, 1999; Normark, 2000). 3) The
major fimbrial subunits that build up the “body” of the fimbriae. These subunits are
the most prominent polypeptide seen on sodium dodecyl sulphate (SDS)-gels after
electrophoresis of purified fimbriae. In some fimbrial systems, such as F2, F4, F5
and others, the major subunits display carbohydrate-binding activity. 4) Minor fim-
brial subunits, which may be involved in initiation and termination of subunit poly-
merization, in regulation of the extent of fimbriation, in determining the length of
fimbriae, in carbohydrate-binding, etc.
Assembly of the fimbriae structures described in table 1 follows the highly conserved
so-called “chaperone–usher pathway”, which is schematically depicted in fig. 1 (see e.g.
Soto and Hultgren, 1999; Normark, 2000; Sauer et al., 2000, and references therein). The
model is essentially based on investigations performed with both F1 and uropathogenic
Pap fimbriae. It can be used as a working hypothesis for the other systems.
Newly synthesized subunits are picked up by the chaperone as soon as they
emerge from the inner cytoplasmic membrane through Sec, the general secretion
apparatus. Two subfamilies of chaperones have been described in literature (Hung
et al., 1996), named FGS and FGL, the former being involved in the assembly of
fimbriae with a rod-like architecture (e.g. the thick and rigid F1 and F6 fimbriae, as
well as the thinner and more flexible F4, F5, and F17 fimbriae), the latter being
involved in biogenesis of atypical structures such as very thin fimbriae or afimbrial
adhesins. The chaperone molecules facilitate the release of nascent fimbrial subunits
from the inner membrane, they mediate folding of these subunits into an assembly-
competent shape, and they protect them from premature oligomerization in the
periplasmic space. A unique mechanism of so-called donor strand complementation,
whereby the chaperone donates a β-strand that fits within a groove of the nascent
subunit, was shown to form the basis of the action of these chaperone molecules
(Barnhart et al., 2000). The chaperones further deliver the subunits to the usher,
which is embedded within the outer membrane of the bacterium. The usher is a
pore-forming molecule built up of several protein subunits, presumably presenting
extensive regions towards the periplasm ready to interact with incoming chaperone–
subunit complexes. All fimbriae grow from top to bottom, as more and more
subunits are being delivered and handed over to the “chaperone–usher pathway”.
The incoming subunits pass through the usher’s pore, and get packaged into their
final structure when reaching the bacterial surface. This packaging event was shown
to involve donor strand exchange, whereby the N-terminal extension of the incom-
ing subunit (previously capped by the chaperone) displaces the chaperone’s β-strand
and inserts itself within the groove of the subunit that was most recently incorpo-
rated in the fimbrial structure (Barnhart et al., 2000).
Besides being a pore, the usher is also supposed to play a more active role in fim-
briae formation. Indeed, the usher seems to be able to discriminate between various
incoming chaperone–subunit complexes. This would result in trafficking these
incoming subunits in the right order. This order is assumed to be dictated both by
kinetic parameters and by subunit–subunit surface complementarity. In this respect,
distinct fimbrial minor subunits are thought to have unique structural determinants
required to either initiate or terminate fimbrial growth.
In the absence of chaperone molecules, fimbrial nascent subunits get misfolded
and aggregate in the periplasmic space, thereby activating the Cpx signalling system
that consists of two components, CpxA (a membrane-bound kinase) and CpxR
(a response regulator) (Sauer et al., 2000). Phosphorylation of the latter results in
the induction of a variety of genes encoding periplasmic folding factors on the one
hand, and periplasmic proteases (e.g. DegP) on the other hand.
4. THE POTENTIAL USE OF ENTEROTOXIGENIC E. COLI

FIMBRIAE AS VACCINE COMPONENTS
It is now generally accepted that attachment of ETEC to the intestinal mucosa is an
initial but essential step in pathogenesis. Consequently, any strategy that prevents
attachment will interfere with the development of disease. Because of their size,
architecture and extracellular expression, fimbriae can easily be obtained in a highly
purified state and in appreciable quantities to be used as vaccine components.
The purification procedure generally consists of a solubilization step, ammonium
sulphate precipitation, and, if necessary, eventually followed by gel filtration or ion-
exchange chromatography (Van Driessche et al., 1993, 2000; Van den Broeck et al.,
1999a) (see scheme 1). It should be noticed that the experimental conditions used
for the solubilization may critically affect the following steps required to achieve
Scheme 1. Flow sheet describing the purification procedure for enterotoxigenic E. coli fimbriae.
successful purification. For example, sonication of the bacterial suspension com-

pletely disrupts the cells and sets free all cytoplasmic proteins as well, some of
which may later be difficult to remove. Similarly, heat shock of the bacterial sus-
pension can give quite different degrees of homogeneity of fimbrial lectins after
ammonium sulphate precipitation, even for fimbriae isolated from the same strain
but grown in different conditions. A gentle way to solubilize fimbriae is by shearing
force using a Waring blender or a similar device. Upon using this technique,
fimbriae released from the cells are mostly only slightly contaminated by other
proteins, and the fimbriae can easily be recovered in very pure form using low con-
centrations of ammonium sulphate that will precipitate fimbriae from solution.
Using this procedure, we succeeded, for example, in obtaining F5, F17, F41, and
F18 fimbrial preparations that are completely devoid of contaminating proteins (Van
Driessche et al., 1993). This procedure could even be used to isolate separately F5
and F41 fimbriae from an E. coli strain expressing both types of fimbriae at the same
time, using differential ammonium sulphate precipitation.
Purified fimbriae have proven to be strong immunogens, and humoral antibodies
are readily generated in rodents, chickens and farm animals. Already in the mid
1970s, Rutter et al. (1976) described the antibacterial activity of colostrum of sows
that had been vaccinated with F4 fimbriae. These authors further showed that this
activity is due to the presence of anti-adhesive F4 antibodies. Similarly, Sojka et al.
(1978), Nagy et al. (1986), and Acres et al. (1979) showed that respectively newborn
lambs, piglets and calves are successfully protected by colostral antibodies raised by
vaccination of dams with F5 fimbriae during pregnancy. Colostral antibodies
directed against F6 fimbriae were found by Lösch et al. (1986) to passively protect
piglets against infection by F6 positive E. coli. These examples convincingly
demonstrate that newborns can successfully be protected from being colonized by
pathogens in the small intestine by maternal antibodies secreted in colostrum and
milk upon vaccination of dams, during pregnancy, with fimbriae isolated from the
challenging E. coli. It should be kept in mind, however, that many strains express
more than just one type of fimbriae. The investigations of Contrepois and Girardeau
(1985) and Runnels et al. (1987) showed that in these cases newborns will only be
successfully protected upon suckling colostrum and milk when the dams had been
vaccinated with a mixture of all fimbriae involved. These studies thus clearly
demonstrate the urgent need for retrieving new adhesins, or variants of existing
fimbriae that might appear in the population.
According to Moon and Bunn (1993), the successful protection of newborns that
is achieved upon parenteral vaccination of pregnant cattle, sheep and pigs can be
explained by the facts that: 1) most ETEC infections occur soon after birth, when
colostral and milk antibody titres are high; 2) only a limited number of fimbrial types
are important in farm animals; 3) fimbriae are good immunogens, being present at the
bacterial surface; 4) fimbriae are implicated in an initial but essential step in the devel-
opment of disease, i.e. in the attachment of the pathogens to the mucosal surfaces.
The prophylactic effect of various inactivated E. coli vaccines in the control of

pig colibacillosis has been investigated under large-scale farm conditions by Osek
et al. (1995). These investigators immunized 2472 pregnant sows with eight differ-
ent vaccines containing E. coli fimbrial adhesins and adjuvants. Upon considering
the general health status of the newborn pigs, the percentage of piglets with diar-
rhoea and of dead piglets, as well as the mean body weight gain of weaned piglets,
it was found that vaccination had an overall positive effect, although the vaccines
tested differed in their protective effect. Best results were obtained when pregnant
sows were immunized with a vaccine containing F4, F5 and F6 fimbriae and the
B-subunit of the LT enterotoxin.
At the time of weaning, a new period of high sensibility to ETEC starts because
of the lack of protective maternal antibodies, changes in the diet composition, stress,
new environmental conditions, etc. Two approaches have been used to treat or pre-
vent post-weaning diarrhoea immunologically, namely by active immunization in an
attempt to provoke the secretion of protective antibodies at the level of the intestinal
mucosa, and by passive immunization using egg yolk antibodies. Protection of
piglets against post-weaning diarrhoea was described by Alexa et al. (1995) who
used a combination of parenteral and oral vaccination against ETEC expressing F18
fimbriae. When piglets received an intramuscular injection the day before weaning
and were treated perorally with a live but non-toxic E. coli strain expressing the
same F18 fimbriae one day after weaning, they were protected against a subsequent
challenge with the virulent strain. Field trials confirmed the protective effect of this
immunization scheme. Bianchi et al. (1996), on the other hand, reported that par-
enteral immunization of mice or piglets with E. coli expressing F4 fimbriae or with
purified F4 fimbriae is ineffective in inducing protective immunity at the mucosal
level against a subsequent challenge with live bacteria. These authors reported that
this immunization procedure might even be detrimental by inducing a state of
suppression. Oral vaccination with live bacteria expressing F4 fimbriae induces an
enteric immune response, while killed bacteria were without effect.
Using rabbits as a model, Reid et al. (1993) observed that the colonization fac-
tor F3 isolated from ETEC, when incorporated in biodegradable polymer capsules,
is immunogenic when administered intradermally. After vaccination, Peyer’s patch
cells responded by lymphocyte proliferation to in vitro challenge with F3, and B cells
secreting specific anti-F3 antibodies were detected in the spleen of vaccinated
animals. In human volunteers, the studies of Tacket et al. (1994) showed that F3
encapsulated in biodegradable microspheres stimulated the secretion of s-IgA
anti-F3 in the jejunal fluid. Some volunteers were found to be protected against
challenge with a pathogenic strain expressing F3 antigens. In pigs, however, neither
microencapsulated ETEC nor their isolated fimbriae were found to induce serum
antibodies, or to reduce the intestinal colonization (Felder et al., 2000).
In a series of elegant investigations, Van den Broeck and associates (1999a,b,c,
2000, 2002) showed that purified F4 fimbriae induce both a systemic and an intes-
tinal mucosal immune response in just-weaned pigs that express F4 receptors, and
consequently are susceptible to E. coli F4 infections. No response to the orally

administered F4 antigen could be evidenced in F4 receptor-negative animals. These
observations made these researchers conclude that the expression of specific F4
receptors by the enterocytes is a prerequisite for purified F4 fimbriae to induce an
immune response upon oral immunization. The secretion of IgA antibodies by the
intestinal mucosa of F4 receptor-positive animals prevented a subsequent coloniza-
tion by E. coli F4. The results obtained open new avenues to use oral vaccination for
the protection of animals from being infected by E. coli (and probably other
pathogens that colonize the intestine following initial binding to mucosal receptors
via surface adhesins) that induce post-weaning diarrhoea.
Intestinal infection with F18-expressing E. coli was shown by Imberechts et al.
(1997a) to induce protection against repeated infections with E. coli expressing either
the homologous or the heterologous fimbrial variant of F18 (F18ab or F18ac). This
protection was ascribed by Sarrazin and Bertschinger (1997) to IgA secretion at the
level of the intestinal mucosa. Further studies by Bertschinger et al. (2000) revealed
that oral vaccination of piglets with a fimbriated E. coli F18 strain 10 days before
weaning successfully protected the animals against a subsequent challenge with a
homologous strain, but not with a heterologous strain. When non-fimbriated E. coli
F18 were used for the vaccination, no protective immunity developed. Upon study-
ing the antibody responses in newly weaned pigs following infection with an ETEC
expressing F4 or a verotoxigenic E. coli (VETEC) expressing F18 fimbriae,
Verdonck et al. (2002) reported that F4+ ETEC rapidly colonized the intestine and
provoked a fast F4 specific mucosal immune response, while the VETEC express-
ing F18 fimbriae colonized the intestinal mucosa slower, and also made the animals
respond slower in developing an F18 specific mucosal immune response. The
authors argued that, in view of the susceptibility of piglets to infection soon after
weaning, a quick response at the level of the mucosa is required. In the case of infec-
tion by F4+ E. coli, the immune response already occurred 4 days after infection,
possibly as the result of the adjuvant effect of LT produced by the challenging strain.
Because of the slow colonization and the retarded immune response provoked by the
VETEC F18-producing strain that lacks LT, the same authors suggested that the
immune response might be accelerated by using LT enterotoxin as an adjuvant.
From the examples given above it becomes clear that protective immunity at the
level of the intestinal mucosa can be achieved by oral vaccination with E. coli
expressing fimbriae, and/or by purified fimbriae (as is the case for F4) on condition
that the intestinal receptors that are recognized by the fimbriae are present at the
surface of the enterocytes. In order to deal with post-weaning diarrhoea, instead of
using active immunization schemes, passive immunization has also proven to
successfully prevent animals from being infected by ETEC. In particular, yolk from
the eggs of immunized laying hens was shown on several occasions to be a most
appropriate source of protective antibodies. As will be shown below, egg yolk anti-
bodies can be used to tackle neonatal diarrhoea in cases where colostrum or milk
contain too low specific antibody titres, or no protective antibodies at all. Indeed, it
is known that upon immunization, laying hens produce high titres of specific anti-
bodies in the egg yolk. These antibodies are called IgY, and can readily be isolated
from the yolk if required. Passive immunization experiments were already being per-
formed in 1992 by Yokoyama and co-workers (1992). They showed that colostrum-
deprived piglets were successfully protected against fatal enteric colibacillosis using
powder preparations of specific antibodies obtained by spray-drying the water-soluble
fraction of yolk from the eggs of laying hens that had been immunized against F4,
F5 and F6 fimbriae. Scanning electron microscopy of intestinal sections showed that
animals fed with egg yolk powder containing specific anti-fimbriae IgY were devoid
of adhering E. coli, while in control animals adherent bacteria could be evidenced.
Moreover, when the specific yolk anti-fimbriae antibodies were removed from the
preparations using a fimbrial immunosorbent, the protective activity of the yolk
preparations was significantly reduced, proving that the protective effect is indeed
due to the anti-fimbriae antibodies. Similarly, O’Farrelly et al. (1992) showed that
rabbits were protected from developing diarrhoea when fed egg yolk from hens
previously immunized with E. coli that produced heat-labile enterotoxin and colo-
nization factor antigen-I. Ikemori et al. (1992) reported that neonatal, colostrum-
deprived calves could be protected against ETEC-induced diarrhoea by the protein
fraction (containing anti-fimbriae antibodies) of egg yolk from hens vaccinated with
heat-extracted antigens from F5-fimbriated E. coli. Whereas control calves that only
received milk with egg yolk powder from non-immunized hens suffered from severe
diarrhoea and died within 72 h after challenge, the calves fed milk containing egg
yolk powder from immunized hens all survived and had good weight gain, although
transient diarrhoea occurred. Independently, Imberechts et al. (1997b) and Yokoyama
et al. (1997) described the protective effect of hen egg yolk containing specific anti-
bodies against F18 fimbriae. It was also shown that the yolk antibodies interfere
with the attachment of the E. coli to the intestinal mucosa. Similar results were
reported by Zuniga et al. (1997), and these authors also concluded that the oral
application of egg antibodies is a promising approach for the prevention of infec-
tious diseases of the digestive tract. The prophylactic effect of yolk antibodies
directed against F4, F5, F6 and rotavirus has also been reported by Erhard et al.
(1996). When egg yolk containing these antibodies was included in the diet of
piglets, a significant decrease was observed in the number of piglets with diarrhoea,
as well as a decrease in the severity of the symptoms, when compared to piglets fed
on a diet containing yolk of non-immunized hens, or without any egg yolk at all.
Using in vitro competition and displacement tests, Jin et al. (1998) showed that
egg yolk antibodies inhibit the attachment of E. coli F4 to piglet small intestinal
mucus, but that displacement of attached E. coli is not possible. The in vivo experi-
ments of Marquardt et al. (1999) on the other hand revealed that E. coli F4 can
induce neonatal diarrhoea in 3-day-old piglets, and that animals that were treated
with egg yolk antibodies were cured within 24 hours, while animals that received
egg yolk powder of non-immunized hens continued to suffer from diarrhoea, most of
them dying from the infection. Also 21-day-old weaned piglets, fed with egg yolk
containing specific fimbriae antibodies, successfully survived the infection, whereas
control animals developed severe diarrhoea, suffered from dehydration, and some
animals eventually died. These authors further reported that, in a field trial, the incidence
and severity of diarrhoea of 14–18-day-old weaned piglets fed egg yolk antibodies
were much lower than in piglets fed a commercial diet containing an antibiotic.
From this study, it is convincingly clear that egg yolk antibodies are able to protect
both neonatal and weaned piglets from being infected by ETEC.
Carlander et al. (2000) favourably advocated the use of IgY to establish passive
immunity against gastrointestinal pathogens such as bacteria and viruses in both
humans and animals, because these antibodies do not activate mammalian comple-
ment or interact with mammalian Fc receptors that can mediate an inflammatory
response in the gastrointestinal tract. These authors also considered the risk of toxic
side effects very limited, since eggs are part of the diet.
Although it might be expected that, because of the intensive use of fimbrial
vaccines, a rapid selection would occur in favour of previously rather infrequently
occurring fimbrial antigens, this does not seem to be the case (Moon and Bunn, 1993).
5. ANTI-ADHESION THERAPY BASED ON RECEPTOR ANALOGUES

As mentioned above, the susceptibility of animals and humans towards the colonization
of the small intestinal mucosa depends on the presence of mucosal receptors (glyco-
proteins and/or glycolipids) that are recognized by surface lectins of E. coli. In several
instances, the temporal susceptibility of the host to infection could be correlated with
the presence of soluble receptors in the intestinal lumen and mucus. Since the oligosac-
charides of the bacterial lectin receptors are the structures that are recognized,
researchers have attempted to use receptor analogues to prevent binding of E. coli to
the intestinal surface by blocking the carbohydrate-binding sites of the bacterial lectins.
Already in 1979, Aronson and co-workers (1979) showed the potential of this approach
by demonstrating that methyl-α,D-mannopyranoside is able to prevent the in vivo col-
onization of the urinary tract of mice by uropathogenic E. coli expressing F1 fimbriae.
Similarly, Neeser et al. (1986) used short oligomannoside-type glycopeptides
of ovalbumin and oligomannoside-type glycopeptides derived from legume
storage proteins to inhibit the agglutination of erythrocytes caused by E. coli
expressing F1 fimbriae. The studies of Mouricout and co-workers (Mouricout, 1991,
1997; Mouricout and Julien, 1987; Mouricout et al., 1990, 1995) boosted the investi-
gations on the applicability of receptor analogues by showing that glycoprotein glycans
obtained from bovine plasma protect colostrum-deprived newborn calves against lethal
challenge by E. coli F5. In vitro investigations by Sanchez et al. (1993a,b) conclusively
demonstrated that glycoproteins such as fetuin, ovomucoid, submaxillary gland mucin,
hen egg white glycoproteins as well as plasma glycoproteins, especially those of cow,
prevent E. coli F17 from attaching to mucus and brush border membranes isolated from
different parts of intestines from calves. Subsequent in vivo studies of Nollet et al.
(1996) revealed that non-immune plasma powders from cows protected newborn and
colostrum-deprived calves from developing diarrhoea upon infection with E. coli
expressing F5 or F17 fimbriae. All control calves infected with E. coli expressing either
F5 or F17 fimbriae developed signs of lethargy, loss of appetite, fever, severe watery
diarrhoea and dehydration from the first day after infection, and eventually died
between 1 and 7 days after infection. In one control calf infected with E. coli F17+, the
challenge strain could be isolated not only from the small and the large intestine, but
also from the spleen and liver, indicating that the strain used was septicaemic.
Septicaemia was never observed with E. coli F5. Unlike the controls, animals that were
fed milk supplemented with 25 g/litre plasma powder remained completely healthy,
showed no loss of appetite and did not develop diarrhoea during the first 5 days after
infection with either E. coli F17 or E. coli F5. When the dose of plasma powder was
reduced to10 g/litre milk, mild disease symptoms developed in animals infected with
E. coli F17 cells, but none of the animals developed septicaemia. At this lower dose of
plasma supplementation, the E. coli F5 infected calves on the other hand got fever and
also developed more severe diarrhoea. Remarkably, unlike E. coli F5 infected calves,
animals challenged with E. coli F17 continued to excrete the pathogen, indicating that
these bacteria might colonize the colon, which is not inhibited by plasma powder. After
some time, this strain behaves like a commensal that is no longer harmful for the
carrier. All these studies show that plasma glycoproteins are perfectly able to act as
receptor analogues for both F17 and F5 fimbriae, since the plasma preparations used
were shown to be devoid of antibodies directed against these fimbriae. Similar results
were obtained by Deprez et al. (1996), who used swine plasma components as adhesin
inhibitors in the protection of piglets against E. coli enterotoxaemia. It was shown that
the amount of plasma powder to be administered can be reduced when using plasma
from pigs that had been vaccinated with fimbriae isolated from the challenge strain. The
investigations of Nollet et al. (1999) also conclusively show that non-immune porcine
plasma powder protects just-weaned piglets against infection with an E. coli strain
expressing F18 fimbriae isolated from a clinical case of oedema disease. The best
results were obtained when the piglets received a daily supplementation of feed with
45 g plasma powder. When the amount of plasma powder was increased to 90 g per
day, the animals developed diarrhoea, which was ascribed to biogenic amines released
from excessive protein in the diet.
As well as plasma, hen egg white and milk have been shown to be rich sources of
oligosaccharides that can be applied in receptor analogue therapy. In 1983, Wadström
and co-workers (1983a,b) reported milk to be an excellent source of oligosaccharide
structures that mimic intestinal receptors for E. coli adhesins. Also milk fat globule
membranes were shown by Schroten et al. (1992, 1993) to have great potential for
use as a source of receptor analogues. It should also be kept in mind that, besides
oligosaccharides and milk fat globules, milk also contains other antimicrobial active
components such as lysozyme, the lacto-peroxidase system, macrophages, as well as
secretory IgA. The protective action of IgA is not exclusively attributed to its specific
antigen-binding properties, but also to its covalently linked oligosaccharides that may
interact with bacterial surface lectins (Wold et al., 1990, 1994). Consequently, IgA
displays antibacterial activity not only because of its action as an antibody, but also
as a glycoprotein that can prevent intestinal mucosal colonization by blocking sur-
face lectins of bacteria. Other glycoproteins present in milk, such as lactoferrin, may
be acting as attachment inhibitors or as inhibitors of toxins that display carbohydrate-
binding properties (Shida et al., 1994; Newman, 1995).
Lindahl (1989) showed that E. coli expressing F41 and F5 fimbriae bind glycopro-
teins present in porcine and bovine colostrum, the former being a richer source. Non-
immunoglobulin fractions of human milk and colostrum were shown by Ashkenazi and
Mirelman (1987) to inhibit enterotoxigenic E. coli expressing F2 and F3 from attach-
ing to the intestinal tract of guinea pigs. No effect was noticed on the binding of E. coli
expressing F1 fimbriae. Since the inhibitory activity was not affected by boiling or by
trypsin digestion, but was sensitive to metaperiodate oxidation, the authors concluded
that carbohydrate residues of the inhibitor were involved. Similar results were obtained
by Holmgren et al. (1981), who showed that the non-immunoglobulin fraction of
human milk and colostrum inhibited the haemagglutination provoked by E. coli
expressing F2, F3, or F4 fimbriae, as well as the haemagglutination by Vibrio cholerae.
Moreover, these authors reported that the non-immunoglobulin fraction interfered with
binding of cholera toxin and the LT enterotoxin to GM1 ganglioside. Consequently, the
non-immunoglobulin fraction of milk and colostrum may contribute to the protective
effect of milk against enteric infections. Giugliano et al. (1995) identified lactoferrin
and free secretory components (fsc) in human milk to be inhibitors of E. coli F2 adhe-
sion, as monitored by haemagglutination. At least in the case of F1 fimbriae, Teraguchi
et al. (1996) showed that inhibition of haemagglutination is due to the glycan part of
bovine lactoferrin. In addition to inhibiting bacterial attachment, some glycoconjugates
from milk may also interfere with binding of enterotoxins to their intestinal receptors.
From the examples given above, it has been shown that receptor analogues can
successfully be used to protect calves and pigs against neonatal and post-weaning diar-
rhoea by interfering with the colonization of the intestinal mucosa by these pathogens.
On the basis of the results obtained in animals, the use of carbohydrates and oligo-
saccharide drugs to prevent the attachment of pathogens to human tissues, and/or to
revert attached microorganisms, opens new perspectives for combating diseases that
are initiated as a result of lectin-mediated attachment. The major advantage of using
carbohydrate-based anti-adhesion drugs, when compared to antibiotic or chemothera-
peutic approaches, is to be found in the fact that saccharides are not bactericidal and,
consequently, selection of resistant strains is unlikely to occur (Zopf and Roth, 1996;
Sharon and Ofek, 2000). At present, high production costs of obtaining the required
oligosaccharides may be a limiting factor, but the further development of techniques
that allow the enzymatic tailoring of anti-adhesive oligosaccharides could make them
available at reasonable costs (Sharon and Ofek, 2000).
6. IN VIVO MODIFICATION OF BACTERIAL LECTIN RECEPTORS

An alternative to blocking the E. coli surface lectins might consist of interfering with
the lectin-binding properties of host receptors for bacterial lectins. To achieve this goal,
Pusztai and co-workers (1993a,b,c) investigated the effect of the non-toxic mannose-
specific lectin from Galanthus nivalis, and showed that it prevented the overgrowth of
the small intestine of rats by F1 fimbriated E. coli. Plant lectins are indeed potential
candidates to block intestinal mucosal E. coli lectin receptors, because most plant
lectins are rather resistant to proteolytic degradation in the stomach and small intestine,
and, as such, can bind to glycoconjugates present in the intestinal mucosa. It should be
kept in mind, however, that many plant lectins, when present in appreciable quantities,
can adversely affect the structure and functional properties of the small intestine
(Pusztai et al., 1993a,b,c). Also, because many plant lectins themselves are glycopro-
teins, they could act as “neo-receptors” for bacterial surface lectins. Moreover, in view
of the fast turnover of the small intestinal epithelium, and also because plant lectins,
once bound to intestinal receptors, are often internalized, a constant supply of huge
amounts of plant lectin molecules, for instance present in food and feed, would be
required, making this approach hardly feasible for prophylactic or therapeutic purposes.
A more promising approach to, at least temporarily, modify intestinal glycocon-
jugates might be offered by the studies of Mynott et al. (1996) and Chandler and
Mynott (1998). These investigators showed that bromelain, a proteolytic enzyme
present in pineapple juice, can prevent attachment of E. coli F4 to the small intes-
tinal mucosa, without causing adverse effects. Although this approach deserves fur-
ther investigation, for other E. coli fimbrial systems, possible adverse effects should
be carefully looked for. Indeed, the glycoconjugates that we designate as “E. coli
lectin receptors” have of course other roles to fulfil in the intestine, bacteria just ben-
efit from their presence to attach to and colonize the intestinal lining. Consequently,
proteolytic modification of these glycoconjugates could eventually interfere with
the endogenous function(s) of these intestinal receptors.
7. PROBIOTICS: TOWARDS THE USE OF HEALTH PROMOTING

BACTERIA TO COMBAT PATHOGENS
As mentioned earlier in this chapter, because of the ever increasing explicit demand
of the consumer for meat from animals that have not been treated with antibiotics,
but mainly because of the concern of the appearance of bacteria that are resistant or
even multiresistant towards currently used antibiotics, the use of probiotics to
achieve “organic food” has gained a lot of popularity during recent years. Probiotics
can be described as microorganisms, often Lactobacillus spp., Streptococcus spp.,
and Bacillus spp., that are administered either on their own, or included in food or
feed, in order to achieve a favourable intestinal flora and to prevent digestive disor-
ders and/or to increase performance.
Kyriakis and co-workers (1999) reported that viable spores of Bacillus licheniformis
or Bacillus toyoi added to the feed can be used to successfully control post-weaning
diarrhoea caused by enterotoxigenic E. coli. Moreover, these probiotics improved the
general health status and performance of the piglets considerably. The beneficial effect
of probiotics may be due to competition with pathogens for available receptors to estab-
lish themselves in the small intestine, but also to their possible immunomodulatory
effects. Herias and co-workers (1999) reported that in gnotobiotic rats, a mannose-
binding Lactobacillus plantarium strain not only competes with F1-fimbriated E. coli
for colonization sites, especially in the small intestine, but also influences both systemic
and intestinal immunity.
Several groups investigated the effect of Lactobacillus spp. on the colonization
of the small intestine by ETEC. Spencer and Chesson (1994) reported that some
Lactobacillus strains isolated from the gastrointestinal tract of piglets at the time of
weaning attach to enterocytes. On the basis of the number of bacteria that attached
to the enterocytes, strongly and non-to-weakly attaching isolates could be discrimi-
nated. However, even the strongly adherent strains had no effect on the attachment
of ETEC. Fimbriated E. coli F4 were shown to co-aggregate with some
Lactobacillus strains. The same authors concluded that the Lactobacilli under inves-
tigation could have a beneficial in vivo effect by aggregating pathogens and pre-
venting them from binding to the mucosa. Rojas and Conway (1996) showed that
Lactobacilli colonize the mucus layer of the small intestine. It was further shown
(Ouwehand and Conway, 1996) that Lactobacillus fermentum 104R releases a com-
pound (or compounds) in the culture fluid that inhibits (inhibit) the adhesion of
E. coli F4. On the basis of their properties, these compounds were identified as cell
wall fragments released from dead cells in the culture fluid. Upon examining the
effect of including Lactobacillus reuteri BSA131, isolated from pig faeces, in the
feed of 1-month-old piglets, Chang et al. (2001) reported a beneficial effect on
liveweight gain and feed conversion, as well as a decrease in the number of entero-
bacteria in the faeces, and they considered the strain as a potential probiotic for
piglets after weaning.
In addition to Lactobacillus species, also Enterococcus faecium 18C23 has been
shown by Jin et al. (2000b) to inhibit in vitro the attachment of E. coli F4 to the
mucus of the small intestine of piglets. These authors showed that the mucus recep-
tor for Enterococcus faecium is a glycoprotein, since treatment of the mucus with
metaperiodate decreased the adhesion of the bacteria. The inhibitory effect on
E. coli F4 attachment is most probably not the result of competition for the same
mucus receptors, since treatment of mucus with pronase reduced adhesion of E. coli
F4 but increased the adhesion of the probiotic bacteria. From these results, the
authors concluded that the inhibition of adhesion of E. coli F4 by E. faecium is due
to steric hindrance. According to Jin et al. (2000b), also the spent culture super-
natant contains some substances that might contribute to the inhibitory effect
displayed by the E. faecium cells.
In conclusion, the beneficial effect of probiotics in protecting the host from intes-
tinal disorders and/or colonization of the intestine is supposed to be due to one or a
combination of the following effects (Rolfe, 2000): 1) stimulation of the immune
system, possibly by cell wall fragments that act as adjuvants; 2) competitive inhibi-
tion for bacterial adhesion sites on the intestinal surface; 3) degradation of intestinal
toxin receptors; 4) production of inhibitory substances such as bacteriocins, hydro-
gen peroxide or organic acids. Some organic acids, e.g. lactic acid and others, were
indeed shown by Tsiloyiannis et al. (2001) to reduce both the incidence and sever-
ity of post-weaning diarrhoea caused by ETEC in piglets. Bacteriocins produced by
lactic acid bacteria, on the other hand, are a large group of small antimicrobial
peptides varying in spectrum of activity and molecular structure (De Vuyst and
Vandamme, 1994; Nissen-Meyer and Ness, 1997).
8. CONCLUSIONS
Enterotoxigenic E. coli are an important group of pathogens that cause diarrhoea in
humans and livestock. These bacteria are able to colonize the small intestinal epithe-
lium by virtue of expressing adhesins (lectins) in the form of long proteinaceous
appendages that protrude from their surface, and that are called fimbriae. Fimbriae
are multimeric structures consisting of several types of subunits, referred to as major
and minor subunits. In most instances, it is a special type of minor subunit that
displays carbohydrate-binding activity. Sometimes, also the major subunits can bind
saccharides, such as is the case in F4 fimbriae. On the basis of their carbohydrate
specificity, fimbriae are classified as F1 (type-1) fimbriae that cause haemaggluti-
nation inhibitable by mannose and mannosides, and host-specific fimbriae that
cause haemagglutination not inhibitable by mannose or mannosides. For some
fimbriae such as F6, no erythrocytes have been described that are agglutinated either
by isolated fimbriae or by the E. coli expressing them. The expression of non-
haemagglutinating fimbriae can be investigated by in vitro adhesion systems using
for example enterocytes, Eupergit, or another solid support to which glycoproteins
or glycolipids have been linked or adsorbed.
Whereas the overall structure, the expression and its regulation of several impor-
tant fimbriae, as well as the genes that encode them, have been studied in great detail,
our knowledge of the structure of the fimbriae receptors present at the surface of the
enterocytes, and often in the mucus layer that covers mucosal surfaces, is lacking.
However, whether mucosae will be colonized by ETEC depends not only on the
expression of fimbriae, but also on the presence of receptors, i.e. glycoproteins or
glycolipids. It is the oligosaccharides of the latter macromolecules that are recognized
by the fimbriae. Tissue-specific, developmentally regulated and species-specific glyco-
sylation explains the tissue-dependent, age-dependent and species tropism of ETEC.
During the past decades, antibiotics have successfully been used to combat ETEC
infections. However, the intensive use of these chemicals, not only as therapeutics but
also as growth promoters, has finally cumulated in the selection of E. coli strains
that are resistant to the action of these compounds. As such, we will have to face in
the future the situation where it will be impossible to cure some bacterial infections
with antibiotics and, consequently, alternatives should become available. Also, the
attitude of consumers has changed quite dramatically. The demand for “organic food”
is not just a romantic reflex to go back to the practices of “good-old-days”, but is rather
the consequence of our growing concern about the quality and safety of food. Both the
appearance of resistant and even multiresistant bacteria, and consumer demand for
meat and meat products derived from animals not fed antibiotics are the driving force
in the search for alternatives to reduce antibiotic use as much as possible.
As mentioned earlier in this chapter, modern husbandry practices create the opti-
mal conditions to make animals susceptible to all kinds of infections and allow disease
to spread quickly. Besides management measures, several strategies are available
today that allow for the protection of neonates as well as animals at the age of
weaning and thereafter, from infection by ETEC-causing diarrhoea. Nature itself
has come up with solutions to prevent the colonization of the small intestine by
ETEC, where they find the best environment to reproduce massively and to release
their toxins in the immediate vicinity of the toxin receptors. In this chapter, several
strategies have been described, each of them illustrated with examples that show
how the colonization of the small intestine by ETEC can be prevented.
Neonates can successfully be protected by maternal antibodies directed against
E. coli fimbriae and secreted in colostrum and milk. This type of passive immu-
nization can easily be achieved by vaccinating dams during pregnancy with purified
fimbriae, sometimes in combination with the heat-labile toxin that acts as an adju-
vant. At the age of weaning and soon thereafter, animals can be passively protected
by egg yolk antibodies mixed in the feed. Eggs from hens immunized with fimbriae
are indeed an excellent source of protective antibodies for animals at an age where
active immunization does not provide adequate protection in the short term. More
recently, oral immunization with purified fimbriae such as F4 has been shown to
protect animals from post-weaning diarrhoea by stimulating an immune response at
the level of the small intestinal mucosa.
Since a successful attachment of ETEC to the mucosa is required for these
bacteria to cause disease, blocking the fimbrial carbohydrate sites by receptor ana-
logues is another alternative to combat ETEC-induced diarrhoea. This strategy,
called “receptor analogue therapy”, is still in its infancy, and further progress can be
expected only when we get a more detailed picture of the structure of fimbrial recep-
tors, and more particularly of the oligosaccharide sequences involved. Tailored syn-
thetic oligosaccharides should make it possible to obtain attachment inhibitors that
optimally fit the carbohydrate-binding sites of the fimbrial lectins, and consequently
bind with much higher affinity than the receptor analogues available today. Since
receptor analogues are not toxic for the bacteria, selection for resistance is highly
improbable.
Still another alternative to antibiotics is probiotics that can compete for ETEC
receptors, prevent pathogens from binding by steric hindrance, and/or stimulate the
mucosal immune system. Finally, temporal modification of fimbrial receptors by
proteolytic enzymes such as bromelain might prevent ETEC from binding to the
small intestinal mucosa.
Although this chapter has mainly dealt with diarrhoeagenic ETEC in pigs and
calves, it should be kept in mind that in the tropics ETEC diarrhoea can still be a
fatal disease in humans, mainly affecting young children. It is hoped that the know-
ledge gained from investigating ETEC in farm animals will be used to deal with
human ETEC.
Especially during the past decade, much progress has been made in understanding
the mechanisms that govern the attachment of ETEC to the intestinal mucosa and
several investigations have shown new ways to combat ETEC-induced diarrhoea.
Promising strategies that have been developed to prevent colonization of the small
intestine have to be proven valid and useful for more types of fimbriae than those
used until now. In particular oral vaccination to achieve local mucosal immunity, as
shown for F4 fimbriae, should be extended to other fimbrial systems. Optimal doses
of fimbriae to be used for vaccination, the effect of adjuvants, optimal time of vacci-
nation, etc., should further be analysed. A continuous search for possible new fim-
briae or non-fimbrial adhesins should be carried out in order to improve the
protective effect of existing vaccines. Also elucidation of the fine structure of fim-
briae receptor and the changes in the glycosylation pattern during development, and
further development of enzymatic synthesis procedures that allow the production of
tailored receptor analogues at a reasonable cost, will make the use of receptor ana-
logues as therapeutics more feasible. Further investigations are also required to find
out whether temporal enzymatic receptor modification is a safe and general way to
prevent intestinal infection. Finally, further proof of the often only presumed bene-
fits of probiotics, and their mode of action, deserve more attention.
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Index
Adhesin receptors, 159, 163 Bacillus spp., 382, 383, 386t, 388, 391, 396t, 400,
Adhesins, 159, 168 402, 404, 405, 408
Adhesion, 474, 476, 485, 487, 489, 490 Bacteria, 57, 60, 65, 69, 70, 79, 81, 83, 85, 86,
Adhesion-receptor, 360 88–96, 119
Advances in age, 106 Bacterial adhesins, 210, 215
Aeromonas hydrophila, 209, 222 Bacterial adhesion, 211, 359, 360
Aeromonas salmonicida, 209, 211, 214, 216, environmental factors, 210
221, 226, 227 mechanisms, 212
S-layer, 211 Bacterial flora
AFA (afimbrial adhesin), 174, 176 autochthonous, 217
Age, 112 stability, 217
Agglutination, 474, 485 Bacterial invasion, 218
Agrin, 296, 308 Bactericidal activity, 302
Ammonia, 60, 65, 69, 70 Bacteriocins, 419, 423, 424, 426, 427, 431, 433, 434
Amplified fragment length polymorphism (AFLP), 126 Bacteroides, 124, 126, 130, 131
Amplified ribosomal DNA restriction analysis thetaiotaomicron, 130
(ARDRA), 126 vulgatus, 124
Anaerobes, 25 Batch culture, 144
Anguilla japonica, 225 Bifidobacteria, 274–275
Antibiotic, 473, 484, 487, 488, 490, 491 Bifidobacterium, 126, 131
Antibiotics, 277, 278 Biochemical fingerprinting, 32
Antibody (-ies), 474, 481–484, 486, 494 Biochip, 136
Antibody-dependent cell-mediated cytotoxicity Biosynthesis (fimbriae), 473, 477, 479
(ADCC), 298 Blocker (attachment), 477
Antigen presenting cells (APC), 296, 299, 362 B-lymphocytes, 318, 319
Antimicrobial activity, 427 Bovine milk, 302, 305
Antwerp database, 130 Brevoortia patronus, 225
Apoptosis, 307 Bromelain, 488, 492
Appearance of ciliates, 54, 62, 63 Bronchus-associated lymphoid tissues (BALT), 295
Aquatic animals, 418, 423, 424, 427, 433, 434, 437 Brush border, 476, 477, 485
ARB, 130
Arctic, 75, 78–81, 86, 88, 90 Caco-2, 151
Arctic charr, 216 Caco-2 cells, 303
Associated immune cells, 361 Caecum, 86, 90, 91
Attachment, 472–477, 480, 481, 484, 487, 488, Calf, 473, 481, 484, 485, 487, 492
491, 492 Calves, 54, 56, 61–63, 65, 69, 70
Autoantigens, 357 Campylobacter, 114, 265–267
Autochthonous strains, 358 Campylobacter jejuni, 211, 16
Autochtonic microflora, 296 Capsule, bacterial, 210
Carbohydrate-binding, 476, 477, 479, 485, 487,
B cells, 357, 359, 369, 370t 490, 491
B cells (lymphocytes), 298 Carbohydrate fermentation, 55, 56, 65
Bacillus, 488 Carbohydrates, 60, 65
499
500 Index
Carnobacterium, 418, 420, 430, 431, 434, 442 Dynamic

Carnobacterium piscicola, 209 conditions, 146, 149
Carnobacterium spp., 390, 391, 393, 394, 396t, model, 142, 144, 148
403, 407
Celiac disease, 299 E. coli, 260–263
Cellulose, 60 EAF (EPEC adherence factor), 169, 171
Chaperone, 479 Edwardsiella ictaluri, 209, 211, 222, 226
Chemokines, 296, 299 pili, 211
Chlamydia, 127 Edwardsiella tarda, 209, 222
psittaci, 127 Egg yolk, 482–484, 491
CHO cells, 300 EHEC (enterohaemorrhagic E. coli), 169
Chromophore, 135 Electron microscopy, scanning (SEM), 211, 214
Ciliate fauna, 54, 55, 62, 63, 70 Electron microscopy, transmission (TEM), 211
Ciliates, 61, 79, 87, 89 Elemental ration, 107
Clostridia, 268, 269 EMBL, 130
Clostridium, 130 Enterobacteriaceae, 126
coccoides, 130 Enterobacteriaceae, 353, 355
leptum, 130 Enterococcus, 272, 420, 436, 489
perfringens, 127 Enterococcus sp., 225
Clostridium difficile, 114 Enterocyte, 474–477, 483, 489, 490
Clostridium perfringens, 108 Entero-mammary link, 302, 303
CNF (cytotoxic necrotizing factor), 173 Enteropathogenic, 363
Colibacillosis, 473, 482, 484 Enterotoxigenic, 473, 477, 480, 487, 489, 490
Coliforms, 27, 32, 33, 36, 42, 44 Enterotoxins, 160, 300, 473, 482–484, 487
Colon, 111 EPEC (enteropathogenic E. coli), 169
Colonization, 258, 259, 472, 477, 482–485, 487, Epithelial barriers, 361
489, 490, 491, 492 Epithelial cells, 361–363
Colonization factor, 482, 484 Epithelium, 472, 474, 476, 488, 490
Colostrum, 295, 305, 306, 481, 483–485, 487, 491 Epitopes, 369
Commensal microflora, 302 Erysipelothrix rhusiopathiae, 272
Common mucosal antigen, 360 Escherichia
Competitive exclusion, 275–277 coli, 125, 127, 133
ConA, 250 Escherichia coli, 210, 214, 218
Continuous model, 142 attaching and effacing, 108
Continuously Stirred Tank Reactor (CSTR), 145 enteroinvasive, 215
Cryptdins, 297 enteropathogenic (EPEC), 214
Cytochalasin D, 220, 222, 224 enterotoxigenic, 108
Cytokines, 296, 299–303, 367, 368 Escherichia coli (E. coli), 472, 474–477,
Cytostatic factor, 306 480–492
Cytotoxic effect (CTE), 300 Establishment of ciliates, 54, 61, 63, 65, 70
Cytotoxic T lymphocytes (CTL), 296 ETEC, 472, 473, 480, 481–485, 489, 490, 491
Cytotoxins, 300 ETEC (enterotoxigenic E. coli), 158
Dam, 481, 491 Eubacterium, 120
Dam’s milk, 104 rectale, 130
Delivery, 328–330, 333, 335, 341, 343, 346 Eupergit-C, 476, 490
Denaturing gradient gel electrophoresis (DGGE), Expression vector, 337, 338, 345
121, 128
Dendritic cells, 367 Factors affecting establishment of ciliates, 63
DGGE, 151, 152 FAE cells, 364, 365
Diagnosis, 177 Family Ophryoscolecidae, 57, 60
Diarrhoea, 41, 42, 114, 472, 473, 482, 483, 484, Fat globule, 486
486, 487, 489–492 Fc receptors (FcR), 305
Dicentrarchus labrax, 222 Fermenter, 150
Diet, and disease resistance, 226 Fermentative capacity, 34–36, 38, 39
Digestive tract, 4–6 Fibre degradation, 60
Disease control, 381, 383, 385, 404, 409 Fibrinogen, 214
DNA, 120–125, 129, 134 Fibronectin, 214, 222
Index 501
Fimbriae, 158 Growth rate, 67, 69

Fimbriae, 210, 262, 472–492 Gut-associated lymphoid tissues (GALT), 297, 299
E. coli type 1, 208, 210
E. coli type 4, 210, 214 Haemagglutination, 474, 487, 490
E. coli type 5, 210 Helicobacter infection, 103, 106
Fimbrial lectin, 473–477, 480, 485, 487, 488, Helper oligonucleotides, 133
490, 491 Hippoglossus hippoglossus, 221
Fish, 314–325, 386t, 389t, 395t, 396t Human, 473, 482, 485, 487, 492
FISH, 151 Humic compounds, 123
Fish larvae, 385, 394, 398, 399, 405 Hybridization, 121
Fish pathogens, 424, 431–433, 436, 442, 445 dot-blot, 121, 123, 131, 132
Fish, See Chapter 17 slot-blot, 121, 124
Flagella, 210, 211, 262 touch-blot, 124
Flexibacter maritimus, 211 whole cell-in situ, 121, 132
Fluorescence-in situ-hybridization (FISH), 121, 132
Fluorophore, 135 Ictalurus punctatus, 222, 226
Food conversion efficiency, 67, 68t IgA, 297–299, 302
Fry, 383, 385, 388, 391, 393, 395t, 399, 406 IgA, 482, 487
Fungi, 55, 57, 61, 79, 89 IgG1, 302
Fusobacterium, 130 IgM, 298
prausnitzii, 130 IgY, 484, 485
Immune complexes (Ic), 305
Gastrointestinal (GI) tract, 294, 296, 297, 299, Immune escape, 302
302, 303 Immune memory, 324
Gastrointestinal tract, 119–121, 123, 125, 136 Immune paradox, 305
Gastro-Intestinal Tract (GIT) Immune response, 354, 358, 361
metabolites/products, 142, 144, 145, 149, 152 Immunity, 5, 314–316
analysis of, 11 Immunization, 481–485, 491
Genbank, 130 Immunoglobulin A, 362
Genome sequencing, 227 Immunosuppression, 116
Genomic fingerprint, 125 “In-feed” vaccines, 180
GIT, 142 In vitro model, 143, 144, 150–152
bile, 144, 146, 149 Infection routes, 209, 223
conditions, 143 Infectious diseases, 108
crop, 143 Infectious haematopoietic necrosis (IHN)
digestive processes, 144 virus, 225
gastric emptying, 147, 149 Infectious pancreatic necrosis (IPN) virus, 225
gizzard, 143 Infectious salmon anaemia (ISA) virus, 225
monogastric animals, 144 Inhibitor (attachment), 474, 486, 487, 489, 491
morphology, 143 Integrin, 220
parameters Intergenic spacer regions (ISR), 122
composition, 150 Internal transcribed spacers (ITS), 127
digestive juices Intestinal microflora, 363, 370, 371
composition, 142 Intestinal mucosa, 472, 473, 480, 481–484, 485,
residence time, 142 487–489, 491, 492
secretion, 142 Intestine
pH, 142 large, 144, 150
residence time, 143 small, 144, 146, 149
secretion, 146, 149 Intimin, 169, 214
rumen, 144 Intracellular bacteria, 218
ruminants, 143, 144 Intracellular killing, 300
Glycolipid, 476, 477, 485, 490, 491 Intraepithelial lymphocytes (IELs), 297, 300, 302
Glycoprotein, 476, 477, 485–490 Invasion of eukaryotic cells, 220, 222
GNA, 250 Ity, 241
Gnotobiotic animals, 3–8 Lactation, 303
Goblet cells, 296 Lactic acid bacteria (LAB), 382, 383, 390–392,
Green fluorescent protein, 222, 227 399, 400, 403, 407, 418, 427, 436–445
502 Index
Lactic acid bacteria (Lactobacilli), 273, 356, 363, Native flora, 272, 273
364, 367, 370t Neonatal, 477, 483, 484, 487, 491
Lactobacillus, 33, 39, 46, 127, 131, 328–330, Neurotoxins, 300
332–336, 338, 340–346, 418, 420, 427, NK cells, 298, 302
430, 434, 436, 439, 440, 488, 489 Nodavirus, 225
Lactobacillus casei, 218 Non-specific defence, 316
Lactococcus garviae, 209 Normal microflora, 25, 28
Lambs, 6, 7, 8, 56, 61, 62, 65, 67, 69, 70 NTEC (necrotoxigenic E. coli), 173
Lectin, 473, 474, 476, 477, 481, 485, 487, 488,
490, 491 Oligonucleotide probes, 29, 122, 131–133
Lectins, 250 Oligosaccharide, 474, 477, 485–487, 490, 491
LEE (locus of enterocyte effacement), 169 Oncorhynchus keta, 224
Length heterogenicity PCR (LH-PCR), 126 Oncorhynchus kisutch, 224
Listeria monocytogenes, 272 Oncorhynchus tshawytscha, 224
Lymphoid system, 317 Ontogeny, 321, 324
Lysozyme, 209 Oral cavity, 104
Oral immunization, 328, 329, 333
M cells, 218, 361, 363, 364, 369 Oral tolerance, 358, 360, 367
Macrophages (MΦ), 300, 303, 357, 360, 362, 367 Oral vaccination, 482–484, 490, 491
Major histocompatibility complex (MHC), 299, Outer membrane proteins, bacterial, 210
300, 302, 359, 363
Major subunit (fimbriae), 477, 490 P (Pap) fimbriae, 174
Mammary gland, 295, 302–304 paa (porcine attaching effacing associated)
Mannose, 214, 224, 474, 488, 490 factor, 172
Mastitis, 303, 304 Paneth cells, 296, 297
Maternal antibodies, 481, 482, 491 Panleukopenia virus, 116
Maternal influences, 315, 316 Pap fimbriae, 479
Metabolic fingerprinting, 29 Parvovirus, 108
Methanogens, 79, 81 Pasteurella, 271
Microbiota, 302 Pasteurella piscicida, 212
Micro-flora, 258, 260 Pathogens, 92, 93, 191, 196, 197, 202, 203
Microflora PCR, 151, 476
composition, 142, 150–152 PE (phosphatidylethanolamine), 171
Milk, 106, 481, 483, 484, 486, 487, 491 Peyer’s patches (PP), 297, 357, 358, 362, 364, 369
Minor subunit (fimbriae), 480, 491 pH, 54, 63
Molecular beacons, 135 Phagocytic system, 316
Monogastric animals, 295, 302 Phagocytosis, 300, 305
Moraxella, 271 Photobacterium damselae subsp. piscicida, 212,
Moritella marina, 209 214, 223
Moritella viscosa, 209 PhPlates, 30
Morone saxatilis, 225 Phylogeny, 130
Mucosa, 472, 473, 480, 481–485, 487–489, Phylogenetic markers, 120
490–492 Pig, 44, 473, 476, 477, 481–484, 486, 487, 489, 492
Mucosal immune system (MIS), 293, 307 Piglets, 7, 13, 14
Mucosal immunization, 329 Piscirickettsia salmonis, 209, 223, 227
Mucosal lymphoid system, 318 Plant lectin, 488
Mucosal mast cells (MMC), 298 Plasma powder, 486
Mucosal-associated lymphoid tissues (MALT), Plasmid, 122, 123
295, 297 Polygastric animals, 302
Mucus, 215, 216, 296, 476, 477, 484, 485, 489, 490 Polymerase chain reaction (PCR), 121, 122, 125,
as bacterial nutrient source, 216 127, 130, 134
composition, 215 Arbitrarily primed PCR (AP-PCR), 125
functions, 215 Length heterogenicity PCR (LH-PCR), 126
Mugil cephalus, 225 Repetitive extragenic palindromic sequences
Mycobacterium, 270 (REP-PCR), 127
Mycoplasma, 269–270 Reverse transcription PCR (RT-PCR), 134
Nasal-associated lymphoid tissues (NALT), 295 Polymorphism, 125
Index 503
Polymorphonuclear neutrophils (PMNs), 300, Salmonella in the mouse, 241, 243

303, 304 Salmonella in the pig, 246, 247
Postweaning, 22, 23, 23, 35, 37–39, 41–43, 45, 46 Salmonella in the rat, 238–241
Post-weaning, 482, 483, 487, 489, 490 Salmonella typhi, 236
Potentiated probiotics, 454, 458–459 Salmonella typhimurium, 116, 237
Prebiotics, 217, 457–458, 459, 467 Salmonellosis, 103, 114
Prevotella, 126 Salmonid rickettsial septicaemia (SRS), 223
Probiotics, 2, 23, 24, 25, 28, 30, 351, 382–383, Salvelinus alpinus, 214, 216, 221
384t, 385, 390–394, 399, 400, 402, 407, Scophthalmus maximus, 216, 221, 222, 224
409, 454–457, 467 Seasonal changes in body mass, 78, 85, 87, 94
Protection, 323 Sec, 479
Protein tyrosine phosphatase, 222 Seriola spp. (yellowtail), 225
Protozoa, 119 Shigella, 220
Pseudomonas spp., 382, 386t, 387, 388, 389t, 392, adhesins, 220
395t, 396t, 401, 404 invasin, 220
Pulse-field gel electrophoresis (PFGE), 123 Single-strand-conformation polymorphism
Purification (fimbriae), 473, 480 (SSCP), 127
Skin immune system (SIS), 293
Radiotoxicity, 5 S-layer, bacterial, 211
Receptor, 472, 474, 476, 482, 483, 485, 486, Small intestine, 91, 108, 472, 473, 476, 481, 483,
486–492 485, 488–492
Receptor analogue, 472, 485–487, 491, 492 Sparus auratus, 222
Receptors, 159, 162 SPF pigs, 39
Rectum, 111 Spirochaetes, 267, 268
Regulation of expression, 161 Staphylococcus, 271
Renibacterium salmoninarum, 209 Staphylococcus aureus, 105, 214
Repetitive extragenic palindromic sequences Static model, 146
(REP-PCR), 127 Stomach, 105, 144, 146, 147
Resistance, 472, 476, 477, 491 kittens, 106
Restriction fragment length polymorphism puppies, 105
(RFLP), 125 Streptococcosis, 225
Ribonuclease protection assays (RPA), 134 Streptococcus difficile, 209
Ribosomal Database project (RDP), 130 Streptococcus iniae, 209
Ribosomal intergenic spacer analysis Structural carbohydrates, See also cellulose and
(RISA), 127 hemicellulose, 54, 55, 60
Rickettsia, 221, 223 Substrate-tracking autoradiographic fluorescent-
Rickettsia prowazekii, 227 in situ-hybridization (STARFISH),
RNA, 120–123, 127, 129–131, 133, 136 133
Rumen, 54–56, 60, 61, 62, 63, 65, 70, 76, 79–83, Suckling, 22, 23, 33, 35, 36, 38, 40, 42, 44, 46
85–87, 89, 90, 92, 95 Synbiotics, 457–458
Rumen ciliates, 58, 61
Rumen fauna, 55, 61 T cells, 367, 369, 370t
Rumen microflora, 6, 11 T lymphocytes, 318
Rumen protozoa, 57, 59, 70 T-cells (lymphocytes), 298, 299, 301
Ruminant(s), 54, 55, 56, 60, 63, 65, 67, 69, 70 Temperature gradient gel electrophoresis
Ruminants, 76, 78, 79, 81, 82, 84, 86–89, 93–95 (TGGE), 128
TIM-1, 148, 149
S fimbriae, 175 TIM-2, 148
Salmo trutta, 221 Tir (translocated intimin receptor), 169
Salmonella, 220, 263–265 Tolerance, 321
Salmonella, 127 Toxin, 473, 476, 483, 484, 487, 490, 491
enterica, 127 Turbot, see Scophthalmus maximus, 216
Salmonella and malnutrition, 249 Type 3 secretory system, 214
Salmonella enteritidis, 236 Tyramide System Amplification, 133
Salmonella fimbriae, 241 Tyrosine kinase, 220, 222
Salmonella flagellin, 241
Salmonella in calves, 247 Usher, 477, 479, 480
504 Index
V. anguillarum, 211 Viral Encephalopathy and Retinopathy

Vaccinations, 179, 325, 472, 481–484, 491, 492 (VER), 225
Vaccine, 329, 330, 333, 337, 341, 342, 346, 493, Viral haemorrhaghic septicaemia (VHS) virus, 225
480, 481, 485, 492 Virulence factor, 474
Verotoxigenic, 483 Volatile fatty acids (VFA), 6, 11, 12, 56, 60, 65, 67t
VETEC, 483 VTEC (verotoxigenic E. coli), 167
Vibrio anguillarum, 209, 211, 214, 216, 224, 226
Vibrio cholerae, 211, 218 Weaning, 472, 482, 483, 487, 489–491
Vibrio harveyii, 224
Vibrio ordalii, 209, 224 Yersinia, 271
Vibrio salmonicida, 209, 211, 214, 226 Yersinia sp., 215, 220
Vibrio spp., 381, 382, 387, 391, 394, 395t, Yersinia ruckeri, 209
398–400, 402–405 α-defensins, 297
Vibrio viscosus, 209, 211 α-lactalbumin (α-LA), 303, 305
Vibrio vulnificus, 224 β-lactoglobulin (β-LG), 303

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Microbial Ecology

Biology of Growing Animals

Edinburgh • London • New York • Oxford • Philadelphia •

MICROBIAL ECOLOGY IN GROWING ANIMALS

dominating bacterial populations of different animal types. Reference is also made

Aschfalk A. – Section of Arctic Veterinary Medicine, Department of Food Safety

Holzapfel W.H. – Institute of Hygiene and Toxicology BFE, D-76131 Karlsruhe,

Pouwels P.H. – Toegepast Natuurkundig Onderzoek (TNO) Prevention and Health,

A. Bomba, R. Nemcová and S. Gancarčíková

University of Veterinary Medicine,

Gnotobiology is the science of gnotobiotic animals. Such animals have a precisely

Microbial Ecology in Growing Animals

2. CONTRIBUTION OF GNOTOBIOTIC TECHNIQUES AND

Gnotobiotic animals typically display remarkable morphological and physiologi-

display increased resistance to irradiation (Mandel et al., 1979). In infectiology,

3. THE USE OF GNOTOBIOTIC ANIMALS IN STUDIES INTO THE

morphological and functional development in germ-free, gnotobiotic and conven-

4. DEVELOPMENT OF THE DIGESTIVE TRACT IN

4.1. Morphological development of the digestive tract in

the oesophagus, abomasum, caecum or intestines, nor in the consistency of the

4.2. Physiological development of the digestive tract in gnotobiotic lambs

Butyrivibrio fibrisolvens and Selenomonas ruminantium at a concentration of 106

A complex microflora presents a requisite condition of optimum development of the

5. DEVELOPMENT OF THE GASTROINTESTINAL TRACT OF

5.1. Morphological development of the digestive tract in gnotobiotic

5.2. Intestinal metabolism in gnotobiotic pigs

Table 1. The influence of continuous application of Lactobacillus casei on colonization and

Lactobacilli (content) Lactobacilli (mucosa)

Jejunum O 0 0 7.49 ± 0.22*

Table 2. The influence of continuous application of Lactobacillus casei on colonization and

Lactobacilli (content) Lactobacilli (mucosa)

Jejunum O 0 0 7.12 ± 0.06**

knowledge obtained, the mode of action of probiotic microorganisms upon digestive

M. Katoulia and P. Wallgrenb

Uppsala S-751 89, Sweden

Microbial Ecology in Growing Animals

3. PHYSIOLOGY AND ANATOMY OF THE GASTROINTESTINAL

4. DIET REGIMES AND ALTERATIONS OF FOOD

5. INDIGENOUS INTESTINAL MICROFLORA OF PIGS

Stomach Duodenum Ileum Caecum Rectum

Lactobacilli 7−8 6–7 7−8 8−9 6−9

suppressed by other groups of microorganisms, which are in turn suppressed by new

5.1. Interference and protection

6. METHODS OF ANALYSING INTESTINAL MICROFLORA

6.1. Quantitative analyses

6.2. Qualitative analyses

6.3. New methods to analyse the intestinal microflora

6.3.2. Metabolic fingerprinting

7. IN VIVO MODELS AND SAMPLE COLLECTION STRATEGIES

9. MICROFLORA OF DIFFERENT REGIONS OF

complex flora dominated by anaerobic species is established in the gut.

10. DYNAMICS OF THE INTESTINAL MICROFLORA IN

10.1. Intestinal microflora of sows

Veillonella, Fusobacterium and Peptostreptococcus can be isolated from different

10.2. Intestinal microflora of piglets during the suckling period

10.3. Intestinal microflora of piglets following weaning including

between 2 to 3 weeks after weaning. However, if invaded by pathogenic strains of

10.4. Intestinal microflora of pigs during the fattening period

11. INTESTINAL MICROFLORA OF SPECIFIC

12. ALTERATIONS OF THE INTESTINAL MICROFLORA

12.1. Intestinal coliforms during the suckling and post-weaning periods

12.2. At-risk situations

12.3. Diarrhoea pre-weaning