Book Program and Abstracts

PROGRAM AND ABSTRACTS
Eleventh Congress
of the Bulgarian Microbiologists
with International Participation
International House of Scientists

Frederic Joliot-Curie
St. Constantine, Varna
October 5-7, 2006
1
CONGRESS AGENDA
Congress October 5, 2006 October 6, 2006 October 7, 2006

Topics 9.00 – 13.00 h 14.00 – 18.30 h 9.00 – 13.00 h 14.00 – 18.30 h 9.30 – 11.30 h
Plenary Tuberculosis Actual Problems of the BioScience
Hall 1 Hall 1
GAM Sessions I and II Sessions III and IV
Hall 1 Hall 1
MM Sessions I and II Sessions III and IV
Hall 2 Hall 2
2
VM Sessions I and II
Hall 3
V Session I Session II Session III
Hall 1 Hall 3 Hall 3
PM and SM Session I
Hall 2
II and P Sessions I and II
Hall 4
Workshop Hall 4 Hall 4
Posters Session I Session II

Lobby of IHS Lobby of IHS
Trade Exposition
Lobby of IHS Lobby of IHS Lobby of IHS Lobby of IHS Lobby of IHS
Table of Contents
Congress Organizers ……………………………………………..

Congress Organizing Committee……………………………………..
Congress Sponsors
Congress Sections
Social Program
FINAL PROGRAM
Opening Ceremony
ORAL PRESENTATIONS
Thursday, October 05
Hall1: Memorial Session Dr. Stamen Grigorov
Tuberculosis
Hall1: Virology, Session I
Hall2: Medical Microbiology, Sessions I and II
Hall3: Veterinary Microbiology, Sessions I and II
Hall4: Infectious Immunology and Parazitology, Sessions I and II
Friday, October 06
Hall1: General and Applied Microbiology, Sessions I, II, III and IV
Hall2: Medical Microbiology, Sessions III and IV
Hall2: Plant and Soil Microbiology, Session I
Hall3: Virology, Sessions II and III
Saturday, October 07
Hall1: Plenary Session “Actual Problems of the BioScience”
POSTER PRESENTATIONS
Lobby: Poster Session I:
General and Applied Microbiology
Veterinary Microbiology
Friday, October 06
Lobby: Poster Session II:
Medical Microbiology
Virology
Infectious Immunology
Parazitology
Plant and Soil Microbiology
ABSTRACTS
First Authors’ Index
3
CONGRESS ORGANIZERS
Union of Scientists in Bulgaria –
Bulgarian Society for Microbiology
with associated
Bulgarian Society of Medical Microbiology

Bulgarian Society of Medical Virology
Bulgarian Society of Plant Virology “Prof. Dr. D. Atanasoff”
and
The Stephan Angeloff Institute of Microbiology,

Bulgarian Academy of Sciences
4
CONGRESS ORGANIZING COMMITTEE
President: Prof. Angel S. GALABOV, DSc,

Corr. Member of BAS
Secretary General: Assoc. Prof. Hristo NAJDENSKI, PhD
Members:
Assoc. Prof. Angel ANGELOV, PhD Assoc. Prof. Mira KOJOUHAROVA, PhD
Prof. Maria ANGELOVA, DSc Assoc. Prof. Rossica KOTSEVA, PhD
Prof. Radka ARGIROVA, DSc Angel KUNCHEV, PhD
Assoc. Prof. Zheko BAICHEV, PhD Assoc. Prof. Rossitsa KURDOVA, PhD
Assoc. Prof. Milyana CHUCHKOVA, PhD Prof. Jordanka KUZMANOVA, DSc
Assoc. Prof. Svetla DANOVA, PhD Prof. Ivan MITOV, DSc
Assoc. Prof. Stefan DENEV, PhD Prof. Ivan MURGOV, DSc
Prof. Raycho DIMKOV, PhD Assoc. Prof. Nedelcho NEDELCHEV, PhD
Assoc. Prof. Lyuba DOUMANOVA, PhD Prof. Plamen NENKOV, DSc
Assoc. Prof. Elka EMANUILOVA, PhD Prof. Bogdan PETRUNOV, DSc,
Assist. Prof. Elica GOLKOCHEVA, PhD Corr. Member of BAS
Prof. Stoyan GROUDEV, DSc Assoc. Prof. Maria SHISHINIOVA, PhD
Assoc. Prof. Veneta GROUDEVA, PhD Assoc. Prof. Penka SOTIROVA, PhD
Assoc. Prof. Irina HAYDUSHKA, PhD Assoc. Prof. Maria SREDKOVA, PhD
Assoc. Prof. Tsonka HRISTOZOVA, PhD Assist. Prof. Antoniy STOEV, PhD
Prof. Iskra IVANOVA, DSc Tencho TENEV, MD
Assoc. Prof. Todor KANTARDJIEV, PhD Assoc. Prof. Pavel TEOHAROV, PhD
Assoc. Prof. Rossica VATCHEVA, PhD
Local organizing committee:
Assoc. Prof. Velina YONKOVA, PhD Assoc. Prof. Vesselin RUSSEV, PhD
(Coordinator)
Branimir SHMATOV, MD (Secretary)
Secretariat:
Liliana HARALAMPIEVA, MS, Assist. Prof. Ralitca NEDKOVA, MS

(Coordinator) Krassya NENOVA, Clerk
Assist. Prof. Radoslav ABRASHEV, MS Assist. Prof. Lubomira NIKOLAEVA-
Assist. Prof. Nelly SLAVOVA- GLOMB, PhD
AZMANOVA, MD Emilia POPOVA-AYVAZOVA, BS
Elka GENOVA, BS Dessislava TODOROVA, MS
Ivailo GEORGIEV, MS Eng. Ralitsa VASSILEVA, MS
Mihail ILIEV, MS
5
ORGANIZING SECRETARIAT
The Stefan Angeloff Institute of Microbiology,

Bulgarian Academy of Sciences
26, Acad. G. Bonchev Str.
1113 Sofia, Bulgaria
Tel.: +359 2 8701081, fax: +359 2 8700109
e-mail:congress11@microbio.bas.bg
www.microbio.bas.bg/congress11
6
CONGRESS SPONSORS
GENERAL SPONSORS
БИОКОМ Tрендафилов ЕООД
IDEXX - USA
представлявана за България от фирма КРОСПОЙНТ ЕООД

чрез фирма HATO Handelsgesellschaft mbH - Germany
SPONSORS
Medical Technics Engineering Ltd.

GlaxoSmithKline
Roche Bulgaria Ltd.
BioSystems Ltd.
AQUAlab
Ecopharm Ltd.
TECOM Analytical Systems Ltd.
Аквахим ЕООД
Anti-sell / Selidis Bros Bulgaria Ltd.
L.K.B. Bulgaria Ltd.
БУЛ БИО-НЦЗПБ ЕООД
BIOLAB Ltd.
ФОТ ООД - официален представител на SIGMA-ALDRICH за България
Sevex Pharma
ЕЛТА’ 90М ООД
Dr. Herbert Knauer Ltd. Berlin, Germany
5040 Сървисиз ООД
Thermo Electron LED GmbH Germany
Диамед ООД
ИНФОМЕД ЕООД (OLYMPUS)
DMG Clinic Ltd.
Лаборбио ООД
CONTRIBUTORS
Национална ветеринарномедицинска служба, МЗГ
Институт по микробиология „Стефан Ангелов”, БАН
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CONGRESS SECTIONS
Tuberculosis (Tb)
General and Applied Microbiology (GAM)
Medical Microbiology (MM)
Veterinary Microbiology (VM)
Virology (V)
Parasitology (P)
Infectious Immunology (II)
Plant and Soil Microbiology (PSM)
Actual Problems of the BioScience (BS)
SOCIAL PROGRAM
Thursday, October 05, 19:00

Welcome Party
Friday, October 06, 20:00

Congress Banquet
8
FINAL PROGRAM
Registration: Wednesday, October 04 16:00 – 19:00

Thursday, October 05 08:00 – 18:00
Friday, October 06 08:00 – 14:00
Opening Ceremony
Hall 1
Chairpersons: Angel S. Galabov

Hristo Najdenski
9:00 Welcoming Addresses
President of the Organizing Committee

Mayor of the City of Varna
President of the Dr. Stamen Grigorov Foundation
Others
9
10
ORAL PRESENTATIONS
11
12
Thursday , October 05
Hall 1
9:00 – 9:20 Opening Ceremony
Memorial Session Dr. Stamen Grigorov

Tuberculosis
Chairpersons: A. Engibarov, Sofia
H. Najdenski, Sofia
M. Chouchkova, Sofia
9:20 Tb1 Vitae of Dr. Stamen Grigorov

A. Galabov, Sofia
9:40 Tb2 BCG vaccines
M. Chouchkova, T. Stefanova, Sofia
10:05 Tb3 Renaissance of tuberculosis. Actual problems
Z. Yankova, Plovdiv
10:30 Break
11:00 Tb4 Control of tuberculosis and introduction of DOT strategy
in Bulgaria
D. Stefanova, Sofia
11:25 Tb5 Tuberculosis among the children in the past and today
P. Minchev, Sofia
11:50 Tb6 Contemporary microbiological diagnostics of
tuberculosis and methods for epidemiological tracing
T. Kantardjiev, St. Panaiotov, E. Bachiiska, Sofia
12:15 Tb7 Molecular and epidemiologycal characteristics of
Mycobacterium tuberculosis strains originating from
different regions of Bulgaria
V. Valcheva, I. Mokrousov, O. Narvskaja, N. Markova,
Sofia, St. Petersburg
12:45 Tb8 Intra-species determination of mycobacteria by PCR
M. Bonovska, H. Najdenski, Sofia
13:00 Confocal microscopy and microbiology applications
J. Pala, Leica Microsystems, Brno, Czech Republic
13:30 Session end
13
Thursday, October 05, 2006
Hall 1: Virology
Session I
Chairpersons: A. S. Galabov, Sofia
F. Wild, Lyon
L. Doumanova, Sofia
14:00 V1 Henipaviruses, a new family of paramyxoviruses,

Plenary which have emerged in Asia and Australia
F. Wild, Lyon, France
14:30 V2 Structural basis for antiviral drug-resistance of a

Plenary VP1 mutated enterovirus
A. S. Galabov, I. Nikolova, R. Petkova,
S. Chakarov, B. Atanasov, Sofia
15:00 V3 The hierarchical QSAR technology for effective

Plenary virtual screening and molecular design of
potential antiviral agents
V. E. Kuzmin, A. G. Artemenko, E. N. Muratov,
L. N. Ognichenko, A. I. Hromov, A. V. Liahovskij,
P. G. Polischuk, Odessa, Ukraine
15:30 Break
16:00 V4 Christo Russeff Memorial Lecture

Plenary Oxoglaucine: a new highly potent antienteroviral
compound
L. Nikolaeva-Glomb, S. Filipov, A. S. Galabov,
Sofia
16:20 V5 Rational treatment course schedule effective

against enteroviral neuroinfection in mice
R. Vassileva-Pencheva, A. S. Galabov, Sofia
14
16:35 V6 Еffects of picornavirus replication inhibitors against
calicivirus FCV
J. D. Tumbarski, A. S. Galabov, Sofia
16:50 V7 In vivo effective anti-flu combination of antivirals

L. Simeonova, G. Gegova, A. S. Galabov, Sofia
17:05 V8 Must monesine preparative be used for prophylaxis of

bird flu
S. Dundarov, Sofia
17:20 V9 In vitro anti-influenza virus effect of a protease inhibitor

from Streptomyces chromofuscus 34-1
J. Serkedjieva, L. Angelova, I. Roeva, I. Ivanova, Sofia
17:35 V10 Anti-herpes activities of Pseudomonas sp. S-17

rhamnolipid and its complex with alginate
M. Remichkova, I. Roeva, D. Galabova, A. S. Galabov, Sofia
17:50 V11 Antioxidant effects of plant polyphenols quercetin and

rutin in influenza virus infected mice
M. Mileva, A. S. Galabov, Sofia
18:05 V12 A plant polyphenol extract reduces oxidative stress in

the livers of influenza virus-infected mice
E. Krumova, S. Abarova, L. Tancheva, M. Angelova,
J. Serkedjieva, Sofia
18:20 Session end
15
Thursday , October 05
Hall 2: Medical Microbiology
Session I
Chairpersons: G. Terziiski, Sofia

M. Petrovska, Skopie, R. Macedonia
M. Sredkova, Pleven
14:00 MM1 Polyphase identification analysis of clinical

Plenary isolates from Burkholderia cepacia complex
(BCC)
M. Sredkova, S. Mihailova, E. Moor, Pleven,
Goteborg, Sweden
14:30 MM2 Study of vaginal lactobacilli from Bulgarian

Plenary women
G. D. Stoyancheva, S. P. Dimitonova,
P. M. Petrova, R. N. Aleksandrova, I. Tzvetkova,
S. T. Danova, Sofia
14:50 MM3 Frequency of the triggering infection at patients

Plenary with reactive arthritis and unidentified
oligoarthritis and necessity for their
examination
R. Stoilov, Sofia
15:10 Real-Time PCR in molecular microbiology
V. Kardjeva, Biosystems Ltd., Sofia
15:30 Break
16
Session II
Chairpersons: G. Terziiski, Sofia
M. Petrovska, Skopie, R. Macedonia
M. Sredkova, Pleven
16:00 MM4 Microbiological investigation of imported

Brucella infection among Bulgarian citizens
R. Nenova, T. Kantardjiev, I. Ivanov, I. Tomova,
B. Popov, Vl. Novkirishki, Sofia
16:15 MM5 Characteristics of the cultivation of

Bifidobacterium bifidum 1 in media with
palatinose
D. Blazheva, Z. Denkova, A. Krastanov, Sofia
16:30 PCR-DIAPOPS method for detection of

Legionella in water
E. Domínguez, ZEU-INMUNOTEC, Zaragoza, Spain
17:00 MM6 Contamination of hotels from Bulgarian Black

Sea Coast with different branches of Legionella
– ecological precondition for colonization
I. Tomova, U. Helbig, V. Levterova, R. Nenova,
I. Ivanova, Sofia
17:15 MM7 Normal flora is found in human blood

E Kalfin, Sofia
17:30 MM8 Q-fever – an insufficiently known and

underestimated infection in Bulgaria.
V. Serbezov, Sofia
17:45 MM9 Clinical aspects of lung mycotic infections

V. Pencheva, D. Petrova, O. Georgiev,
T. Kantardjiev, Tz. Mondeshki, Tz. Terzieva,
D. Valchev, Sofia
17
18:00 MM10 PCR-based methods for diagnosis of systemic
mycoses and genotyping of pathogenic fungi
P. Angelov, T. T. Kantardjiev, V. Levterova,
E. Zamfirova, M. Leseva, R. Vacheva, E. Shopova,
E. Bobcheva, Sofia
18:15 Session end
18
Hall 3: Veterinary Microbiology
Session I
Chairpersons: I. Ivanov, Sofia

H. Najdenski, Sofia
14:00 Microbiological methods for detection of

antibiotic residues in food
E. Dominguez, ZEU-IMMUNOTEC, Zaragoza,
Spain
14:20 VM1 Development and application of rabbit polyclonal

Plenary monospecific affinity purified antibody against
synthetic sequence from peptide P32 to detect
sheep pox virus by immunohistochemical polymer
staining technique on frozen tissue sections
V. Bumbarov, R. Eligulashvili., H. Yadin,
Beit Dagan, Izrael
14:40 VM2 Findings and serotyping of Listeria

monocytogenes in meat of chicken broilers
R. Karakolev, Veliko Tarnovo
15:00 VM3 Optimization of PCR-DGGE for direct detection

Plenary and molecular typing of Campilobacter jejuni and
Campylobacter coli in bird cecal samples
H. Najdenski, Sofia
15:30 Break
19
Session II
Chairpersons: R. Karakolev, Veliko Tarnovo
N. Korudjiisky, Sofia
16:00 Rapid solutions for food safety – A review of methods

and industry trends
F. Martens, Hygiena International Ltd., Hertfordshire,
United Kingdom
16:30 Reducing the risk of antibiotic drug-residue

contamination and ensuring milk safety and quality with
the reliable IDEXX SNAP® test kits
G. Trifonov, IDEXX-USA, Sofia
16:50 VM4 Comparative researching of pH in some mussels

from sacrified animals
A.Kuzelov; O. Kirovska Cigulevska, Sveti Nikole,
Skopje, R. Macedonia
17:10 VM5 Ecological alternatives of antibiotic prophylactics

and therapy
H. Arnaudov, R. Karakolev, P. Kalcheva, Veliko Tarnova
17:35 VM6 Drug resistant bacteria from the genus

Moraxella, isolated from rabbits with pneumonia
S. Ivanova, N. Korudjiisky, T. Galabinova, B. Mitov, Sofia
17:55 VM7 Disinfection of objects in the apiculture contaminated

with Paenibacillus alvei and Ascosphaera apis
K. Gurgulova, D. Ilieva, J. Hristov, S. Karadjov, E. Iliev, Sofia
18:25 VM8 Microbiological investigation on pseudotuberculosis in

sheeps and goats
N. Korudjiisky, S. Ivanova, B. Gurov, D. Todorov,
Zv. Dikova, Sofia
18:40 Session end

20
Hall 4: Infectious Immunology
Session I
Chairpersons: I. Smirnov, Moscow, Russia

T. Vassilev, Sofia
14:00 Intro of IDEXX Lab Inc main subject WATER

CLT / CLT 18
S. Herterich, G. Trifonov, IDEXX EUROPE B.V.,
Schiphol-Rijk, Netherland; Sofia
14:30 II1 Quality of analysis in clinical microbiology

Plenary I. Smirnov, Moskow, Russia
14:50 II2 Improved pooled IgG prevents death in

Plenary experimental sepsis
T. Vassilev, J. Dimitrov and N. Ivanovska, Sofia
15:10 II3 Effect of Yersinia pseudotuberculosis YopK

Plenary protein on invasion mediated endocytosis from
HELA cells
E. Ivanova, H. Najdenski, A. Vesselinova,
F. Deleuil, H. Wolf-Watz, Sofia, Umea, Sweden
15:30 Break
21
Session II
Hall 4: Parazitology
Chairpersons: R. Kurdova-Mintcheva, Sofia

P. Petrov, Sofia
16:00 Conversation of the EUDWD into National Law

M.G. Czapp, HATO Handelsgesellschaft mbH,
Achim, Germany
16:45 P1 Emerging and reimurging parasitic diseases in

Plenary Bulgaria
R. Kurdova-Mintcheva, D. Jordanova,
T. Marinova, M. Ivanova, N. Tsvetkova,
I. Marinova, R. Harizanov, Sofia
17:15 Session end
22
Friday, October 06
Hall 1: General and Applied Microbiology
Session I
Chairpersons: E. Emanuilova, Sofia

V. Groudeva, Sofia
9:00 GAM1 Mycological studies in Continental

Plenary Antarctica: an overview
S. Tosi, Pavia, Italy
9:30 GAM2 Microbiological control of detoxification

bioremediation technologies
Y. Topalova, R. Dimkov, C.V.Keer,
C. Y.Cheng, C.Y.Cheng, M. Nunes, Sofia,
Gent, Belgium; Porto, Portugal
9:45 GAM3 Diversity of Synechococcus community in

freshwater fake George, NY
D. Gouliamova, Sofia
10:00 GAM4 Microbial status of 7 Thracian vaults near

to Kasanlak, Bulgaria and methods for
preventation
Tz. Groudeva, A. Doycheva, Sofia
10:15 GAM5 Taxonomical identification of arsenic

resistant and arsenic transforming sulfate
- reducing bacteria
K.S. Krumova, V.I. Groudeva, Sofia
10:30 Break
23
Session II
Chairpersons: S. Groudev, Sofia

Z. Alexieva, Sofia
11:00 GAM6 Low-temperature biodegradation of

Plenary phenol
R. Margesin, Innsbruck, Austria
11:30 GAM7 In situ bioremediation of soil polluted with

crude oil and heavy metals
V.I.Groudeva, A.S.Doycheva and
S.N.Groudev, Sofia
11:45 GAM8 Comparative characteristics of oil-

degrading activity of microorganisms with
different taxonomic status
V. Groudeva, I. Ivanova, A. Doycheva,
M. Dumitru, A-R. Vasculesku, Sofia,
Bucharest, Romania
12:00 GAM9 Comparative characteristics of toxicity

assessment of heavy metal polluted soils
with different microorganisms
I. Ivanova, D. Dimova, A. Stojanova,
V. Groudeva, Sofia
12:15 GAM10 Study for intensification of the biogas

production from agricultural and agro-
industrial wastes
D. Galabova, D. Karakashev, A. Mirkov,
L. Nikolov, I. Simeonov, Sofia
12:30 Lunch
24
Session III
Chairpersons: R. Dimkov, Sofia

I. Ivanova, Sofia
14:00 GAM11 Molecular biomarkers of oxidative stress in

filamentous fungi
M. Angelova, Sofia
14:15 GAM12 New mitochondrial catalase in yeast

Saccharomyces cerevisiae
V. Petrova, Sofia
14:30 GAM13 Proteomics in target-specific antibacterial drug

discovery based on UMP kinase
N. Slavova-Azmanova, C. Evrin, L. Assairi,
H. Najdenski, O. Bârzu and A.- M. Gilles, Sofia,
Paris, France
14:45 GAM14 Heat shock response of Streptococcus

thermophilus industrial strains
P. Petrova, D. Gouliamova and G. Stoyancheva,
Sofia
15:00 GAM15 Second generation of glucooligosaccharides with

prebiotic properties, synthesied by
glycosiltransferases from Leuconostoc
mesenteroides LM 286
I. Iliev, T. Vassileva, Plovdiv, Sofia
15:15 GAM16 Isolation and properties of extracellular â-

xylosidase produced by Aspergillus niger B03
G. Dobrev, I. Pistijski, L. Koleva, Plovdiv
15:30 Break
25
Session IV
Chairpersons: Ch. Chomakov, Sofia

I. Abrashev, Sofia
16:00 GAM17 Polyhydroxyalkanoic acids (PHA) –

Plenary interesting bacterial polymers and aspects
of their production
J-U. Ackermann, G. Mothes, Dresden,
Leipzig, Germany
16:20 GAM18 Peculiarities of biofilm system

Plenary implementation in microbial
biotechnologies
L. Nikolov, Sofia
16:45 GAM19 Evolution modeling of bacterial oxidation

of ferrous ions in biofilm system
L. Nikolov, E. Petrova , V. Mamatarkova,
Kl. Mladenov, St. Stoytchev, Sofia
17:00 GAM20 Precondition for producing of organic food

in Macedonia
A. Kuzelov, O. Cigulevska, Skopie,
R. Macedonia
17:15 GAM21 Heterogeneity of L. plantarum isolates

from artisanal white brined cheese
R. Aleksandrova, S. Dimitonova,
I. Ivanovaand S. Danova, Sofia
17:30 GAM22 Comparison of fermentative capacity of

Lactobacillus bulgaricus cultivated on
media with oligosaccharides
Tz. Ignatova, I. Ivanova, I. Iliev, Shumen,
Sofia, Plovdiv
26
17:45 GAM23 Amino acids use and production - current
status and prospects
A. Ratkov, Sofia
18:00 GAM24 Mathematical identification of L-valine

fed-batch fermentation process
K. Todorov, I. Dimov, Tz. Georgiev,
J. Kristeva, V. Ivanova, Al. Ratkov, Sofia
18:15 GAM25 Carcinogen-induced transposition of

Saccharomyces cerevisiae Ty1 transposon
depends on mitochondrial function
T. Stoycheva, P. Venkov, Ts. Tsvetkov, Sofia
18:30 Session end
27
Friday , October 06
Hall 2: Medical Microbiology
Session III
Chairpersons: I. Mitov, Sofia

T. Kantardjiev, Sofia
E. Keuleyan, Sofia
8:30 MM11 Etiological structure of drug resistance

Plenary and consumption of antibiotics in
Bulgaria
T. Kantardjiev, M. Petrov, Ts. Velinov,
A. Bachvarov, P. Angelov, Sofia
8:55 MM12 Investigation of the virulence factors and

Plenary antibiotic resistance of Moraxella
catarrhalis
I. Mitov, R. Gergova, V. Ouzunova,
R. Markovska, D. Kuncheva, Sofia
9:20 Resistance of Streptococcus pneumoniae

to β-lactam antibiotics. New dose form of
Amoxicillin/Clavulanate in the therapy of
respiratory and ORL infections-
AugmentinES 600
B. Markova, GlaxoSmithKline, Sofia
9:50 MM13 Species affiliation and antibiotics

resistance of clinical isolates from
hemocultures
D. Rukanova, E. Staikova, K. Rachkova,
I. Dukova, H. Djeneva, M. Baicheva,
G. Lazarova, Stara Zagora
28
10:00 MM14 Study on the species affiliation and
antibiotic sensitivity of strains isolated
from urine of patients with chronic
pyelonephritis
V. Grigorova, V. Todorov V. Edreva,
K. Dragoev, Pleven
10:15 MM15 Antimicrobial susceptibility of urine

pathogenes isolated from patients with
benign prostatic hyperplasia treated with
urostim .
V. Grigorova, S. Stratev, F. Shargabi,
N. Kolev, P. Panayotov, R. Kostov,
O. Mihaylov, Ts. Georgiev, V. Dunev,
B. Atanasov, Pleven
10:30 Break
Session IV
Chairpersons: I. Mitov, Sofia

T. Kantardjiev, Sofia
E. Kyolean, Sofia
11:00 MM16 Susceptibility of S. pneumoniae clinical isolates

to some antibiotics
K. Bojkova, T. Stoeva, V. Kaludova, V. Kamenova,
V. Russev, Varna
11:15 MM17 Antibacterial and antifungal activities of a

polyphenol-Rich extract from Geranium
sanguineum L.
M. Gulluce, F. Sahin, A. Sokmen, A. Teodosieva,
J. Serkedjieva; Erzurum, Sivas, Turkey; Sofia
29
11:30 MM18 Investigation of the inhibitory effect of lactic acid
bacteria on the cells of Escherichia coli
NBIMCC 8739
P. Nedelcheva, Z. Denkova, R. Nikolova, Plovdiv
11:45 MM19 Probiotics and probiotic foods in the protection of

human health
I. Murgov, Z. Denkova, Plovdiv
12:00 MM20 Sepsis associated with central venous catheters

at patients in critical situation – causative agents
and frequency
D. Terziiski, N. Petrov, N. Mladenov, Sofia
12:15 MM21 Molecular diagnostics of the causative agents of

atypic pneumonia
N. Brankova, T. Kantardjiev, V. Levterova,
E. Georgieva, I. Tomova, S. Panajotov, Sofia
12:30 Lunch
30
Friday, October 06
Hall 2: Plant and Soil Microbiology
Session I
Chairpersons: N. Bakardjieva, Sofia

D. Sakalieva, Plovdiv
14:00 PSM1 Spread and detection of phytoplasma

Plenary diseases
D. Sakalieva, Plovdiv
14:45 PSM2 Manifestation of tolerance to sharka

Plenary (plum pox) virus of plum cultivars
imported in Bulgaria
A. Stoev, P. Iliev, Kostinbrod, Dryanovo
15:15 PSM3 Proposals for improvement of the variety

Plenary testing of wheat and barley accordingly
their reaction to the most important in
Bulgaria viruses
N. Bakardjieva, A. Stoev, Kostinbrod
15:45 Break
31
Friday, October 06
Hall 3: Virology
Session II
Chairpersons: S. Dundarov, Sofia

R. Argirova, Sofia
R. Kotseva, Sofia
8:30 V13 SARS-coronavirus interaction with ACE2

Plenary receptor; implications for virus adaptation and
vaccine design
A.P. Andonov, Winnipeg, Canada
9:00 V14 Evidence supporting the replicative homeostasis

Plenary hypothesis concerning HIV replication in cell
culture
R. Argirova, Sofia
9:30 V15 Strain diversity and mechanisms of evolution of

rotaviruses
Z. Mladenova, N. Korsun, S. Gyurova, Sofia
9:45 V16 Diagnostic of the first suspected patients in

Bulgaria for avian influenza virus subtype
А(H5N1).
T. Hadjiolova, S. Pavlova, R. Kotseva, Sofia
10:00 V17 Diagnostic studies of etiological role of

respiratory-syncytial
virus in hospitalized children.
S. Pavlova, T. Hadjiolova, R. Kotseva, Sofia
10:15 V18 Diagnostic aspects of haemorrhagic fevers

D. Velcheva, Sofia
32
10:30 Break
11:00 V19 Studies on CCR5, CXCR4 And CCR2 Genetic

Polymorphism in HIV-Infected Bulgarians
K. Borisov, A. Savov, I. Kremensky, S. Raleva,
L. Froloshka, R. Markova, V. Terzieva, K. Kostov,
R. Argirova, Sofia
11:15 V20 An experimental study of HIV-1 epitope

structure changes under inhibition of
glycosylation
R. Gavazova, S. Ivanov, D. Ivanov, P. Genova,
S. Raleva, L. Froloshka, D. Dundarova,
R. Argirova, Sofia
11:30 V21 Spread of the genetic forms of HIV-1 circulating

in Bulgaria
M. Peneva, D. Beshkov, I. Aleksiev, V. Georgieva,
K. Kostov, I. Elenkov, T. Varleva, I. I. Elenkov,
Sofia
11:45 V22 Study of the spread of human T-lymphotropic

viruses HTLV-I and II among Bulgarian
population
I. Aleksiev, D. Beshkov, V. Georgieva, M. Peneva,
S. Bakalova, M. Genova, M. Atanasova,
I. Elenkov, Sofia
12:00 V23 HIV-1 variants (mutants) after long-term

passaging in presence of newly synthesixed
4-hydroxicoumarins (4-Hc)
S. Raleva, A. Yordanova, Y. Gradinarova,
A. Savov, L. Froloshka, P. Genova, I. Manolov,
D. Dundarova, R. Argirova, Sofia
33
12:15 V24 Antiretroviral resistance of HIV-1 among
Bulgarian seropositive patients (2002-2006)
D. Beshkov, I. Aleksiev, M. Peneva, V. Georgieva,
K. Kostov,I. Elenkov, T. Varleva, I. I. Elenkov, Sofia
12:30 Influenza and influenza pandemic: epidemiology

and current possibilities for prophylactic and
treatment
М. Kamenova (Roche Bulgaria Ltd.), Sofia
13:00 Lunch
Hall 3: Virology
Session III
Chairpersons: P. Teoharov, Sofia

V. Serbezov, Sofia
Z. Kalvachev, Sofia
14:00 V25 Diagnostics of the viral hepatitis -

Plenary achievements and problems
P. Teoharov, Sofia
14:30 V26 Molecular characteristics and pathogenetic

Plenary potential of human polyomaviruses
Z. Kalvachev, Sofia
15:00 V27 Rapid identification of polyomavirus hominis-1

(ВКV) in the urine of patients with kidney
transplantation
S.. Slavov, I. Nenkov, A. Petrova, L. Hristova,
P. Simeonov, D. Dimova, Z. Kalvachev, Sofia
34
15:15 V28 Polyomavirus hominis-2 (JCV) in the urine of
patients with kidney transplantation
I. Nenkov, S. Slavov, A. Petrova, L. Hristova,
P. Simeonov, Z. Kalvachev, Sofia
15:30 V29 Clinical features and serological verification of
acute EBV infection
A. Gotseva, T. Kuzmova, D. Velcheva, Sofia
15:45 V30 Bird flu – the new global threat: epidemiology,

spread and possible use as biological weapon
K. Mekouchinov, Sofia
16:00 Session end
35
Saturday, October 07
Hall1
Plenary Session Actual Problems of the BioScience
Chairpersons: Angel S. Galabov, Sofia

Hristo Najdenski, Sofia
9:30 BS1 Science funding in Bulgaria

A. Vutsova, Ministry of Education and Science, Sofia
10:15 BS2 Bioinformatics resources in the microbiology: challenges

and perspectives
D. Vassilev, AgroBio Institute, Sofia
11:00 Congress Adjourn
36
POSTER PRESENTATIONS
37
38
Poster session I
16.00 – 18.30
General and Applied Microbiology
GAM26 Phenol hydroxylase dependance on the variouse hydroxy

phenols utilized as substrates by Trichosporon cutaneum
R57
Z. Alexieva, M. Gerginova, B. Atanasov, Sofia
GAM27 Creating oligonucleotide primers for pcr analysis and
phyA gene sequencing in Trichosporon cutaneum R57
strain
Y. Manasiev, T. Primov, M. Gerginova, N. Peneva,
Z. Alexieva, Sofia
GAM28 Dot-blot analysis by biotin labeled probe for identifying
PHYA gene in microbial strains
M. Gerginova, Y. Manasiev, P. Petrova, A. Krastanov,
Z. Alexieva, Sofia, Plovdiv
GAM29 Influence of toxic phenolic compounds on the phenol
hydroxilase activity in Trichosporon cutaneum
M. Gerginova, Y. Manasiev, N. Shivarova, Z. Alexieva, Sofia
GAM30 Microflora in a natural wetland used fpr treatment of acid
mine drainage
V.I. Groudeva , S.G. Bratkova and S.N. Groudev, Sofia
GAM31 Biodegradation of mixed phenol compounds by microbial
association of Aspergillus awamori and Thermoascus
aurantiacus
I. Stoilova, A. Krastanov, H. Bui, V. Stanchev, Plovdiv
GAM32 Benzonitrile and 4-cyanopiridine degradation by
immobilized cells of Bacillus sp. UG-5B in a column
bioreactor
L. Kabaivanova, E. Dobreva, E. Emanuilova, B. Samuneva,
G. Chernev, Sofia
39
GAM33 Biological properties of biosurfactant-complex from
Pseudomonas sp. PS-17
A. Sotirova, D. Spasova, E. Vasileva-Tonkova, D. Galabova,
Sofia
GAM34 Decolorization of the acid orange 7 by resting Alcaligenes
faecalis and Rhodococcus erythropolis cells: a
comparative study
T. Avramova, L. Stefanova, B. Angelova and S. Mutafov, Sofia
GAM35 pH–Related equilibrium study on copper biosorption by
Penicillium cyclopium
M. Ianis, K. Tsekova, P. Marinov and D. Todorova, Sofia
GAM36 Immobilization of Penicillium cyclopium cells in PVA
hydrogels for heavy metal ions biosorption applications
D. Christova, K. Tsekova, S. Ivanova, M. Ianis, S. Ganeva,
Sofia
GAM37 Biosorption of binary mixtures of copper and cobalt by
Penicillium brevicompactum
K. Tsekova, M. Ianis, V. Dencheva, S. Ganeva, Sofia
GAM38 Sensitivity of Saccharomyces cerevisiae yeast to
arsenate
T. Todorova, S. Vuilleumier, A. Kujumdzieva, Sofia,
Strasbourg, France
GAM39 Transport of arsenat in yeast cells
S. Shilev, Sofia
GAM40 Isolation, identification and selection of arsenic and
cadmium resistant yeast
N. Krumov, V. Gotcheva, Ts. Hristozova, C. Posten,
A. Angelov, Sofia, Karlsruhe, Germany
GAM41 Characterisation and identification of bacterial community
isolated from metalworking fluids
S. Bakalova, P. Hristova, R. Dimkov, Veneta Groudeva, Sofia
GAM42 Taxonomic identification of Xenorhabdus
(Enterobacteriaceae) species by Restriction analysis of
PCR-Amplified 16S rDNA genes
J.H. Georgieva, V.I. Groudeva, M.D. Shishiniova, Sofia
GAM43 Preliminary investigations of thermal sources biodiversity
from Rupite region
40
A. Terziyska, R. Mandeva, D. Lyutzkanova,
M. Stoilova-Disheva, G. Radeva, M. Kambourova, Sofia
GAM44 Biodiversity of carbohydrate degrading cultivable bacteria
from Bacillus group, isolated from bulgarian hot springs
A. Derekova, C. Sjøholm, R. Mandeva, M. Kambourova,
Sofia;
GAM45 Spatial pattern of microbial numbers in the free water of
the Srebarna lake
S. Naumova, A. Petrova, Sofia
GAM46 Comparison of bacterioplankton development between
control and fertilized fish ponds
H. Kalcheva, D. Tersiisky, R. Kalchev, Sofia
GAM47 Adaptive stress response of Humicola lutea 103to copper
exposure
E. Krumova, Y. Gocheva, A. Dolashki, P. Dolashka,
S. Stefanovic, W. Voelter, M. Angelova, Sofia, Tuebingen,
Germany
GAM48 Physiological response of Antarctic fungi to long-term
temperature stress
Y. Gocheva, E. Krumova, L. Slokoska, J. Miteva,
M. Angelova, Sofia
GAM49 Cellular response and antioxidants enzymes in Aspergillus
niger strain against temperature stress
R. Abrashev, A. Dolashki, S. Stevanovic, P. Dolashka,
W. Voelter, L. Stefanova, S. Pashova, R. Hristova,
M. Angelova, Sofia, Tuebingen, Germany
GAM50 Biosynthesis of proteolytic enzymes from Antarctic
actinomycete strains
D. Dimitrova, P. Dorkov, B. Gocheva, Sofia
GAM51 Biosynthesis of antimicrobial substances from Antarctic
GAM52 Biosynthesis of proteinase inhibitor from Antarctic
GAM53 Cyclodextrin glucanotransferase production by
magnetically responsible Bacillus circulans atcc 21783 cells
41
M. Safarikova, N. Atanasova, V. Ivanova, S. Engibarov,
I. Safarik, A. Tonkova, Ceske Budejovice, Sofia, Plovdiv
GAM54 Molecular analysis of a thermostable gellan lyase by
MECC/HPLC
M. Atanassova, J.I.G. Sanchez, A. Terziiska, A. Derekova,
R. Mandeva, M. Kambourova, Sofia, A Coruña, Spain
GAM55 Purification and characterisation of keratinolytic
proteases produced by Streptomyces albidoflavus
V. Stefanova, N. Kirilov, K. Tsiroulnikov, M. Dalgalarrondo,
J-M. Chobert, I. Ivanova, T. Haertlé, Sofia, Moskow, Russia;
Nantes, France
GAM56 BNMPK: a web based suite of bacterial nucleotide mono
phosphte kinases
Ajay Bikumandla, Sai Guduru, Paulina Daalova,
Alexandra Shosheva, Petya Christova, Petras Kundrotas,
Emil Alexov, Sofia
GAM57 Screening of phytase producing yeasts
D. Georgiev, S. Gargova, Plovdiv
GAM58 Mutagenic treatment of strain Aspergillus awamori K-1,
producer of xylanase
Y. Evstatieva, S. Ilieva, D. Nikolova, V. Savov, A. Atev,
Sofia
GAM59 Studying of bioactive metabolites from Arctic cold-adapted
streptomycetes
D. Lyutskanova, M. Stoilova-Disheva, , M. Kolarova,
K. Alexieva, V. Peltekova, V. Ivanova, Sofia
GAM60 Properties and immobilization of fungal cellulase on
polyamide
G. Delcheva, I. Pistijski, G. Dobrev, Plovdiv
GAM61 Primary characterization of a newly discovered
bacteriocin-like substance duracin produced from
Enterococcus durum M-3
S.G. Dimov, Sofia
GAM62 Novel bacteriocin from Enterococcus faecium 3587 –
spectrum of activity and some molecular
characteristics
V.M. Peeva, P.M. Ivanova, S.G. Dimov, Sofia
42
GAM63 Lactococcus lactis subsp. Lactis HV219 – a probiotic?
S.D. Todorov, M. Botes (neé Brink), S.T. Danova, L.M.T.
Dicks, Sofia, Stellenbosch, South Africa
GAM64 Effect of medium components on production of
bacteriocins JW3BZ and JW6BZ by Lactobacillus
plantarum isolated from boza
J.W. von Mollendorff, S.D. Todorov, L.M.T. Dicks,
Stellenbosch, South Africa, Sofia
GAM65 Factors affecting the adsorption of bacteriocin ST194BZ
to Lactobacillus sakei and Enterococcus faecium
S.D. Todorov, M. Meincken, LMT Dicks, Stellenbosch, South
Africa, Sofia
GAM66 Deformation of bacterial cells as a result of bacteriocins
produced by lactic acid bacteria
M. Meincken, S.D. Todorov, J.W. von Mollendorff, L.M.T.
Dicks, Stellenbosch, South Africa, Sofia
GAM67 Partial characterization of a bacteriocin produced by
Lactobacillus curvatus isolated from cervelat salami
A. van den Worm, S.D. Todorov, L.M.T. Dicks, Stellenbosch,
South Africa, Sofia
GAM68 Sanionins: antiinflammatory and antibacterial agents with
weak cytotoxicity from the Antarctic moss Sanionia
georgico-uncinata
V. Ivanova, K-J. Dornberger, A. Haertl, U. Moellmann,
H-M. Dahse, M. Kolarova, K. Aleksieva, N. Chipev, Sofia,
Jena, Germany
GAM69 Malonyl,4-5-dihydroniphimycin: new polyol macrolide
antibiotic, produced by Streptomyces hygroscopicus
V. Ivanova, M. Kolarova, K. Aleksieva, Sofia
GAM70 Diphenylether and macrotriolides occurring in a fungal
isolate from the antarctic lichen Neuropogon
V. Ivanova, U. Graefe, B. Schlegel, M. Kolarova,
K. Aleksieva, Sofia, Jena, Germany
GAM71 Microbiaeratin, a new natural indole alkaloid from a
Microbispora aerata strain, isolated from Livingston
island, Antarctica
V. Ivanova, U. Gräfe, H-M. Dahse, M. Kolarova,
43
K Aleksieva, H Laatsch, Sofia, Jena, Göttingen, Germany
GAM72 An investigation on the opportunities for increasing
Streptomyces ambofaciens biosynthetic activity through
induced mutagenesis
N. Hristova, V. Baloutzov, S. Karafizova, Sofia
GAM73 Permeabilization of yeast cells for Α -keto acid production
D.D. Kostova, A. Kujumdzieva, Sofia
GAM74 Study of effect of oxygen mass transfer on biosynthesis of
exopolysaccharide P-45
A.V. Atanassova, K. Pavlova, G. Atanassova, A.I. Tonchev,
V.V. Lossev, V.G. Nasarov, Pestera, Plovdiv
GAM75 Scaling-up of amino acid biosynthesis using oxygen mass
transfer
A.V. Atanassova, A.I. Tonchev, V.V. Lossev,S.B. Petkov,
V.G. Nasarov, Pestera
GAM76 Quantitative assay for gentamicin in blood plasma by
microbiological method
P. Yankova, A. Varssanova, Pestera
GAM77 Designing a whole-cell biotransformation double-phase
oxidation system with Streptomyces roseochromogenes
B. Marinov , D. Koleva, I. Kostova, Razgrad
GAM78 Biophysical characteristics of bacterial biosurfactants
E. Stoimenova, E. Vassileva-Tonkova, M. Ivanova,
Ch. Petkova, A. Jordanova, A. Sotirova, D. Galabova, Z.
Lalchev, Sofia
GAM79 Combination of methods for species identification of
Lactobacillus delbrueckii ssp. bulgaricus
T. Savova, Z. Urshev, M. Spassova, I. Petrova,
D. Ishlimova, P. Alexieva, Sofia
GAM80 Selection of Streptomyces thermophilus strains for
improvement of starter cultures for direct application
K. Pashova-Baltova, N. Ninova, Z. Urshev, M. Michailova,
Sofia
GAM81 Valuation of proteolytic activity in cultures of
Lactobacillus delbrueckii ssp. bulgaricus
Z. Urshev, N. Fachikova, K. Pashova-Baltova, I. Petrova,
Sofia
44
GAM82 Antimicrobial activity of lactic acid bacteria isolated from
Bulgarian rye acid dough
L. Iovcheva, G. Dobreva, R. Vassileva,
S. Antonova-Nikolova, Sofia
GAM83 Milk-clotting enzymes from submerge cultivated
basidiomycetes
T. Dmitrieva, I. Feist, M. Shamtsyan, St. Petersburg, Russia
GAM84 Intensification of yeast biomass accumulation and ethanol
fermentation processes
I. Koltyga, T. Dmitriyeva, M. Shamtsyan St. Petersburg,
Russia
GAM85 Ballistic disintegration of biomass from Lactobacillus
delbrueckii subsp. bulgaricus BTCC 50
D. Nikolova, V. Savov, Y. Evstatieva, S. Ilieva, P. Dalev,
A. Atev, Sofia
GAM86 Hybridization of Lactobacillus plantarum 226-15 and
Lactobacillus casei subsp. casei C
A. Slavchev, I. Murgov, Z. Denkova, Plovdiv
GAM87 Investigation of the inhibitory effect of lactic acid bacteria
on the cells of Escherichia coli NBIMCC 8739
P. Nedelcheva, Z. Denkova, R. Nikolova, Plovdiv
GAM88 The effect of DNA replication on the repair of damaged
plasmids in Saccharomyces cerevisiae
M. Koprinarova, A. Gospodinov, G. Russev, Sofia
GAM89 Ageing in brewing yeast
T. Ginova, S. Mileva, Sofia
GAM90 Growth inhibitory properties of chalcones against various
yeast species
K.L. Lahtchev, D.I. Batovska, St. Parushev, V. Bankova, Sofia
GAM91 Electro-optical method as a new approach of investigation
and discrimination of two strains Esherichia coli
A. Gurova-Chausheva, E. Velichkova, R. Aleksandrova,
S. Danova, S.P. Stoylov, Sofia
GAM92 Application of co-polymer microparticles for
immobilization of trypsin
T. Ivanov, M. Kamburov, V. Ivanova, J. Hristov, Sofia
45
GAM93 Immobilization of trypsin on co-polymers of acrylonitrile
and maleinic anhydride
T. Ivanov, M. Kamburov, V. Ivanova, J. Hristov, Sofia
Veterinary Microbiology
VM9 Comparative studies on the methods for detection of

Mycobacterium bovis in animals
M. Bonovska, S. Milashki, A. Abass, T. Savova, E. Gjurova, Sofia
VM10 Influence of karbofuran on capability of Fusarium
moniliforme to produce fumonisins upon maize
L. Borisova, Y. Tasheva, V. Vrabcheva, Sofia
VM11 Determination of Pseudomonas aeruginosa in bull and
boar semen by reaction co-agglutination
G. Vassilev, Stara Zagora
VM12 Experiments of induction of toxigenous mutants of
Clostridium perfringens type C
E. Iliev, D. Ilieva, M. Petkov, N. Korudgiisky, Sofia
VM13 Changes in the pathogenic potential of Yersinia
enterocolitica during storage of contaminated pig meat
M. Iliev, H. Najdenski, Sofia
VM14 Detection of pathogenic serotypes of Yersinia
enterocolitica in contaminated milk
M. Iliev, H. Najdenski, Sofia
VM15 Exactingness bacteria in etiology of endometritis
puerperalis cow and possibility for therapy
N. Korudjiiski, Sofia
VM16 Drug resistant bacteria of the genus Alcaligenes, isolated
from rabbits with intestinal disorders
T. Galabinova, B. Mitov, N. Korudjiiski, S. Ivanova,
L. Angelov, Sofia
VM17 Neoplasms in farm-raised Russian sturgeon
V. Chikova, V. Kolarova, A. Tsekov, Sofia, Varna
VM18 Occurrence of aeromonosis infection among cultured
Paddlefish (Polyodon spathula)
V. Chikova, T. Hubenova,V. Kolarova, R. Atanasova,
A. Zaikov, Sofia, Varna
46
Friday, October 06
Poster session II
16:00-18:30
Medical Microbiology
MM22 Emm Sequence typing of clinical isolates Streptococcus

pyogenes recovered in Bulgaria
A. Decheva, V. Karjeva, D. Beshkov, I. Alexiev, Sofia
MM23 Recombinant Borrelia Burgdorferi proteins as antigens
for serologic diagnosticof lyme borreliosis
I. Christova, M. Lesseva, G. Miloshev, Sofia
MM24 Detection of Borrelia burgdorferi sensu lato, Anaplasma
phagocytophilum and Francisella tularensis in wild
rodents from an endemic focus of tularemia in Bulgaria
T. Gladniska, I. Christova, E. Taseva, R. Nenova, Sofia
MM25 New focus of tularemia established in North-East Bulgaria
during 2004 – 2005
N. Gotev, K. Mladenov, Tz. Tzvetanov, E. Penkov,
N. Korudjiysky, S. Ivanova, V. Doicheva, Sofia
MM26 Fenotypic characteristics of Francicella tullarensis
isolated in Bulgaria during 1961 – 1965 and 1998 – 2005
N. Gotev, K. Mladenov, Sofia
MM27 Treatment of experimental tularemia infection
K. Mladenov, H. Najdenski, Tz. Tzvetanov, N. Gotev, Sofia
MM28P Rapid identification and biodiversity of Lactobacillus
species in vaginal samples
S. Dimitonova, P. Grozdanov,A. Galabov, B. Bakalov,
R. Aleksandrova, G. Stoyancheva and S. Danova, Sofia
MM29 Virulence determinants of Aeromonas spp. isolated from
food, drinking water and patients in Bulgaria
P. Orozova, I. Abrashev, Sofia, Bulgaria
MM30 Determination of expiry term of antimicrobial disks
Cefoxitin and Ceftriaxon and their introduction for
47
manufacturing
D. Chankova, D. Pencheva, Sofia
MM31 Laboratory methods for drug susceptibility testing of
medically important yeast and moulds
A. Kouzmanov, T. Kantardjiev, Z. Ivanova, L. Boyanova,
Sofia
MM32 Study of hypocholesterolic effect of Higher
Basidiomycetes
A. Popov, A. Panchenko, O. Chistova, N. Petrishchev,
N. Denisova, M. Shamtsyan, St. Petersburg, Russia
MM33 Resistance and genotypic diversity of multidrug resistant
Acinetobacter baumanii in university hospita
R. Vatcheva-Dobrevski, E. Savov, A. Bernards,
van den Barselaar, Sofia, Ceiden, Netherlands
MM34 Immunomodulating and antitumour action of higher fungi
V. Spiridonova, P. Tsvetkov, A. Panchenko,
A. Korchmaryova, N. Petrischev, M. Shamtsyan,
St. Petersburg, Russia
MM35 Antioxidant properties of higher mushrooms
N. Dubyago, I. Shugaley, E. Tozik. M. Shamtsyan,
St. Petersburg, Russia
MM36 Indirect immunofluoroscence method for detection of
Helicobacter pylori directly from stomach biopsy
K. Ivanova, Tz. Ilieva, M. Mariana, I. Mitov, B. Vladimirov,
J. Churchev, Sofia
Virology
V31 Effects of some antivirals against rhinovirus H-14

I. Georgieva, A. S. Galabov, Sofia
V32 Study of virus RNA synthesis in cell cultures infected with

bovine viral diarrhea virus (BVDV) using hypertonic
NaCl solutions
Yu. P. Abashev, L. Mukova, L. Wassilewa, A. S. Galabov,
Sofia
48
V33 Biological activity of extract from Orthosiphon stamineus
Benth
S. Shishkov, K. Kostova, E. Georgieva, V. Kapchina-
Toteva, Zh. Iordanova, V. Chipeva, S. Trandeva, Sofia
V34 Virological surveillance of acute flaccid paralysis in

Bulgaria during 2001-2006 period
N. Korsun, S. Gyurova, Z. Mladenova, Sofia
V35 Effect of thiosemicarbazone on HSV-1 and HSV-2

replication in vitro
N. Vilhelmova, M. Gacovska, S. Shishkov, P. Souza, Sofia;
Madrid, Spain
V36 Seroepidemiological studies of influenza in Varna for

1990-2004 period
V. Rusev, L. Ivanova, A Shtilianova, R. Ivanova,
B. Shmatov, Varna
V37 Influence of osmotic swelling on the dhpc lipid bilayers

containing ndv glycoproteins
P. Borissova, V. Neitchev, L. Doumanova, Sofia
V38 Detection of canine parvovirus serotype 2 in fecal

samples from dogs by polymerase chain reaction
C. Filipov, P. Grozdanov, Z. Raikov, H. Haralambiev,
A. S. Galabov, Sofia
V39 Molecular diagnostic of the Lewandowsky-Lutz syndrome

(Еpidermodysplasia Verruciformes)
B. Boneva, M. Sajedg, G. Mateev, Z. Kalvachev, Sofia
V40 Varicella zoster virus (VZV) and pregnancy

L. Ivanova, Varna
V41 Demonstration of rabbit myxoma virus in cell cultures by

direct immunoperoxidase method
49
R. Bostandjieva, Z. Dimitrova, R. Peshev, Sofia
V42 Evaluation of the efficacy of the broad-spectrum

disinfectant on some viruses pathogenic for the fishes
D. Ilieva, Sofia
V43 Comparative studies of whales for diagnostic of some

virus infections in fishes
V. Chikova, D. Ilieva, Sofia
Infectious Immunology
II4 Rapid immunohistochemistry method for detection of TSE

prion protein on frozen brain tissue sections
E. Lubashevsky, H. Yadin, S. Perl, N. Adry, A. Gorochov,
V. Bumbarov, Beit Dogan, Israel
II5 “Respivax” modulates the expression of CD86 on
different circulating APC subsets
D.Stankulova, A. Mihova, H. Taskov, Vl. Maximov,
M. Nikolova, Sofia
II6 Skin test for assessment of post-vaccine BCG allergy.
Quantity of Bulgarian PPD tuberculin for human use
E. Sapungieva, E. Jordanova, Sofia
II7 Fifty-year immunoprophylactics of tetanus with Bulgarian
vaccine
E. Sapungieva, I. Todorova, E. Jordanova, J. Hristova,
M. Demireva, R. Malchanova, Sofia
II8 Monitoring of cytotoxic CD8 T lymphocytes in HIV-1+
patients subjected to HAART
M. Muhtarova, M. Nikolova, S. Magaev, H. Taskov, Sofia
II9 Protection against whlooping cough in children between 0-
6 years old
R. Alexiev, K. Hadjiisky, S. Malchanova, V. Demireva,
Pl. Nenkov, Sofia
II10 Investigation on the immune status of the population
against diphtheria during the period 2001 – 2005
50
R. Alexiev, K. Hadjiiski, S. Malchanova, V. Demireva,
Pl. Nenkov, Sofia
II11 Determination of minimal sensitizing doze of BCG vaccine
(substrain Sofia SL222) in guinea pig
T. Stefanova, M. Chouchkova, S. Nikolaeva, Sofia
II12 Personal experience for diagnostics and differential
diagnostics of avian flue
V. Lupke, B. Yonkova, V. Lyoutzkanova, Veliko Tarnovo,
Varna
II13 Chlamydia trachomatis antibodies in serum and genital
fluids in infertile couples
V. Yonkova, V. Lyoutzkanova, V. Savouleva, Y. Yonkov,
Varna
II14 Immunoblot analysis of antibody response to plasmid
encoded released proteins of Yersinia enterocolitica in
patients with reactive arthritis
E. Golkocheva, H. Najdenski, R. Stoilov, Sofia
II15 Attenuation and preserved immunogenic potential of
Yersinia pseudotuberculosis mutant strains evidenced in
oral pig model
H. Najdenski, E. Golkocheva, E. Ivanova, V. Kussovski,
A. Vesselinova, S. Garbom, H. Wolf-Watz, Sofia, Umea,
Sweden
II16 Hemocyanins as immunostimulators
R. Toshkova, E. Ivanova, M.-D. Nastke, L. Velkova,
S. Stevanovic, R. Hristova, A. Dolashki, M. Gardeva,
I. Dimitrov, W. Voelter, P. Dolashka-Angelova, Sofia,
Tuebingen, Germany
Parazitology
P2 Epidemiological aspects of human trichinellosis in

Bulgaria (2001 – 2005)
M. Ivanova, R. Kurdova, D. Jordanova, N. Tsvetcova, Sofia
P3 Study on the diagnostic value of specific IgE antibodies in
human echinococcosis
51
I. Marinova, G. Nikolov, A. Mihova, R. Kurdova,
B. Petrunov, Sofia
P4 Detection of cross-reactive bands in cystic fluid by
western blot analysis
I. Marinova, I. Rainova, A. Tchernov, R. Kurdova, Sofia
P5 Application of IgG аvidity for diagnosis of acute
toxocarosis
I. Rainova, Sofia
P6 Antibodies against Toxoplasma gondii in human Ig
preparations
I. Rainova, J. Nacheva, A. Tchernov, Sofia
P7 Identification of free-living amoebae by PCR.
N. Tsvetkova, R. Kurdova, Sofia
P8 Visceral leishmaniasis in Bulgaria
R. Harizanov, G. Filipov, D. Yordanova, R. Kurdova, Sofia
P9 Echinococcosis distribution among children and
adolescents in Bulgaria (1990 – 2005)
D. Yordanova, R. Kurdova, Sofia
P10 Clinical forms and chemotherapy of trichinosis
D. Vuchev, K. Anichina, K. Eneva, V. Blagoeva, A. Russinova,
M. Darakchieva, G. Stancheva, Sofia, Plovdiv, Smoljan
P11 Epidemiological features of trichinosis in central southern
Bulgaria (Plovdiv, Pazardjik and Smolian regions)
D. Vuchev, K. Eneva, V. Blagoeva, A. Russinova,
M. Darakchieva, G. Stancheva, N. Paliiska, Plovdiv,
Smoljan, Pazardjik
Plant and Soil Microbiology
Chairpersons: A. Stoev, Sofia

R. Donkova, Sofia
N. Kaloianova, Sofia
PSM4 Morphologic and cultural variation in Phomopsis

foeniculiI
R. Rodeva, J. Gabler, Sofia, Aschersleben, Germany
52
PSM5 In vitro and in planta interaction between three sclerotial
plant pathogens
R. Rodeva, R. Pandeva, Sofia
PSM6 Biological properties and frees-drying of apple chlorotic
leave pop virus isolates from fruit tree species in
Bulgaria
A. Borisova, A. Yordanova, Kjustendil, Sofia
PSM7 Effect of inoculation with Azospirillum and AM fungi on
the plant biomass of white thistle (Sylibium marianum
L.)
E. Jonova, N. Kaloyanova, Sofia
PSM8 Effect of inoculation with local phosphate decomposing
bacterial strains on the rye-grass
K. Nedjalkova, Sofia
PSM9 Taxonomical identification of arsenic resistant and
arsenic transforming sulfate – reducing bacteria
K. Krumova, V. Groudeva, Sofia
PSM10 Effect of protein hydrolysate from keratin wastes on the
soil microflora
M. Nustorova, A. Gushterova, D. Braikova, E. Vasileva,
Sofia
PSM11 Response of soybean genotypes to inoculation with
Bradyrhizobium japonicum
A. Markova, R. Altimirska, Y. Kirkova, G. Stoimenov, Sofia
PSM12 Microbiological activity in soybean rhizosphere at
different soil moisture
R. Altimirska, A. Markova, Hr. Stoykov, K. Chachev, Sofia
PSM13 The influence of herbicide Relay on the main properties of
Bradyrhizobium japonicum
R. Donkova, Sofia
PSM14 Microbiological characteristic of soils in the area of non-
ferrous metals factory, town of Plovdiv, Bulgaria
R. Donkova, N. Dinev, Sofia
PSM15 Cadmium influence on microbiological activity of
calcareous chernozem
G. Petkova, R. Donkova, Sofia
17:30 General discussion
53
54
ABSTRACTS
55
56
TUBERCULOSIS
Tb1
VITAE OF DR. STAMEN GRIGOROV
Angel S. Galabov
The Stephan Angeloff Institute of Microbiology, BAS
Tb2
BCG VACCINES
M. Chouchkova, T. Stefanova
BB-NCIPD Ltd., Sofia
The BCG vaccines celebrate the 100th anniversary of their discovery in a decade at the
beginning of 21 century since Albert Calmette and Camille Guérin had presented it before
the Academie des Sciences in 1908. Over a period of 13 years, from 1908 to 1921, the both
researchers produced ever less virulent subcultures by cultivating bovine tubercle bacilli
over and over again. More than three billion doses of BCG vaccine have been given over
the past 80 years. We would again emphasize the immense role played by BCG immunization
in the struggle against tuberculosis in children.
The evolution of BCG strains and the diversity of strains used for production as well
as a need to explore their potential impact in efficacy and safety of BCG vaccines in
humans have been considered at the WHO meetings in the last few years. WHO had
identified the need for better characterization of BCG substrains in the presently used
BCG vaccines. The Bulgarian BCG substrain is one of the three substrains used in the
world for the production of BCG vaccines, which have been prequalified by WHO for the
UN agencies. The moderate residual virulence, the adequate postvaccinal tuberculin
sensitivity and the genetic stability of the strain as well as the consistency of the production
have been confirmed in the recent studies.
57
Tb3
РЕНЕСАНС НА ТУБЕРКУЛОЗАТА. АКТУАЛНИ
ПРОБЛЕМИ
Зл. Янкова
Клиника по пулмология - МУ, гр. Пловдив
Може да се твърди, че туберкулозата е в “топлистата” на 10-тте заболявания,

завършващи най-често със смърт. СЗО наблюдава туберкулозната епидемия в
повечето страни на Света.
В последните години честота на туберкулозата е около 6,3 - 11,1 милиона
болни ежегодно. Смив положителните са 2,8 - 4,9 милиона. Фаталният изход е
23%. Заболяването поразява млади хора и за високо рисковите райони – често се
среща при родилки. В 22 страни са съсредоточени 80% от всички заболели -
Индия, Китай, Пакистан, Бангладеш; Африка - южно от Сахара и др. За период
от 1 година /1997-1998/ 36500 инфектирани с НІV са починали от туберкулоза.
Бедните страни имат средно 2,6 пъти по-често туберкулоза от богатите.
Няма тясна корелация между отделяните средства за борба с туберкулозата и
ефективност на Националните програми /СЗО/. Има 4 фактора, с които е свързана
епидемиологията:
1.Разрушаване на принципите на туберкулозния контрол и появата на
резистентност в Източна Европа.
2.Инфектиране с HIV в Африка. Появяване на асоциирани инфекции от двете
заболявания.
3.Миграция на инфектирани с ТБК от бедните - в индустриализираните страни.
4.Нарушението на туберкулозния контрол в Латинска Америка и Азия.
У нас Национална програма за борба с туберкулозата действува от 1950
год., когато са създадени първите диспансери. От 400 на 100 хиляди население,
туберкулозата се редуцира до 28 на 100х. За съжаление след 1992 година
заболяването зачести и сега се движи между 42 – 45 на 100 хиляди за известната
туберкулоза, но има хронично болни с недоказана туберкулоза. Един важен и
нерешен проблем остава доказването на Латентната туберкулозна интоксикация.
Предлагаме за обсъждане диагностичен алгоритъм за ежедневната клинична
практика.
Tb4
КОНТРОЛ НА ТУБЕРКУЛОЗАТА И ВЪВЕЖДАНЕ НА DOТ
СТРАТЕГИЯ В БЪЛГАРИЯ
Д. Стефанова
Увеличаването на заболеваемостта от туберкулоза в глобален мащаб през
58
последните две десетилетия стана един от водещите проблеми, ангажиращи
световната общественост. В резултат на съчетанието на множество
неблагоприятни фактори в някои страни туберкулозата взе застрашителни
размери. Затова целите на Туберкулозния контрол са насочени към :
1. Намаляване заболеваемостта и смъртността от туберкулоза
2. Ограничаване на лекарствената резистентност
Единственно рационалната химиотерапия може да спре развитието на
туберкулозния процес. Навременното започване на комбинирана, продължителна
и непрекъсната терапия е гаранция за ефективно лечение. През 1990 год.
Световната Здравна Организация изрази тревогата си от нарастване на
разпространението на туберкулозата и започна въвеждането на DOTS стратегията
– Директно наблюдавано лечение в съкратени срокове.DOTS се превърна в
повратна точка в контрола на туберкулозата. През 1998 г. започна въвеждането
на стратегията у нас, което продължи до 2003 год. Лечебните режими на DOTS
стратегията се основават на най-ефективни съчетания от противотуберкулозни
препарати и синергичното им действие върху Туберкулозния микобактерий. Тъй
като лечението при всички болни не може да се провежда само с краткосрочни
режими СЗО ревизира стратегията и въведе DOT /директно наблюдавано лечение/
. DOT позволява и индивидуализирани програми с вариации в комбинациите при
тежки прогресиращи форми на туберкулоза.
След започването на модерната химиотерапия Туберкулозният микобактерий
показа резистентност към различните медикаменти, която компрометира
Националните програми за контрол и лечение на туберкулозата. От съществено
значение е мултирезистентността, която се определя от резистентност към
основните туберкулостатици – Тубоцин и Римицид при наличие или отсъствие на
резистентност към другите туберкулостатици. В България мултирезистентността
варира в последните години. Най-висока е тя през 1998 г. – 7,3% , а за 2005 г. е
4.1% . Намаляването на процента на мултирезистентност се дължи на включване
на стратегията DOT+ с етиоамиди, макролиди, нови генерации аминоглюкозиди,
респираторни хинолони и въвеждането на хирургическо лечение при болни без
дисеминация на туберкулозния процес и съхранени кардио-респираторни резерви.
Традиционно резистентността се разделя на първична и придобита.
Първичната резистентност се развива при болни, при които няма анамнеза за
провеждано туберкулостатично лечение. Първичната резистентност се задържа
висока до 40%, което показва, че в страната циркулират вирулентни
мултирезистентни щамове. Вторичната резистентност се развива когато се
провежда лечение с неефективни лекарствени режими или лечението е прекъснато
преди определените срокове.
За първи път у нас ще се представи и новата стратегия на СЗО / 2006- 2011 / за
Контрол на туберкулозата.
59
Tb5
ТУБЕРКУЛОЗАТА СРЕД ДЕЦАТА – В МИНАЛОТО И
ДНЕС
П. Минчев
Университетска Детска Клиника по Белодробни Болести
Медицински Университет – София
Разпространението на туберкулозата в България сред детското население след

войната е изключително голямо – 90 на 100 000 души. За период от 45 години
заболеваемостта намалява и през 1990 година е в границите на 8 на 100 000
души. В структурата на заболяемостта настъпват промени, като значително
намаляват относителните дялове на хематогенно – дисеминираните форми и
туберкулозния менингит. Основният фактор за благоприятните тенденции в
епидемиологията на първичната туберкулоза е провеждането на специфична
имунопрофилактика с ваксината БЦЖ. България заедно с Хонг – Конг въвежда
задължителна БЦЖ ваксинация при децата още през 1951 година.
След 1990 година започва неблагоприятна тенденция в епидемиологията и
структурата на първичната туберкулоза. Причините за това са комплексно
обусловени. През 2005 година заболяемостта от туберкулоза сред децата е вече
25,8 на 100 000 души. Съществен проблем е и латентната туберкулозна инфекция,
която налага контролирана химиопрофилактика.
Лечението на туберкулозата при децата се извършва с въведените у нас
терапевтични режими за всяка форма на първична туберкулоза. DОТS е
неприемлива за деца в целия свят, поради различията между туберкулозата у
възрастни и деца. Основната обективна разлика е, че бацилоотделянето при деца е
в ниски граници /6-10%/ и не е единствения критерии за поставянето на диагнозата.
Туберкулозата сред децата и възрастните е заболяване, което не може да бъде
изцяло инактивирано. Абсолютно сигурно е че е възможно да бъде поставено под
непрекъснат контрол.
Tb6
СЪВРЕМЕННА МИКРОБИОЛОГИЧНА ДИАГНОСТИКА
НА ТУБЕРКУЛОЗАТА И МЕТОДИ НА
ЕПИДЕМИОЛОГИЧНО МАРКИРАНЕ
Т. Кантарджиев, Ст. Панайотов, Ел. Бачийска,

НЦЗПБ, София
Представени са възможностите на НРЛ по туберкулоза и НРЛ по молекулярна

микробиология към Националния център по заразни и паразитни болести в
60
съвременната микробиологична диагностика на туберкулозата. Посочени са
основните – класически методи, както и нови, не конвенционални методи в
култивирането на M.tuberculosis. Изтъква се голямото диагностично значение на
методите за епидемиологично маркиране на туберкулозните щамове – RFLP,
AFLP, сполиготипиране.
Tb7
МОЛЕКУЛЯРНО-ЕПИДЕМИОЛОГИЧНА
ХАРАКТЕРИСТИКА НА ЩАМОВЕ MYCOBACTERIUM
TUBERCULOSIS ОТ РАЗЛИЧНИ РЕГИОНИ НА БЪЛГАРИЯ
Виолета Вълчева1, Игорь Мокроусов2, Ольга Нарвская2, Надя Маркова1

Институт по Микробиология, БАН ,София1;Санкт Петербургский Институт
Пастьор, Русия2
В България броят на новооткритите случаи на туберкулоза се е увеличил от

41.0/100,000 през 2000г. до 42.4/100,000 през 2004г. Цел на изследването беше
молекулярно епидемиологично охарактеризиране на популационната структура
на M. tuberculosis в България и анализ на мутациите, свързани с резистентността
към противотуберкулозни препарати. Въз основа на метода spoligotyping, 113
щама бяха подразделени на 35 сполиготипа: 13 уникални профили и 15 профили,
включващи от 2 до 29 щама; индексът на Хънтър-Гастон беше 0.9. Сравнението
със световната база дани SpolDB 4.0 показа присъствието на два глобални
сполиготипа ST53 (25.7%) и ST47 (6%). 19 (16.9%) и 7 (6%) щама принадлежаха на
ST125 и ST41. 8 (7%) сполигопрофили не бяха намерении в SpolDB 4.0. Щамове с
генотип Beijing не бяха открити в изследваната колекция. Типирането с методите
IS6110-RFLP, MIRU-VNTR и DRE-PCR позволи диференциране на щамовете вътре в
ST53, ST47, ST41 и ST125. 17 (15%) щама бяха генотипно резистентни към
рифампицин, въз основа на анализ на rpoB гена. 13 от тях имаха мутация в rpoB531
TCG>TTG докато един щам беше хетерорезистентен. Мутация в katG315 се
установи в 9 щама. 8 щама имаха мутация в embB306, а 3 от тях - мутации
едновременно в rpoB и katG. При сравнение с фенотипните тестове, генотипната
детекция на резистентността даде 80% чувствителност за рифампицин (анализ на
rpoB) и само 30% за изониазид (анализ на katG315). Въз основа на получените
резултати, беше установено, че изследваната популация на M. tuberculosis е доста
хетерогенна и в нея преобладават няколко глобално разпространени и балкански
сполиготипа. Трансмисията на мултирезистентни щамове в България (21.6% в
колекцията) не е свързана с глобалното распространение на генотип Beijing, който
очевидно все още не е достигнал страната ни.
61
Tb8
ВЪТРЕВИДОВО ОПРЕДЕЛЯНЕ НА МИКОБАКТЕРИИ
ЧРЕЗ PCR
Магдалена Боновска1, Христо Найденски2

1
Централен Научноизследователски Ветеринарномедицински Институт, София,
2
Институт по микробиология “Стефан Ангелов” – БАН
Различни представители на род Mycobacterium са диференцирани успешно с

мултиплексен формат на PCR, прилагайки две и три двойки праймери в различни
количествени съотношения. Използвани са двойките праймери ТВ15/ТВ19, IS41/
IS43 и PT1/PT2 и геномна ДНК от M. bovis, M. bovis BCG, M. tuberculosis, M. avium,
M. kansasii, M. intracellulare, M. malmoense, M. phlei, M. chelonae и M. scrofulaceum.
При мултиплексна PCR с праймери ТВ+РТ в съотношение1:1, се наблюдава
PCR продукт с големина 600 bр при M. bovis, M. avium, M. kansasii, M. intracellulare,
M. malmoense и M. chelonae. При ТВ+РТ (2:1,7) само M. bovis, M. avium и M.
malmoense амплифицираха PCR продукт от 600 bр, а при ТВ+РТ (2:1,5) - само M.
bovis. M. tuberculosis амплифицира два фрагмента (400 и 600 bр) при всички
количествени съотношения на праймерите. Комбинацията ТВ+IS (2:1)
амплифицира два фрагмента от 300 и 600 bp с M. bovis, M. bovis BCG и M.
tuberculosis. Подобни амплификати се наблюдаваха при M. bovis, M. bovis BCG и
един щам M. tuberculosis и при съотношение на праймерите 1,7:1. Другите 2 щама
M. tuberculosis показаха един фрагмент от 300 bp. При ТВ+IS (1,5:1) двуфрагментни
амлификати показаха M. bovis и M. bovis BCG, а всички M. tuberculosis бяха
еднофрагментни. При амплификация с три двойки праймери ТВ+IS+РТ (2:1:2) се
наблюдават три фрагмента - 300, 400 и 600 bp при M. tuberculosis, два (300 и 600
bp) при M. bovis и M. bovis BCG и един (600 bp) при M. avium, M. phlei и M.
scrofulaceum. При ТВ+IS+РТ (2:1:1,5) амплификати се установяват само при M.
bovis и M. bovis BCG (двуфрагментни) и при M. tuberculosis (трифрагментни).
Получените резултати показват, че мултиплексната PCR с 2 и 3 двойки
праймери, при подходящо съотношение между тях, може да се използва успешно
за специфично и бързо вътревидово разделяне на представителите на M.
tuberculosis complex от една страна, а от друга между тях и осталите микобактерии,
което не винаги е възможно с нормална PCR, използваща за амплификация една
двойка специфични праймери.
62
VIROLOGY
V1
HENIPAVIRUSES, A NEW FAMILY OF PARAMYXOVI-
RUSES, WHICH HAVE EMERGED IN ASIA AND AUSTRALIA
F. Wild
INSERM U404, Immunity and Vaccination, CERVI, IFR 128, Lyon, France.
During the past ten years in Southern Asia and the Western pacific a number of
viruses have emerged in which the natural host is the fruit bat (Pteropus). Amongst these
were two closely related viral pathogens Hendra and Nipah, which appeared in
geographically distinct regions. The viruses were shown to belong to the Paramyxovirus
family. In 1994 in Australia, Hendra virus crossed the species barrier infecting horses and
eventually man. In 1998 in Malaysia, Nipah virus was found in pigs and subsequently man
became infected. Since this time, it has been shown that a high proportion of the fruit bat
population in Asia and Australia are infected. Further studies have identified epidemics in
humans in India and Bangladesh. Infection in pigs gives mainly an acute respiratory
disease with approximately 10% mortality, whereas humans develop encephalitis with up
to 70% mortality. For these reasons, the virus has been classified as a P4 virus i.e. can only
be handled in laboratories with the highest security levels.
In order to analyse the various processes during infection, we have developed the
hamster as an animal model. The infected animals develop fatal encephalitis and the
pathology is similar to that observed in humans. Immunisation of hamsters with either the
G or F glycoproteins protected the animals from a clinical infection. Further, either polyclonal
or monoclonal antibodies directed against either the G or F glycoprotein when given
passively protected animals against infection. Thus, we have defined the main actors in
both preventive (vaccination) and treatment (passive) of the virus infection.
V2
STRUCTURAL BASIS FOR ANTIVIRAL DRUG-RESISTANCE

OF A VP1-MUTATED ENTEROVIRUS
Angel S. Galabov1, Ivanka Nikolova1, Roumena Petkova2, Stoyan Chakarov2 and

Boris Atanasov3
1
Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria;
2
Scientific Biological Service, Ltd., Sofia, Bulgaria; 3Institute of Organic Chemistry,
Bulgarian Academy of Sciences, Sofia, Bulgaria
63
Twenty years ago was found that antiviral WIN-compounds (such as arildone,
pleconaril, disoxaril and etc) inhibit virus uncoating. Based on direct crystal X-ray analysis
of virus-inhibitor complexes it was shown that the primary target structure of this action is
the VP1 capsid protein: inhibitors, inserted into a twisted â-sheet formed hydrophobic
cleft, increased structural rigidity of VP1 subunit and thus prevent virus “undressing”.
Following Darwinian evolution all WIN-treated wild viruses are blocked and the only
inhibitor-resistant generation survived – a phenomenon also well desribed.
Using “Darwinian selsction” approach two disoxaril-resistant mutant strains of the
coxsackievirus B1 from a wild-type disoxaril-sensitive (Connecticut 5) were obtained. One
of them was produced in FL cells and the other one isolated from brains of newborn mice,
infected with coxsackievirus B1 and treated with disoxaril. They were object of further
molecular genetic studies.
Analysis of the RNA sequence of an RT-PCR assay which primer sets selected from a
region of the coxsackievirus B1 genome coding for the capsid protein VP1 was carried out.
A parallel comparative analysis of the sequences of resulting fragments from the disoxaril
mutant studied and the Gen-Bank sequence of origin of the VP1 gene of coxsackievirus B1
was performed with the BLAST alignment tool. Distinct alternations in the VP1 locus of
the disoxaril-resistant compared to the sequence of origin from the Gen-Bank (namely, a
deletion of UUG at ntt. 2749-2751 and an insertion of UUU at nt. 2769) were observed. The
resistant mutant obtained in mice was found to be very similar to the strain, dependent in
cell cultures. A crucial important change in disoxaril-resistant strain was two point mutations
– M213H and F237L – both in ligand-binding pocket. As general, amino acid sequences in
a large VP1 peptide 195-255 is highly different in comparison with the corresponding
positions of the wild protein.
A putative 3D-models of coxsackievirus B1 VP1 protein – wild (Sofia variant) and its
disoxaril-resistant mutants – was constructed using as template known X-ray structure of
coxsckievirus B3 (pdbcov.ent file) with “Composer-4” and “MOLIDE” programming
packets. Also palmitate at B3-VP1 virus complex was replaced to disoxaril using GROMOS-
96 molecular dynamics program at very restricted degree of freedom. Generated tetrameric
proteins of wid and resistant mutant forms (both with myristilate as amide bond to VP4 N-
terminal group) was studied in terms of their intra-/inter molecular electrostatic and
hydrophobic interactions. Specific stabilizing effect of VP1 on tetramer assembling; a
cooperative effect of ligand binding supported by all chains and sterically forbidden
access to VP1-binding cavity in the resistant mutant were obtained. A mechano-chemical
hypothesis explaining vital difference between palmitate and disoxaril complexes will be
discussed.
64
V3
THE HIERARCHICAL QSAR TECHNOLOGY FOR EFFEC-

TIVE VIRTUAL SCREENING AND MOLECULAR DESIGN OF
POTENTIAL ANTIVIRAL AGENTS
V. E. Kuz’min, A. G. Artemenko, E. N. Muratov, L. N. Ognichenko, A. I. Hromov,

A. V. Liahovskij, P. G. Polischuk
A. V. Bogatsky Phys.-Chem. Institute of the Ni=ational Academy of Sciences of
Ukraine, 86 Lustdorfskaya doroga, Odessa 65080, Ukraine; E-mail:
victor@farlep.net
Hierarchical QSAR (Quantitative Structure Activity Relationship) technology (HT) is

destined for optimization of new effective antiviral agents creation process. HT allows to
solve the QSAR task not ab ovo, but with the use of information received from a previous
stage by mean of the system of improved solutions. The unique and principle feature of
the HT consists in multiple-aspects hierarchical strategy that related to: models of molecular
structure descripion; models of atoms description in molecular simplexes; scales of activity
estimation; mathematical methods of analysis the structure-activity relationship; final
aims of QSAR research (prediction, interpretation, structure optimization, molecular design).
The set of different QSAR models that are supplementing each other is the result of
application of HT. These models all together, in complex, solve the problems of virtual
screening, evaluation of structural factors influence on activity, modification of known
molecular structures and design of new high-performance potential drugs. Innovative
aspect and main advantages of HT: simplex representation of molecular structure, that is
providing universality, diversity and flexibility of description of compounds related to
different structural types; HT that depending on the concrete aims of research allows to
construct the optimal strategy of QSAR models generation. HT does not have the
restrictions of such well-known and widely used approaches as CoMFA and HASL, usage
of the lasts is limited in the structurally homogeneous set of molecules and only one
conformer. The efficiency of the HT shown on the example of compounds that possessing
antiviral activity. If in an initial set there were only 12% of active compounds, after
application of the HT 75% of new designed compounds turned out perspective antiviral
agents.
65
V4
CHRISTO RUSSEFF MEMORIAL LECTURE:
OXOGLAUCINE: A NEW HIGHLY POTENT
ANTIENTEROVIRAL COMPOUND
Lubomira Nikolaeva-Glomb1, Stephan Filipov2, Angel S. Galabov1

1
Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria;
2
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy
of Sciences, Sofia, Bulgaria
A series of aporphinoid alkaloids isolated From Glaucinum flavum L. or obtained

synthetically were tested in vitro for antiviral activity against viruses belonging to picorna-,
orthomyxo-, paramyxo- and herpesviruses. One of them, oxoglaucine, manifested a well
pronounced inhibitory effect on poliovirus 1 replication in FL cells measured by the semi-
quantitative agar-diffusion plaque-inhibition test. In virucidal activity testing the compound
did not show direct virucidal effect on the extracellular virus. Oxoglaucine’s 50% inhibitory
concentration for poliovirus 1 (Mahoney) was found to be 0.188 µg/ml in the CPE-inhibition
test and 0.041 µg/ml in the classical plaque-inhibition test. Similar values were obtained for
the vaccinal poliovirus type 1 strain, LSc-2ab. The antiviral effect of oxoglaucine on the
replication of viruses belonging to another enteroviruses was tested, i.e. coxsackie and
echoviruses (HEV-B group). Coxsackievirus A-9, the six coxsackie B viruses and 6
echoviruses were tested for their sensitivity against the antiviral effect of oxoglaucine by
the end-point dilution method in the multi-cycle CPE inhibition set-up in FL cells.
Oxoglaucine revealed a marked inhibitory effect on all tested enteroviruses. The
concentrations that reduced virus titer by 1 lg ranged from 0.01 µg/ml to 1.0 µg/ml. Selectivity
index was greater than 100 and even greater than 1000 for some of the viruses tested.
Time-of-addition study showed that the susceptible period to oxoglaucine’s effect is the
latent and lag phase of the virus replication cycle.
V5
RATIONAL TREATMENT COURSE SCHEDULE EFFECTIVE

AGAINST ENTEROVIRAL NEUROINFECTION IN MICE
Ralitsa Vassileva-Pencheva and Angel S. Galabov

The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences,
Sofia, Bulgaria; E-mail: galabov@microbio.bas.bg
Enteroviral infections are a basic indication for the application of antiviral chemotherapy.
The lack of an effective therapeutic registered for clinical use, in spite of the substantial
66
number of enteroviral replication inhibitors found in vitro, is due mainly to the rapid
development of resistance in vivo. This phenomenon is a consequence of an accumulation
of resistant population of quasispecies as a result of countless number of point mutations.
One of the main possible approaches for an attempt of preventing the occurrence of
resistance is the method of combined aplication of antiviral inhibitors. Because of the
multiple viral replication cycles in the presence of the partners in the combination, the
usual scheme of administration of antiviral drugs – all partners are administered at once in
one day, give no guarantee that the problem with the resistance would be resolved. In
order to find a solution to this relevant question of present interest we propose another
scheme for combined administration of inhibitors - consecutive administration of the
partners, as in double combinations they are applied every other day, in triple combinations
–every third day, and in quadriple combination –every fourth day. In previous study of
our team we found out that two of the triple combinations – Dis/Oxo/PTU-23 and Dis/Oxo/
Guan show significant effect of protection. In order to optimize this combined course of
treatment we studied the influence of chronology of the arrangement of inhibitors. In the
experiments that we carried out, three substances were applied in a combination – disoxaril
(WIN compound), oxoglaucine (a new antiviral drug, developed in our laboratory) and
guanidine-hydrochloride (a classic enteroviral inhibitor), on the model of a neurotropic
infection with coxsackievirus B1 in newborn mice. As a result of this investigation, it
could be concluded that the start of the treatment course with disoxaril has certain priorities,
especially when disoxaril is followed by guanidine-hydrochloride. The effect of the triple
combination starting with oxoglaucine, followed by guanidine-hydrochloride is moderate.
The combination in which guanidine-hydrochloride is the first of the partners to be applied,
proved to be ineffective.
V6
EFFECTS OF PICORNAVIRUS REPLICATION INHIBITORS

AGAINST CALICIVIRUS FCV
Julian D. Tumbarski and Angel S. Galabov

Sofia, Bulgaria; E-mail address: galabov@microbio.bas.bg
The search of substances suppressing replication of caliciviruses is of special interest

due to their particular role in the human infectious pathology. Calicivirises belonging to
Norovirus genus are among the principal causative agents of viral gastroenteritides (in
children predominantly, and in all age groups in the developed countries). Caliciviridae is
a comparatively recently differentiated family, initially (till 1979) included as a separate
genus in Picornaviridae. Caliciviruses possess a (+) RNA genome; they are close in
structure, but reveal a markedly different genome strategy. Application of anti-calicivirus
67
chemotherapy is indicated in contrast to the lack of systematic search for antivirals
efficient vs. caliciviruses.
The aim of present report is the testing anti-calicivirus effects of several highly efficient
inhibitors of picornavirus replication. Study was carried out on the feline calicivirus (FCV),
F9 strain, a surrogate norovirus, grown in the Crandell’s feline kidney cell line (CrFK),
highly susceptible to FCV replication. The antiviral screening carried out included the
following compounds: arildone, disoxaril and S-7 (inhibitors of early stages of the
picornavirus replication cycle), guanidine hydrochloride, PTU-23 and HBB (picornavirus
specific RNA synthesis inhibitors), ribavirin (a broad-spectrum antiviral agent) and
oxoglaucin (a recently described in this laboratory enterovirus replication inhibitor). Anti-
norovirus activity was tested through the CPE inhibition test in the microplate monolayer
cell cultures vs. virus inoculation doses ranging within 1 and 10 000 CCID50. The neutral
red uptake and the routine visual microscopic methodical variants were used for
measurement of both compound antiviral effect and cytotoxicity. Results obtained manifest
a pronounced efficacy of HBB (IC50 of 7.0 ìM), a marked activity of PTU-23 (IC50 67.4
ìM), ribavirin (IC50 6.6 ìM) and oxoglaucin (IC50 0.076 ìM). Inhibitors of early
stages in picornavirus growth cycle (arildon, disoxaril and S-7) and guanidine
hydrochloride did not show an anti-norovirus effect.
V7
IN VIVO EFFECTIVE ANTI-FLU COMBINATION OF

ANTIVIRALS
Lora Simeonova, Galina Gegova and Angel S. Galabov

The combination effect of rimantadine hydrochloride and oseltamivir phosphate

(prodrug of the active compound oseltamivir carboxylate) on experimental infection in
mice with influenza A/Aichi/2/68(H3N2) virus was studied. Compounds were administered
simultaneously in a 5-day-treatment course, starting on the day of virus inoculation, 4
hours before intranasal infection of animals with 10 or 20 viral 50% mouse lethal doses
(MLD50). Initially, we tested a series of compound combinations in a checkerboard order,
using oseltamivir at daily doses of 0.05, 0.1 and 0.2 mg/kg/day and rimantadine – 2.5, 5.0
and 7.5 mg/kg/day. Mortality rate dynamics was followed till the 14th day post infection,
mean survival time (MST) and protection index (PI) being evaluated. It was established: (i)
PI values of all combinations of oseltamivir with 5.0 and 7.5 mg/kg rimantadine reaching
87%, while the individual effect of oseltamivir - between 0 and 10% only, and of rimantadine
– 0% at 5 mg/kg and 18.7-29.6% at 7.5 mg/kg; (ii) markedly lengthened MST in combination-
treated groups. The combination effect was characterized as synergistic by the three-
68
dimensional method of Prichard and Shipman, modified for in vivo studies. In further
experiments, we studied in more details the antiviral activity of oseltamivir at a daily dose
of 0.05 mg/kg (200 times lower than the compound optimal effective dose of 10 mg/kg) in
combinations with rimantadine 5 mg/kg (8-16 times lower than the optimal effective dose
of 40-80 mg/kg). Measurement of mouse pneumonia parameters (lung virus titer in MDCK
cells, lung index and consolidation score) proved the high effectiveness of this combination
for treating of influenza virus A(H3N2) infection. At the 48-60th hour post infection (the
peak of lung virus growth) a 2.8 log10 CCID50 lower titer was recorded in the combination-
treated group, compared to that in the placebo group, in contrast to 0.1-1.0 log10 and 1.1-
1.4 log10 in the groups treated with oseltamivir and rimantadine, respectively. These data
emphasize the high anti-influenza A potential of the combination. As a next step we tested
the combination selected activity following a therapeutic treatment course: onset on days
1, 2 etc. post virus inoculation. Moreover, efficiency of higher doses of combination
partners (the ratio rimantadine/oseltamivir 99:1 been preserved) was studied.
V8
ТРЯБВА ЛИ ДА СЕ ИЗПОЛЗВА ПРЕПАРАТА

МОНЕНЗИН ЗА ПРОФИЛАКТИКА НА ПТИЧИЯ ГРИП
С. Дундаров
Монензинът е полиетерен йонофорен антибиотик. За него са известни следните

данни: 1.Притежава изключително широк противоинфекциозен спектър, в който
са включени много и различни бактерии, фунги, паразити и вируси ( американски
патент); 2. Широко се използва от над 30 години в цял свят като антикокцидиално
средство и за угояване на птици и добитък (американски патент); 3. Притежава
способност да преминава през клетъчните мембрани като променя обмена на соли
и образува микрофисури. Това му позволява да улеснява вътреклетъчния достъп
на препарати с противораков ефект (френски патент). 4. От над 20 год. успешно
се използва като препарат за локално приложение при лечение на повърхностни
заболявания, причинени от HSV1, HSV2 ,VZV и аденовирусни конюнктивити
(български патент на препарат Модувир); 5. Противохерпесният му ефект се
обуславя от комплексна инхибиция на синтезата на вирусните белтъци и коствено
въздействие върху репликацията на вирусната ДНК. Продукцията на
инфекциозен вирус е редуцирана в над 99%; 6. Доказан е като мощен индуктор на
интерферон (българско авторско свидетелство); 7. Според някои автори може да
предотврати събличането на грипния вирус в ендозомите като алкализира средата
им и ограничава ефекта на ензима клатрин; 8. Проведени наскоро у нас
изследвания показаха, че Монензин инхибира развитието на грипните вируси
А(H1 N1 ) и A(H7 N1 ) в клетъчна линия МДСК (резултатите ще бъдат докладвани
69
от авторите). Тези предпоставки ни дават основание да го препоръчаме като
хранителна добавка за профилактика срещу заразяване от птичи грип на
рисковите групи птици.
V9
IN VITRO ANTI-INFLUENZA VIRUS EFFECT OF A PRO-

TEASE INHIBITOR FROM STREPTOMYCES
CHROMOFUSCUS 34-1
Julia Serkedjieva1, Lidiya Angelova1,2, Ivana Roeva1, Iskra Ivanova2

1
Institute of Microbiology “Stefan Angelov”, Bulgarian Academy of Science, 26,
Acad. Georgy Bonchev St.; 2Department of Microbiology, Sofia University, 8
Dragan Tzankov Blvd., Sofia, Bulgaria
Twenty three Streptomyces strains, producers of typical serine protease inhibitors,

were tested for inhibitory activity on the replication of influenza virus A/Germany/34,
strain Rostock (H7N1) (A/Rostock) in Madin-Darby canine kidney cells. Eleven of them
(52.2%) significantly inhibited viral reproduction. The most effective was the inhibitor,
produced by Streptomyces chromofuscus 34-1 (SS 34-1). Its in vitro anti-influenza virus
effect was studied in more detail. As a first approach we assessed the susceptibility of
representative influenza viruses to the inhibitory action of SS 34-1; most sensitive to
inhibition were A/Rostock and A/PR8/34 (H1N1). By the use of complementary virological
assays it was demonstrated that the expression of the viral haemagglutinin on the surface
of infected cells, the virus-induced cytopathic effect and the infectious virus yields, used
as measures of A/Rostock virus growth, were all reduced at non-toxic concentrations of
SS 34-1. In addition the preparation protected mice from mortality in the experimental
influenza A/Aichi virus infection. The isolated novel protease inhibitor (PISC-2002) was
purified by anion-exchange chromatography and reversed phase-HPLC analysis. It was a
hydrophobic and a termostable protein, had a molecular mass of 11.2 kDa, isoelectric
point of 7.5 and a high content of hydrophobic amino acids and proline. The N-terminal
sequence demonstrated its homology to the Streptomyces subtilisin inhibitors family.
V10
ANTI-HERPESVIRUS ACTIVITIES OF PSEUDOMONAS SP.

S-17 RHAMNOLIPID AND ITS COMPLEX WITH ALGINATE
Mimi Remichkova, Ivana Roeva, Danka Galabova, Angel S. Galabov
70
Acad. Georgi Bonchev str. 26, 1113 Sofia, Bulgaria
Pseudomonas sp. S-17 produces rhamnolipid (biosurfactant) and alginate

(polysaccharide). In the present study, we investigated the antiviral activity of rhamnolipid
and its complex with alginate against herpes simplex virus (HSV) types 1 and 2. Rhamnolipid
and its complex with alginate significantly inhibited the herpes virus cytopathic effect
assessed in MDBK cell line. The suppressive effect of the compounds on HSV replication
was dose-dependent and occurred at concentrations lesser than critical micelle
concentration (CMC) of the surfactant. The 50% inhibitory concentration (IC50) of
rhamnolipid was 14.5ìg/ml for HSV-1 and 13ìg/ml for HSV-2. The IC50 values of the
complex were 435ìg/ml for HSV-1 and 428ìg/ml for HSV-2. Similar results were
obtained from the virus yield reduction assay. We observed that the antiviral properties
of rhamnolipid in a complex with a alginate were more pronounced than rhamnolipid alone.
V11
ANTIOXIDANT EFFECTS OF PLANT POLYPHENOLS
QUERCETIN AND RUTIN IN INFLUENZA VIRUS INFECTED
MICE
Milka Mileva1, Angel S. Galabov2

1
Department of Medical Physics and Biophysics, Medical University, 2 Zdrave Str.,
Sofia-1431, Bulgaria; 2Institute of Microbiology, Bulgarian Academy of Sciences,
Akad. G. Bonchev Str. 26, Sofia-1113 Bulgaria
In this study an investigation and comparison of the effects of plant flavonoid

polyphenols quercetin ant its sugar-containing homologue (rutinoside) rutin on the
“oxidative stress” in liver, lung as well as in blood plasma isolated from influenza virus A/
Aichi/2/68 (H3N2) (2.0 of LD50) inoculated mice, is carried out. It was found that experimental
influenza virus infection is accompanied with a significant increase of lipid peroxidation
products and a decrease of natural antioxidants vitamin E, an inhibition of cytochrome c-
reductase and liver monooxygenases (analgin-N-demethylase and amidopyrine-N-
demethylase) as compared to control (non-infected) animals. The preliminary (5 days)
supplementation of mice with rutin, quercetin or its combination and their subsequent
virus inoculation was accompanied with accumulation of lower levels of malondialdehyde
(MDA) and fluorescent lipofuscine-like products and of higher levels of endogenous
vitamin E in comparison with animals, subjected to influenza virus infection only. The
protective effect of rutin against influenza virus-induced lipid peroxidation and activities
of CYP and liver monooxygenases was better expressed than the effect of quercetin may
be due to containing of rutinoside part or difference of its metabolism.
71
V12
A PLANT POLYPHENOL EXTRACT REDUCES OXIDATIVE

STRESS IN THE LIVERS OF INFLUENZA VIRUS-INFECTED
MICE
Ekaterina Krumova1, Silviya Abarova2, Lyubka Tancheva2, Maria Angelova1, Julia

Serkedjieva1
1
Institute of Microbiology, 2Institute of Physiology, Bulgarian Academy of Sciences,
Akad. G. Bonchev Str. 26, Sofia-1113, Bulgaria
Plant polyphenols aroused considerable interest because of their broad pharmacological

potential which has been ascribed to their antioxidant and free-radical scavenging
capacities. We have found that a polyphenol extract, obtained from the medicinal plant
Geranium sanguneum L. (PC), was highly inhibitory to influenza virus replication in vitro
and protected mice from mortality in the experimental influenza infection (EIVI, Manolova
et al., 1986). At the same time it was shown that PC significantly restored and stimulated
the antioxidant activities in the lungs of virus-infected mice (VIM, Abarova et al., 2004). In
the present study we followed the effect of PC on the total antioxidant activity (AOA), on
the levels of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) as
well on the lipid peroxidation (LPO) in the 9000x g liver supernatant of albino mice on the
2, 6 and 9 days after the challenge with influenza A/Aichi/2/68 (H3N2) virus. The free–
radical character of EIVI (Tancheva et al., 2001) was confirmed clearly. The high
concentrations of LPO products as markers of the oxidative stress, correlated proportionally
with decreased AOA, particularly on day 9 p.i. (180% and 60% of healthy control, HC,
resp.). The preventive nasal treatment with 10 mg/kg of PC successfully normalized both
parameters. EIVI induced also a significant increase in SOD and CAT activities in the
livers of VIM on day 2 p.i.; their values reached 280% and 160% of HC resp. PC-treatment
reduced to normal the elevated enzyme levels. The virological parameters of the infection
were followed in parallel. PC-application to VIM led to noticeable reduction of mortality
rates (index of protection = 77.8%) and marked prolongation of mean survival time (+ 5.2
days). The protective effect of PC on EIVI is related to both the antioxidant activity and
the specific antiviral effect of the extract.
V13
SARS-CORONAVIRUS INTERACTION WITH ACE2 RECEP-

TOR; IMPLICATIONS FOR VIRUS ADAPTATION AND
VACCINE DESIGN
Anton P. Andonov, University of Manitoba, Winnipeg, Canada
72
V14
EVIDENCE SUPPORTING THE REPLICATIVE HOMEOSTA-

SIS HYPOTHESIS CONCERNING HIV REPLICATION IN
CELL CULTURE
R. Argirova
Lab. for Retroviruses, Dept. of Virology, National Center of Infectious and Parasitic
Diseases, Sofia, Bulgaria
The replicative homeostasis hypothesis, launched in 2005 by R. Sallie deals with

viruses replicating via RNA intermediates and capable for persistent infection like hepatitis
C (HCV), hepatitis B (HBV) virus and Human Immunodeficiency Virus (HIV). Their extreme
genetic and antigenic diversity is paradoxically combined with their stability. The
hypothesis reviews the auto-regulation of replication of these viruses and presents a
mechanism mediating this auto-regulation – namely replicative homeostasis. It proposes
a rational basis for all observed viral behaviors and host responses during chronic
infections caused by these viruses. Here we present in vitro data confirming the fact that
the HIV-1 replication is autocontrolled and independent of cellular or humoral immune
function. Studying newly derived HIV-1 mutants after serial passages of the virus in the
presence of increasing concentrations of IM-7 (designed to be HIV-1 integrase inhibitors)
an over-accumulation of 2-LTR DNA over linear cDNA (normally integrated) and un-
integrated DNA was observed in cell culture. This fact, similarly to emergency of viral
mutants presents strong evidence in support of autoregulation of HIV-1 replication – the
center of replicative homeostasis hypothesis.
V15
ЩАМОВО РАЗНООБРАЗИЕ И МЕХАНИЗМИ НА
ЕВОЛЮЦИЯ ПРИ РОТАВИРУСИТЕ
З. Младенова, Н. Корсун, Сн. Гюрова

Национална референтна лаборатория по Ентеровируси -
Национален център по заразни и паразитни болести, София
Човешките ротавируси от група А са главен причинител на тежки

дехидратиращи гастроентерити при децата на възраст под 5 години в целия свят.
По данни на СЗО те са отговорни за 20-70% от хоспитализациите и за 20% от
смъртните случаи, дължащи се на диария, в тази възрастова група. Понастоящем
единствената стратегия за контрол и превенция на тежките ротавирусни инфекции
е ваксинацията, тъй като мерките, целящи подобряване на хигиената и
73
водоснабдяването не са ефикасни за ограничаване на заболяемостта.
Използването на съвременните антигенни методи за характеризиране на
серотипа, генотипирането чрез RT-PCR и нуклеотидното секвениране през
последните 10 г. показа, че над 90% от циркулиращите по света ротавирусни
щамове са от типове G1, G2, G3 и G4, а също така позволи идентифициране на
над 42 комбинации от G/P типове. Днес проучването на щамовото многообразие
при ротавирусите е задача от първостепенно значение в световните програми за
надзор над заболеваемостта от ротавирусни гастроентерити и хвърля светлина
върху потенциалните механизми на еволюция и разпространение на нови щамове
ротавируси.
Разработването на ротавирусни ваксини се основава на създаване на
хомотипен и хетеротипен имунен отговор срещу един или няколко от най-
разпространените G и Р типове ротавируси. Лицензирането на две ротавирусни
ваксини в началото на 2006 г. налага мониториране на циркулацията на
ротавирусните щамове във всяка страна, откриване на нови щамове с глобално
или регионално разпространение, което е определящо за успеха на ротавирусните
ваксинални програми.
В доклада ще бъдат представени актуални данни за типовото разнообразие
сред ротавирусите, евентуалните механизми на тяхната еволюция и поява на
нови типове, както и съвременните методи за диагностика на ротавирусните
гастроентерити.
V16
ДИАГНОСТИКА НА ПЪРВИТЕ СУСПЕКТНИ ЗА ПТИЧИ

ГРИПЕН ВИРУС ПОДТИП А/H5N1/ ПАЦИЕНТИ У НАС
Т. Хаджиолова, С. Павлова, Р. Коцева

Отдел Вирусология, НЦЗПБ
Направени са първите диагностични изследвания за доказване етиологичната

роля на птичия грипен вирус подтип А/H5N1/ у нас. За периода януари – март
2006 г. в лабораторията по “ Грип и ОРЗ” са изследвани 26 суспектни пациенти
в тесен контакт с болни или умрели птици и последваща симптоматика на
респираторно заболяване. Приложени са специфични тестове за доказване на
птичия вирус А/H5N1/-бърз антиген откриваща тест GeNet Bio и RT-PCR кит на
фирма Sacace. За вирусна изолация са използвани два лабораторни модела-
клетъчна линия MDCK и кокоши ембриони. Вирусът не беше доказан като
етиологичен причинител в нито един от изследваните пациенти.В три от пациентите
бе изолиран човешки грипен вирус идентифициран чрез РЗХА и RT-PCR като
подтип А/H1N1/.
74
V17
ДИАГНОСТИЧНИ ПРОУЧВАНИЯ ВЪРХУ

ЕТИОЛОГИЧНАТА РОЛЯ НА РЕСПИРАТОРНО-
СИНЦИТИАЛНИЯ И ГРИПНИТЕ ВИРУСИ ПРИ
ХОСПИТАЛИЗИРАНИ ДЕЦА
С. Павлова, Т. Хаджиолова, Р. Коцева

Отдел Вирусология, НЦЗПБ
Ролята на РСВ като причинител на тежки заболявания на дихателната система

в малки деца е приоритетен проблем в световен мащаб. Ежегодното епидемично
разпространение на грипните вируси също дава отражение върху заболеваемостта
в тази възрастова група. През периода октомври 2005г.-март 2006г. в
лабораторията по “Грип и ОРЗ” са проведени сравнителни изследвания за РСВ и
грипни вируси на общо 359 проби от хоспитализирани деца до 5 г. възраст.
Няма паралелизъм между изследванията при едни и същи деца. При
вирусологичното изследване (изолация, бързи имуносорбетни тестове и ИФМ)
на 174 проби 14 (8,04%) са положителни за РСВ, а 10 (5,7%) за грипни вируси тип
А. При изследванията по ELISA IgG на 185 единични проби серуми са получени
42 (22,7%) положителни резултата за РСВ и 52 (28,1%) за грипни вируси тип А.
Получените резултати потвърждават литературните данни за едновременната
циркулация на РСВ и грипните вируси сред посочената възрастова група.
Изследването обогатява диагностичните проучвания за етиологичната роля на
тези вируси в детска възраст със съвременни данни и поставя началото на добра
колаборация между вирусолози и педиатри.
V18
ДИАГНОСТИЧНИ АСПЕКТИ НА ХЕМОРАГИЧНИТЕ

ТРЕСКИ
Д. Велчева
НЦЗПБ, отдел Вирусология, София
Показани са основните диагностични методи за диагностика на Конго

Кримска хеморагична треска и Хеморагична треска с бъбречен синдром, които
се използват в лаборатория „Арбовируси” – изолация, типизация и серологична
диагностика, както и диагностичната стойност на всеки методл Проследена е
заболеваемостта за периода 1997-2005 година. Сравнена е и заболеваемостта от
75
Конго Кримска хеморагична треска преди и след въвеждането на специфична
ваксинопрофилактика.
V19
STUDIES ON CCR5, CXCR4 AND CCR2 GENETIC POLY-

MORPHISM IN HIV-INFECTED BULGARIANS
K. Borisov1, A. Savov3, I. Kremensky3, S. Raleva1,

L. Froloshka1, R. Markova2, V. Terzieva2, K. Kostov4, R. Argirova1
1
Laboratory for Retroviruses, Natl. Center of Inf. And Paras. Dis.,2Dept. of Immu-
nology, Natl. Center of Inf. And Paras. Dis., 3National Lab. of Genetics, Med.
Uni.,Sofia, 4Inf. Dis. Hospital, Sofia
Although intensive studies of chemokine receptor genes have been done globally , it
would be of interest to study genetic polymorphism of co-receptor genes in HIV-infected
Bulgarians, as well as the link to clinical course of HIV-infection. Here we present 177
HIV(+) Bulgarians infected within 1986 – 2004 studied for genetic polymorphism of CCR5.
63 out of them were also studied for CCR2 and SDF-1 (CXCR4) genetic polymorphism. The
analyses were performed by PCR (CCR5) and restriction analysis + PCR using blood
spots on Gutrie cards with preliminary DNA extraction. Concerning the course of the
disease 9 out of all persons studied were long-term non-progressors (LTNPs) and the rest
had moderate course of HIV-infection. Three LTNPs showed CCR5 heterozygous pattern,
the other 6 LTNPs and 174 progressors had wild type CCR5. Separately, the frequency of
CCR2-64I in healthy Bulgarians as control was measured (6,9%) – one of the lowest in
Europe. No clear link between CCR2-64I genotype and disease progression has been
found. In 100 non-infected Bulgarians SDF-1-3’A frequency has been studied – 26,5% -
one of the highest in Europe. There was no significant difference in SDF-1-3’A frequencies
in healthy and HIV(+) individuals. In LTNPs SDF-1-3’A homozygosity was significantly
higher (44%) compared to the group with moderate progression to AIDS (5,5%). The data
obtained show that CCR5 heterozygotic pattern was not the only one linked to LTNP
status. In conclusion, multiple polymorphism of genes coding for chemokine receptors
were observed. CCR5, CCR2 and SDF polymorphism frequencies alone and in combination
suggest a number of HIV-infected individuals should be genetically tested to predict the
course of infection, the prognosis and the response to highly active anti-retroviral therapy
(HAART).
76
V20
AN EXPERIMENTAL STUDY OF HIV-1 EPITOPE STRUC-

TURE CHANGES UNDER INHIBITION OF
GLYCOSYLATION
R. Gavazova1, S. Ivanov1, D. Ivanov1, P. Genova3, S. Raleva2,

L. Froloshka2, D. Dundarova3, R. Argirova2
1
Section of Biochemistry, Institute of Experimental Pathology and Parasitology,
Bulgarian Academy of Sciences, G. Bonchev Str.,
Building 25, 1113 Sofia, Bulgaria; 2Laboratory for Retroviruses, 3Laboratory for
Cell Culture, Dept.of Virology, National Center of Infectious and Parasitic Diseases,
44A Boul.,Stoletov, 1233 Sofia, Bulgaria
To further understand changes of epitope structures conferring viral escape in HIV-

infected individuals we studied the Sgp profile of HIV-1LAI in MT-2 infected cells treated
with tunicamycin (Tu) – a well known inhibitor of glycosylation, as well as corresponding
ST activies. A study of N-acetyl-[C14]-mannosamine (14C-NAcMan) incorporation in
cytosols of HIV-1LAI acutely infected and uninfected MT-2 cells treated with Tu (0,5µg/
ml) was carried out. Fractions derived after isoelectrofocusing (IEF) were measured for
14C-incorporation, protein content and reverse transcriptase (RT) activity (Cavidi, Sweden).
ST-activity was detected by Serafini-Cessi F. method. Higher incorporation of 14C-NAcMan
for MT-2/HIV + Tu compared to MT-2 + Tu was observed. MT-2 IEF profile compared to
that of MT-2 + Tu showed a shift of the latter to the basic zone and only two common pI
peaks. On the contrary, a number of common pI peaks in MT-2/HIV and those treated by
Tu were seen. ST activity was reduced by 33% for MT-2 + Tu compared to MT-2 v. 20,6 %
for MT-2/HIV + Tu compared to MT-2/HIV (inhibition of glycosylation 27,8 and 14,2%
resp.).
As a result from these experiments it could be stated that the higher inhibition of
glycosylation by Tu in MT-2 cells compared to MT-2/HIV probably confers higher
inhibition of sialylation and the changes in Sgp profiles of Tu treated cells. The differing
effect of equal concentrations of Tu on glycosylation in MT-2 and MT-2/HIV under
experimental conditions might at least partially explain already described viral escape in
HIV-infected persons by changes in glycosylation.
77
V21
РАЗПРОСТРАНЕНИЕ НА ГЕНЕТИЧНИТЕ ФОРМИ НА

HIV-1, ЦИРКУЛИРАЩИ В БЪЛГАРИЯ
Пенева М.1, Бешков Д.1, Алексиев И.1, Георгиева В.1, Костов К.2, Еленков И.2,
Върлева Т.3, Еленков И.И.4
1
Национална Потвърдителна Лаборатория по HIV, НЦЗПБ, София; 2СБАЛИБ
„Проф. И. Киров”, София; 3Министерство на Здравеопазването, София; 4СУ
„Св. Климент Охридски”, Биологически факултет, София
Известна е извънредно голямата генетична разнородност на HIV, както и

непрекъснатото възникване на нови HIV варианти. Голямото разнообразие от
различни форми на HIV може да има влияние върху диагностиката,
антиретровирусната терапия, прогресията до СПИН, предаването на вируса и
създаването на ваксина срещу него.
Целта на настоящето изследване беше да установим разпространените
субтипове и циркулиращи рекомбинантни форми (CRF) на HIV-1 в България.
Нуклеотидните последователности, използвани за субтипирането, бяха получени
чрез секвениране на RT-PCR продуктите на регионите PR и RT от гена pol на
HIV-1 чрез диагностичните набори TrugeneTM и ViroSeqTM. Субтипирането беше
извършено според база данните на Standford University, CA, USA; Los Alamos
National laboratory, CA, USA и National Center for Biotechnology Information
(NCBI), NIH, MD, USА.
Бяха изследвани 80 пациента, инфектирани с HIV-1, които представляват 13%
от регистрираните HIV серопозитивни лица в България. Установихме, че най-
разпространен е субтип В, който циркулира в 41 (51.25%) от тях, следван от
CRF01_AE в 24 пациента (30%), субтип C – в 6 (7.5%), субтип H – в двама (2.5%),
CRF02_AG (2.5%) – в двама, субтип F – в двама (2.5%), субтип A – в един (1.25%),
G – в един (1.25%) и D – в един (1.25%) пациент.
Това изследване представя първи данни за разпространението на
циркулиращите генетични форми на HIV-1 в България. Беше установено
присъствието на девет генетични форми на HIV-1, от които преобладават субтип
B и CRF01_AE. Установеното голямо разнообразие на генетични варианти на
HIV-1 в България вероятно се дължи на географското разположение на страната
и засилените миграционни процеси през последните 20 години.
78
V22
ПРОУЧВАНЕ НА РАЗПРОСТРАНЕНИЕТО НА ЧОВЕШКИ

Т-ЛИМФОТРОПНИ ВИРУСИ HTLV-І И ІІ СРЕД
БЪЛГАРСКОТО НАСЕЛЕНИЕ
Алексиев И.1, Бешков Д.1, Георгиева В.1, Пенева М.1, Бакалова С.2, Генова
М.2, Атанасова М.3, Eленков И.4
1
Национална Потвърдителна Лаборатория по НІV, НЦЗПБ, София;
2
Национален Център по Хематология и Трансфузиология, София ; 3Медицински
Университет – Пловдив; 4СУ “Св. Климент Охридски”, Биологически Факултет
Цел на настоящото изследване е да се проучи разпространението на HTLV

сред различни групи от българското население, както и да се оцени
необходимостта от въвеждане на скрининг за HTLV при кръводарители и донори
на тъкани и органи. HTLV–I и II са човешки ретровируси, които могат да причинят
онкохематологични и неврологични заболявания. Те се предават по кръвен, полов
и вертикален път. Понастоящем скрининг на кръводарители за HTLV се прави в
Япония, САЩ и някои европейски страни.
Материали и методи: Проведоха се серологични изследвания за наличие
на анти-HTLV I и II антитела с ELISA тестове на Murex и Bio-Rad, както и
потвърдителни изследвания с Western blot на Genelabs Diagnostics. Бяха
изследвани 1446 кръвни проби от различни групи от населението: кръводарители
- 380, пациенти с болести предавани по полов път - 180, венозни наркомани - 99,
пациенти с хематологични заболявания: таласемия - 20, хемофилия А и В - 70,
левкози и лимфоми - 21, лица от КАБКИС - 676.
Резултати: Серологичните изследвания на 1446 серумни проби показаха,
че 11 от тях (0,76%) са реактивни в ELISA тест. Нито една от тях не бе потвърдена
в Western Вlot.
Изводи: В настоящото проучване не бе установено нито едно
серопозитивно лице за HTLV І и ІІ. Имайки предвид циркулацията на тези вируси
в съседните ни страни, както и свободното движение на големи групи хора, такива
серологични проучвания следва да се провеждат периодично. По този начин ще
се осигури мониторинг на тази инфекция и актуализиране на показанията за
скрининг на донорската кръв за HTLV в България.
79
V23
HIV-1 VARIANTS (MUTANTS) AFTER LONG-TERM PAS-

SAGING IN PRESENCE OF NEWLY SYNTHESIZED
4-HYDROXICOUMARINS (4-HC)
S. Raleva1, A. Yordanova3, Y. Gradinarova3, A. Savov3, L. Froloshka1, P. Genova2,

I. Manolov4, D. Dundarova2, R. Argirova1
1
Lab. for Retroviruses, 2Lab. for Cell Culture, Dept. of Virology, National Center of
Infectious and Parasitic Diseases, Sofia, 3 National Lab. of Genetics, Med. Uni.,
Sofia
4
Faculty of Pharmacy, Med. Uni., Sofia, Bulgaria
A number of studies has been published about the anti-HIV potency of some 4-
hydroxycoumarins (4-hc) (Zhao, H. et al., 1997). Stimulated by these findings mostly
targeting HIV-1 integrase, in Faculty of Pharmacy, Med. Uni – Sofia 19 novel 4-hc were
synthesized by I. Manolov and 3 out of them – IM-7, IM-8 and IM-10 showed anti-HIV
effect in MT-2 cells using HIV-1LAI. Further, no effect on both early (reverse transcription)
and late (protease, budding and release) phases of HIV-1 replication has been observed.
To target the anti-HIV-1 activity of studied 4-hc, serial passages (20 – 30) to yield mutant
HIV-1 variants in presence of increasing concentrations of 4-hc were undertaken. Culture
supernatants were concentrated 20x by Polyethyleneglycol (PEG) method. Supernatants
were tested for reverse transcriptase (RT) activity and the viral concentrates – for linear
cDNA (integrated) and 2-LTR rings by routine PCR. The results obtained showed an
abundance of 2-LTR rings compared to linear and unintegrated DNA. The latter finding is
typical for HIV-1 integrase mutants and was observed in viral concentrates after the 20th
passage of HIV-1LAI in presence of increasing concentrations of IM-7. The mutation(s)
are not reversible after the next 17 passages without IM-7 pressure. The mutant(s) obtained
are subjected to further analysis.
V24
АНТИРЕТРОВИРУСНА РЕЗИСТЕНТНОСТ НА HIV-1 СРЕД

БЪЛГАРСКИ СЕРОПОЗИТИВНИ ПАЦИЕНТИ (2002-2006)
Бешков Д.1, Алексиев И.1, Пенева М.1, Георгиева В.1, Костов К.2, Еленков И.2,
Върлева Т.3, Еленков И.И.4
1
Национална Потвърдителна Лаборатория по HIV, НЦЗПБ, София, 2СБАЛИБ
„Проф. И. Киров”, София, 3Министерство на Здравеопазването, София, 4СУ
„Св. Климент Охридски”, Биологичестки факултет, София
80
Високоактивната антиретровирусна терапия (HAART) е въведена в България
през 1999 г. За първи път се съобщават резултати от изследвания за
антиретровирусна резистентност (АРВР), като са обхванати пациенти с
регистиран неуспех при провеждане на HAART и наивни за HAART пациенти.
За периода 2002 г.- април 2006 г. са проведени изследвания на 101 кръвни
проби от 80 български ХИВ-1 серопозитивни лица, като 65 от тях са получавали
HAART, а 15 са наивни пациенти. При всеки пациент вирусът е генотипиран,
чрез сенвениране на RT-PCR продуктите на регионите RT и PR на pol гена на
HIV-1. Използвани са генотипиращите тестове TrugeneTM и ViroSeqTM за
установяване на мутации, отговорни за АРВР.
При 49 (75,38%) от 65 пациента, показали неуспех в хода на приложение на
HAART е установена АРВР към трите групи препарати по отделно и в различни
комбинации от тях. Към NRTI тя е 69.39%, към NNRTI-36.73% и към PI -59.18%.
Към трите групи препарати АРВР е установена при 10.20% от пациентите, към
NRTI и NNRTI при 14.29%, към NRTI и PI при 24.50% и към NNRTI и PI при
6.12%. АРВР само към една група лекарствени средства е устновена както следва:
към NRTI при 20.41%, към NNRTI при 6.12% и към PI при 18.37% от пациентите.
Изследванията при 15 наивни пациенти не показаха данни за АРВР.
Получените резултати показват, че при 75.38% АРВР е отговорна за неуспеха
при HAART. Тези данни корелират с прилаганата терапия. Това налага
рутинното приложение на изследванията за АРВР, имащи основна роля при избора
на нови терапевтични режими.
V25
DIAGNOSTICS OF THE VIRAL HEPATITIS -

ACHIEVEMENTS AND PROBLEMS
P. Teoharov
Infection of the liver with hepatotropic viruses is a serious public health problem. The
burden of disease from acute and chronic viral infections is staggering. Each of the five
hepatotropic viruses belongs to different virus family and three of them - HAV, HBV and
HCV are the leading cause of the acute and chronic liver diseases in Bulgaria.
A combination of biochemical, serological and virological tests and histological features
have been used to diagnose and classify infections from hepatotropic viruses. Assays
for HAV, HBV and HCV antigens and antibodies are widely available and standardized.
There are a highly specific and sensitive kits for detection of viral markers. The most
important method for laboratory diagnosis of the viral hepatitis is immuno-ensyme assay
/EIA /, but molecular techniques for determination of nucleic acids become widely use
also. Different molecular assays have been developed with different sensitivity and range
81
of linearity. There are some disadvantages, mostly connected with the specificity of the
more sensitive molecular methods based on amplification of the viral nucleic acids. Some
of the techniques, like determination of the genotype of HBV remains a research tool, but
genotyping of HCV is important step from the therapy. The polymerase chain reaction /
PCR/ is now accepted as a gold standard for detecting nucleic acids and it has become an
essential tool in the research laboratory. Real-time PCR has wider acceptance due to its
imprived sensitivity, rapidity, reproducibility and the reduced risk of carry-over
contamination.
V26
МОЛЕКУЛЯРНИ ХАРАКТЕРИСТИКИ И
ПАТОГЕНЕТИЧЕН ПОТЕНЦИАЛ НА ЧОВЕШКИТЕ
ПОЛИОМНИ ВИРУСИ
Зл. Кълвачев
Лаборатория по молекулярна вирусология
Национален Център по Заразни и Паразитни Болести (НЦЗПБ)
Наскоро (2000 г.) формираното семейство Polyomaviridae обединява 13 вируса

паразитиращи по различни млекопитаещи, вкл. човек, някои видове примати,
гризачи, зайци и птици. Огромният научен и медицински интерес, който
предизвикват тези вируси се дължи на високият им онкогенен потенциал и
възможните последствия за човек. За някои неврологични заболявания
(прогресиращата мултифокална левкоенцефалопатия) се счита за доказана
етиопатогенетичната роля на човешкия полиомен вирус JC. Освен това
пациентите, на които се извършват тъканни или органни трансплантации се
подлагат на имуносупресивна, при което често се активират инфекции,
предизвикани от другият човешки полиомен вирус - BK. Молекулярните
механизми, по които се развиват тези инфекции са в процес на интензивни
проучвания. Представеният материал обобщава съвременната информация
относно основните биологични свойства на човешките полиомни вируси BK и
JC, както и данни за епидемиологията, клиниката, диагностиката и възможностите
за терапията при тези инфекции.
82
V27
БЪРЗА ИДЕНТИФИКАЦИЯ НА POLYOMAVIRUS

HOMINIS-1 (ВКV) В УРИНАТА НА ПАЦИЕНТИ С
БЪБРЕЧНА ТРАНСПЛАНТАЦИЯ
С. Славов1, И. Ненков1, А.Петрова1, Л. Христова2, П. Симеонов2, Д. Димова3,

З. Кълвачев1
1
Национален Център по Заразни и Паразитни Болести, Отдел по вирусология,
Лаборатория по молекулярна вирусология; 2Медицински Университет-София,
Университетска болница-„Александривска”, Клиника по нефрология и
трансплантации; 3САГБАЛ-„Шейново”, Лаборатория по патологична
хистология
Polyomavirus hominis-1, по-известен като BK вирус (сем. Polyomaviridae) е

патоген с широко разпространение сред човешката популация. Инфекцията се
придобива още в ранна детска възраст, като остава латентна в генито-уринарния
тракт. Реактивация се наблюдава често,особено при пациенти с бъбречна
трансплантация. Поради подтиснатите имунни механизми те развиват симптоми
като хеморагичен цистит, уретрална обструкция, ретинит, хепатит, като често се
стига до отхвърляне на трансплантанта. Поради това, своевременното откриване
на вируса е от съществено значение при прогнозата от трансплантацията и
съставянето на подходяща терапевтична схема.
За бързо диагностициране на вируса в урина на пациенти с бъбречна
трансплантация бяха използвани уринарно-цитологичен метод и полимеразно-
верижна реакция (PCR) в реално време (TaqMan real-time PCR). Изследвани бяха
общо 50 проби. В 36 от тях, бяха наблюдавани характерните за полиомавирусната
инфекция клетки с вирусни включвания, известни като „Decoy cells”. От седимента
на всичките 50 проби беше екстрахирана ДНК, която изследвахме чрез TaqMan
real-time PCR. Наличие на специфични вирусни геномни секвенции бяха доказани
при 48 проби. Обсъждат се клиничните и лабораторните находки, което е във
връзка с прогнозата на състоянието след трансплантация. Използваният от нас
вариант на PCR e подходящ за скриниране на BK полиомната инфекция при
пациенти с бъбречна трансплантация, и би послужил като допълнение към
класическите цитологични изследвания.
83
V28
POLYOMAVIRUS HOMINIS-2 (JCV) В УРИНАТА НА

ПАЦИЕНТИ С БЪБРЕЧНА ТРАНСПЛАНТАЦИЯ
И. Ненков1, С. Славов2, А. Петрова2, Л. Христова3, П. Симеонов3 , З.

Кълвачев2
1
Софийски университет ,,Св. Климент Охридски”, Биологически факултет,
катедра Биохимия; 2Национален Център по Заразни и Паразитни Болести;
Лаборатория по молекулярна вирусология, 3Медицински Университет - София;
УМБАЛ „Александровска”, Клиника по нефрология и трансплантации
Polyomavirus hominis-2 или JC вирусът (JCV) принадлежи към сем.

Polyomaviridae. Според литературните данни, около 80% от населението по света
има антитела към този вирус. Счита се, че става още в ранна детска възраст, но
инфекцията протича безсимптомно. По време на първичната инфекция се
наблюдава виремия и вирусът се транспортира до мозъка, бъбреците и/или други
органи, където остава да персистира в латентно състояние. При
имунокомпроментирани индивиди (такива с трансплантации или други
имунодефицитни състояния) се наблюдава реактивация на вируса и вирурия.
JCV е невротропен вирус и е причинител на прогресивната мултифокална
левкоенцефалопатия (progressive multifocal leucoencephalopathy, PML). При
бъбречно-трансплантирани пациенти може да се развият тежки усложнения и дори
да се стигне до отхвърлянето на трансплантанта. Поради това, своевременното
откриване на вируса е критично за прогнозата от трансплантацията и
назначаването на подходяща терапевтична схема.
Ние изследвахме 50 уринарни проби получени от пациенти с бъбречна
трансплантация и с помощта на полимеразно-верижната реакция (PCR) проведена
в реално време (TaqMan real-time PCR) доказахме наличието на JCV при 43 от
тях. TaqMan real-time PCR e ефективен метод за бързо скриниране на JCV инфекция
при пациенти с бъбречна трансплантация. Установено беше, че най-подходяща
за изследване е втората сутрешна урина. Обсъжда се връзката между получените
резултати, клиничните данни и вероятният бъдещ ход на състоянието.
84
V29
КЛИНИЧНИ ОСОБЕНОСТИ И СЕРОЛОГИЧНА

ВЕРИФИКАЦИЯ НА ОСТРАТА ЕВV ИНФЕКЦИЯ
А.Гоцева1,Т.Кузмова1, Д.Велчева2
1
УМБАЛ ”Св.Иван Рилски”, София
2
НЦЗПБ, отдел Вирусология, София
Проследени са 24 пациенти с мононуклеозен синдром, лекувани в Клиника по

инфекциозни, паразитни и тропически болести- УМБАЛ ”Св.Иван Рилски”, София
и изследвани в лаборатория “Херпесни вируси “към НЦЗПБ.Всичките бяха
изследвани чрез ELISA за наличие на антитела от клас IgM към капсидния антиген
на ЕВV(маркер за активна инфекция).При 17 от тях бяха доказани специфични
антитела , докато при останалите 7- резултатът беше негативен. П р и
всички пациенти основни клинични прояви бяха интоксикацията,фебрилитета,
лимфоаденопатията, възпалителните промени в гърлото и хепатомегалията. При
17 от тях ( всички позитивни за anti EBV VCA IgM) клиничната картина се
утежняваше от наличието на лекарственоиндуциран екзантем, ранен и преходен
двустранен оток на горните клепачи, обструкция на въздухоносните пътища,
спленомегалия и повишени стойности на чернодробните ензими.При останалите
7 лица описаните клинични симптоми не бяха регистрирани, което съответстваше
на липсата и на серологично потвърждение.
V30
ПТИЧИ ГРИП (H5N1)-НОВАТА ГЛОБАЛНА ЗАПЛАХА:

ЕПИДЕМИОЛОГИЯ, РАЗПРОСТРАНЕНИЕ И ВЪЗМОЖНО
ИЗПОЛЗВАНЕ КАТО БИОЛОГИЧНО ОРЪЖИЕ
Красимир Мекушинов
Вирусологична лаборатория, Военномедицинска академия, София-1606,
ул. Г. Софийски, 3
Цел: Да се опише специфичната епидемиология, разпространение и главните

особености на вируса, които го определят като потенциален агент за Биологично
оръжие:
1.Начин на предаване: заразена вода, храна и почва
2.Клинично протичане: къс инкубационен период, внезапно начало, тежка
пневмония, респираторен дистрес синдром
85
3. Вирулентност: смъртността при заразените хората е над 50%, а у чувствителни
птици може да достигне до 100%, причинявайки огромни икономически загуби.
V31
EFFECTS OF SOME ANTIVIRALS AGAINST RHINOVIRUS

H-14
Irina L. Georgieva, Angel S. Galabov

Department of Virology, The Stephan Angeloff Institite of Microbiology, Bulgarian
Academy of Sciences, 26 G. Bonchev St., 1113 Sofia
Human rhinoviruses (HRVs) are the predominant cause of viral respiratory tract
infections, particularly common cold, as well as acute otitis media, sinusitis, bronchitis
etc. A great number of picornavirus replication inhibitors in vitro have been described but
until now there are no specific antiviral chemotherapy to prevent or treat diseases caused
by rhinoviruses.
Here are presented data of a pilot study on the effect of several antiviral substances
with different mode of action on the replication of HRV-14. Monolayer cultures of human
cervical carcinoma (HeLa Ohio-I) cells in 96-well tissue culture plates were used. The viral
CPE inhibition test was applied. The neutral red uptake (NRU) assay was done to quantitate
the antiviral activity and the cytotoxicity of the compounds. Absorbance values of the
drug-treated samples were expressed as percentage of the untreated controls and the
dose-response curves of each compound were drawn. The compound action at various
viral inoculation doses was studied. The following compounds have been tested: ribavirin
(large-spectrum viral inhibitor, mostly of RNA viruses), arildone, disoxaril, S7, PTU-23,
HBB and oxoglaucine (a newly characterized in our laboratory antiviral efficient against
enteroviruses).Two of these compounds - HBB and oxoglaucine show highest activity,
characterized with: (i) lowest values of IC50; (ii) highest values of selectivity ratio and (iii)
effectivity against all viral inoculation doses tested. Ribavirin and disoxaril occupy
intermediate position by their antiviral effect followed by PTU-23, active against 100 and
1000 CCID50. Arildone and S7 manifested a border-line effect.
86
V32
STUDY OF VIRUS RNA SYNTHESIS IN CELL CULTURES

INFECTED WITH BOVINE VIRAL DIARRHEA VIRUS (BVDV)
USING HYPERTONIC NACL SOLUTIONS
Angel S. Galabov, Yurii P. Abashev, Lucia Mukova and Lilia Wassilewa

Bovine viral diarrhea virus (BVDV) is a positive-strand RNA virus, member of the
family Flaviviridae, genus Pestivirus. It causes infections in cattle with significant economic
losses. BVDV is often used as a surrogate hepatitis C virus in screening tests for anti-HCV
antivirals. The exact study on the mechanism of action of such antivirals insists the well
known synthesis of viral RNA and proteins. The problem of pestiviral RNA synthesis
study is the lack of a significant shut-off of the cellular RNA and protein synthesis in the
caurse of pestivirus infection until the later step when the cytolytic changes are evident.
In order to overcome this problem the antibiotic actinomycin D in concentration of 5
ìg/ml is usually applied. Unfortunately, addition of actinomycin D in such
concentration to some cell lines (e.g. calf trachea cell line) even at short time interval
resulted in a great degree of cell toxicity. The hypertonic NaCl solution used for the first
time by Nuss et al. (1875) blocks selectively the host cell RNA and protein syntheses. Our
experiments using hypertonic NaCl solution (0.15 – 0.3 M/l) and calf trachea (CT) cell line
showed a significant (98.9% inhibition of cellular RNA and 98.0% inhibition of the cellular
protein synthesis. Addition of the same NaCl concentration to CT cells infected with
BVDV, CC strain, with multiplicity of infection 10 resulted in a well documented viral RNA
synthesis with a maximum at 9th hour after virus adsorption in parallel with a marked shut-
off of the cellular RNA synthesis. The maximum BVDV infectious titre (107.5 CCID50) was
found at 18th hour after viral adsorption.
V33
БИОЛОГИЧНА АКТИВНОСТ НА ЕКСТРАКТ ОТ

ORTHOSIPHON STAMINEUS BENTH.
Стоян Шишков1, Калина Костова1, Елена Георгиева1, Венета Капчина-

Тотева1, Женя Йорданова1, Валентина Чипева2, Станислава Трандева2,
1
Биологически факултет, СУ “Св. Кл. Охридски”, бул. Драган Цанков №8,
София 1164
2
НБПМКК, бул. “Цариградско шосе” № 125, бл. 2, София 1113
87
Проучен за биологична активност е етанолов екстракт от азиатското
медицинско растение Orthosiphon stamineus Benth. Растението е отгледано в
условия in vitro. Установена е ясно изразена инактивираща активност спрямо
екстрацелуларен вирус херпес симплекс тип 1 (ВХС-1). Инфекциозният вирусен
титър е понижен с 2 log (99%) само след 5 минутен контакт на вирионите с
екстракта. Ефектът се запазва за целия интервал на изследване. Вирусната
репликация не се повлиява при апликиране на изследвания извлек.
С прилагане на дифузионен метод е изследвана антибактериалната активност.
Резултатите показват наличие на активност спрямо референтни бактериални
щамове от Bacillus, Staphylococcus, Klebsiella. Изледван е ефектът и върху широк
набор от клинични грам-отрицателни и грам-положителни бактериални изолати.
V34
ВИРУСОЛОГИЧЕН НАДЗОР НАД ОСТРИТЕ ВЯЛИ

ПАРАЛИЗИ В БЪЛГАРИЯ ПРЕЗ ПЕРИОДА 2001-2006
ГОДИНА
Н. Корсун, Сн. Гюрова, З. Младенова

Национална референтна лаборатория по Ентеровируси -
Национален център по заразни и паразитни болести, София
В педиода на близка ерадикация на полиомиелита опасността от възникване

на паралитични заболявания, причинени от внос на диви полиовируси от
останалите четири полиоендемични страни, както и от появата на вируси деривати
на живата полиоваксина, все още съществува. Надзорът над острите вяли
парализи е ключов елемент в контрола над полиомиелита, поради което тази
система трябва да продължи да функционира до пълно прекъсване на циркулацията
на диви полиовируси в глобален мащаб. Висококачественият, отговарящ на
сертификационните стандарти надзор гарантира бързо откриване на вирулентни
полиовируси и предприемане на спешни противоепидемични мерки.
Националната референтна лаборатория по Ентеровируси към НЦЗПБ, София
е част от Глобалната лабораторна мрежа на СЗО и като такава осъществява
вирусологичен надзор над случаите на остри вяли парализи при деца на възраст
под 15 години в цялата страна. В доклада са представени резултатите от
извършените вирусологични изследвания в лабораторията в това направление
през периода 2001-2006. След епидемичния взрив от полиомиелит у нас през
2001г., причинен от вносен див полиовирус І тип, у нас диви и ваксино-дериватни
полиовируси не са изолирани. Представени са и актуалните данни относно
ситуацията по отношение на полиомиелита по света.
88
V35
EFFECT OF THIOSEMICARBAZONE ON HSV-1 AND HSV-2
REPLICATION IN VITRO
N. Vilhelmova1, M. Gacovska1, S. Shishkov1, P. Souza2

University of Sofia”St. Kl. Ohridski”, Sofia 1164, Bulgaria, 2Universidad
1
Aoutonoma de Madrid, Madrid 28049, Spain
Thiosemicarbazones are big group of organic molecule with prove activity against
HSV. We tested the effect of two bisthiosemicarbazone - H2L1 and H2L2 and theirs complexes
with Zn(II), Pd(II) and Cd(II) on HSV-1 (Victoria) and HSV-2 (BJ) in cell culture MDBK. We
determined maximal nontoxic concentration (MNC) and concentration required to inhibit
cell viability by 50% (CC50) of compounds tested. The complexes with Zn(II) and Pd(II) are
with the same value of MNC such as its ligands - 1x10-8 µM. The cadmium complexes in
respect to cell viability are most cytotoxic: Cd2/H2L1 – MNC=1x10-10µM and Cd/H2L2 -
MNC=1x10-9µM.
The ligands and theirs zinc complexes expresse activity against HSV-1 and HSV-2.
Toward strain Victoria Zn2/H2L1 is most active with effective concentration required to
inhibit virus yield by 50% (IC50) - 0.001x10-8µM. The ligands and Zn/H2L2 indicate the
same activity - IC50b= 0.01x10-8 µM. Against strain BJ the strongest effect exhibit H2L2 and
Zn2/H2L1 - IC50 = 0.001x10-8 µM both. IC50 of H2L1 and Zn/H2L2 have value 0.01x10-8 µM. The
most highly selectivity toward both strains have H2L1 и Zn2/H2L1.
V36
СЕРОЛОГИЧНО-ЕПИДЕМИОЛОГИЧНИ ПРОУЧВАНИЯ
НА ГРИПА В ГРАД ВАРНА ЗА ПЕРИОДА 1990-2004 г.
В. Русев, Л. Иванова, А. Щилянова, Р. Иванова, Бр. Шматов
Серологично-епидемиологичните проучвания са от голямо научно-теоритично

и приложно значение. За периода серологично-епидемиологичните проучвания
са от голямо научно – теоретично и приложно значение.
За периода 1990-2004 година ние изследвахме общо 16 000 единични проби
серуми от здрави лица от различни възрастови групи от град Варна и региона.
Проучванията ни са проведени по РЗХА с 4 антигена от щамове на грипен
вирус тип А с антигенна формула H1N1 и H3N2.
Основната цел на това проучване е определянето на средните геометрични
титри на антихемаглутинините към различните щамове на грипен вирус тип А.
При проследяване динамиката на средните геометрични титри на антителата
към изследваните щамове грипни вируси бе установено, че титрите варират от 2
89
до 20. Тези резултати в едни случаи отразяват епидемичната обстановка– при
вирусите А (H3N2), а в други – се тълкуват като резултат на анамнестични реакции
към по-стари щамове, под влияние на заболявания, причинени от по-нови щамове
(H1N1).
V37
INFLUENCE OF OSMOTIC SWELLING ON THE

DHPC LIPID BILAYERS CONTAINING NDV GLYCOPRO-
TEINS
Petrana Borissova, Vassil Neitchev, Lyuba Doumanova1

Institute of Biophysics, 1 The Stephan Angeloff Institute of Microbiology, Bulgarian
Academy of Sciences, 1113 Sofia, Bulgaria, e-mail: vas@polymer.bas.bg
The osmotic stress is one of the ways to achieve high membrane tension which can
result in strong adhesion between membranes of fixed volume. There is increasingly data
indicating a possible effect of osmotic stress on membrane fusion. The NDV (Newcastle
disease virus) glycoproteins play a critical role in limiting membrane lipids in the virus-cell
fusion. It has been found that they both interact with the lipid bilayers (liposomes) to
change the spontaneous membrane curvature, as well as the lipid hydration and surface
tension, with minimum osmotic work required for the lipid phase transition. These
glycoproteins, when associated with phosphatidylcholine liposomes, may affect the
osmotic permeability of lipid bilayers under positive osmotic (higher inside) gradients
across the membrane. Lipids thus participate in this process, and their structure should be
considered of interest for examination of membrane fusion.
We investigated the influence of the osmotic stress on the structure of 1,2 dihexadecyl-
sn-glycero-3-phosphatydilcholine (DHPC) vesicles under transition from lamellar gel to
fluid state in the presence of both HN and F glycoproteins in the vesicles. Although the
lamellar bilayer structures do not promote the lipid fusion, it is of interest to examine how
these structures are influenced in such conditions. Our assumption is that the lamellar
interdigitated gel structure of DHPC membranes is more sensitive to external osmotic
stress than the normal gel phase. The last is also influenced by the presence of viral
glycoproteins in the membrane which yield strikingly different molecular shapes of the
lipid. X-ray study was performed in this report to observe the changes in the structure of
HN-F/lipid associations under osmotic stress conditions.
The results obtained show the appearance of a swollen gel phase in pure DHPC
vesicles at the boundary between the normal gel phase and liquid crystalline phase. The
nature of the swollen phase is similar to the anomalous swelling reported in literature. The
addition of HN-F to swollen vesicles suppresses the formation of any gel phase at
temperature below 20 0C. Above Tm (42.3 0C) the presence of HN-F leads to formation of
different lamellar and non-lamellar structures which can exist simultaneously. The structural
90
changes due to the presence of HN-F in the vesicles are almost limited to the outer
hydrophilic regions of the bilayer structure. This infers that the hydrophobic chains are
only peripherally influenced by the forces acting on the polar headgroups. Probably, the
HN-F molecules occupy the free water layer, where their location is thermodynamically
more favourable. This effect is more pronounced in the liquid crystalline phase. The
process is assisted by the positive osmotic gradient across the lipid membrane.
V38
DETECTION OF CANINE PARVOVIRUS SEROTYPE 2 IN

FECAL SAMPLES FROM DOGS BY POLYMERASE CHAIN
REACTION
Chavdar Filipov1, Petar Grozdanov2, Zahari Raikov2, Haralambi Haralambiev1

and Angel S. Galabov2
1
Faculty of Veterinary Medicine, University of Forestry, Sofia, 2 The Stephan
Angeloff Institute of Microbiology, BAS
There are two serotypes of parvoviruses, circulating among canine populations and
the most dangerous of them, causing diarrhea, is canine parvovirus serotype 2 (CPV-2). In
this study we have worked out parameters of polymerase chain reaction (PCR) for canine
parvovirus detection. A reference strain of CPV-2 was used as a control. It was found that
the samples tested contained reaction inhibitors, possible reasons for false negative
results. Therefore, we have used two pairs of specific for CPV-2 primers following the
standard procedure for DNA isolation from fecal samples. The optimal temperature
conditions for each pair of primers were determined. Four PCR positive out of the total 27
fecal samples tested of dogs with enteritides were recorded with both pairs of primers. We
consider this method is very sensible and precise for detection of parvoviral infection in
dogs.
Key words: canine parvirus serotype 2, polymerase chain reaction (PCR), fecal samples
V39
МОЛЕКУЛЯРНА ДИАГНОСТИКА НА СИНДРОМ НА
LEWANDOWSKY-LUTZ (ЕPIDERMODYSPLASIA
VERRUCIFORMES)
Б. Бонева1, М. Сайедж1, Г. Матеев2, З. Кълвачев1

1
Национален център по Заразни и Паразитни Болести, Отдел по Вирусология,
лаборатория по Молекулярна Вирусология;
91
Медицински Университет-София, Университетска Болница” Александровска”,
2
Клиника по Дерматология и Венерология, Централна Лаборатория по Микози
Открити са повече от 100 типа човешки папиломни вируси (HPV). Около 30

типа се предават при сексуален контакт със заразен парнтньор и инфектират
гениталните области, като вагина, вулва, цервикс, скротум, пенис и ректум. Други
типове HPV се предават при орален секс и причиняват поява на брадавици в
устна кухина и фаринкс. Симптомите при папиломната инфекция се появяват от
няколко седмици до месеци или години след първична инфекция и започват със
сърбеж или парене около половите органи, дискомфорт и болки при полов контакт.
Ако не се лекуват, гениталните брадавици се разрастват гроздовидно и
причиняват усложнения.За диагностиката HPV вируса се изолира от
повърхността или вътрешността на ректум, гърло, уретра и вагина. Синдрома
на Lewandowsky-Lutz (Epidermodysplasia verruciformes) е автозомно-рецесивна
болест, която отслабва Т-клетъчния и хуморален имунитет към HPV антигени.
Открити са около 15 вирусни типа, като HPV 5, 8, 9, 12, 14, 15, 17, 19, 25, 36, 38,
47, 50.Това заболяване започва в детството и се среща в 10 дo 20 % от пациентите,
обикновено млади индивиди. Води до злокачествена трансформация, сравнима
цитологично с плоскоклетъчен карцином. Лечението е хирургично или с
химиотерапия с Acitretin (25 mg/j).
Case report: В лаборатория по Молекулярна вирусология бе изследван
материал от пациент със синдром на Lewandowsky-Lutz. По метода на
конвенционалната полимеразно верижна реакция (PCR) бе открит HPV-6, като
бяха използвани праймерни системи за HPV- 6, 11, 18, 16 и 33. HPV-6 не се счита
за етиологичен агент при съответния синдром, но това изследване показва, че в
етиопатогенезата на епидермодисплазията може да участват и други HPV- типове.
Key words : Epidermodysplasia verruciformes, HPV, PCR
V40
ВАРИЦЕЛА ЗОСТЕР ВИРУС (VZV) И БРЕМЕННОСТ
L.Ivanova
Dept of Microbiology and Virology, University hospital St Marina, MU - Varna ,
BULGARIA
Варицела и херпес зостер по време на бременността могат да прчинят

конгенитални дефекти, неонатална варицела, херпес зостер на новороденото дете.
Цел на настоящото проучване е да се определят последиците за новородените
деца от майки, контактни на варицела по време на бременността. Изследвана
популация: 56 бременни жени с несигурна анамнеза за преболедуване от варицела
в детството, в тесен контакт с болни от варицела деца, бяха изследвани за
определяне на специфични IgG анти VZV антитела в CFT и ELISA. Единадесет
92
(20%) бременности бяха усложнени с варицела и 4 (7%) – с херпес зостер.
Интраутеринна варицела зостер вирусна инфекция беше идентифицирана по
клинични критерии (синдром на конгенитални аномалии, остра варицела при
раждането, херпес зостер на новороденото дете) въз основа на история на
раждането. Четири деца с неонатална варицела бяха изследвани серологично в
сравнителен аспект с техните майки. Резултати: Серонегативни в CFT бяха 19
(34%) от бременните жени и 10 (18%) в ELISA. Синдром на конгенитални дефекти
и херпес зостер на новороденото дете не бяха регистрирани. Новородените деца
от майки с херпес зостер нямаха клинични данни за заболяване. Три деца с
неонатална варицела бяха родени от майки с варицела в трети триместър на
бременността и ниски титри на антитела спрямо VZV. Едно дете от майка,
контактна на варицела непосредствено преди раждането, серонегативна в CFT
и ELISA почина от неонатална варицела. Заключение: Варицела по време на
бременността, като резултат от първична инфекция с VZV може да предизвика
увреждания на развиващия се плод и заболяване на новороденото дете.
V41
DEMONSTRATION OF RABBIT MYXOMA VIRUS
IN CELL CULTURES BY DIRECT
IMMUNOPEROXIDASE METHOD
R. Bostandjieva1, Zl. Dimitrova2, R. Peshev1

1
Central Diagnostic and Research Veterinary Institute – Sofia, 2National Center for
Contagious and Parasitic Diseases –Sofia
A direct immunoperoxidase assay was developed for direct and quickly indication of
the rabbit myxoma virus /VMZ/ in cell cultures. The method was modified on the basis of
the peroxidase conjugate VMZ for ELISA obtained.
he viral reproduction of cytopathic VMZ strains in the stable RK13 cell line was
studied.
V42
EVALUATION OF THE EFFICACY
OF THE BROAD-SPECTRUM DISINFECTANT ON SOME
VIRUSES PATHOGENIC FOR THE FISHES
Darinka Ilieva
National Diagnostic Research Veterinary Institute “Prof. Dr. D. Pavlov”
The antiviral activity of Aldekol Des FF “in vitro” against a field strain of the Infectious
93
pancreatic necrosis (IPN) of Salmonids and a reference strain of the Spring viremia of carp
(SVC) have been evaluated. The experiments were made under laboratory conditions with
various concentrations and expositions.
The treatment of tested viral suspensions with the disinfectant at the lowest
concentration 1:3200 resulted for 90 min complete inactivation of the two viral strains.
V43
СРАВНИТЕЛНИ ПРОУЧВАНИЯ НА КИТОВЕ ЗА
ДИАГНОСТИКА НА НЯКОИ ВИРУСНИ ЗАБОЛЯВАНИЯ
ПО РИБИТЕ
В. Чикова, Д. Илиева
Национален диагностичен научноизследователски ветеринарномедицински
институт, София
Проведено е сравнително изследване на стандартни китове за откриване и

идентификация на VHSV, SVCV, IHNV и IPNV. Установено е, че използваните
търговски китове на база индиректен имунофлуоресцентен тест (IFAT) са с по-
ниска специфичност и чувствителност за доказване на VHSV и IPNV. Китовете
не дават възможност за отдиференциране на серотипове IPNV - Sр и IPNV - Ab,
които се характеризират с различна патогенност. По аналогичен начин SVC
китовете не позволяват диференциране на SVCV и PFRV. Предлаганите ELISA
китове и имунопероксидазни тестове се характеризират с висока чувствителност
и специфичност и могат да бъдат прилагани с успех за бърза диагностика на
различни вирусни заболявания по рибите.
94
MEDICAL MICROBIOLOGY
MM1
ПОЛИФАЗЕН ИДЕНТИФИКАЦИОНЕН АНАЛИЗ НА
КЛИНИЧНИ ИЗОЛАТИ ОТ BURKHOLDERIA CEPACIA
COMPLEX (BCC)
Мария Средкова1, Сашка Михайлова1, Edward Moore2

Медицински университет – 1 Плевен, 2 Culture Collection University Goteborg
Цел на проучването е идентифициране на клинични изолати от BCC до ниво

вид или геномоварова група чрез използване принципите на полифазната
таксономия. Двадесет и един щама, изолирани в УМБАЛ – Плевен, се изпитаха
чрез конвенционални фенотипни методи (57 теста), използване на автоматизирана
система (32 теста), секвениране на гените за 16S рРНК и анализ на recA гена
(ПВР със специфични праймери и секвениране). Резултатите от конвенционалните
фенотипни тестове позволиха всички изолати да се идентифицират до ниво BCC
и да се определят като сборна група I/III/VII/VIII геномовар. Автоматизираната
система идентифицира изолатите като B. cepacia (геномовар I). Частичното
секвениране на гените за 16S рРНК доказа принадлежност на изолатите към
комплекса (степен на сходство с типови щамове 98-100%). Деветнадесет
секвенции показаха най-близко родство с щам BCC с неизвестна геномоварова
принадлежност, а две секвенции имаха най-изразено подобие с щам от геномовар
IX (B. pyrrocinia). Анализът на recA гена потвърди отнасянето на изолатите към
комплекса. В допълнение се установи, че те не принадлежат към нито един от
известните геномовари. Изолатите от Плевен се обособиха в нова група BCC –
тип АТ, която изисква по-нататъшно таксономично уточняване. Класическите
фенотипни тестове позволяват идентифициране на изолати от BCC до ниво група
от геномовари. Секвенирането на гените за 16S рРНК е подходящ метод за
разграничаване на бактериите от комплекса от BCC подобните микроорганизми,
но не притежава достатъчна дискриминираща сила за видово диференциране.
Изследването на recA гена дава възможност за по-прецизен таксономичен анализ.
Необходими са нови методи в полифазния подход за окончателно идентифициране
на изолатите.
95
MM2
STUDY OF VAGINAL LACTOBACILLI FROM BULGARIAN
WOMEN
G. D. Stoyancheva, S. P. Dimitonova, P. M. Petrova, R. N. Aleksandrova, I.
Tzvetkova, S. T. Danova
Department of Microbial Genetics, Institute of Microbiology, Bulgarian Academy
of Sciences, 26, Acad. G. Bontchev, str, 1113 Sofia, Bulgaria,
e-mail: galinadinkova@abv.bg
The human vagina is a complex dynamic ecosystem containing an abundance of

microorganisms. In women of childbearing age this system is dominated by Lactobacillus
spp., producing a variety of metabolites including lactic acid which maintains the acidic
conditions (pH<4.0), hydrogen peroxide (H2O2), bacteriocins and other antimicrobial
substances as important inhibitors of the pathogenic organisms in the vagina. The use of
probiotics to control certain infections and to reestablish the human bacterial microbiota
is gaining acceptance as an alternative to conventional antibiotic therapy.
The present study summarised results from tree-years research project on
Lactobacillus microflora of sixty reproductive-age Bulgarian women. Forty eight strains
isolated from collected vaginal samples were determined as Lactobacillus sp. Antagonistic
activity of all vaginal lactobacilli were evaluated by different in vitro methods. Active
strains inhibiting the growth of pathogens were selected. Qualitative tests for H2O2 and
exopolysaccharides production was made. Some of these exopolysaccharides promoted
Lactobacillus adhesion to epithelial cells and it is important for vagina colonization. In
order to revealed the biodiversity of studied Lactobacillus strains six molecular methods
(ribotyping, ARDRA, rep-PCR, PCR with species-specific primers, hybridization with
species-specific probes and sequencing analysis) were applied. The predominant species
in the studied group was L. fermentum. Three strains, possessing high antimicrobial
activity were studied by sequencing analysis of 16S rDNA. After additional research,
these strains could be used as therapeutic agents for the treatment and prevention of
urogenital infections. This work was financially supported by National Council for
Scientific Research of Republic of Bulgaria. (Grant МУ-Л-1406/2004).
MM3
ЧЕСТОТА НА ОТКЛЮЧВАЩАТА ИНФЕКЦИЯ ПРИ
ПАЦИЕНТИ С РЕАКТИВЕН АРТРИТ И
НЕДИФЕРЕНЦИРАН ОЛИГОАРТРИТ И
НЕОБХОДИМОСТТА ЗА ТЯХНОТО ИЗСЛЕДВАНЕ
Р. Стоилов
МУ, София – МБАЛ “Св. Иван Рилски”
Клиника по ревматология
96
Реактивният артрит, предизвикан от някои грам-негативни бактерии (Chlamidia
trachomatis, Yersinia enterocolitica, Salmonella, Shigella, Campylobacter jejuni) е с
много висока степен на асоциация с HLA-B27 молекулата от клас І на главния
комплекс за тъканна съвместимост. Въпреки че тези микроорганизми имат
интрацелуларен начин на живот, отделни техни фрагменти могат да бъдат
представени чрез HLA-B27 молекулата. Цитотоксичните Т клетки разпознават
тези бактериални пептиди или други, индуцирани от самия гостоприемник като
реагират кръстосано със собствените пептиди, представени в ставата. По този
начин се отключва автоимунна възпалителна реакция, чиято клинична изява е
синовита.
Реактивният артрит (РеА) е основна диференциална диагноза при пациенти с
недиференциран олигоартрит. В 55 – 60% от случаите при болните, отговарящи
на клиничните критерии за РеА може да бъде идентифициран отключващия
патоген. При изследване на пациенти с недиференциран олигоартрит, липса на
клинични симптоми за чревна или урогенитална инфекция и изключено друго
ревматично заболяване в 45 – 50% от случаите може да бъде доказана инфекция.
Това означава, че приблизително в половината от пациентите с вероятен или
възможен РеА може да бъде доказана отключващата инфекция в случай, че те
бъдат изследвани и тестовете са достатъчно надеждни.
MM4
MICROBIOLOGICAL INVESTIGATION OF IMPORTED
BRUCELLA INFECTION AMONG BULGARIAN CITIZENS
R. Nenova, T. Kantardziev, I. Ivanov, I. Tomova, B. Popov, Vl. Novkirishki
Brucellosis in Republic of Bulgaria has been officially eradicated since 1958. Despite
this, the closeness of the Mediterranean region, endemic for this zoonotic infection and
also the yearly presence of human cases in the neighbor countries creates a real possibility
of importing this infection in Bulgaria.
The aim of this study is the microbiological investigation of clinical samples from
suspected for brucellosis bulgarian citizens, who have been worked in a neighbor country
which proved to be endemic for the disease.
We use methods in consistent with the guidelines of FAO/WHO Expert Committee of
Brucellosis and the CDC Basic Diagnostic Tseting Protocols for the Presumptive
Identification of Brucella species.
529 investigations of 164 materials from 83 patients are made – 21 haemocultures, 429
serological and 79 genetic. All haemocultures are negative (45days of cultivation)
According to the serological tests there are three groups of sera samples:
- Negative
- Serological data for active Brucella infection
- Serological data for chronic Brucella infection
97
In 28 of the patients Brucella DNA was found in the sera samples.
The golden standard for proving the infection is the isolation of Brucella from the sera
samples. In our case the haemocultures were negative because of the delayed time for
testing the blood and the previous antibiotic therapy.
Detection of a four-fold or greater change in the serologica titres against Brucella
MM5
CHARACTERISTICS OF THE CULTIVATION OF
BIFIDOBACTERIUM BIFIDUM 1 IN MEDIA WITH
PALATINOSE
D. Blazheva, Z. Denkova, A. Krastanov
The cultivation of Bifidobacterium bifidum 1 was investigated in nutrient media,

containing palatinose, which was obtained through the transformation of sucrose with
immobilized cells of Serratia plymuthica ATCC 15928.
It was determined, that Bifidobacterium bifidum 1 assimilates palatinose as a single
carbohydrate source.
It is shown, that during the cultivation of this Bifidobacterium strain in a laboratory
bioreactor with constant stirring in a medium with palatinose, a higher concentration of
viable cells was reached for a shorter period of incubation and a moderate titratable acidity.
The redox potential of the medium (Eh) decreased during the lag-phase (from +26 to -
11) and during the transition to the stationary phase increased (from -11 to +42).
MM6
КОНТАМИНАЦИЯ НА ХОТЕЛИ ПО БЪЛГАРСКОТО
ЧЕРНОМОРИЕ С РАЗЛИЧНИ КЛОНОВЕ ЛЕГИОНЕЛИ-
ЕКОЛОГИЧНИ ПРЕДПОСТАВКИ ЗА КОЛОНИЗАЦИЯ
И. Томова, Ю. Хелбиг, В. Левтерова, Р. Ненова, И. Иванова
През последните три години зачестиха сигналите от Надзорната мрежа на

Европейската Работна Група по Легионелни Инфекции за Легионерската болест
при пътуване (ЛБП) в България. В тази връзка бяха изследвани води от
водоснабдителни системи (ВС) на хотели по черноморието за легионелни бактерии
(Лб) и се събраха данни за особеностите на изследваните ВС, отговорни за
намножаване на Лб. Проучването се извърши по методите описани в
Европейското методично указание за превенция на ЛБП. Обследвани бяха общо
12 хотела в различни курортни комплекси. Контаминацията им с култивируеми
Лб варираше от <101 до 104 CFU/L. Изолатите, принадлежащи към вида Legionella
98
pneumophila бяха субтипирани, при което се установиха серогрупи: L.р.Sg1 (с
моноклонални субтипове Allentown, Knoxville, Philadelphia и Oxford), Sg 3, Sg5, Sg6,
Sg8 и единадесет добре разграничени (независимо от серогруповата
принадлежност) генетични варианти. Физико-химичните показатели на ВС
варираха в широки граници: t0C студена H2O от 9- 29.70C , t0C топла H2O от 38.6-
61 0C, остатъчен Cl 2 <0.05-0.28 mg/L. Осем от обследваните хотели бяха
новопостроени, с ВС на 4 месеца до 3 години. При всички хотели ВС бяха с
разнороден дизайн и материали. Липсата на нормативна база и информация по
проблема на ЛБП бе причината за проектантски грешки, използване на
неподходящи материали и устройства, неправилната им експлоатация, неволно
създаване на слепи точки във ВС, отсъствие на профилактика в мъртвите точки и
програми за превенция на ЛБП. Комбинацията от изброените фактори е в основата
на създаването на подходящи за намножаване на Лб екологични ниши в хотелските
ВС. Резултатите доказват необходимостта от мониториране на антропогенните
водни екосистеми за Лб и спазване на международните норми за превенция на
ЛБП.
MM7
NORMAL FLORA IS FOUND IN HUMAN BLOOD
E. Kalfin
Abstract: Normal flora microorganisms, living in human’s blood, are unknown to

scientific literature. They were isolated only because instead of sheep blood, 10% of
native human blood was added to the nutrient medium, and the cultivation period continued
for 5 months, instead for 7 days. Routine cultivation requires a 3-4-week period, at 37°C.
The newly discovered microorganisms have no cell nuclei. They have no Gram-positive-
or Gram-negative microorganisms cell walls. Their surface is covered with very fine pili,
clearly expressed in young cells, as demonstrated by electron microscopy During their
reproductive cycle, they penetrate through erythrocyte walls and settle inside the
erythrocytes as in nests. Some formation resemble EB -/elementary bodies/ Outside
erythrocytes, they show well-expressed mimicry, but differ in size-0.3µm-2.6µm /
microorganisms / versus 3.5µm-7.5µm / erythrocytes / and pili presence. Pure cultures of
CLM was isolate in BAS and in NCIPD, but DNA analysis failed in both institutions
MM8
Q-FEVER – AN INSUFFICIENTLY KNOWN AND UNDERES-
TIMATED INFECTION IN BULGARIA.
V. Serbezov
99
The Q-fever is cause by Coxiella burnetii – ricketsiosis which is spread all over the
world. It is known on the Balkan Peninsula and in Bulgaria since the World War II.
Our investigations in the 90-ties of the XXth century indicated that the social and
economic changes in Bulgaria during the last 15-17 years (the vast areas of deserted land,
the small farms, the primitive stock-breeding, the increase of goat-breading, lower levels
of veterinary service, etc.) lead to increase in the natural and anthropurgical focuses of the
Coxiella.
From the clinical forms of the acute human Q-fever in our country is diagnosticated as
Q-fever only the one which presents like athipical pneumonia during endemic outbreaks.
The most frequent – influence-like, also the hepatitis-like and other forms of the disease,
including the variety of sporadic forms usually are not recognized. The inappropriate
treatment leads to chronization of the infection. Almost nothing is known and presented
about the Q-fever in childhood.
Severe chronical forms like: endocarditis, hepatitis and so on, are also not familiar to
the general practitioners, despite that in 1.5 to 5% of the people who had passed the acute
forms of Q-fever the infection becomes chronic.
The unsterile immunity of the Q-ricketsiosis has negative effect on the pregnancy of
women who had been ill with Q-fever: late abortions, premature birth, still-born children.
In spite of the bad demographic situation in our country this problem is seriously
underestimated.
In the moment there is discussion about the problems and the situation with Q-fever in
our country.
MM9
КЛИНИЧНИ АСПЕКТИ ПРИ МИКОТИЧНИ ИНФЕКЦИИ
НА БЕЛИТЕ ДРОБОВЕ
В. Пенчева* , Д. Петрова*, О. Георгиев*, Т. Кантарджиев**, Ц. Мондешки*,

Ц. Терзиева*, Д. Вълев***
*Катедра по Пропедевтика на Вътрешните болести, УМБАЛ”Александровска”
; **Национален институт по заразни и паразитни болести-София;
***СБАЛББ”Св.София” - бронхологично отделение
С широкото използване на антибиотичните средства в амбулаторната и

болнична практика се създават условия за увеличаване на микотичните инфекции.
При 73% от пациентите постъпили в клиниката е било приложено перорално
антибиотично лечение в амбулаторни условия. След последващата
хоспитализация в 8% от случаите персистират общите оплаквания, без динамика
в рентгеновия образ и лабораторните показатели. При всички тези пациенти се
касае за подостро протичане на заболяването с водещи симптоми суха дразнеща
кашлица, фебрилно- интоксикационен синдром, бронхиална обструкция и в част
от случаите придружаваща патология - ХОББ, бронхиална астма, захарен диабет,
100
белодробна туберкулоза. С оглед диагностичното уточняване бяха приложени
допълнителни неинвазивни и инвазивни методи на диагностика - ФБС /
фиброоптична бронхо скопия/ с БАЛ /бронхоалвеоларе лаваж/, катетърбиопсия,
фиброщипкова биопсия, серологични проби, компютър томография. В резултат
на проведените изследвания се позитивираха резултати за налични микотични
инфекции. При 22 пациента се изолира Candida spp.,при 5 пациента Аspergillus
spp., при 1 болен Coccidiomycosis. След антимикотична терапия оплакванията
отзвучаха, лабораторните показатели се нормализираха. В 36% от случаите
рентгенологичните промени претърпяха обратна резорбция, като при трима от
болните се наложи последващо хирургично лечение.
Микотичните инфекции на белите дробове се увеличават по честота, което
създава значителни диагностични проблеми по отношение изолиране на
етиологичния причинител. Използването на инвазивни техники на изследване в
комбинация със серологични тестове в значителна степен повишава възможността
за верификация на инфекциозния агент и последващ правилен терапевтичен
подход.
MM10
PCR-БАЗИРАНИ МЕТОДИ ЗА ДИАГНОСТИКА НА
СИСТЕМНИ МИКОЗИ И ГЕНОТИПИРАНЕ НА
ПАТОГЕННИ ГЪБИЧКИ
П. Ангелов, Т. Кантарджиев, В. Левтеровa, Е. Замфирова, М. Лесева, Р.

Вачева, Е. Шопова, Е. Бобчева
Честотата на системните микози нараства непрекъснато през последните 20

години, представлявайки сериозен проблем за съвременната клинична
микробиологична диагностика.
На базата на изследването на 40 клинични материала от пациенти със системни
микози, чрез разработен от нас PCR-базиран генетичен метод беше доказана
специфична за патогена ДНК, като методът би могъл с успех да се използва за
ранното установяване на микозен сепсис и назначаването на подходяща
антимикотична терапия.
Установяването на сродство между различните щамове гъбички, причинители
на инфекции посредством генетични методи (генотипиране), се характеризира с
висока дикриминативност и е от основно значение за ефикасния епидемиологичен
надзор на различните типове микози. Чрез използуване на различни генетични
маркери е проведено генотипиране посредством техниките AFLP, RAPD-PCR, PCR-
RFLP.
101
MM11
ЕТИОЛОГИЧНА СТРУКТУРА НА ЛЕКАРСТВЕНА
РЕЗИСТЕНТНОСТ И КОНСУМАЦИЯ НА АНТИБИОТИЦИ
В БЪЛГАРИЯ
Т. Кантарджиев, М. Петров, Ц. Велинов, А. Бъчварова, П. Ангелов

Отдел по микробиология – НЦЗПБ, София
На базата на данните от BulSTAR за изминалата година се прави анализ на

етиологичната структура на вътреболничните инфекции, инфекции, придобити в
обществото, пневмонии, сепсиси и уроинфекции, като получените данни са
сравнени с тези, получени при аналогични проучвания в Европа и САЩ.
Въз основа на данните от 150 микробиологични лаборатории в страната и
извършени 200 000 антибиограми се прави анализ на лекарствената резистентност
на най-важните патогенни микроорганизми.
Анализира се лекарствената резистентност на основните причинители на
бактериални менингити, пневмонии, уроинфекции и сепсиси – S. aureus, S.
pneumoniae, H. influnzae, E. coli, K. pneumoniae, E. fecalis, както и данни за тяхната
антибиотична резистентност
На базата на данните за надзора и консумацията на антибиотици в болничната
и извънболничната помощ, се правят изводи и препоръки за национална стратегия
и научно обоснована антибиотична употреба в клиничната медицина.
MM12
INVESTIGATION OF THE VIRULENCE FACTORS AND
ANTIBIOTIC RESISTANCE OF MORAXELLA
CATARRHALIS
I. Mitov1, R. Gergova1, V. Ouzunova1, R. Markovska1, D. Kuncheva2

Chair of Microbiology, Medical University of Sofia, Bulgaria1; Microbiology
laboratory, Capital City Regional Inspectorate of Public Health Protection and
Control, Sofia, Bulgaria2
During the last decade in Bulgaria the etiology role of Moraxella catarrhalis has
increased. omp B2, omp CD and omp E genes, which M. catarrhalis demonstrates in vivo
as outer membrane proteins (OMPs), as well as bro1 and bro2 genes coding beta-lactamase
production were detected in 103 clinical isolates and 4 control strains from CCUG and CIP
collections by PCR. The first group of 52 strains was isolated from patients with pulmonary
infections. The second group of 41 isolates M. catarrhalis comprises patients with
otorhinolaryngological infections. The third group of 10 strains consists of healthy children.
The strains with the OMP factors in the first group were more than 60%, with prevalence
102
of omp B2 and omp E. The role of OMP B2 is important to human serum resistance. It
enhances pulmonary clearance and inhibits iron acquisition from lactoferin and transferin.
omp E is coding OMP E – major adhesin, which plays role in serum resistance and transport
of fatty acids porin. In the second group the strains having the three OMP factors were
nearly 30%. The prevailing OMP factors in this group were omp CD and omp E – 86% and
96%, respectively. Adhesin coding omp CD gene binds to middle ear mucin, It is connected
with resistance towards complement killing. 50% of the strains from healthy children
possessed only omp E and in 40% no factors were detected. Since 1996 more than 90% of
clinical strains in Bulgaria possess bro1 gene determining high level resistance to penicillin,
aminopenicillins without inhibitors and first generation cephallosporins. This resistance
mechanism and virulence factors studied by PCR in M. catarrhalis isolates are reason for
the increase of the infections caused by this microorganism.
MM13
ВИДОВА ПРИНАДЛЕЖНОСТ И АНТИБИОТИЧНА
РЕЗИСТЕНТНОСТ НА КЛИНИЧНИ ИЗОЛАТИ ОТ
ХЕМОКУЛТУРИ
Руканова Д., Е. Стайкова, К. Рачкова, И. Дукова, Х. Дженева, М. Бойчева, Г.

Лазарова
Тракийски университет, Медицински факултет, катедра Микробиология, Стара
Загора, България
Изследването на хемокултури е от изключителна важност за диагнозата,

терапията и прогнозата на редица инфекциозни заболявания и инфектни процеси.
Целта на настоящото проучване е да установи видовото разпределение на
изолати от хемокултури и честотата на фенотиповете на резистентност.
Материали и методи: За периода 2002-2005 година са изследвани хемокултури
от болни, лекувани в Университетска болница-Ст.Загора. Използвана е системата
Bactec, BBL, както и среди за хемокултури на Бул Био,София. Идентификацията
е извършена чрез автоматизирани системи Sceptor и Crystal и рутинни методи, а
антибиотичната чувствителност е определяна по дифузионния-дисков метод на
Bauer-Кirby.
Резултати: Положителните хемокултури са 10.9%. Преобладават Грам-
положителните бактерии-61.8%, срещу 37.0% Грам-отрицателни и Candida
albicans 1.2%. Най-чести са CNS (32.8 %), E. coli (13.9%), S .aureus (11.3%), P.
aeruginosa (10.1%) и E. faecalis (7.5%).
Устойчиви на Oxacillin са 51.3% от CNS и 28.6% S. aureus. Ampicillin-
резистентни са 5.6% E. faecalis и 60.0% E. faecium. HLAG резистентност е намерена
при 11.1% E. faecalis и при 80.0% E. faecium. Не са регистрирани PNSSP и
Vancomycin–устойчиви Грам-положителни бактерии. ESBLs cа открити при 23.7%
от сем. Enterobacteriaceae. Резистентността на P. aeruginosa се движи в широки
103
граници: 7.1% към Imipenem, 25.0% към Ceftazidime, 30.0% към Amikacin, 52.9%
към Piperacillin и 60.9% към Ciprofloxacin.
MM14
ПРОУЧВАНЕ НА ВИДОВАТА ПРИНАДЛЕЖНОСТ И
АНТИБИОТИЧНА ЧУВСТВИТЕЛНОСТ НА ЩАМОВЕ,
ИЗОЛИРАНИ ОТ УРИНА НА ПАЦИЕНТИ С ХРОНИЧЕН
ПИЕЛОНЕФРИТ
В. Григорова 1, В. Тодоров 2, В. Едрева 1, К. Драгоев 1

1
МДЛ по Микробиология и вирусология,
2
Клиника по нефрология; „УМБАЛ д-р Г. Странски” Плевен, ЕАД, България
Въведение:Хроничният пиелонефрит е една от най-честите причини за

рецидивиращи инфекции на уринарния тракт.
Цел: Да се проучи видовата принадлежност и антибиотичната чувствителност
на микроорганизми, изолирани от урина на пациенти с хроничен пиелонефрит.
Материали и методи: проведени бяха микробиологични изследвания на урини
от 218 пациенти с хроничен пиелонефрит, лекувани в Клиниката по нефрология
при „УМБАЛ д-р Г. Странски” Плевен, ЕАД за едногодишен период (2005). От
116 пациенти със сигнификантна бактериурия и пиурия бяха изолирани 213 щама
уропатогени, идентифицирани с конвенционални методи. Щамовете E.coli бяха
скринирани за продукция на ESBLs. Чувствителността към антибиотици беше
определена чрез дисково-дифузионен метод на Bauer-Kirby, съгласно NCCCLS.
Резултати: Най- често изолираните микроорганизми бяха: E.coli (44.1%),
E.faecalis (26.8%), и P.aeruginosa (10.8%). Продуценти на ESBLs сред щамовете
E.coli бяха 18.3%. Щамовете E.coli продуценти на широкоспектърни â-лактамази
(ESBLs) бяха резистентни към gentamicin (72.7%), ciprofloxacin и TMP/SMZ. Щамовете
E.faecalis бяха резистентни към ampicillin (61.4%), gentamicin (70.5%) и ciprofloxacin
(77.3%). Щамовете P.aeruginosa бяха резистентни към gentamicin и ciprofloxacin
(33.3%). Всички щамове E.coli и P.aeruginosa бяха със запазена чувствителност
към imipenem и meropenem, а E.faecalis към vancomycin и teicoplanin.
Лечението на пациенти с хроничен пиелонефрит е необходимо да се провежда
съобразно чувствителността на изолираните микроорганизми към антимикробни
средства, за да се постигне максимален терапевтичен ефект и да се ограничи
разпространението на полирезистентни щамове.
104
MM15
ANTIMICROBIAL SUSCEPTIBILITY OF URINE
PATHOGENES ISOLATED FROM PATIENTS WITH BENIGN
PROSTATIC HYPERPLASIA TREATED WITH UROSTIM .
V. Grigorova 1, S. Stratev 2, F. Shargabi 2, N. Kolev 2, P. Panayotov 2, R. Kostov 2,

O. Mihaylov 2, Ts. Georgiev 2, V. Dunev 2, B. Atanasov
1
Laboratory of microbiology,2 Clinic of Urology, University Hospital - Pleven,
Bulgaria
Introduction:
Benign prostatic hyperplasia (BPH) is one of the most frequent reasons for recurring
infections of the urinary tract.
Aim:
To determine the antibiotic susceptibility of microorganisms isolated from urine of
patients with BPH and the results of immunotherapy with urostim.
Material and Methods:
A total number of 126 urine samples of patients with BPH treated in the Clinic of
Urology during 2005, were examined. Isolated strains were identified by conventional
methods. E. coli and K. pneumoniae strains were screened for ESBLs production.
Susceptibility to antibiotics was determined by using disc diffusion method of Bauer-
Kirby, according to NCCLS. Immunotherapy with urostim for a 3-month period was applied
to 36 patients and the urine was monitored on a monthly basis.
The patients were divided into two groups: Group I – 12 patients with asymptomatic
bacteriuria treated with urostim only, and Group II – 24 patients with symptomatic bacteriuria
treated with urostim and antibiotic.
Results:
Fifty strains were isolated from 40 patients with significant bacteriuria and pyuria.
The most frequently isolated microorganisms were: E. coli (42.0%), K. pneumoniae (18.0%)
and E. faecalis (12.0%). Producers of ESBLs among E. coli strains were 61.9%, and among
K. pneumoniae - 77.8%. The E. coli and K. pneumoniae strains remained susceptible to
imipenem and meropenem. The E. faecalis strains were susceptible to vancomycin and
teicoplanin.
After conducted treatment liquidation of the uroinfection were registered in 10
(83.3%) patients from Group I and in 18 (75.0%) patients from Group II. The bacteriuria
remained persistent in 2 (16.7%) patients from Group I and in 6 (25.0%) patients from
Group II.
Conclusions:
The immunotherapy with urostim does not exclude treatment with antibiotics in
order to achieve highest therapeutic results
105
MM16
ЧУВСТВИТЕЛНОСТ НА БОЛНИЧНИ ИЗОЛАТИ S.
PNEUMONIAE КЪМ НЯКОИ АНТИБИОТИЦИ
К. Божкова, Т. Стоева, В. Калудова, В. Каменова, В. Русев
Проучени са общо 69 етиологично значими щамове S. pneumoniae, изолирани

от различни клинични материали (секрети от горни и долни дихателни пътища,
ликвор, хемокултури) в Лабораторията по клинична микробиология и
вирусология на УМБАЛ “Св. Марина” – Варна за периода 01.01.2005 – 1.06.2006.
Определена е чувствителността им in vitro към няколко антимикробни препарата
(penicillin, erythromycin, clindamycin, tetracyclin, rifampin, vancomycin, linezolid,
trimethoprim/sulfamethoxasol). От изследваните изолати делът на пеницилин-
нечувствителните S. pneumoniae, определен чрез дисково-дифузионния метод е
76,8% (53 щама). Определянето на MIC, с Е тест, установява, че 95,4% от тях са
резистентни на пеницилин. Относително резистентни с МIC 0.12 до 1 µg /ml са
44,2% (23 щама), а 55,8% (29 щама) са абсолютно резистентни. Резистентността
към останалите антибиотици е както следва: V, Lzd – 0%, R – 4%, Cli – 10%, Ery, T, ST
– 22%. Един от изолатите демонстрира MLS индуцибелен фенотип на
резистентност.
Предвид нарастващата резистентност към penicillin, определянето на MIC за
изолати S. pneumoniae, особено при тежки инвазивни инфекции, е от критична
важност за правилно терапевтично поведение.
MM17
ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF A
POLYPHENOL-RICH EXTRACT FROM GERANIUM
SANGUINEUM L.
Medine Gulluce1, Fikrettin Sahin2, Atalay Sokmen3, Ani Teodosieva4, Julia

Serkedjieva4
1
Atatьrk University, Faculty of Art and Science, Department of Biology, Erzurum,
Turkey, 2Atatьrk University, Biotechnology Application and Research Centre,
Erzurum, Turkey, 3Cumhuriyet University, Faculty of Art and Science, Department
of Biology, Sэvas, Turkey, 4Institute of Microbiology, Bulgarian Academy of
Sciences, Sofia, Bulgaria
With the appearance of bacterial and viral variants, resistant to “classical”

chemotherapeutic agents, the search of microbial inhibitors of plant origin becomes a
promising approach in the development of new drugs. A polyphenol extract (PC) has been
obtained from the aerial roots of the Bulgarian medicinal plant Geranium sanguineum L.
106
The extract inhibited the reproduction of a range of viruses (influenza, herpes simplex,
vaccinia, HIV-I) in cell cultures. Its anti-influenza effect was most pronounced; PC reduced
the infectivity of various influenza virus strains in vitro and protected mice in the
experimental influenza infection. The present study sought to evaluate the antibacterial
and antifungal properties of the preparation using routine assays. PC was tested against
a panel of microorganisms including a total of 56 microbial cultures, belonging to 32
bacteria and 24 fungi and yeast species. However the preparation showed a marked
antifungal potential. Five strains - Fusarium pedis, Alternaria solani, Trichophyton
rubrum*, Trichophyton mentagrophytes* and Aspergillus niger* were resistant to the
PC-antifungal activity. Most susceptible to the inhibitory action of the extract was
Rhizoctonia solani (MIC 15.62 µg/ml). There was no activity in terms of bacteria tested,
the only exception being Staphylococcus aureus 209 (MIC 1000 µg/ml). Based on the
results from the present and previous investigations we believe that the Geranium
sanguineum extract possesses some interesting pharmacological properties. Its
phytochemical analysis revealed the presence of flavonoids, catechins, gallotannins and
carboxilic acids; some of them were identified by chromatographic methods (PC, TLC,
HPLC).
MM18
INVESTIGATION OF THE INHIBITORY EFFECT OF LACTIC
ACID BACTERIA ON THE CELLS OF ESCHERICHIA COLI
NBIMCC 8739
P. Nedelcheva, Z. Denkova, R. Nikolova
The inhibitory effect of growing cultures of Lactobacillus plantarum 226-15,

Lactobacillus casei subsp. casei C, Lactococcus lactic subsp. lactis l4 and Pediococcus
cerevisiae 2P on the cells of Escherichia coli NBIMCC 8739 was investigated during their
mixed cultivation in skimmed milk at temperatures 23±1°С and 37±1°С.
It was established, that during the first 6 h the viable cell counts of both the lactic acid
bacteria and Escherichia coli NBIMCC 8739 increased. During the next 12-24 h the growth
of Escherichia coli NBIMCC 8739 gradually slowed up and held up. This effect was more
distinct during the mixed cultivation of lactic acid bacteria and E. coli at 37°С.
MM19
PROBIOTICS AND PROBIOTIC FOODS
IN THE PROTECTION OF HUMAN HEALTH
I. Murgov, Z. Denkova
107
A series of probiotic preparations ‘Enterosan’ was developed on the basis of selected
Lactobacillus and Bifidobacterium strains from the national gene fund, which survive the
conditions in the stomach and the intestines, exhibit high antimicrobial activity and
resistance to most of the applied in the clinical practice antibiotics.
Tested in leading clinics in Bulgaria and abroad th eprobiotics ‘Enterosan” are applied
successfully in the complex therapy of diseases of the digestive, endocrine, secretory,
vascular, bone, neural and other systems.
The necessity was reasoned for the inclusion of probiotic foods and drinks in the diet
of the contemporary human.
MM20
СЕПСИС, СВЪРЗАН С ЦЕНТРАЛНИ ВЕНОЗНИ КАТЕТРИ,
ПРИ БОЛНИ В КРИТИЧНО СЪСТОЯНИЕ –
ПРИЧИНИТЕЛИ И ЧЕСТОТА
Димитър Терзийски, Николай Петров, Николай Младенов

КАРИЛ, ВМА, София, е-mail: dterziiski@yahoo.com
УВОД: Системните нфекции, свързани с централни венозни катетри (ЦВК), са

сред най-тежко протичащите вътреболнични инфекции и причина за значителна
смъртност и увеличени разходи при лечението на болни в критично сътояние.
ЦЕЛ: Да се установят честотата и причинителите на сепсис, свързан с ЦВК,
при болни в критично състояние.
МЕТОД: Болни: Проспективно изследване на болни, лекувани в КАРИЛ,
ВМА, за периода март 2001 – април 2004 г. ЦВК: метод на въвеждане, престой,
локализация и условия на поставяне. Микробиологични методи: полу-
количествено изследване на катетера по метода на Maki; определяне на
диференциално време до позитивиране на хемокултури с кръв, взета през ЦВК и
от периферна вена.
РЕЗУЛТАТИ: Болни: 95 (от 18 до 81год) и ср.сбор по APACHE II – 20 т. Значима
колонизация на ЦВК – 52 (44 %); локална инфекция, свързана с ЦВК – 26 (22 %);
сепсис, свързан с ЦВК – 25 (21 %). Микробиологични изследвания : (1) свързани с
ЦВК – изолирахме 89 щама, грам-положителни –53 щама, грам-отрицателни 31
щама, Candida spp – 5 щама; (2) несвързани с ЦВК изследвания – изолирахме 221
щама, грам-отрицателни – 161 щама, грам-положителни коки 44 щама и Candida
spp – 16.
ИЗВОДИ : Установената от нас висока честота на сепсис, свързан с ЦВК,
може да се дължи на спецификата на изследваните болни, както и на
поливалентната употреба на катетрите. Най-чест причинител на сепсис, свързан
с ЦВК, е S. aureus, следван от K. pneumoniaе. Броят на щамове S. epidermidis,
S.aureus и Enterococcus spp, изолирани от катетрите и от другите области е сходен,
което показва значимата им роля за възникване на сепсис, свързан с ЦВК, при
108
изследваните болни.
MM21
МОЛЕКУЛЯРНА ДИАГНОСТИКА НА ПРИЧИНИТЕЛИ НА
АТИПИЧНА ПНЕВМОНИЯ
Надя Бранкова, Тодор Кантарджиев, Виктория Левтерова, Елина Георгиева,

Искра Томова и Стефан Панайотов
Национален център по заразни и паразитни болести, Отдел „Микробиология”,
Янко Сакъзов 26, София - 1504
Coxiella burnetii, Chlamydophila pneumoniae, Legionella pneumophila и

Mycoplasma pneumoniae причиняват остри респираторни инфекции като атипична
пневмония придобита в обществото, хронични бронхити, астма и с по-малка
честота инфекции на горните дихателни пътища. Настоящето изследване
представя обобщение на разработени съвременни методи за диагностика на C.
burnetii, C. pneumoniae, Legionella pneumophila и M. pneumoniae в различни
респираторни материали. Методите са разработени на базата на видово-
специфични IS1111A, omp1, mip и P1 гени. Всички клинични материали бяха
анализирани чрез метода на полимеразно-верижна реакция. Бяха потвърдени
епидемични взривове на Ку-треска в гр. Ботевград (2004) и атипична пневмония
сред деца, причинена от M. pneumoniae в с.Ключ (2002) и гр. Павликени (2005).
Положителните резултати са от носогърлени секрети и храчки. PCR методите се
отличават с висока специфичност и чувствителност и могат да бъдат използвани
в рутинната лабораторна диагностика на C. burnetii, C. pneumoniae, Legionella
pneumophila и M. pneumoniae.
MM22
Emm SEQUENCE TYPING OF CLINICAL ISOLATES
STREPTOCOCCUS PYOGENES RECOVERED IN BULGARIA
A. Decheva , V. Karjeva , D. Beshkov , I. Alexiev

National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria
Streptococcus pyogenes (group A streptococci – GAS) is an important bacterial

pathogen for humans. Its surface M protein is the main pathogenic factor with antiphagocitic
activity. More than 120 M-serotypes have been registered until now. Production of rabbit
typespecific antisera has become quite expensive and time consuming. Sequence typing
of the emm gene encoding the M protein has proved to be more specific, sensitive, quick
and convenient method for epidemiological typing of S. pyogenes. We performed emm
sequence typing of 69 clinical isolates (48 from upper respiratory tract, 15 from skin lesions,
109
1 vaginal isolate, 3 from invasive infections and another 2 from noninvasive wound infection
but epidemiologically related with the three invasive isolates). The group of upper
respiratory tract isolates was heterogenous but the most frequent genotypes were emm 12
and emm 1. The group of skin isolates was also heterogenous with emm 1, emm 44/61 and
emm 117 being most common. Overlapping of genotypes was observed for these two
groups of isolates. The three invasive isolates and two wound isolates were recovered
from epidemiologically related cases. All of them expressed emm 65 which confirmed the
relation. To our knowledge emm 65 has not been reported in association with invasive
infections until now.
MM23
RECOMBINANT BORRELIA BURGDORFERI PROTEINS AS
ANTIGENS FOR SEROLOGIC DIAGNOSTICS OF LYME
BORRELIOSIS
I. Christova1), M. Lesseva2), G. Miloshev2)

1)
National Center of Infectious and Parasitic Diseases; 2) Institute of Molecular
Biology, Bulgarian Academy of Sciences
Application of purified or recombinant antigens, instead of whole-cell Borrelia

burgdorferi antigens, has been suggested to increase specificity of serological diagnostics
of Lyme borreliosis. In addition, using recombinant antigens, dynamics and variation of
the main serological markers in the course of the disease could be investigated. The
problem is, that at least three B. burgdorferi species – B. burgdorferi sensu stricto, B.
garinii and B. afzelii, cause Lyme disease in Europe and respectively in Bulgaria. In an
attempt to obtain recombinant B. burgdorferi proteins, which could be used to study on
specificity of humoral immune response in Lyme disease, we made a comparative analysis
of genes for the major outer surface protein antigens, OspA and OspC, as well as for the
gene of immunodominant flagellar antigen, p41, of the three species Borrelia that cause
Lyme disease. The highest level of homology was detected in the gene FlaB – 93-94%,
lower but also high was detected in the genes OspA and OspC, respectively 87-88% and
83-88%. Next step was cloning and expression of these proteins. The proteins obtained
were confirmed by immunoblot with specific monoclonal antibodies. Applied as antigens
for ELISA, OspA showed higher sensitivity in the late stages of the disease, OspC -
higher sensitivity in the early stages, and FlaB – higher sensitivity but at the same time
low specificity. The optimal combination of recombinant antigens for detection of different
stages of Lyme disease, should be elucidated.
110
MM24
DETECTION OF BORRELIA BURGDORFERI SENSU LATO,
ANAPLASMA PHAGOCYTOPHILUM AND FRANCISELLA
TULARENSIS IN WILD RODENTS FROM AN ENDEMIC
FOCUS OF TULAREMIA IN BULGARIA
Gladniska T., Christova I., Taseva E., Nenova R.

Microbiology Department, National Center of Infectious and Parasitic Diseases
Lyme disease, tularemia and human granulocytic anaplasmosis (HGA) are among the
most common tick-borne bacterial infections in Bulgaria. Reservoirs of the infections are
wide spectrum of wild and domestic mammals. The most important reservoirs among wild
mammals are rodents – Mus musculus, Rattus rattus, Apodemus agrarius. The aim of this
study was to establish prevalence of infections with Borrelia burgdorferi sensu lato,
Anaplasma phagocytophilum and Francisella tularensis, the etiological agents of Lyme
disease, HGA and tularemia, in wild rodents from an endemic focus of tularemia in west
Bulgaria, Slivnitza region. Serologic methods (ELISA, agglutination) detected infection
with B. burgdorferi in 14% of the rodents and in 21% infection with F. tularensis. Genetic
methods (PCR) established infection with B. burgdorferi in 26% of the rodents, with F.
tularensis in 21% of the samples and with A. phagocytophlim in 7% of the rodents. The
higher percentage of infected rodents, detected by PCR is due to the higher sensitivity of
the methods. This is also probably the explanation of the discrepancy detected between
some of the serologic and PCR – positive results in different stages of the diseases.
Contemporary PCR methods are adequate tools for fast and reliable surveillance of
circulations of different tick borne pathogens among rodent populations. Combination of
PCR and serologic methods reveal current incidence of rodent infections.
MM25
НОВОУСТАНОВЕНО ОГНИЩЕ НА ТУЛАРЕМИЯ В
СЕВЕРОИЗТОЧНА
БЪЛГАРИЯ ПРЕЗ 2004 – 2005 ГОД.
Н. Готев, К. Младенов, Ц. Цветанов, Е. Пенков, Н. Коруджийски,

Сн. Иванова, В. Дойчева
През 60-те години на миналия век беше наблюдавано огнище на туларемия в

района на гр. Силистра – езерото Сребърна, а след 1997 год. и до сега се
наблюдава такова в района западно от София.
През 2004-2005 год. са установени данни за “ново” огнище в района на гр.
Русе – Разград – Търговище. От намерен умрял заек е изолиран причинителя на
туларемията – Francicella tullarensis и у двама заболели е потвърдена положителна
111
за туларемия серологична проба.
Тези данни говорят за наличие на ензоотична туларемия сред дивите зайци и
разпространенние на инфекцията и сред хората в този район.
Фенотипната характеристика на изолираната култура Francicella tullarensis е
характерна за тип В на този бактерий, както в установените преди това две огнища.
Тези данни показват, че е необходимо д0а се провеждат системно наблюдения
предписани в Националната програма за борба с кърлежо-преносимите инфекции.
MM26
ФЕНОТИПНА ХАРАКТЕРИСТИКА НА ПРИЧИНИТЕЛЯ
НА ТУЛАРЕМИЯТА – FRANCICELLA TULLARENSIS
ИЗОЛИРАНИ В БЪЛГАРИЯ ПРЕЗ 1961 – 1965
И 1998 – 2005 ГОД.
Н. Готев, К. Младенов
Досегашните лабораторни изследвания показват, че причинителя на

туларемията F. tullarensis проявява характерни фенотипни белези, което позволява
неговата индентификация и разграничаване на две разновидности – тип А и тип
В.
Тип А е установен досега в Северна Америка, а тип В и в Европа и Азия.
В нашата страна бяха изолирани голям брой щамове на F. tullarensis в
наблюдавани огнища през периода 1961 – 65 год. в района на езеро Сребърна,
Силистренско и 1998 – 2004 год. западно от София.
Щамовете са изолирани от различни източници – от гризачи (ондатри, зайци,
мишевидни), артроподи, вода и човек.
Фенотипната характеристика на всички изследвани щамове съответства за F.
tullarensis – тип В, разпространен в Европа.
MM27
ЛЕЧЕНИЕ НА ЕКСПЕРИМЕНТАЛНА ТУЛАРЕМИЙНА
ИНФЕКЦИЯ
К. Младенов1, Х. Найденски2, Ц. Цветанов1, Н. Готев1

1
Военномедицинска академия, София, България
2
Институт по микробиология “Стефан Ангелов” - БАН
Туларемията е зоонозна инфекция, към която човек показва висока

възприемчивост. Заразяването е възможно по всички пътища на попадане на
възбудителя в организма – през дихателния тракт, слизестите обвивки,
112
конюнктивата, през увредена кожа и парентерално.
Туларемийният бактерий има висока инфекциозност по отношение на човека
и редица дребни и едри гризачи, което го прави подходящ за използване като
агент за биологично поразяване.
Целта на разработката е да се проучи чувствителността на възбудителя към
някои антибиотици и продължителността на третиране на модела на
експериментални животни.
Използвани са опитни животни, отнасящи се към I група по отношението си
към инфекцията – морски свинчета, чрез парентерално заразяване в доза 1000
микробни клетки на туларемийния щам “Сребърна-19”.
Третирането е извършено с два антибиотика: доксициклин (Doxycyclin), в доза
6 mg на един прием с продължителност 5, 10 и 15 дни per os.
Tetracyclin – depo, в денонощна доза 15 и 30 mg, дадени на два приема с
продължителност 10 дни.
Установени са положителни резултати и очистване на организма от
причинителя на инфекцията. Данните от количественото натрупване и
персистиране на бактериите в различните органи са определени в динамика и при
двата антибиотика.
Пероралното третиране с Doxycyclin – еднократно-6 mg иTetracyclin-depo –
двукратно - 30 mg, осигуряват добър защитен ефект на експерименталните
животни от туларемийната инфекция.
MM28
RAPID IDENTIFICATION AND BIODIVERSITY OF LACTO-
BACILLUS SPECIES IN VAGINAL SAMPLES
S. Dimitonova1, P. Grozdanov2, A. Galabov2, B. Bakalov3, R. Aleksandrova1, G.

Stoyancheva1 and S. Danova1
1
- Department of Microbial Genetics and 2- Department of Virology, Institute of
Microbiology, Bulgarian Academy of Sciences, 26, Acad. G. Bontchev, str, 1113
Sofia, Bulgaria.
3
- Department of General and Applied Microbiology, Biological faculty, Sofia
University, 8, Dragan Tzankov, bvd, 1113 Sofia, Bulgaria
Lactobacilli are commonly present in the human vagina and critically important to
women’s vaginal health. They have received a considerable attention as a result of their
significant properties. The application of modern genomic analyses has advanced their
correct species identification and taxonomy. In this study the Polymerase chain reaction
(PCR) was demonstrated to be suitable tools for the analysis of Lactobacillus community
of reproductive age Bulgarian women. It allow the detection of species very quickly and
reliable. The results showed the presence of L. fermentum, L. crispatus, L. acidophilus
and species from L. plantarum group in vaginal smears collected from healthy volunteers
113
and patients with HPV. We did not find a correlation between HPV infection and disturbance
of Lactobacillus microbiota, by PCR and classical microbiological analysis. Combined
molecular analyses revealed the presence of two or three different Lactobacillus species
originated from a single vaginal sample. The study accomplishes the scarce information
existed in Bulgaria on the real identity of Lactobacillus species in relation of women’s
healthy status.
MM29
VIRULENCE DETERMINANTS OF AEROMONAS SPP. ISO-
LATED FROM FOOD, DRINKING WATER AND PATIENTS IN
BULGARIA
P. Orozova, I. Abrashev
Bulgarian Academy of Sciences, Institute of Microbiology, Department of Microbial

Biochemistry, 26 Acad. G. Bonchev str., Sofia 1113, Bulgaria
In the present study 45 Aeromonas spp strains isolated from food, drinking water and
patients in Bulgaria were tested for pathogenicity by studding its hemolysis, neuraminidase
activity and cytotoxicity. The drug resistance profiles were also evaluated and all strains
displayed multiple drug resistance.
Hemolysis, tested on sheep erythrocytes, was more frequently seen with water isolates
and human isolates than with food isolates. Neuraminidase activity was detected in all
isolates and it was the highest to Aeromonas hydrophila strains. Cytotoxicity, evaluated
on Vero cells was frequently observed with food (90%), with water isolates (70%) and
with human isolates (62%). It was found that negative pyrazinamidase activity was
associated with Aeromonas sobria and positive pyrazinamidase activity was associated
with Aeromonas hydrophila and Aeromonas caviae. All isolates exhibited multiple drug
resistance.
These results indicate that the occurrence of Aeromonas spp. in the environment
represents a potential risk for humans. These results also suggest that Aeromonas species
are potential enteric pathogens in our geographical region.
MM30
ОПРЕДЕЛЯНЕ СРОК НА ГОДНОСТ НА АНТИМИКРОБНИ
ДИСКОВЕ ЦЕФОКСИТИН И ЦЕФТРИАКСОН И
ВЪВЕЖДАНЕТО ИМ В ПРОИЗВОДСТВО
Даринка Чанкова, Даниела Пенчева

“Бул Био – НЦЗПБ” ЕООД
114
Цефокситинът принадлежи към втора генерация цефалоспорини. Отличава се
с висока стабилност към ß-лактамазите и подчертана активност към облигатните
анаероби и гонококите. При изпитване ин витро чувствителността на
микроорганизмите се налага залагането му като отделен диск. Според CLSI/NCCLS
2006 докладването на оксацилиновата резистентност се извършва на базата на
цефокситиновия диск. Препоръчва се да се използва за изпитване резистентността
към пеницилиназа стабилните пеницилини при S.aureus и S.lugdunensis.
Цефокситиновият дисков тест има по-голяма специфичност и еквивалентна
чувствителност от оксацилиновия дисков тест към коагулаза негативните
стафилококи.
Цефтриаксонът има най-широк спектър на действие в сравнение с останалите
цефалоспорини от трета генерация, като към него са чувствителни и S.pneumoniae,
стрептококи група А и С, H.influenzae, гонококите (включително ß-лактамаза
продуциращите), менингококите. От всички цефалоспорини неговият полуразпад
е най-бавен, което позволява да се прилага веднъж на 24 часа. Според CLSI/
NCCLS 2006 е задължително залагането в рутинните антибиограми на
цефтриаксоновия диск при наличие на резистентност на семейство
Enterobacteriaceae и Acinetobacter към антимикробните дискове от група А.
Точното определяне срока на годност на антимикробните дискове е от голямо
значение за получаване на достоверни и възпроизводими резултати от
антибиограмите, а от там и за правилното лечение на пациентите.
MM31
LABORATORY METHODS FOR DRUG SUSCEPTIBILITY
TESTING OF MEDICALLY IMPORTANT YEASTS AND
MOULDS
Kouzmanov A., T. Kantardjiev, Ivanova Z., L. Boyanova

National Center of Infectious and Parasitic Diseases, Sofia, Bulgaria
Introduction: During the last two decades the number of patients with serious fungal
infections has increased dramatically. The risk group includes: immunocompromized
patients, especially those infected with HIV, those receiving immunosuppressive therapy
for organ transplantation and cancer. The long-term use of broad-spectrum antifungals
for prevention and therapy of mycosis has led to the detection of increased drug resistance
of fungal pathogens. Therefore there is a prominent necessity of standard susceptibility
testing methods.
Aim: Determination of Fluconazole susceptibility of strains from genus Candida with
the use of standard disk diffusion method M44-A. Additional testing of preliminary chosen
resistant to Fluconazole strains and determination of their susceptibility to five antifungals:
Itraconazole, Ketoconazole, Voriconazole, 5-Fluorocitosine, Amphotericine B
Materials and Methods: In the study were used clinical isolates collected by the Referent
115
Mycology Laboratory of National Center of Infectious and Parasitic Diseases, Sofia. They
are strains from genus Candida and some moulds. All were identified by conventional
biochemical methods API 20C AUX, AUXACOLOR 2 or automated system Vitek and
direct microscopy combined with the fungal culture. For the susceptibility testing were
used 5 methods: disk diffusion method (DDM) NCCLS M44-A; reference microdilution
method NCCLS (currently CLSI); agar dilution E-test; commercial kit ATB FUNGUS 2 INT
and MICRONAUT-AM (Merlin).
Results: We detected high pro cent of resistant strains, witch can be explained with
their isolation from patients with HIV, under a long-term azole therapy. High resistance
was detected in the strains C. glabrata and C. krusei. There is a good correlation between
the 5 methods in the testing of Fluconazole resistance. Voriconazole showed good
effectiveness but the results received for Itraconazole and Ketoconazole were not so
good. For the determination of 5-Fluorocitozine were used ATB FUNGUS 2 INT and
MICRONAUT-AM with only one resistant strain detected. There was a sufficient
correlation between the results received for Amphotericine B.
Conclusions: The most important benefit of antifungal susceptibility testing is the
determination of the minimal inhibitory concentration of a certain drug. This allows an
accurate dozing and adequate therapy of the patient. The introduction of standard methods
for antifungal susceptibility testing can stop the aimless use of drugs.
MM32
STUDY OF HYPOCHOLESTEROLIC EFFECT OF HIGHER
BASIDIOMYCETES
Popov A.1, Panchenko A.2, O. Chistova1, Petrishchev N.2, Denisova N.3, Shamtsyan
M.1
1
. St. Petersburg State Technological Institute (Technical University), Moskovsky
prospect, 26, St. Petersburg, Russia. E-mail: shamtsyan@yahoo.com
2
. I.P. Pavlov State Medical University, 6/8 L.Tolstoy Str., 197022 Saint Petersburg,
Russia
3
. V.L. Komarov Botanical Institute, Russian Academy of Sciences, ul. Professora
Popova, 2, St. Petersburg, 197376, Russia
It is known, that hypercholesterolemia increases the risk of heart disease. Elevated

levels of circulating cholesterol cause deposits to form inside blood vessels. These
deposits can result in a disease process called atherosclerosis, which can cause blood
clots to form that will ultimately totally stop blood flow. If this happens in the arteries
supplying the heart, a heart attack will occur. If it happens in the brain, the result is a stroke
where a portion of brain tissue dies. Atherosclerosis causes more deaths from heart disease
than any other single condition. The most common cause of elevated serum cholesterol is
eating foods that are rich in saturated fats or contain high levels of cholesterol.
Cholesterol has been divided into two major categories: low-density lipoprotein (LDL),
116
the so-called “bad” cholesterol, and high-density lipoprotein (HDL), the so-called “good”
cholesterol.
We studied the influence of mushroom additives to hypercholesterolic diets on the
levels of total cholesterol and high-density lipoproteins in rats. The obtained results are
demonstrating, that the addition of mushrooms to hypercholesterolic diets is significantly
decreasing the levels of LDL, leading to increase of HDL, and almost normalizing the
coefficient of atherogenity.
MM33
RESISTANCE AND GENOTYPIC DIVERSITY OF
MULTIDRUG RESISTANT ACINETOBACTER BAUMANII IN
UNIVERSITY HOSPITAL
Vatcheva-Dobrevski*, Rossi, Savov* Encho, Bernards** Alexandra, van den

Barselaar** M., van den Broek** Peterhans, Dijkshoorn** Lenie
*Military Medical Academy, Dept. Clinical Microbiology, Sofia BULGARIA
**Laiden University Medical Center, Dept.Medical Microbiology, Leiden,THE
NETHERLANDS
OBJECTIVE: To evaluate the frequency, antimicrobial resistance evolution and

appearance of carbapenem-resistant Acinetobacter baumanii isolates. To investigate
genotypic diversity during reccurrent epidemic episodes.
MATERIALS&METHODS: Between 2000-2004 a total of 326 multidrug resistant
(MDR)A.baumanii isolates, investigated by Vitek 2 System(Bio Merrieux ,France) were
analysed. The MICs of imipenem(Imp),meropenem(Mp), amikacin(Amk), cefoperasone/
sulbactam(Cps),ciprofloxacin(Cl),ceftazidime(Caz),and metallo-beta lactamase production
were determined by E-test(AB,Biodisk,Sweden). The genotype method was AFLP genomic
fingerprinting.
RESULTS: The isolates were recovered from bronchial aspirates(42,6%), surgicalsite
(18,3%),catheters(12,7%),bloodcultures(8,1%)in ICU.The susceptibility to antimicrobials
decreased as follow from/to: ticarcillin 76%-59%,piperacillin 73-42%,Caz 52%-32,4%,Cp
46%-23%,tobramycin 96%-81%.The most common MDR patterns for 2000-2002:isolates
were resistant to multiple antibiotics,but most were susceptible to Tob and colistin,all
susceptible to Imp and Mp.From 2003-2004 very common MDR patterns were
observed:i.ImpR,MpR, CpsS(11%),ii.CpsS,TobS,AmkS ImpR,MpR(17%),iii.only ImpS,MpS
(56%).The resistance to carbapenems increased from 0% to 16%.
CONCLUSIONS:The Acinetobacter baumanii nosocomial isolates during last five years
in our hospital presents a high level antimicrobial resistance. Four different genotypes
were distinguished, one of which (type 1) was observed among 13 isolates indicating the
prevalence of an epidemic strain.The emergence of MDR isolates require controlled
carbapenem prescription and infection control measures for prevention of untreatable
infections by Acinetobacter baumanii.
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MM34
IMMUNOMODULATING AND ANTITUMOUR ACTION OF
HIGHER FUNGI
V. Spiridonova(a), P. Tsvetkov(a), A. Panchenko(b), A. Korchmaryova(a),

N. Petrischev(b), M. Shamtsyan(a)
(a)
St. Petersburg State Institute of Technology (Technical University), Russia, St.
Petersburg, 198013, Moskovsky prospect, 26 E-mail: shamtsyan@yahoo.com
(b)
St. Petersburg State I. P. Pavlov Medical University , Russia, St. Petersburg,
197089, Leo Tolstoy str., 6/8
Aqueous extracts from fruit bodies and submerged mycelia of various higher
Basidiomycetes were studied in search for reliable biological effects. In vitro and in vivo
experiments were conducted.
The results showed that the aqueous extracts demonstrated various types of marked
biological actions: an increased production of reactive oxygen forms by neutrophil cells
of human peripheral blood; a significant mitogenic activity in a wide range of concentrations;
stimulation on production of inflammatory cytokines. Administered orally mixed with daily
food they cause a decrease in average tumor size in mice with transplanted melanoma B16
and Ehrlikh’s ascit carcinoma and a prolongation in the survival rate of such mice.
MM35
ANTIOXIDANT PROPERTIES OF HIGHER MUSHROOMS
N. Dubyago,, I. Shugaley, E. Tozik. M. Shamtsyan

St. Petersburg State Technological Institute (Technical University), Moskovsky
Ageing of an organism, influence of negative environmental factors, development of

many pathological processes, are connected with formation of oxygen active forms in
superfluous quantities, that leads to infringements in functioning of living systems, so-
called “free-radical pathologies”, to processes of the peroxide damages of proteins, lipids,
DNA, carbohydrates.
One of the prospective ways to control related diseases as well as, for their prophylaxis
is use of antioxidants. However, correction of each type of pathology by means of
antioxidants requires detailed studies of suggested preparations, determination of dozes
and introduction methods. At the same time it must be considered, that supposed
antioxidants are far from being universal and at different disorders of free-radical processes,
their influence is not identical.
Food antioxidants possessing curative - prophylactic action are thought to be the
most preferable and perspective ones. Substances isolated from higher basidiomycetes
are also representing a perspective groups of natural antioxidants.
118
Our studies show that fruit bodies, mycelia and cultural filtrates of various
basidiomycetes possess pronounced antioxidant activity. For some of edible or non toxic
basidiomycetes SOD-like activity was also detected.
Antioxidants of mushroom origin can be used in various fields, such as pharmacy,
food industry and cosmetics. Being added to common, daily food products they will also
contribute in preservation of food from spoiling, and thus increasing food safety.
MM36
ИНДИРЕКТЕН ИМУНОФЛУОРЕСЦЕНТЕН МЕТОД ЗА
ОТКРИВАНЕ НА HELICOBACTER PYLORI ДИРЕКТНО В
СТОМАШНИ БИОПСИИ
К. Иванова1, Цв. Илиева1, М. Марина1, И. Митов3, Б. Владимиров2, Й.

Чурчев2
1
Национален център по заразни и паразитни болести; 2Клиника по
гастроентерология, ДУБ“Царица Йоанна”, София; 3Медицински университет,
София
Проучени са възможностите на индиректен имунофлуоресцентен метод /

ИИФМ/, с използването на четири моноклонални антитела /собствено
производство/, за откриване на
Helicobacter pylori директно в стомашни биопсии на хора.
Изследвани са 95 стомашни биопсии, взети от пациенти с различни оплаквания
от страна на гастроинтестиналния тракт. Чувствителността и специфичността
на ИИФМ беше сравнена с културелния метод, директен микроскопски препарат
и бърз уреазен тест.
Чувствителността и специфичността на ИИФМ беше съответно 93% и 95% в
сравнение с другите методи, което го прави сравнително точен метод за
диагностика на H.pylori.
119
PRESENTATIONS OF FIRMS:
ZEU-INMUNOTEC
PCR-DIAPOPS METHOD FOR DETECTION OF

LEGIONELLA IN WATER
Dr. Elena Domínguez- ZEU-INMUNOTEC, Maria de Luna 11, Zaragoza, Spain
Legionella is naturally found in environmental water sources. Hot water systems,

cooling towers, and water distribution systems found in large buildings, such as hotels
and hospitals, are susceptible to legionella colonization. The presence of high
concentrations of Legionella in aerosols from contaminated waters, are associated with
respiratory diseases that can cause deaths. Since the first identification in 1977 numerous
cases have been described in different countries causing loss of lifes and economical
damages. Therefore, monitoring of susceptible water systems is of a paramount importance.
Traditionally, microbiological techniques have been used for Legionella identification
in water samples. However, these methodologies can be tedious and time consuming,
taking over a week to generate results. ZEU-INMUNOTEC presents a molecular biology
test kit -Microline-Legionella- for screening of Legionella in water.
Microline-Legionella is based on a polymerase chain reaction (PCR) connected with
an immunoenzymatic assay for colourimetric detection of the amplificated product
(DIAPOPS). The test allows a qualitative detection of Legionella spp and/or Legionella
pneumophila in water samples within 24 hours, allowing quick decisions in events of
outbreak.
BIOSYSTEMS LTD., SOFIA, BULGARIA
REAL-TIME PCR IN MOLECULAR MICROBIOLOGY
Velichka Kardjeva, Biosystems Ltd., Sofia, Bulgaria
In the past ten years molecular biology techniques became a routine for identification
of pathogen and non pathogen viruses, bacteria and fungi. Widely applied technologies
are PCR, real-time PCR and sequencing. Genotyping and mutation analysis are both
powerful tool for identification of any microorganism and discovery of resistant strains to
antibiotics, different substances and physical factors.
Applied Biosystems provides the latest innovation in TaqMan assays for genotyping
/ subtyping and quantitation of different microorganisms, which are applicablenot only to
genomics studies but also in medicine for detection, identification and quantification of
pathogens, in ecology, taxonomy and etc. Real-Time PCR instruments from Applied
120
Biosystems are well known in Bulgaria.
Stay tuned with the latest developments in molecular genetics.
Visit us at www.appliedbiosystems.com, or contact officebiosyst@mbox.contact.bg
121
GENERAL AND APPLIED MICROBIOLOGY
GAM1
MYCOLOGICAL STUDIES IN CONTINENTAL
ANTARCTICA: AN OVERVIEW
Solveig Tosi
Sezione di Micologia, Dipartimento di Ecologia del Territorio e degli Ambienti
Terrestri, Universitа di Pavia, via S. Epifanio 14, 27100, Pavia, Italy.
stosi@et.unipv.it
The geographic isolation and the relatively low anthropogenic impact are two of the
main reasons why the study of the Antarctic microbiota involved a great interest since the
beginning of the XX century. The majority of papers dealing with mycoflora published
until today were carried out in Victoria, Wilkes, Princess Elizabeth, McRobertson, and
Enderby Lands, areas that can be considered biologically “rich” thanks to periodically
unfrozen sites, presence of mosses and freshwater habitats, lichens, and animal remains.
After more then one century of investigations it is possible to identify a continental
Antarctic mycoflora mainly composed of anamorphic fungi (filamentous and yeasts, 66%),
some Zygomycetes, and very few species of Ascomycetes and Basidiomycetes. In
continental Antarctica 1634 fungal or pseudofungal records, belonging to 146 genera and
253 species and infraspecific taxa, were reported. They are present as psychrophiles,
psychrotrophs, thermotolerants and thermophiles . These last two ecological groups were
found mainly in heated sites on active volcanoes. The Italian Program for Antarctic
Research (PNRA) has investigated on fungi since 1987 and several studies have been
conducted in the Victoria Land concerning the mycoflora of different substrates (mosses,
lichens, soil, rocks, feathers, dung) as well as taxonomy, ecology, adaptations to extreme
conditions, production of antimicrobial compounds, extracellular enzymes, and molecular
biology of the fungal isolates. Particular attention has been paid to the fungal species that
are supposed to be endemic for the Continental Antarctica such as those belonging to the
cryptoendolithic community and the new taxa described from Antarctic sites. The main
results of these researches are presented here.
GAM2
MICROBIOLOGICAL CONTROL OF DETOXIFICATION
BIOREMEDIATION TECHNOLOGIES
Y. Topalova*, R. Dimkov*, C.V.Keer**, C. Y.Cheng***C.Y.Cheng***, M.

Nunes***
*Biological Faculty, Sofia University, Dragan Tzankov str. 8,
122
1164 Sofia, Bulgaria, Email: topalova@biofac.uni-sofia.bg
**KAHO Sint-Lieven, KIHO, Dept. Biochemistry, Gebr. Desmetstraat 1, 9000
Gent, Belgium
*** University of Porto,Faculty of Engeneering, 4200-465,Porto, Portugal, Email:
drcheng@mail.telepac.pt
The microbiological indicators are important element of the control and management
of the bioremediation technologies. In the model conditions several bioremediation
technologies are accomplished – aerobic, anaerobic, two-step anaerobic/aerobic, hybrid
technologies for the detoxification of xenobiotics of the group of penthachlorophenols
(PCP). In parallel a microbiological pre-bioremediation research of the adaptive ponds of
Luck-Oil, Bourgas has been carried out. The sediments of these ponds are heavily
contaminated with crude oil and polycyclic aromatic compounds (PAHs). The specific
design of microbiological control has been elaborated according to the type of technology
and features of the microbial communities.
The key taxonomical and physiological groups have been selected. The microscopic
and electronic microscopic analyses have been applied for diagnostic of the structural
changes of the biological systems in the course of the bioremediation technologies. The
obtained results from one hand connect the effectiveness of detoxification with quantitative
microbiological indicators and biodiversity, but from the other – with the localization of
the various processes occurring in bioremediation niches. As a third statement our results
additionally reveal new intermicrobial relationships on the base of microorganisms
relationships like co-metabolism, modulation and synthrophy.
GAM3
DIVERSITY OF SYNECHOCOCCUS COMMUNITY
IN FRESHWATER LAKE GEORGE, NY
Dilnora E. Gouliamova
Rensselaer Polytechnic Institute, Department of Biology 110 8-th street Troy, New
York 12180.e-mail: dilnorag@gmail.com
Members of the genus Synechococcus together with Prochlorococcus contribute

significantly to the primary production in aquatic ecosystems. In the present study
phylogenetic relationships of Synechococcus clones retrieved from Lake George and
Synechococcus clones and strains isolated from marine and freshwater environments
were studied. The results shown that Synechococcus clones from Lake George (USA),
Lake Loosdrecht (The Netherlands), Lake Biwa (Japan) and Synechococcus strains from
Lake Zurich (Switzerland) all group outside of the marine picoplankton clade and form a
separate freshwater clade. The clade is subdivided into two clusters. The first cluster
included: the clones from Lake George (LGTI1, LGBH2, LHBH3, LGBH4, LBP1),
Synechococcus rubescens and Synechococcus strain BO8807. The second cluster
123
included: strains of Synechococcus PCC6307 and Synechococcus PCC7001 and clones
LGTI5, LGBH6, LD9. Sequence similarity between clones of the first cluster (LGTI1, LGBH2,
LHBH3, LGBH4, LBP1) ranged from 97.9-99.4%; sequence similarity between clones of the
second cluster (LGTI5, LGBH6, LD9) ranged from 99.7-99.9%. Sequence similarity between
the clones of the two clusters ranged from 96.6-97.5%. The differences in 16S rRNA
sequences of 2.5-3.4% could correspond to ecologically significant physiological diversity
as it was previously demonstrated for isolates of Prochlorococcus. Our results show
existence two genetically close freshwater populations of Synechococcus which are globally
distributed despite the geographic distance separating Lake George, Lake Loosdrecht,
Lake Biva, and Lake Zurich.
GAM4
MICROBIAL STATUS OF 7 THRACIAN VAULTS NEAR TO
KASANLAK, BULGARIA AND METHODS FOR
PREVENTATION
Tzvetanka Groudeva, Ana Doycheva

Sofia University ‘St. Kl. Ohridski”, Faculty of Biology, 8 Dragan Tzankov ,
Department of General and Industrial Microbiology, Sofia, Bulgaria
As one of the most active deteriogens, microorganisms have a significant role in

destruction of cultural monuments. They act in different ways, causing a variety of
damages. The interactions between microorganisms (bacteria, actynomycetes and fungi)
and monuments can be direct or indirect (production of different microbial metabolites).
The object of this research is the investigation of the microbial community of seven
Thracian vaults near to Kasanlak in Bulgaria.
Determination of the prevalent physiological groups of microbial community as well as
their eventual role in the process of destruction was under investigation.
Eight physiological groups have been examined, considered that they are potential
biodeteriogenes. Different taxonomical and biochemical tests have been carried out using
pure cultures, isolated from the predominant physiological groups. Classical taxonomy
was used for investigation of the most important isolates.
The results obtained will be good basis for development of effective program for
conservation of each vault.
124
GAM5
TAXONOMICAL IDENTIFICATION OF ARSENIC
RESISTANT AND ARSENIC TRANSFORMING SULFATE -
REDUCING BACTERIA
Krasimira S. Krumova, Veneta I. Groudeva

Department of General and Industrial Microbiology, Lab. ”Geomicrobiology”,
1164 Sofia, Bulgaria
Twenty eight bacterial strains, which are able to transform arsenic compounds, were
isolated and characterized in the laboratory of ”Geomicrobiology” at the Department of
“General and Industrial Microbiology”, SU “St. Kliment Ohridski”. It was determined, that
11 of these isolates are able to efficiently oxidize the high mobile and high toxic arsenite
[As (III)] to less mobile and less toxic arsenate [As (V)]. The others 17 strains are able to
reduce arsenate [As (V)] to arsenite [As (III)].
Оn the basis of the determination via classical methods, the isolated bacteria were
related to different genera within the big group of sulphate-reducing bacteria.
The confirmation of the taxonomical belonging of these strains was done on basis of
the DNA amplification with SRB specific primers for each genus.
GAM6
LOW-TEMPERATURE BIODEGRADATION OF PHENOL
Rosa Margesin, Institute of Microbiology, Leopold Franzens University,

TechcnikerstraЯe 25, 6020 Innsbruck, Austria
Phenol and phenolic compounds are widely distributed in nature and as environmental
pollutants. In cold climatic regions, wastewater temperature can decrease to 10°C and
below. The activity of mesophilic degraders is limited at these temperatures. It was the
objective of this study to investigate the potential of cold-adapted bacteria and yeasts to
degrade phenol at low temperatures. Psychrophilic and cold-tolerant bacterial and yeast
strains were isolated from various alpine habitats (soils, caves, glacier cryoconite) and
were characterized with regard to their growth temperature profile and their ability to
degrade high amounts of phenol.
Using fed-batch cultivation in mineral medium with phenol as the sole carbon source,
high amounts of phenol were degraded at 10°C. Bacterial strains were representatives of
the genera Rhodococcus, Arthrobacter (including the novel species A. psychrophenolicus)
and Pseudomonas; they utilized up to 12.5 mM phenol as the sole carbon source. All
yeast strains investigated were basidiomycota (Cryptococcus, Rhodosporidium,
Rhodotorula, Mastigobasidium, Sporobolomyces, Trichosporon); they degraded up to
15 mM phenol. Investigations on the toxicity of phenol and phenol-related monoaromatic
125
compounds showed that Rhodotorula creatinivora strains were characterized by higer
IC50 values than other species, while Sporobolomyces roseus was the most sensitive
species. Yeasts were characterized by a substantially lower optimum temperature for growth
and phenol biodegradation compared to the bacteria.
GAM7
IN SITU BIOREMEDIATION OF SOIL POLLUTED
WITH CRUDE OIL AND HEAVY METALS
V.I. Groudeva 1, A.S. Doycheva 1 and S.N. Groudev2

1 – Department of General and Industrial Microbiology, Faculty of Biology,
University of Sofia, Sofia Bulgaria
2- Department of Engineering Geoecology, University of Mining and Geology,
Sofia, Bulgaria
An experimental plot of soil contaminated with crude oil and heavy metals (cooper,
zinc and cadmium) was subjected to in situ bioremediation using the activity of the
indigenous soil microflora, which contained different oil-degrading and metal-solubilizing
microorganisms. The oil was light, rich in paraffins and with a very low content of
asphaltene-resinous substances. The heavy metals were present mainly in forms
susceptible to biological leaching.
The microbial activity was enhanced by suitable changes in the levels of some essential
environmental factors such as water, oxygen and nutrient content in the soil. This was
achieved by mixing the top soil horizon with solid biodegradable organic substrates (cow
manure, plant compost, straw), by applying irrigation with water solutions of organic
sources of carbon and energy (lactate and acetate) and by adding zeolite saturated with
ammonium, phosphate and potassium ions. The soil was subjected to periodical flushing
by means of the above-mentioned water solutions to remove the products from the oil
degradation and dissolved heavy metals.
As results of such treatment the oil content in the soil was decreased from the initial 14
g/kg dry soil to 1.70 g/kg within a period of 8 months. Simultaneously, the contents of
copper, zinc and cadmium were decreased below the relevant permissible levels for soil of
such type.
126
GAM8
COMPARATIVE CHARACTERISTICS OF OIL-DEGRADING
ACTIVITY OF MICROORGANISMS WITH DIFFERENT
TAXONOMIC STATUS.
Veneta Groudeva1, Iliana Ivanova 1, Ana Doycheva 1, Mihail Dumitru2, Anca-

Rovena Vasculesku2
1 - Department of General and Industrial Microbiology,
Faculty of Biology, University of Sofia, Sofia, Bulgaria
2 - National Research and Development Institute for Soil Science,
Agrochemistry and Environmental Protection, Bucharest,Romania
The decontamination of soil polluted with oil, petroleum products and petroleum
residues could be realising through different physical and chemicals and very expensive
methods, reasons for these are inaccessible to economic agents, which are responsible to
soils pollution. The alternative is in situ bioremediation because of their relative low cost
and not disturbing the natural soil structure in the process time. Among microorganisms,
bacteria and fungi contain the enzymatic equipment necessary petroleum hydrocarbons
break down in soil. For applying of bioremediation techniques is necessary to know very
well the fate and the effects of pollutant compound on the ecosystems, how could be
optimise the biodegradation process and, mainly, finding some microorganisms with high
degrading abilities.
The main objective of this work is to establishing a efficient methodology for laboratory
testing of the capacities for oil degradation as well as to compare the degrading ability of
different isolates from polluted soils with different taxonomic status.
GAM9
COMPARATIVE CHARACTERISTICS OF TOXICITY
ASSESSMENT OF KEAVY METAL POLLUTED SOILS WITH
DIFFERENT MICROORGANISMS
Iliana Ivanova, Damiana Dimova, Ana Stojanova,Veneta Groudeva

Sofia University “Snt.Kl.Ohridski”, Faculty of Biology, Dept. of Microbiology
8 Dragan Tzankov, 1164 Sofia, Bulgaria
E-mail: ilivanova@biofac.uni-sofia.bg
Two samples from heavy metal polluted soils in West part of Bulgaria were investigated.
Toxicity assessment was processed with two microorganisms: Bacillus cereus and
Pseudomonas putida. International standard with growth multiplication inhibition [ISO
10712, 1994] was used. Two bacterial tests with Bacillus cereus - growth inhibition and
dehydrogenase activity inhibition for toxicity evaluation of environmental samples were
127
used. The 5-th hour of cultivation was the most suitable for measuring the growth inhibition
of Bacillus cereus. Dehydrogenase activity inhibition was measured at 6th h. This way the
Bacillus cereus tests are much shorter than international standard with Pseudomonas
putida growth inhibition [ISO 10712, 1994], which duration is 10-16 h and had higher
sensitivity.
GAM10
ИЗСЛЕДВАНИЯ ВЪРХУ ВЪЗМОЖНОСТИТЕ ЗА
ИНТЕНЗИФИКАЦИЯ ПОЛУЧАВАНЕТО НА БИОГАЗ ОТ
ОТПАДЪЦИ В СЕЛСКОТО СТОПАНСТВО И
ХРАНИТЕЛНТА ПРОМИШЛЕНОСТ
Данка Гълъбова, Димитър Каракашев, Асен Мирков,

Людмил Николов, Иван Симеонов
Институт по микробиология “Aкад. Ангел Стефанов”, БАН
Биодеградацията на органичната материя, образуваана вследствие човешката

производителна дейност, има тристранен ефект: опазването на околната среда
от много вредни змърсители, получаването на възобновяем енергиен източник
(биогаз) и производстево на естествен органичен тор. В практично отношение
това е известен биотехнологичен процес с множество реализации в голям мащаб.
Въпреки сравнителната нестабилност на метановата ферментация, пораждаща
се от сложните взаимодействия на голям брой различни видове микроорганизми,
интензификацията и стабилизацията на процеса могат да подобрят значително
икономическите покзатели на някои от приложенията й.
Целта на проведените изследвания в лабораторни биореактори с различни
видове субстрат (говежда тор, суроватка и спиртна шлемпа) е да се проследи
влиянието на вида на органичните отпадъци и техни комбинации върху растежа
и хидролитичната ензимна активност на анаеробнте бактериални култури.
Изследвано е също влиянието на вещества стимулиращи микробния метаболизъм
с оглед интензификацията на получаването на биогаз. Тези субстанции могат да
бъдат определени в две основни групи: растежни фактори (дрождев екстрат,
пептон) и повърностно активни вещества (тритон 100-Х и биосърфактант PS-17).
С оглед намаляване степента на замърсеност на отпадните води от процеса са
проведени опити в каскада от два анаеробни биорактори.
Натрупан е ценен експериментален материал, който може да послужи за
основа за математическо моделиране на процеса след обработване н
експерметалите даннни. Направени са важни изводи за практиката.
128
GAM11
MOLECULAR BIOMARKERS OF OXIDATIVE STRESS
IN FILAMENTOUS FUNGI
M. Angelova
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences
Aerobic metabolism entails the production of reactive oxygen species (ROS), including
superoxide anion radical (yO2-), H2O2 and the hydroxyl radical. These ROS cause oxidative
damage to important biomolecules, such as lipids, proteins, and DNA. Under normal
conditions, ROS are cleared from the cell by the action of superoxide dismutase (SOD) and
catalase (CAT). Oxidative stress is imposed on cells as a result of increase in ROS
generation, decrease in antioxidant protection, or failure to repair oxidative damage. A
state of oxidative stress can be induced by a number of environmental factors, including
chemical compounds, heavy metals, temperature treatment etc. The scientific validity of
this hypothesis should be confirmed. Present study was designed to evaluate the effects
of paraquat, hydrogen peroxide, copper toxicity, as well as heat- and cold-stress treatment
on fungal cells by biomarkers, reflecting specific toxicity mechanism. Several oxidative
stress biomarkers were validated: cyanide-resistant respiration; direct assessment of ROS
production; oxidative damaged proteins; synthesis of reserve carbohydrates and changes
in antioxidant enzyme defence (SOD and CAT). As a model microorganisms were used
fungal strains belonging to the genera Humicola, Aspergillus and Penicillium.
Our results indicate that exposure of fungal spores or mycelia to different stressors
promoted oxidative stress, as evidenced by stimulation of cyanide-resistant respiration,
overproduction of ROS, accumulation of oxidative modified proteins, and acceleration of
trehalose and glycogen synthesis. Cell responses include enhanced expression of SOD
and catalase, however, the extent was different: treatment with PQ, Cu ions and temperature
increased mainly SOD, whereas exogenous H2O2 led to enhanced CAT. We also found
that glucose-6-phosphate dehydrogenase has a relevant role in the mechanism of protection
against superoxide and peroxide stresses.
GAM12
NEW MITOCHONDRIAL CATALASE IN YEAST
SACCHAROMYCES CEREVISIAE
Ventsislava Petrova
In all eukaryotic cells, peroxisomes and mitochondria share a great variety of enzymatic
reactions that are catalyzed by isoenzymes present in both organelles. As catalase and
superoxide dismutase are essential enzymes in the decomposition of intracellular ROS
(reactive oxygen species), their activities have been explored in the yeast Saccharomyces
cerevisiae during batchwise growth experiment. A mitochondrial fraction from three type
129
strains of Saccharomyces cerevisiae has been isolated. Then the catalase, peroxidase,
Mn and Cu/Zn superoxide dismutase activities were evaluated in the mitochondrial fraction.
PAGE separations allowed identifying a new mitochondrial catalase which differed from
the known catalase A and T in S. cerevisiae. A positive correlation between the activity of
mitochondrial catalase and Mn superoxide dismutase have been proved. To identify which
of the two catalase isoenzymes in yeast cell contributes to this mitochondrial activity a
Cta1p co-targeting was studied in a catalase A null mutant. A Cta1p–GFP (green fluorescent
protein)-fusion protein or a Cta1p derivative containing either a c-Myc epitope (Cta1pmyc)
or a SKF-extended tag (Cta1pmyc-SKF) was constructed and their expression was followed
up after growth of S. cerevisiae on different carbon sources. In the present study we
demonstrated that Cta1p can also enter mitochondria, although the enzyme lacks a classical
mitochondrial import sequence. Efficient Cta1p import into peroxisomes was observed
when cells were cultivated under peroxisome-inducing conditions (i.e. growth on oleate),
whereas significant co-import of Cta1p–GFP into mitochondria occurred when cells were
grown under respiratory conditions that favor oxygen stress and ROS accumulation within
this organelle.
GAM13
PROTEOMICS IN TARGET-SPECIFIC ANTIBACTERIAL
DRUG DISCOVERY BASED ON UMP KINASE
Neli Slavova-Azmanova, Cecile Evrin, Liliane Assairi, Hristo Najdenski, Octavian

Bвrzu and Anne-Marie Gilles
Bacterial uridine monophosphate (UMP) kinases are homohexamers whose primary

structure diverges from that of other nucleoside monophosphate kinases and from
eukaryotic UMP/CMP kinases. They are rather specific for UMP as substrate and are
allosterically regulated by GTP (activator) and UTP (inhibitor). Being essential for bacterial
cell survival, UMP kinases are appropriate candidates for designing new antimicrobial
agents.
The aim of the present study was to identify common characteristics and/or differences
between UMP kinases from Gram-positive and Gram-negative bacteria regarding some
pathogenic microorganisms. The pyrH genes encoding UMP kinase in S. pneumoniae and
H. influenzae were cloned into pET expression vectors and overexpressed in E. coli. The
recombinant proteins were purified and studied with respect to kinetic characteristics,
temperature and chemical stability. Despite several common properties including activation
by GTP and inhibition by UTP, important differences between these enzymes were
discovered. It was found that activation by GTP in H. influenzae UMP kinase occurs by
the increase of Vm and K0.5, whereas in S. pneumoniae activation is due both to a decrease
in K0.5 and to an increase in Vm. In the case of H. influenzae and E. coli the inhibition by
UTP was strongly dependent on MgCl2 and ATP concentrations. The strongest activation
of UMP kinase from H. influenzae was achieved by cGMP, whereas the S. pneumoniae
130
enzyme was activated to the highest level by 3’-antraniloil-3’-dGTP (Ant-dGTP).
UMP kinases from S. pneumoniae and H. influenzae are a reliable model for examination
of activity’s regulation. The initial characterization of the biochemical properties of these
enzymes is an important step towards target-specific drug design in the era of increasing
microbial resistance.
GAM14
HEAT SHOCK RESPONSE OF STREPTOCOCCUS
THERMOPHILUS
INDUSTRIAL STRAINS
Penka M. Petrova, Dilnora E. Gouliamova and Galina D. Stoyancheva

of Sciences, 26, Acad. G. Bontchev Str, 1113 Sofia, Bulgaria, e-mail:
pepipetrova@yahoo.com
Streptococcus thermophilus is the most frequently used in dairy industry species

among all lactic acid bacteria (LAB). It takes part not only in the production of yogurt, but
also in the cheese making (Mozzarella, Cheddar). During different stages of industrial
fermentations S. thermophilus is subjected to the harsh heat treatment - up to 50-70ЪС.
That is why heat resistant starters would be preferable in use under “industrial stress”
conditions.
The response of S. thermophilus strain ST2980, containing 3.3 kb plasmid and its
plasmid free derivative (ST2980*) to heat-shock was studied. The percentage of survived
cells was determined by counting of colony forming units. Significant differences in survival
between strain variants were observed, especially when the cells were transferred from
42°C directly to 62°C. After 1 hour only 1% of ST2980* cells were still alive, compared to
50% of ST 2980. The prior incubation at 52°C (pre-shock) enables the plasmid-cured cells
to survive more efficiently. However, after 2 hours of exposure to the increased temperature,
about 7% of ST 2980 cells were alive, compared to 0% of ST2980*. The analysis of growth
curves allows suggesting that the role of small hsp is the fast reaction to changes in the
environment. It is possible that the 16.4 kDa Hsp works as a signal molecule, inducing the
expression of other (60 – 100 kDa) heat-shock proteins.
This work was financially supported by National Council for Scientific Research of
Republic of Bulgaria. (Grant K 1307/2003).
131
GAM15
ВТОРА ГЕНЕРАЦИЯ ГЛЮКООЛИГОЗАХАРИДИ С
ПРЕБИОТИЧНИ СВОЙСТВА, СИНТЕЗИРАНИ
ПОСРЕДСТВОМ ГЛИКОЗИЛТРАНСФЕРАЗИ ОТ LEU-
CONOSTOC MESENTEROIDES LM 286
И. Илиев1, Т. Василева1 и И. Иванова2

1
- Катедра “Биохимия и микробиология”, Биологически факултет, Пловдивски
Университет, ул. “Цар Асен” № 24, стая 17, 4000 Пловдив, България,
ilievini@abv.bg
2
- Катедра “Обща и индустриална микробиология” Биологически факултет,
Софийски Университет, бул. “Драган Цанков №8”, 1113 София, България
Неразградимите олигозахариди са обект на интензивни научни изследвания

поради приложението им като пребиотици. Потенциалът на олигозахаридите като
пребиотици, представен като пребиотичен индекс, включва свойства като
бифидогенен ефект, синтез на късоверижни мастни киселини, рН-устойчивост,
имуностимулатор.
В представените изследвания се демонстрира възможността за ензимен синтез
на глюкоолигозахариди от гликозилтрансферази, получени от мутантен щам
Leuconostoc mesenteroides Lm 286. Изследваният мутант Lm 286 секретира два
типа гликозилтрансферази – декстранзахараза и алтернанзахараза и допълнително
леванзахараза. Гликозилтрансферазите, предварително изолирани и частично
пречистени, синтезират олигозахариди с преобладаващи á-(1,3) и á-(1,4)
гликозидни връзки. Синтезираните олигозахариди показаха висока степен на
резистентност при хидролиза с декстраназа и амилоглюкозидаза, съответно за
глюкоолигозахаридите 47% и за изомалтоолигозахаридите 36%. След
допълнително тестуване ин витро на пребиотичните свойства на синтезираните
олигозахариди със силно разклонена верига се установи подчертан бифидогенен
ефект, както и стимулиране на растежа на някои пробиотични щамове L.
bulgaricus, проявяващи антибактериален ефект срещу E. coli и L. innocua и
увеличена продукция на късоверижни мастни киселини.
GAM16
ИЗОЛИРАНЕ И СВОЙСТВА НА ИЗВЪНКЛЕТЪЧНА
В-КСИЛОЗИДАЗА
ПРОДУЦИРАНА ОТ ASPERGILLUS NIGER B03
Георги Добрев, Иван Пищийски, Лидия Колева
Изолирана е извънклетъчна â-ксилозидаза, продуцирана от Aspergillus niger
132
B03. За целта е използвана колонна хроматография с Sephadex G 75 и Sephadex G
100. Определени са оптималните рН и температура за действие на ензима,
съответно рН 3.5 и t= 70 °C. Изследвани са рН и температурната стабилност на
пречистеният ензим. Определени са кинетичните параметри на изолирания ензим
спрямо субстрат ñ-нитрофенил-â-D-ксилопиранозид, съответно Km= 0.35 µmol/ml и
Vmax= 3.03 µmol/(ml.min). Изследвано е влиянието на някои метални йони върху
активността на изолираната â-ксилозидаза.
GAM17
PECULIARITIES OF BIOFILM SYSTEM IMPLEMENTATION
IN MICROBIAL BIOTECHNOLOGIES
Ludmil N. Nikolov
Biological Faculty of Sofia University “St. Kl. Ohridski”
Implementation of the biofilms in microbial biotechnologies leads to rationalization of

the industrial line structure, which reflects to serious economical advantages and makes
easier the maintenance of production systems. The efficiency of this implementation is
based on the manifold increase of the bioagent concentration in the bioreactor working
zone, rationalization of the separation processes and realization of the bioprocess in high
performance bioreactors.
In the presented study is shown that these attractive advantages have to be used
having in mind the properties and the structure of the biofilms as self-organizing living
systems. Special attention is paid to the specificities of the biofilm applications like their
high sensitivity to the intensification of hydrodynamics in the vicinity to the biofilm
surface, the influence of the biofilm thickness on the bioprocess development as well as
the changes in biofilm properties during the bioprocess course. Experience is shared in
the use of this knowledge for development of suitable bioprocess systems both for biofilm
studies as self-organizing living systems in laboratory scale and realization of their
potentialities in microbial biotechnologies in industry. On the concrete examples it is
shown the increasing of the biofilm bioprocess systems productivity in comparison with
the suspended culture cultivation of the same microorganisms. Some considerations about
the applicability of the intensity working regimes in the high performance biofilm reactors
like two and three phase fluidization in the bioprocess systems with fixed films are discussed.
133
GAM18
EVOLUTION MODELING OF BACTERIAL OXIDATION OF
FERROUS IONS IN BIOFILM SYSTEM
L. Nikolov1, E. Petrova2 , V. Mamatarkova1, Kl. Mladenov2, St. Stoytchev2

1
Biological Faculty, Sofia University, “St. Kl. Ohridski”,
2
Institute of Mechanics, BAS
At recent time the biofilm systems are also studied using mathematical modeling,
which is the subject of the present work. This research presents the development of the
authors previous experience in the problems of formulation and improvement of the
mathematical model of a biofilm system, formed by Acidithiobacillus ferrooxidans, based
on information about the subsystems and on verbal model of oxidation of ferrous ions in
biofilm reactors. The mathematical description of the bioprocess system consists of five
ordinary differential equations, at the following assumptions: oxidation proceeds through
homogenous-heterogenity mechanism; all the processes proceed without diffusion
limitation; the kinetics of the sedimentation of the oxidated ferrous ions is of zero order;
the economical coefficients of the biofilm and in the swimming cells are one and the same;
as well as the maximal specific velocities of the biomass growth. Our investigations are
realized by evolution strategy, which consists in gradual complicating of the mathematical
description through adding some equations and members. The model sensitivity is tested
with respect to its parameters.
The numerical experiments show that the dimension of the identification task increases
significantly during the model complication because of increasing the number of the
kinetic constants. Here an attempt is made of decreasing the dimensions of the identification
task using experimental data, obtained by homogeneity cultivation. The oxidizing dynamics
in fed–batch culture is investigated in parallel. The obtained results show that the used
software is effective and helps to get a better understanding of the experimental results,
which come from the starting phases of forming and functioning of biofilm systems at
different bioreactors.
GAM19
POLYHYDROXYALKANOIC ACIDS (PHA) – INTERESTING
BACTERIAL POLYMERS
AND ASPECTS OF THEIR PRODUCTION
Jцrg- Uwe Ackermann1, Gisela Mothes2

1
University of Applied Sciences Dresden (HTW), Department of Chemical Engineer-
ing, F.-List-Platz 1, D-01069 Dresden, Germany
2
Saxon Institute of Applied Biotechnology (SIAB), Permoserstr. 15, D-04318
Leipzig, Germany
134
PHAs (polyhydroxyalkanoid acids) are intracellular thermoplastic reserve polyesters
occurring in a wide variety of bacteria. These polymers became commercially attractive
due to their special physico-chemical properties together with biodegradability and
biocompatibility. Because PHA can be produced from renewable carbon sources they are
also interesting from the viewpoint of sustainability. Currently different applications,
especially in the fields of pharmacy and medicine, are in the focus of interest. A wide
spectrum of polymer properties can be adjusted by the possibility to synthesize copolymers
and, additionally, by the ability of post-biosynthetic modifications.
Poly(3-hydroxybutyrate-co-4-hydroxybutyrate), (P(3HB/4HB)), is one of the most
suitable absorbable materials for implantable medical applications, e.g. for the use as
scaffolds. In contrast to the multitude of potential producers of polyhydroxybutyric acid
(PHB) only a few bacteria are able to accumulate copolymers of P(3HB/4HB). Some results
of production with Delftia acidovorans will be presented.
The overall process of PHA production comprises three stages, the synthesis of cells,
the synthesis and accumulation of the polymer and the isolation of the product from the
biomass. The biosynthetic steps are commonly performed as batch or fed batch processes.
However, continuous regimes of PHA production have several advantages and, moreover,
a two-stage continuous process is more favourably than a single chemostat, especially
for the production of copolymers. For a better usability and understanding of the process
we developed mathematical models.
GAM20
PRECONDITIONS FOR PRODUCING OF ORGANIC FOOD IN
MACEDONIA
A. Kuzelov; O. Kirovska Cigulevska

MIK Sv. Nikole; Sveti Nikole, Macedonia
Zavod za zdravstvena zastita – Skopje; Treta Makedonska Brigada 18, 1000
Skopje, Macedonia; e-mail: kicok38@hotmail.com
Our purpose in this work will be to explain conditions and preconditions for producing
organic food in Macedonia. The main reason for that kind of farm – producing is to
purchase helthier food without presence of food contaminants. It will be very complex
process because we have to avoid pollutions from the air, water and soil to get healthy
organic food.
Our government is preceding a low for organic food producing and in that low will take
place preparing, producing, marketing, labeling and inspection of organic food products.
But, to get organic meat products, we have to purchase:
• healthy organic row material – meat from healthy animals,
• organic way of animal farming,
• marking of healthy animals,
• using of organic food for cows feeding,
135
• human way of transporting and sacrificed cows,
• sanitary and veterinary inspection of animals (row materials)
• using proposed additives for that kind of producing – organic meat products.
GAM21
HETEROGENEITY OF L. PLANTARUM ISOLATES FROM
ARTISANAL WHITE BRINED CHEESE
R. Aleksandrova1, S. Dimitonova1, I. Ivanova2and S. Danova1

1
- Dept of Microbial Genetics, Institute of Microbiology, Bulgarian Academy of
Sciences, 26, Acad. G. Bontchev, str, 1113 Sofia, Bulgaria.
2
- Dept. of General and Applied Microbiology, Biological faculty, Sofia University,
8, Dragan Tzankov, bvd, 1113 Sofia, Bulgaria
L. plantarum is ubiquitous, commonly isolated from foods of both animal and plant
origin, and is one of the main non-starter lactic acid bacteria (NSLAB) contributing to the
final sensorial properties of different kind of cheeses. In this study 30 strains were isolated
from Bulgarian artisanal white brined cheese (WBAC) after a 2 months ripening period in
brine with 10% NaCl. A phenotypically homogenous group of 21 isolates were identified
as L. plantarum according to polyphasic taxonomy. The repetitive PCR analyses allowed
studying the species and intra-species polymorphism within the phylogenetic group.
With Rep PCR analysis we achieved a confirmation of correct species identification and
also a very good discrimination between closely related species L. plantarum, L. pentosus
and L. paraplantarum. Obtained DNA polymorphic profiles using Rep, Eric and Box
primers, revealed the heterogeneity of L. plantarum strains. The most discriminative and
promising for the strain typing was Box-PCR. The results present new information on
genetic diversity of NSLAB microflora of Bulgarian fermented milk products.
GAM22
СРАВНЕНИЕ НА ФЕРМЕНТАТИВНИЯ КАПАЦИТЕТ НА L.

BULGARICS ПРИ КУЛТИВИРАНЕ НА ХРАНИТЕЛНИ
СРЕДИ С ОЛИГОЗАХАРИДИ
Ц. Игнатова1, И. Иванова2 и И. Илиев3

1
- Катедра “Функционална биология”, Факултет “Природни науки”, Шуменски
Университет, ул. “Университетска №115”, 9712 Шумен, България
2
- Катедра “Обща и индустриална микробиология” Биологически факултет,
Софийски Университет, бул. “Драган Цанков №8”, 1113 София, България
3
- Катедра “Биохимия и микробиология”, Биологически факултет, Пловдивски
136
Университет, ул. “Цар Асен” № 24, стая 17, 4000 Пловдив, България,
ilievini@abv.bg
Неразградимите олигозахариди са обект на интензивни научни изследвания

поради приложението им в симбиотични продукти. Потенциалът на
олигозахаридите като пребиотици, представен като пребиотичен индекс, включва
свойства като бифидогенен ефект, синтез на късоверижни мастни киселини, рН-
устойчивост, имуностимулатор.
В представените изследвания се демонстрира възможността за ензимна
деградация на глюкоолигозахариди, галактоолигозахариди и
фруктоолигозахариди от пробиотични щамове L. bulgaricus. Установена е
щамова специфичност при ферментирането на тестуваните олигозахариди.
Потенциалът на олигозахаридите да стимулират растежа на пробиотични щамове
L. bulgaricus е щамово детерминиран. Доказана е щамова специфичност и по
отношение метаболизирането на олигозахаридите и продуцирането на органични
киселини и късоверижни мастни киселини. Доказан е индуциращ ефект на
тестуваните олигозахариди при секрецията на á-глюкозидаза, леванзахараза и
â-галактозидаза.
GAM23
AMINO ACIDS
USE AND PRODUCTION - CURRENT STATUS
AND PROSPECTS
Alexander Ratkov
Institute of Microbiology, Bulgarian Academy of Sciences
As the building blocks of life, amino acids have long played an important role in both
human and animal nutrition and health maintenance. In the 1950s Corynebacterium
glutamicum was found to be a very efficient producer of L-glutamic acid. Since this time
biotechnological production processes have been used for industrial production of amino
acids. At present these processes are among the most important in terms of tonnage and
economical value. Market development has been particularly dynamic for the flavor-
enhancer glutamic acid and the animal feed amino acids L-lysine, L-threonine, and L-
tryptophan. The growing market for amino acid led to significant improvements in
bioprocess and downstream technology as well as in molecular biology. During the last
decade big efforts were made to increase the productivity and to decrease the production
cost.
This article gives an overview of the world market for amino acids. Improvements in
bioprocess technology, i.e. repeated fed batch and continuous production are summarized.
Attempt and achievements in investigation and development of the technology for amino
acids production at the Institute of Microbiology, Bulgarian Academy of Sciences is
137
summarized too.
GAM24
MATHEMATICAL IDENTIFICATION
OF L-VALINE FED-BATCH FERMENTATION PROCESS
K. Todorov* I. Dimov*, Tz. Georgiev**, J. Kristeva*, V. Ivanova*, Al. Ratkov *

* - Institute of Microbiology, Bulgarian Academy of Science
** -Technical University, Faculty of Automatics,
The paper deals with identification problems of fermentation processes. The

identification problem of biotechnological processes is threefold problems:(a) determination
of generalised stoichiometric reactions, (b) identification of the underlying reaction network,
(c) identification of the kinetics-structure and parameters of reaction rates. This stages are
discussed based on experimental results from amino acids production. In practical case,
biotechnological process for L-valine production by fermentation is used for elucidation
of approach for solving the identification problem. The approach in this article included
the following steps:(a) chose a set of generalised stoichiometric reactions, (b) chosen
reactions are validated by extended autoregressive models (ARX). The adequacy of this
models proved equations within reactions, (c) structural and parametric identification of
the specific rates for discussed process using linear and nonlinear regression, (d)
optimisation of unstructured dynamic model of L-valine production using constrained
optimisation procedure. The adequacy of the proposed modes is proved through
simulation researches.
GAM25
CARCINOGEN-INDUCED TRANSPOSITION OF SACCHARO-
MYCES CEREVISIAE TY1 TRANSPOSON DEPENDS ON
MITOCHONDRIAL FUNCTION
T. Stoycheva, P. Venkov, Ts. Tsvetkov

Institute of Cryobiology and Food Technology, Department of Molecular Ecology,
53A”Cherny vrah”Blvd., Sofia, Bulgaria, tedist80@yahoo.com
Ty1 is a Saccharomyces cerevisiae retrotransposon with life cycle and structure very
similar to the known oncoviruses. In previous studies was found that Ty1 transposition
to new places in the genome is increased by carcinogens but not by mutagens that are not
carcinogenic (Pesheva et al., 2005). The molecular mechanisms underlying the specific
response of Ty1 transposition to carcinogens are unknown. Recently, data accumulating
in the literature evidenced that carcinogens increased the cellular level of reactive oxygen
138
species (ROS). Since the mitochondrial oxidative phosphorylation is the main source for
ROS production, we studied the role of mitochondria in the carcinogen-induced Ty1
transposition. We first found that carcinogen-induced Ty1 transposition does not occur
in S.cerevisiae cells deprived of mitochondria, indicating the role of mitochondria in the
process. Next, we disrupted SCO1 – a nuclear gene involved in mitochondrial oxidative
phosphorylation, and found that such cells do not respond to carcinogen treatment with
enhanced Ty1 transposition. This result evidenced that oxidative phosphorylation, but
not another mitochondrial function, is involved into carcinogen-induced Ty1 transposition.
In order to study in details the role of oxidative phosphorylation we treated cells with two
inhibitors: 1) antimycin which increases ROS production by inhibiting the electron transfer
along the respiratory chain and 2) CCCP inhibiting mitochondrial ATP synthesis without
having effect on ROS formation. The results obtained showed that carcinogen-induced
Ty1 transposition increases after treatment with antimycin while CCCP has no effect on
Ty1 transposition.
Our results indicate the key role of cellular ROS level in the carcinogen-induced
Ty1 transposition. The significance of the results obtained will be discussed in the light of
the cellular response to carcinogens.
GAM26
PHENOL HYDROXYLASE DEPENDANCE ON THE
VARIOUSE HYDROXY PHENOLS UTILYZED AS SUB-
STRATES BY TRICHOSPORON CUTANEUM R57
Z. Alexieva, M. Gerginova, B. Atanasov

The ability of the mono hydroxylated aromatics compounds to affect the level of
intracellular FAD-dependent phenol 2-monooxigenase (phenol hydroxylase, EC 1.14.13.7),
catalyzing the initial step of phenol mineralization in Trichosporon cutaneum strain R57
was studied. When Trichosporon cutaneum was grown on 1 g/l phenol, catechol, resorcinol
or hydroquinone as a sole carbon source in the medium, the highest level of specific
activity of the investigated enzyme was attend with hydroquinone (2 U/mg protein). The
intracellular specific activities of phenol hydroxylase in grown on phenol (0.5 g.l-1) cells of
Trichosporon cutaneum R57 strain with different mono-hydroxyl phenol derivatives
(catechol, resorcinol and hydroquinone) used as substrates in the reaction mixture for
determination of the enzyme activity were measured, too. The data obtained showed that
the investigated enzyme was capable of hydroxylating all applied aromatic substrates.
The level of specific phenol hydroxylase activity in experiments with hydroquinone (1 U/
mg protein) was equal to the data measured with phenol as a substrate in the same
conditions. The enzyme capacity to oxidize catechol or resorcinol in these experiments
was significantly lower (50 – 60 %). All enzyme activities were determined in Triton X100
permeabilized cells. The comparison of all results obtained in both sets of experiments
139
showed that investigated aromatics compounds are easily utilizable and strong inducers
of phenol hydroxylase. Nevertheless, the better expression of the enzyme activity in cells
initially grown on medium comprising each one of tested hydroxylated phenols was
observed.
This work was supported by of the NCSR of the Bulgarian Ministry of Education and
Science under project N K 1205/02.
GAM27
CREATING OLIGONUCLEOTIDE PRIMERS FOR PCR
ANALYSIS AND PHYA GENE SEQUENCING IN TRICHOS-
PORON CUTANEUM R57 STRAIN
Y. Manasiev, T. Primov, M. Gerginova, N. Peneva, Z. Alexieva

The methods for discovering new catabolic genes, as well as genes involved in the
bioaugmentation process of the environment demonstrate the advantages of unique and
easily identified molecular markers for the investigation of natural microbial populations.
The metabolism of aromatic compounds, phenol and its derivatives especially, is being
investigated very intensely in prokaryotic microorganisms The available in the scientific
literature data concerning catabolic genes in yeast species, in particular concerning genes
coding for enzymes of the phenol degrading pathway is quite insufficient. The aim of this
work is to contribute to more exact and complete information about the diversity and
specificity of yeast genes, as a specific response to the growing environmental pollution
with toxic aromatic compounds.
Based on the sequence of the gene encoding phenol hydroxilase synthesis in T.
cutaneum ATCC 46490 strain (TORPHD, GI = 170524, NCBI), the oligonucleotide primers
for phenol degrading yeast were designed using Primer 3 algorithm. Three pairs of primers
were used in accomplished PCR-reactions. The DNA fragments obtained were sequenced.
With a purpose to create primers with a maximum specificity to the products obtained by
the first set of primers we also designed a set of degenerative primers. The initial sequence
TORPHD and PCR products sequences were aligned by using ClastalW algorithm. Two
pairs of primers were selected on the basis of homology (not less then 80%) with TORPHD
sequence.
Science under project N MU-K 1402/04.
140
GAM28
DOT-BLOT ANALYSIS BY BIOTIN LABELED PROBE FOR
IDENTIFYING PHYA GENE IN MICROBIAL STRAINS
M. Gerginova, Y. Manasiev, P. Petrova, A. Krastanov*, Z. Alexieva
The Stephan Angeloff Institute of Microbiology, BAS,
*University of food technologies, Plovdiv
Phenol and its various derivatives, as well as many other aromatic compounds, are
known as hazardous pollutants. Various phenol-degrading prokaryotic microorganisms
have been extensively studied but only some members of yeast genera that can metabolize
phenolic compounds as a sole carbon and energy source are described in the literature.
The utilization of molecular techniques can be useful not only to identify species, but
to discover new genes involved in catabolism of aromatics for the purpose to innovate
and improve the technological processes of biodegradation.
In an attempt to estimate the occurrence of phenol hydroxylase – related gene sequences
in different microbial strains we performed a Dot-blot analysis with DNA from phenol
utilizing eukaryotes. The used oligonucletide was homologous to the 5’ end of phyA gene
in Trichosporon cutaneum ATCC 46490. As a negative controls were used incapable to
degrade phenol microbial strain – Lactobacillus acidophilus 4356.
The results of this investigation showed that both strains Trichosporon cutaneum
R57 as well as Trametes versicolor 1 may carry phyA genes of the high degree of similarity
to the phyA gene from Trichosporon cutaneum ATCC 46490. The Lactobacillus
acidophilus 4356 strain’s DNA used as negative control in the experiments did not reveal
any sequence similarity to the phyA gene under the conditions tested.
Science under project N MU-K 1402/04.
GAM29
INFLUENCE OF TOXIC PHENOLIC COMPOUNDS ON THE
PHENOL HYDROXILASE ACTIVITY IN TRICHOSPORON
CUTANEUM
M. Gerginova, Y. Manasiev, N. Shivarova, Z. Alexieva
The phenol-degrading strain Trichosporon cutaneum R57 utilized various aromatic

and aliphatic compounds as a sole carbon and energy sours. The intracellular specific
activities of phenol hydroxylase [EC 1.14.13.7] in grown on phenol (0.5 g.l-1) and
permeabilized cells of Trichosporon cutaneum R57 strain are measured. Different toxic
phenol derivatives (cresols, nitro-phenols and Cl-phenols) were used as substrates in the
reaction mixture for determination of the enzyme activity. The data obtained showed that
the investigated enzyme was capable of hydroxylating all applied aromatic substrates. It
141
should be point that some of them (o-cresol, p-nitro-phenol) are non-growth substrates
but others have different maximal growth concentrations. The measured specific activity
of phenol hydroxylase with phenol as substrate was 1.0 Umg-1 protein. The analyses of
data from experiments with o-, m- and p- cresols showed high degree of similarity to the
data obtained in experiments with phenol. The same effect could be observed in experiments
done with o-, m- and p-Cl-phenols as well as with o-nitro-phenol. The established specific
enzyme activities with both m- and p- nitro- phenols were rather lower (0.6 Umg-1 protein)
than described above. All results demonstrated in this work confirmed the wide enzyme
specificity of phenol hydroxylase in Trichosporon cutaneum R57 cells. Otherwise we
should suppose the action of more than one hydroxylating enzyme towards different
phenols. The enzyme activity obtained in the experiments with non-growth substrates
indicated the existence of different cause for cell’s inability to assimilate them.
Science under project N K 1205/02.
GAM30
MICROFLORA IN A NATURAL WETLAND USED FPR
TREATMENT OF ACID MINE DRAINAGE
V.I. Groudeva 1, S.G. Bratkova 2 and S.N. Groudev 2

1
– Department of General and Industrial Microbiology, Faculty of
Biology,University of Sofia, Sofia Bulgaria
2
- Department of Engineering Geoecology,University of Mining and Geology, Sofia,
Bulgaria
Acid mine drainage waters generated in a polymetallic ore deposit and polluted with
radionuclides, heavy metals and arsenic were treated by means of a natural wetland located
in the deposit. The wetland was characterized by abundant water and emergent vegetation
and a diverse microflora. The water flow rate through the wetland varied in the range of
about 5-20 m3 / 24 h.
An efficient removal of pollutants was achieved within the wetland during the different
climate seasons, even during the cold winter months at temperature close to the water
freezing point. The removal of pollutants was due to different mechanisms but the microbial
dissimilatory sulphate reduction and biosorption played the main role. The microflora in
the wetland was characterized by a rich variety of sulphate-reducing bacteria and other
metabolically independent microorganisms. As a result of their activity the heavy metals
and arsenic were precipitated mainly as the relevant insoluble sulphides and uranium was
precipitated manly as uraninite (UO2). The wetland effluents contained no pollutants in
concentrations higher than the relevant permissible levels for waters intended for use in
the agriculture and/or industry.
142
GAM31
БИОДЕГРАДАЦИЯ НА СМЕСЕНИ ФЕНОЛНИ
СЪЕДИНЕНИЯ С МИКРОБНА АСОЦИАЦИЯ ОТ
ASPERGILLUS AWAMORI И THERMOASCUS
AURANTIACUS
И. Стоилова, А. Кръстанов, Х. Буи, В. Станчев

Университет по хранителни технологии, Пловдив
Изследвана е биодеградацията на четири смеси от фенолни съединения като

въглеродни източници с микробна асоциация от Aspergillus awamori и Thermoascus
aurantiacus: фенол + 2,6-диметоксифенол, фенол + 2,4-дихлорфенол, фенол +
катехол, фенол + дифениламин. Общата концентрация на фенолните съединения
във всяка смес е 0,4%: 0,2% за фенола и 0,2% за втория фенолен дериват. На
среда съдържаща фенол + 2,6-диметоксифенол се установи положително
взаимодействие между 2-те култури, проявяващо се в увеличен брой конидии в
смесената култура превишаващ броя на конидиите в монокултурите, синергична
продукция на лакказа в смесената среда и 1,7 пъти по-висока биодеградация на
фенолната смес в сравнение с тази в монокултурата T. aurantiacus и 2,48 пъти
повече от A. awamori. На среда съдържаща фенол + 2,4-дихлорфенол двата вида
показаха неутрален тип на взаимодействие, на останалите среди двете микробни
популации проявиха конкурентни взаимодействия.
Постигната е 64% деградация на 4,0 g/l смес от фенол и 2,6-диметоксифенол с
микробна асоциация от A. awamori и T. aurantiacus
GAM32
BENZONITRILE AND 4-CYANOPIRIDINE DEGRADATION
BY IMMOBILIZED CELLS OF BACILLUS SP. UG-5B IN A
COLUMN BIOREACTOR
L. Kabaivanova1, E. Dobreva1, E. Emanuilova1,
B. Samuneva2, G. Chernev2
1
Institute of Microbiology, BAS- Sofia, Bulgaria
2
University of Chemical Technology and Metallurgy- Sofia, Bulgaria
Nitrile compounds are one of the most dangerous pollutants of the environment released
by different industrial processes. The aim of the present investigation is to carry out a
biodegradation process of benzonitrile and 4-cyanopiridine by immobilized cells of Bacillus
sp. UG-5B with thermostable nitrilase activity. Cell suspension with concentration of 30
mg/ml cells and nitrilase activity of 2.25 U/ml was used in the immobilization procedures.
The matrix was synthesized by the sol-gel method at room temperature and 5mol% of the
inorganic precursor (tetramethylortosilicate) was replaced by polyacrylamide gel.
143
The structure of obtained hybrid nanocomposites has been studied by IR Spectroscopy,
XRD, BET, SEM, AFM and Roughness Analysis. The results proved that all samples are
amorphous having pores with average diameter about 1 – 1.6 nm. A self- organized
nanostructure have been observed by AFM. From the AFM images were calculated RMS
roughness and average height of the particles and aggregate on the surface. After
entrapment of the whole bacterial cells in the hybrid matrix, the obtained biocatalyst was
applied in a continuous degradation process in a column bioreactor at 55°C. This fact
could make possible the treatment of hot waste waters, containing nitriles. The process
was followed at three different flow rates of the substrate solution- 0.5 ml.min-1; 0.75
ml.min-1 and 1.0 ml.min-1. The concentration of benzonitrile and 4-cyanopyridine in the
phosphate buffer with pH=7.2 was 20 mM. Degradation of benzonitrile-16.07 mM and 4-
cyanopiridine-38.5 mM was achieved at the optimal flow rate of 0.75ml.min-1 for five hours.
GAM33
BIOLOGICAL PROPERTIES OF BIOSURFACTANT-COM-
PLEX FROM PSEUDOMONAS SP. PS-17
A. Sotirova, D. Spasova, E. Vasileva-Tonkova, D. Galabova

Bulgarian Academy of Sciences, The Stephan Angeloff Institute of Microbiology,
Acad. G. Bonchev str., bl.26, 1113 Sofia, Bulgaria
Although rhamnolipids have been studied for 50 years little is known about their
interaction with bacterial cells and their potential for use in biomedicine. The biosurfactant
isolated from bacterial strain Pseudomonas sp. PS-17 contains an unique natural complex
of a biosurfactant and a biopolymer-alginate with high surface and emulsifying activities.
The low parameters for their surface and interfacial tensions as well as their critical
concentrations for micelle formation (CMC), indicate high surface activity. For successful
application of biosurfactants in biomedicine their effect on the microbial surface needs to
be known.
Our research investigates antimicrobial properties of biosurfactant complex and its
effects on bacterial membrane of Gram positive bacteria, using Bacillus subtilis as a model
system. The product showed excellent antimicrobial properties. Antimicrobial activity
was evaluated according to the minimum inhibitory concentration (MIC), the lowest
concentration of an antimicrobial agent that inhibits development of visible microbial
growth. Antimicrobial activity of biosurfactant-complex against Bacillus subtilis was
observed (55 µg/mL),
Our results demonstrated that the exposure of B. subtilis cells to non-lethal
concentration (50 µg/mL) of biosurfactant-complex did not affect the protein component
of bacterial membrane but caused quantitatively changes in phospholipid headgroup.
This changes lead to a higher proportion of negatively charged phospholipids.
Ultrastructural studies revealed that biosurfactant-complex effects was directed not only
on cell surface structures and on inner cell structures of bacterial cells.The results suggest
144
that the biosurfactant-complex could be used in designing new more effective antimicrobial
preparations.
This work was financially supported by of the NCSR of the Bulgarian Ministry of
Education and Science under project K 1206/02.
GAM34
DECOLORIZATION OF THE ACID ORANGE 7 BY RESTING
ALCALIGENES FAECALIS AND RHODOCOCCUS
ERYTHROPOLIS CELLS: A COMPARATIVE STUDY
T. Avramova, L. Stefanova, B. Angelova and S. Mutafov

Acad G. Bonchev St., 26, 1113 Sofia, Bulgaria, e-mail: mutafov@microbio.bas.bg
The presence of azo group in synthetic dyes renders these compounds resistance to
biological attack. Presented study aims at elucidation the role of cell surface in the microbial
decolorization of the Acid Orange 7 (AO7) carried out by resting Alcaligenes faecalis
6132 and Rhodococcus erythropolis 24 cells. The effect of some surfactants on the dynamics
of the decolorization process was investigated and a preliminary evaluation of the energy
of activation of the decolorization reaction was presented.
The process of the microbial decolorization carried out by resting cells of the two
strains started with an immediate adsorption of the AO7 on the cell surface and proceeded
according to the model of the first order decay typical for processes where surface
phenomena like adsorption took place. The degradation constants calculated were ë
= 0.152 h -1 a n d ë = 0 . 1 9 1 h -1 for A. faecalis and R. erythropolis, respectively.
The presence of cationic and anionic surfactants influenced the rate of the
decolorization process. In concentration above the critical micelle concentration (CMC),
the anionic surfactant sodium N-laurosyl-sarcosine (SLS) inhibited the reaction of
decolorization. The cationic surfactant hexadecyltrimetylammonium bromide (HTAB) in
concentrations above and below CMC accelerated the binding of the AO7 by the cells
causing a rapid staining of the biomass and complete decolorization of the reaction medium.
In this, the colour of the A. faecalis cells faded faster compared to the decolorization of the
R. erythropolis cells, which was most probably a result from the surface peculiarities of
the R. erythropolis.
This study was supported by the Foundation for Scientific Investigations, Ministry of
Education, Science and Technology, Grant B1311/03
145
GAM35
PH – RELATED EQUILIBRIUM STUDY ON COPPER
BIOSORPTION BY PENICILLIUM CYCLOPIUM
Maria Ianisa , Kolishka Tsekovaa, Dessislava Todorovaa and Pencho Marinovb

a
Department of Microbial Ecology, Institute of Microbiology, Bulgarian Academy
of Sciences,
b
Central Laboratory for Parallel Processing, Bulgarian Academy of Sciences
PH – related equilibrium and modeling study on copper biosorption in single metal

systems by resting cells of Penicillium cyclopium was performed. The equilibrium of the
process (at different pH values and biomass concentrations) is well described by the
classical Langmuir sorption isotherm and their modifications. It seems that the process
was a chemical, equilibrated and saturable mechanism which reflected the predominantly
site – specific mechanism on the cell surface. The curves of Scatchard transformation
plots obtained are nonlinear, indicating presence of multiple nonequivalent metal binding
sites on the fungal biomass. The maximum copper uptake was calculated to be 49,84 mg/g
dry cells, when pH of the solution and biosorbent concentration were 4,5 and 1 g/l resp.
GAM36
IMMOBILIZATION OF PENICILLIUM CYCLOPIUM CELLS
IN PVA HYDROGELS FOR HEAVY METAL IONS
BIOSORPTION APPLICATIONS
Darinka Christova*1, Kolishka Tsekova2, Sijka Ivanova1, Maria Ianis2, Sonia

Ganeva3
1
Institute of Polymers – Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
phone: +359 2 979 22 85; e-mail: dchristo@polymer.bas.bg
2
Institute of Microbiology – Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
phone: +359 2 979 31 73; e-mail: kolishka@yahoo.com
3
Faculty of Chemistry – Sofia University, 1164 Sofia, Bulgaria
phone: +359 2 8161277; e-mail: sganeva@chem.uni-sofia.bg
The purpose of this work was to develop hybrid hydrogels by entrapping P. cyclopium
cells into poly(vinyl alcohol) (PVA) network and to characterize the ability of the system
for Cu2+, Co2+ and Fe3+ ions uptake from aqueous solutions. The advantage of the created
system is in the proper combination of an organic polymer with metal binding functional
groups and fungal cells containing effective metal binding groups on the wall surface.
Hybrid hydrogels of P. cyclopium cells immobilized in hydrophilic polymer network
have been obtained in situ by crosslinking the PVA aqueous solutions with glutaraldehyde
when dispersing dried powdered biomass in the media. The influence of the reaction
146
conditions as well as the components’ ration on the water content of the hydrogels has
been investigated. Metal binding abilities of the hybrid hydrogels for Cu2+, Co2+ and Fe3+
ions were determined by using atom absorption spectroscopy. The performance of the
immobilized biosorbent was evaluated by sorption kinetics, sorption capacities for different
metal ions as well as mechanical stability in a range of pH.
GAM37
BIOSORPTION OF BINARY MIXTURES
OF COPPER AND COBALT BY PENICILLIUM
BREVICOMPACTUM
Kolishka Tsekova1, Maria Ianis1, Vera Dencheva1and Sonya Ganeva2

1
Department of Microbial Ecology, Institute of Microbiology, Bulgarian Academy
of Sciences
phone: +359 2 979 3173; e-mail kolishka@yahoo.com
2
Faculty of Chemistry, Sofia University
phone: +359 2 8161277; e-mail sganeva@chem.uni-sofia.bg
This work reports on a study of the biosorption of copper and cobalt, both singly and
in combination (in equimolar concentrations), by the resting cells of Penicillium
brevicompactum. Equilibrium batch sorption studies were carried out at 300C and pH 5,0,
for a contact time of 1 hour to guarantee that equilibrium was reached. The equilibrium
data were analyzed using the Langmuir and Freundlich isotherms. The adsorption of
binary mixtures of heavy metals solution on the fungal biomass was found to be of
competitive type where the adsorption capacity for any single metal decreased in the
presence of the others. The cobalt ions showed a greater affinity for Penicillium
brevicompactum than the copper ions.
Financial support: The Grant B 1407/2004 allocated by the National Fond for Scientific
Research to the Bulgarian Ministry of Education and Science supported this work.
GAM38
SENSITIVITY OF SACCHAROMYCES CEREVISIAE YEAST
TO ARSENATE
Tatina Todorova1, 2, Stephane Vuilleumier2, Anna Kujumdzieva1
1
Sofia University, Department of General and Applied Microbiology, 8 Dragan
Tzankov, 1164, Sofia, Bulgaria
2
Universite Louis Pasteur, UMR 7156 Genetique moleculaire, Genomique et
Microbiologie, 28 rue Goethe, F-67083 Strasbourg Cedex, France
Arsenic is a toxic metalloid present in natural and polluted industrial environments. It
147
is a human carcinogen but is also used in treatment of acute promyelocytic leukemia and
protozoan parasitic diseases. When mammals are exposed to arsenate, it is reduced to
arsenite either by the PNP arsenate reductase or MMA(V) reductase, a member of omega
class of glutathione S-transferases (GST). The later enzyme catalyzes the conjugation of
the electrophilic toxic compounds with the –SH group of glutathione (GSH), therefore
playing a critical role in xenobiotic elimination. Based on sequence, substrate specificity,
structure and immunological properties, GSTs have been grouped into eight distinct
families. In contrast with mammals, plants and even bacteria, little is known about GSTs of
yeasts and fungi but they seem to be especially diverse both structurally and functionally.
In this study we have taken a systematic genetic approach to study the potential role
of GSTs in arsenate toxicity in Saccharomyces cerevisiae. A search in Saccharomyces
Genome Database reveals the presence of 11 genes and ORFs with homology to GST,
which single disruption mutants were tested for their sensitivity to arsenate. The mutant,
disrupted for the gene TEF4, coding for translation elongation factor EF1ã (GST
homologue) was studied. A hypersensitivity of this mutant to AsV has been found,
indicating a possible participation of Tef4 protein in arsenate detoxification.
GAM39
ТРАНСПОРТ НА АРСЕНАТ В ДРОЖДЕВИ КЛЕТКИ
Стефан Иванов Шилев

Катедра „Микробиология и екологични биотехнологии”
Аграрен Университет – Пловдив, бул. Менделеев 12, Пловдив 4000, Е-mail:
stefan.shilev@au-plovdiv.bg
Известно е, че в голяма степен почвеното плодородие и разграждането на

различни замърсителите се дължи на микробиалната дейност. Понастоящем,
замърсяването с метали и металоиди е един от най-важните екологични проблеми,
като за неговото разрешаване все по-голямо внимание се обръща на
биотехнологичните подходи. Нови изследвания показаха, че някои щамове
дрожди изолирани от почвата притежават висока толерантност към наличие на
метали в хранителната среда.
Целта на настоящето изследване е охарактеризиране на транспорта на арсенат
в дрождевите клетки, като причина за толерантността към металоидния йон.
Резултатите от изследванията показаха, че в замърсените с арсен почви се
съдържат дрожди с висока толерантност към изследвания металоид.
Толерантността към наличие на арсен в хранителната среда (Na2HAsO4, 50, 100, 500
mg l-1) е обусловена от системите за транспорт в микробната клетка. Поради
структурното сходство между арсенатния и фосфатния йони, съществени
концентрации As постъпиха в клетките. След приемане на вътреклетъчни
концентрации близки до критичните, концентрацията на металоида намалява
значително благодарение на задействане на механизмите за извеждане на
148
токсичния йон извън клетката, както и на вътреклетъчното му изолиране.
В заключение, толерантността към As при различни видове дрожди е
обусловена от способността на клетките да намаляват вътреклетъчната
концентрация на свободните арсенатни йони.
GAM40
ISOLATION, IDENTIFICATION AND SELECTION OF AR-
SENIC AND CADMIUM RESISTANT YEAST
Nikolay Krumov1, Velitchka Gotcheva1, Tsonka Hristozova2, Clemens Posten3,

Angel Angelov1
1
Department of Biotechnology, University of Food Technologies, 26 Maritza Blvd,
4002 Plovdiv, Bulgaria;
2
26 Maritza Blvd, 4002 Plovdiv, Bulgaria;
3
Department of Bioprocess Engineering, MVM-Institute, University of Karlsruhe,
Kaiserstr. 12, D-76131 Karlsruhe, Germany
In the present scientific work, yeast strains capable of growing in the presence of high
concentrations of arsenic (As) and cadmium (Cd) were isolated and identified, and strains
showing highest As- and Cd-resistance were selected. For the purpose, yeast strains from
the collection of the Institute of Microbiology – Bulgarian Academy of Science (IM-BAS)
and strains isolated from nature were first verified. 144 morphologically different isolates
were obtained from soil samples taken from the surroundings of Umicor Med – Pirdop and
Non-Ferrous Metal Smelter (KCM SA) - Plovdiv. The majority of them were fungi – 73 and
actinomycetes – 43, not being an object of the present research. Based on morphological
and cultural characterization, 28 pure yeast cultures were isolated. Within this group, 3
strains Schizosaccharomyces pombe and 1 strain Saccharomyces cerevisiae were identified
by physiology-biochemical methods. Seven strains showed resistance in concentrations
of arsenic of 50 mmol: Saccharomyces cerevisiae 0505 and 0533, Saccharomyces
ellipsoideus 0112 and 0010, Rhodotorula rubra 3032, Torulopsis alba 2962 and strain S.
pombe NK05/2 isolated from KCM surroundings. These strains will be further used in
research on the production of cadmium-sulfide nanobioparticles and in exploring the cell
response in extreme conditions and the activation of cell’s self-defense enzyme system.
149
GAM41
CHARACTERISATION AND IDENTIFICATION
OF BACTERIAL COMMUNITY ISOLATED FROM
METALWORKING FLUIDS
Stela Bakalova, Petia Hristova, Raycho Dimkov, Veneta Groudeva
Metalworking fluids (MWFs) are widely spread and used for cooling and lubricating
during the machining process. They typically are formulated to include chemicals that
inhibit metal corrosion and microbial activity (biocides), whilst lubricating and cooling the
metal cutting process. It can be an extreme environment with a high alkalinity and high
temperature when the MWFs are in-use. Users of MWFs are faced with the problem with
a microbial contamination during their exploitation witch requires approaches that suppress
microbial growth. On the other hand, the disposal of MWFs needs to develop methods
that promote the biodegradation of the operationally exhausted products. Concerning the
factors mentioned above, the research into the microbiology of metalworking fluids is
very important. In this study samples from different machines operating with metalworking
fluids were taken. 60 bacterial isolates as pure cultures were obtained. Morphological,
cultural and biochemical methods were applied for their identification, as well as sequencing
of 16S rDNA gene. The bacterial isolates were identified as Stenotrophomonas maltopilia,
Agrobacterium larrymoorei, and as Micrococcus sp. Stenotrophomonas maltopilia
consisted the dominant group.
GAM42
TAXONOMIC IDENTIFICATION OF XENORHABDUS
(ENTEROBACTERIACEAE) SPECIES BY RESTRICTION
ANALYSIS OF PCR-AMPLIFIED 16S RDNA GENES
Georgieva1, J.H., Groudeva1, V.I., Shishiniova2, M.D.

1.Department of Microbiology, Faculty of Biology, University of Sofia, Dragan
Tzankov 8 Blvd., Sofia, Bulgaria
2.Department of Zoology and Anthropology, Faculty of Biology, University of
Sofia, Dragan Tzankov 8 Blvd., Sofia, Bulgaria
The entomopathogenic nematodes from family Steinernematidae (Rhabditida) are

symbiontically associated with bacteria from genus Xenorhabdus. The bacteria belong to
family Enterobacteriaceae.
Twenty-one bacterial strains isolated from five nematode species (Steinernema spp.,
S.feltiae, S.kraussei, S.intermedium and S.carpocapsae) belong to genus Xenorhabdus
according to the classical taxonomy metodes we used. They were typed by analyzing 16S
rDNA gene restriction patterns obtained after digestion of PCR-amplified 16S rDNAs.
150
Seven restriction endonucleases were required: Cfo I, Hin fI, Dde I, Sau 3AI, Alu I, Hae III
and Msp I.
Our results indicate that amplified 16SrDNA restriction analyses in an accurate tool for
identifying endonucleases entomopathogenic nematode bacterial symbionts
GAM43
PRELIMINARY INVESTIGATIONS OF THERMAL SOURCES
BIODIVERSITY FROM RUPITE REGION
A. Terziyska, R. Mandeva, D. Lyutzkanova, M. Stoilova-Disheva, G. Radeva, M.

Kambourova
In the present work we initiate examination of phylogenetic biodiversity in Bulgarian

thermal sources. Soil and water samples with temperature gradient 45оС - 70 оС were taken
from two hot springs in Rupite field.
Molecular methods based on 16S rDNA analysis were used for evaluation of the real
microorganism variety. Genomic DNA isolated from the samples and purified by
Nucleobond cartridges was used as template for PCR. The optimal PCR parameters for 16S
rDNA amplification with universal and group specific primers for different bacteria were
established. PCR products ready for cloning were obtained.
The samples were screened for new bacterial and actinomycetes producers of
carbohydrases on twelve AZCL substrates. Four strains belonging to Bacillus group
were found to be carbohydrate active. One of them produced cellulase, gluconase and
xylogluconase, two – arabinoxylanase and one – amylase. The taxonomic affiliation of the
enzyme producers was determined by phylogenetic analysis.
GAM44
BIODIVERSITY OF CARBOHYDRATE DEGRADING
CULTIVABLE BACTERIA FROM BACILLUS GROUP,
ISOLATED FROM BULGARIAN HOT SPRINGS
Anna Derekova1, Carsten Sjøholm2, Rossica Mandeva1, Margarita Kambourova1

1
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Science,
acad. G. Bonchev str. 26, 1113 Sofia, Bulgaria
2
Novozymes, Krogshoejvej 36, 2880 Bagsvaerd, Denmark
The diversity of culturable bacteria from genus Bacillus and related genera in Bulgarian
hot springs was investigated. A total of 77 thermophilic and facultative thermophilic
strains were isolated under aerobic conditions at 60°C. Seventy six of them belonged to
eight species in four genera from Bacillus group. Based on phylogenetic analysis (<98%
151
sequence similarity) eleven isolates represent potentially three novel species or genera.
Producers of carbohydrases, degrading 11 from tested 12 substrates were isolated. Between
them, producers of biotechnologically valuable enzymes like starch and pectin degrading
enzymes were isolated. Producers of both, exo- and endo- amylolytic acting enzymes were
screened. Some of the enzymes were between first reported thermostable enzymes in their
groups, like curdlan lyase, gellan lyase and cellulase.
GAM45
SPATIAL PATTERN OF MICROBIAL NUMBERS
IN THE FREE WATER OF THE SREBARNA LAKE
Svetlana Naumova and Anastassia Petrova

Central Laboratory of General Ecology, BAS
A microbiological investigation of the water of the Srebarna Lake was carried out
aiming to test the representativeness of the biomonitoring site in the center of the free of
macrophytes area. The spatial dynamics of the total bacterial count, the reproduction rate,
and the numbers of carbophilic and ammonifying organotrophs were studied in July and
October 2004. The change of bacterial numbers with depth was examined in the center of
the free water at the surface, at the lower edge of the transparent water column (Secchi
depth), and at the bottom. The horizontal distribution of the parameters values was surveyed
in integral samples from the whole water column in three points of a South North transect
of the free water: at Varban Bozu, in the center, and at the entrance of the Dragayka
channel.
The microbiological parameters characterized the water column in the center of the free
of macrophytes area as fairly homogeneous. The total bacterial count and the reproduction
rate changed with the depth insignificantly, which is normal for a shallow lake with an
active water circulation. The slight vertical dynamics of the carbophils could be related to
the development of phytoplankton, and the numbers of ammonifyers followed the oxygen
concentration. No substantial differences were disclosed by the microbiological indicators
among the three points along the transect. Again, the total bacterial count and the
reproduction rate were conservative, representing the transitory state of the water body
in the two observations. The numbers of the two groups of organotrophs were confined
in the limits of an order.
The spatial uniformity of the microbiological pattern proved the adequacy of the site
in the center of the free water to the objectives of the biological monitoring of the Srebarna
Lake.
152
GAM46
COMPARISON OF BACTERIOPLANKTON DEVELOPMENT
BETWEEN CONTROL AND FERTILIZED FISH PONDS
H. Kalcheva, D. Tersiisky, R. Kalchev.

Institute of Zoology, BAS, Sofia
Two treated with manure of cattle origin and two control carp fish ponds were
investigated for bacterioplankton development by direct counting of bacteria stained
with erythrosine (Razoumov,1932 in its contemporary modification). The experiment was
carried out from the beginning of May till the end of September 2005 at the Institute for
fishery and aquaculture, Plovdiv branch, Bulgaria. General physical variables like water
transparency, temperature and others were measured, accompanied by simultaneous
samplings of nutrients, bacterioplankton and chlorophyll a at biweekly intervals. Bacterial
number and biomass of free living and on detritus attached bacteria were determined
separately. The results of control and treated fish ponds were compared by means of
repeated measure variance and by correlation analyses for significant differences and
relationships.
GAM47
ADAPTIVE STRESS RESPONSE OF HUMICOLA LUTEA 103
TO COPPER EXPOSURE
E. Krumova1, Y. Gocheva1, A. Dolashki2, P. Dolashka2, S. Stefanovic3, W. Voelter3,

M. Angelova1
The Stephan Angeloff Institute of Microbiology1, Institute of Organic Chemistry2,

Bulgarian Academy of Science, Sofia, Bulgaria and University of Tuebingen3, Germany
Copper is an essential element for fungal metabolism, but can be toxic to microorganisms
at high concentrations. It is widely considered that copper ions exert its effect at the
cellular level hrough induction of oxidative stress. Adaptive response to heavy metal
treatment refers to the ability of cells to better resist the damaging effects of the toxic
agent when first preexposed to a lower dose. It is a widespread phenomenon that has been
observed in prokaryotes and eukaryotes. In this study, the effect of pretreatment of the
fungal cells of Humicola lutea 103 with copper salts on growth and antioxidant defense
was investigated.
The changes of biomass production, protein carbonyl content, synthesis of reserve
carbohydrates and antioxidant enzyme activities in the experiments with pretreatment of
H. lutea cells in comparison to the stress conditions were analyzed. Pretreatment with 70
µg/ml Cu2+ resulted in enhanced resistance of the conidiosperes and mycelia taken from
exponential growth phase to higher doses of the heavy metal. Adaptive cell response
153
included restoration of the normal growth, decrease in oxidative damaged proteins and
accelerate in glycogen and trehalose production. Induced copper-tolerance in adaptive
experiments corresponded with enhanced activity of the Cu/Zn-superoxide dismutase
due to de novo protein synthesis. The correlation between immunoprotective protein and
Cu/ZnSOD was confirmed by Western blot analysis. The primary structure of this fungal
enzyme has been determined by Edman degradation of peptide fragments derived from
proteolytic digest. A single chain of the protein, consisting of 152 amino acid residues,
reveals a very high degree (74-85%) of structural homology in comparison to the amino
acid sequences of other fungal Cu/ZnSODs. The difference of the molecular masses of H.
lutea Cu/ZnSOD, measured by MALDI-MS (15935 Da) and calculated by its amino acid
sequence (15716 Da), is attributed to the carbohydrate chain of one mole of N-acetyl-
glucosamine, attached to the N-glycosylation site Asn23-Glu-Ser.
This work was supported by grant K-1302/03 from the NCSI of the Ministry of Education
and Science, Bulgaria.
GAM48
PHYSIOLOGICAL RESPONSE OF ANTARCTIC FUNGI TO
LONG-TERM TEMPERATURE STRESS
Y. Gocheva, E. Krumova, L. Slokoska, J. Miteva, M. Angelova

Acad. G. Bonchev 26, 1113 Sofia, Bulgaria
Harsh living conditions of the continent of Antarctica differ substantially from those
of almost all other parts of the Earth’s biosphere, and its microbial inhabitants have had to
become specifically adapted to the severe conditions. This adaptation includes different
strategies, among which is the activation of antioxidant enzyme defense. The aim of this
study was to obtain new data about the role of oxidative stress and antioxidant enzymes
in cold-adaptation of the filamentous fungi. We compared the effect of different temperatures
(10- 30°C) on cell response of two Antarctic Penicillium sp. strains (Penicillin sp. p14
and Penicillium sp. m12) with European temperate Penicillium sp t35. The changes of
biomass production, protein carbonyl content, synthesis of glycogen and trehalose and
antioxidant enzyme activities in long-term temperature stress conditions were analyzed.
The main finding from this study is that fungal strains from Antarctic region, and that
from temperate European region, may have evolved a specialised physiology allowing
them to survive prolonged low temperatures, and have enzyme systems that function at
low temperatures. Our results demonstrated that growth at low temperature does clearly
induce oxidative stress events in all fungal strains tested, namely enhanced level of oxidative
damaged proteins, accumulation of reserve carbohydrates and increased activity of
superoxide dismutase and catalase. Compared to temperate mesophilic Penicillium sp t35,
Antarctic strains (psychrophilic Penicillium sp p14 and mesophilic Penicillium sp m12)
demonstrated adaptations, which permit survival in low-temperature conditions. Cell
154
response of psychrophilic strain to temperatures above 15-20°C may be characterized as
a heat-shock response, whereas a decreased antioxidant defense in temperate mesophilic
strain at low temperatures may be explained by more profoundly changes in metabolism at
cold temperatures than under heat shock conditions.
This work was supported by grant B-1309/03 from the NCSI of the Ministry of Education
GAM49
CELLULAR RESPONSE AND ANTIOXIDANTS ENZYMES IN
ASPERGILLUS NIGER STRAIN
AGAINST TEMPERATURE STRESS
Abrashev R1, Dolashki A4, Stevanovic S3, Dolashka P2, Voelter W4, Stefanova L1,
Pashova S1, Hristova R2, and Angelova M1
The Stephan Angeloff Institute of Microbiology1, Institute of Organic Chemistry2,
Bulgarian Academy of Science, Sofia, Bulgaria and University of Tuebingen3,
Germany
Temperature is among the most crucial of factors affecting the growth and survival of
microorganisms. Exposure to elevated temperatures (“heat shock”, HS) has been found to
increase oxidative damage in microbial cells, possibly by accelerating the formation of
reactive oxygen species (ROS) and/or by increasing there activity. Fungal cell response
against temperature stress is not very well known.
This work was designed to study some of the physiological and biochemical events
that accompany survival of the fungal strain A. niger 26 under conditions of short-term
heat shock. Exposure of mycelia taken from exponential growth phase to the different
temperature (30 – 45oC) for 6 h caused an enhancement in cyanide-resistance respiration
and ROS generation by temperature dependant manner. As a result of raise in oxidant
level, a significant increase in the amount of oxidative damaged proteins was established.
Our results suggest that the heat treatment dramatically increased the concentration of
trehalose and glycogen and induced expression of antioxidant enzymes, superoxide
dismutase (SOD) and catalase (CAT). Two isoenzyme forms of SOD (Cu/Zn- and Mn-
containing enzyme) were found in crude extract of the fungal cells. The complete amino
acid sequence of Cu/Zn-SOD, determined by a combination of automated Edman
degradation, MALDI-TOF analysis, and sequence alignment was shown in comparison
with SODs from other eukaryotic sources.
This work was supported by grant K-1401/03 from the NCSI of the Ministry of Education
155
GAM50
BIOSYNTHESIS OF PROTEOLYTIC ENZYMES FROM
ANTARCTIC ACTINOMYCETE STRAINS
Darina Bl. Dimitrova, Petar D. Dorkov, Blagovesta T. Gocheva

Biological Faculty of Sofia University “St. Kliment Ohridski” Sofia, Bulgaria
Seventeen actinomycete strains, isolated from Antarctic soil samples of the Livingston
Island, are screened in reference to their biosynthetic proteolytic abilities. These strains
synthesize extracellular nonconstitutive proteolytic enzymes, differing from one another
in their activity. Tendencies for considerable differences are registered between the specific
and general proteolytic activities. The highest specific proteolytic activity is peculiar to
Streptomyces sp. 39 - 1430.8943 KU/mg, followed by Streptomyces sp. 35 (818.6874 KU/
mg) and Streptomyces sp. 36 (698.7757 KU/mg). The general proteolytic activity is maximal
for Streptomyces sp. 35.
The pointed three strains are selected as most perspective for further investigations
as a result of the screening.
GAM51
BIOSYNTHESIS OF ANTIMICROBIAL SUBSTANCES FROM
Darina Bl. Dimitrova, Petar D. Dorkov, Blagovesta T. Gocheva

After screening of 17 actinomycete strains, isolated from soil examples of the

isle Livingston – Antarctida for producing antimicrobial substances active against Bacillus
subtilis 6633 ATCC, the three most perspective ones are selected and their biosynthetic
abilities are studied microbiologically. The strains synthesize antibiotic substances against
the studied test microorganisms with the exception of Escherichia coli and Candida
albicans. The two strains Streptomyces sp. 35 and Streptomyces sp. 36 are active against
41.66% of the analyzed cultures and the strain Streptomyces sp. 39 – against 33.33%. The
strain Streptomyces sp. 35 is remarkable for its considerable antifungal activity against
Aspergillus, Fusarium and Penicillium. The antibiotic complex of the strain Streptomyces
sp. 36 is active against Gram-positive and Gram-negative bacteria, and that of the strain
Streptomyces sp. 39 – against phytopathogenic bacteria (Erwinia amylovora and
Pseudomonas syringae patovar syringae). There is a correlation between the biosynthesis
of proteolytic enzymes and antimicrobial substances within the three Actinomycetes strains.
156
GAM52
BIOSYNTHESIS OF PROTEINASE INHIBITOR FROM
Darina Bl. Dimitrova, Petar D. Dorkov, Vasil N. Atanasov, Blagovesta T. Gocheva

A prescreening of 17 actinomycete strains isolated from soil samples of the isle

Livingston – Antarctida for biosynthesis of extracellular proteinase inhibitor proteins is
done. The most perspective three of them, remarkable for their considerably high activity
- Streptomyces sp. 35, Streptomyces sp. 36 and Streptomyces sp. 39, are selected. Maximum
specific proteolytic inhibitory activity is registered for Streptomyces sp. 36 (1544.6384
ТIU/mg). Some qualitative and quantitative methods for detection of the activity are
compared. It is proved that polypeptones have a strongly inducive effect over the
production of proteinase inhibitors. The dynamic in accumulation of inhibitors is studied
and their biosynthesis is registered to begin after the 48-th hour; their maximal activity is
determined to be at the late exponential phase (96-th hour of the cultivation). So it is
established that these substances are not of primary significance for the growth of cells,
but probably their appearance is attended with regulation of proteolytic activity. Probably
the strains inhabiting extreme conditions need protease inhibitors by reason of the strong
regulative systems for their physiological processes. The protease inhibitor of Streptomyces
sp. 36 is isolated and partially purificated by using appropriate methods. The electrophoretic
profiles of the obtained isolate exhibit homogeneity and molecule weight about 14 kDa.
GAM53
CYCLODEXTRIN GLUCANOTRANSFERASE PRODUCTION
BY MAGNETICALLY RESPONSIBLE BACILLUS
CIRCULANS ATCC 21783 CELLS
M. Safarikovaa, N. Atanasovab, V. Ivanovac, S. Engibarovb, I. Safarika, A. Tonkovab

a
Department of Biomagnetic Techniques, Institute of Systems Biology and Ecology
AS CR, Na Sadkach 7, 370 05 Ceske Budejovice, Czech Republic, bDepartment of
Extremophilic Bacteria, Institute of Microbiology, BAS, 26 Acad. G. Bonchev str.,
1113 Sofia, Bulgaria, cDepartment of Organic Chemistry and Microbiology,
University of Food Technologies, 26 Maritza str., 4002 Plovdiv, Bulgaria. E-mail:
tonkova@microbio.bas.bg
Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is a unique enzyme converting

starch into cyclodextrins (CDs). These compounds possess the ability to form inclusion
complexes with many organic and inorganic molecules, changing their physical and
chemical properties, which determines their wide applications in environmental protection,
157
medicine, food, cosmetic, pharmaceutical and chemical industries.
Four types of magnetic nano- and microparticles were used for the immobilization of
Bacillus circulans ATCC 21783 cells: magnetite microparticles (1-5 ìm), entrapped in
agar gel beads with bacterial cells (AM); silanized magnetite (SM, 20-40 nm) covalent
connected on the cell surface; and alkaline and citrate ferrofluids (FFs, 10-20 nm),
attached on the cell wall by ionic interaction.
The highest CGTase production was achieved after 96 h semi-continuous process
with cells immobilized on SM when the CGTase specific activity was 8.4-fold higher compared
to free cells. The enzyme synthesized by all kinds of magnetically immobilized cells was
18-20% more thermostable than that of free cells. The magnetically responsible Bacillus
circulans ATCC 21783 cells demonstrate convincingly the effect of the used magnetic
carriers for a significant enhance of the CGTase yield, specific enzyme activity and thermo
stability.
This work was supported by a bilateral grant between the Bulgarian and Czech
Academies of Sciences, the Grant Agency of the Czech Academy of Sciences and ISBE
Intention No AV0Z60870520.
GAM54
MOLECULAR ANALYSIS OF A THERMOSTABLE GELLAN
LYASE BY MECC/HPLC
Miroslava Atanassova1, Jose Ignacio Garabal Sanchez2, Anna Terziiska1, Anna

Derekova1, Rositsa Mandeva1, Margarita Kambourova1
1
Department of Extremophiles, Institute of Microbiology “Stefan Angelof”, Bulgar-
ian Academy of Sciences, Sofia, Bulgaria
2
Centro de Investigaciуns Agrarias de Mabegondo (CIAM), Apdo. 10, 15080 A
Coruсa, Galicia, Spain
A scheme for the purification to homogeneity was developed for a thermostable gellan
lyase, produced by a Geobacillus stearothermophilus 98 strain after continuous
fermentation. Stepwise ammonium sulphate precipitation, hydrophobic interactions
chromatography, anion exchange chromatography and size-exclusion chromatography
were applied. The final purity of the enzyme was confirmed both by SDS-PAGE and C18
reverse-phase chromatography on HPLC. Its amino acid composition was obtained by the
Waters AccQ•Tag method. Free-solution capillary electrophoresis with SDS containing
BGE (MECC) of the newly isolated molecule showed that the gellan lyase had very low
mobility at alkaline pH, with tendency to precipitate, therefore, the best conditions
established for CE analysis were 0,2M BGE at pH 2 containing 0,1 % SDS, in untreated
fused-silica capillary of 92 cm/75ìm i.d./375 ìm o.d., 22°C and 25 kV. The enzyme’s
isoelectric point and molecular weight were studied.
158
GAM55
PURIFICATION AND CHARACTERISATION OF
KERATINOLYTIC PROTEASES PRODUCED
BY STREPTOMYCES ALBIDOFLAVUS
Vania Stefanova1,3, Nicolay Kirilov1,3, Kirill Tsiroulnikov 2, Michиle

Dalgalarrondo3, Jean-Marc Chobert3, Iskra Ivanova1 and Thomas Haertlй3.
1
Department of Microbiology, Faculty of Biology, Sofia University “St. Kliment
Ohridski”, Sofia, Bulgaria
2
Laboratory of Proteolytic Enzyme Chemistry, Shemyakin & Ovchinnikov Institute
of Bioorganic Chemistry of RAS, Moscow, Russia
3
FIPL, BIA, Institut National de la Recherche Agronomique, Nantes, France
Streptomyces are known to produce multiple proteases into the culture medium. Some
of these proteases are well characterized. In the recent years, production of proteases with
strong keratinolytic activity by different Streptomyces sp has been investigated (Bressollier
et al.,1999; De Azaredo et al. 2006).
In this study the exogenous keratinase produced by the strain of Streptomyces
albidoflavus strain ATCC25422 was purified and characterized. The strain was cultivated
in mineral medium supplemented with porcine hair 5 g/l as sole carbon and energy source.
At least two proteases with powerful keratinolytic activity were obtained in three
purification steps – anion exchange chromatography than cation exchange chromatography
SP Sepharose Fast Flow gel followed by gel filtration on Sephacryl S 300 HR. Two bands
were observed by zymogram on SDS-PAGE. The molecular weights of these two bands
estimated by SDS-PAGE were 54 kDa and 64 kDa. Substrate specificity of keratinases was
determinated with different synthetic amino acid derivates – Suc-Ala-Ala-Pro-Phe-pNA,
Suc-Ala-Ala-Pro-Leu-pNA and Suc-Ala-Ala-Pro-Lys-pNa. The proteases exhibited activity
with Suc-Ala-Ala-Pro-Phe-pNA and Suc-Ala-Ala-Pro-Leu-pNA. However, no hydrolysis
was detected with Suc-Ala-Ala-Pro-Lys-pNa. The studied proteases were inhibited by
PMSF, which is known serine proteinase inhibitor. EDTA and pepstatin have no influence
on enzyme activity. The studied proteases are stable and active at 50 °C and at pH values
between 7.5 – 9.
GAM56
BNMPK: A WEB BASED SUITE OF BACTERIAL
NUCLEOTIDE MONO PHOSPHATE KINASES
Ajay Bikumandla, Sai Guduru, Paulina Daalova, Alexandra Shosheva, Petya

Christova, Petras Kundrotas and Emil Alexov
The web server is a collection of models of 3D structures of all Bacterial Nucleoside
159
Mono Phosphate Kinases (BNMPK), their pre-computed properties and relevant
information. Nucleotide Mono Phosphate (NMP) kinases convert mono-phosphate
nucleotides to di-phosphate nucleotides. The reaction involves binding of both ATP
(adenosine tri-phosphate) and mono-phosphate nucleotide, post-sequential transfer of a
phosphate from ATP to mono-phosphate nucleotide and then release of ADP (adenosine
di-phosphate) and di-phosphate nucleotide. The web server provides information in regard
to sequence identity, 3D structure, Enzyme Commission (EC) number, pKa, desolvation
penalty and interaction energy with permanent dipoles of all BNMPKs. We also have
implemented a search engine using Java script, MS Frontpage and PERL. The BNMPK
database has a search part where the user can search the proteins based on the family,
name, seqID, pKa, polarity and desolvation energy and has an option to download all or
part of the search results. There are five different families of NMP kinases, categorized by
the nucleotide they bind, each family containing approximately 100 known members. These
include adenylate (AMPK, 118 members), guanylate (GMPK, 87 members), uridylate
(UMPK, 73 members), cytidylate (CMPK, 71 members) and thymidylate (TMPK, 107
members) kinases. However while many sequences exist, only few 3D structures were
experimentally determined. The dearth of 3D evidence is illustrated most markedly in the
UMPK family, which does not have a publicly available representative 3D structure. Thus,
our goal is to predict the structures of all members of these families and to use the
structures to calculate specific Biophysical properties. The results are publicly available
through the Internet link: http://www.ces.clemson.edu/compbio/databases/kinases.
GAM57
СКРИНИНГ НА ДРОЖДИЕВИ ПРОДУЦЕНТИ НА ФИТАЗА
Данаил Георгиев1, Стоянка Гаргова2

1
ПУ ,,Паисий Хилендарски” – катедра ,,Биохимия и микробиология” –
Пловдив
2
Университет по Хранителни Технологии – катедра ,,Биотехнология” –
Пловдив
Фитазата е една фосфомоноестераза (КНЕ 3.1.3.8), катализилаща по

стапаловиден механизъм дефосфорилирането на фитиновата киселина и нейните
соли с получаване на различни миоинозитол фосфати и фосфорна киселина.
Проведена е двуетапна скрининг процедура на дрожди, принадлежащи към
родовете Saccharomyces, Zygosaccharomyces, Candida и Pichia, за продуциране на
дефосфорилиращия ензим фитаза. В първата стъпка, осъществена върху 4 вида
твърди хранителни среди, съдържащи като селекциониращ фон калциев фитат са
изследвани 118 култури. Установено е, че върху PSM среда почти всички щамове
показват зони на просветляване още на двадесет и четвъртия час от развитието
си. Във втората стъпка на скрининга, проведена в течна хранителна среда и
количествено определяне на фитазната активност, са селекционирани няколко
160
щама, показващи значителен потенциал за продуциране на ензима. При най –
добрия щам част от ензима се секретира в културалната среда. В следващите
проучвания ще се търсят условия за по – пълна екскреция на ензима, което
представлява значителен интерес в технологичен и практически аспект.
GAM58
МУТАГЕННО ТРЕТИРАНЕ НА ЩАМ ASPERGILLUS
AWAMORI K-1 ПРОДУЦЕНТ НА ЕНЗИМА КСИЛАНАЗА
Яна Евстатиева, Светла Илиева, Диляна Николова, Валентин Савов, Атанас

Атев
Биологически факултет, Софийски Университет “Св. Климент Охридски”,
катедра Биотехнология
Широкото приложение на ензимите определя необходимостта от получаване

на продуценти с повишени биосинтетични възможности. Методите на индуцирания
мутагенез все още са широко използвани, тъй като са лесно приложими и
ефективни. Отборът на мутантните култури се осъществява по коефициента К,
изразяващ съотношението между диаметъра на микробната колония и диаметъра
на хидролизната зона на ксилан съдържащата скринираща среда.
Проведена е мутагенеза на родителски щам Aspergillus awamori K-1 с химичния
мутаген N-метил-N’-нитро-N-нитрозогуанидин (НГ) с концентрация 250 ìg/ml.
След мутагенното третиране са изолирани мутантни щамове, които показват по-
висока активност на ензимите от ксиланолитичния комплекс при дълбочиното им
култивиране в колби. Проучени са морфологичната, физиолого-биохимичната и
биотехнологичните характеристики на секвенираните мутантни култури.
Изолираните мутантни щамове показват над 1.5 пъти по-висока ендоксиланазна
активност в сравнение с родителския щам Aspergillus awamori K-1, пълен набор
на индивидуални ензими разграждащи ксилановите субстрати и с 12 часа по-къс
ферментационен процес от изходната култура.
Благодарности: Настоящата експирементална работа е осъществена с
финансовата подкрепа на Фонд Научни Изследвания при МОН, по проект №ВУ-
Б-7/О5
GAM59
STUDYING OF BIOACTIVE METABOLITES FROM ARCTIC
COLD-ADAPTED STREPTOMYCETES
D. Lyutskanova, M. Stoilova-Disheva, , M. Kolarova, K. Alexieva, V. Peltekova, V.

Ivanova
161
In the course of investigation of soil samples from Spitzbergen, Arctic, 6 strains were
isolated. Microscopically and morphologically they were identified as Streptomycetes. It
is of great scientific interest to identify taxnomically the isolated strains , to study their
biological characteristics, to perform a screening for some active metabolites produced by
them and reveal their biological action. Hydrolitic activity of the isolated strains was
investigated at 4ºС. The enzymes â-gluconase, arabinoxylanase, celulase, amylase,
xylogluconase and arabinase were identified to present.
From the mycelium and the supernatant of one of the strains a 4 component antibiotic
compex was isolated. It is active against Gram (+), Gram (-) bacteria and yeasts. The
complex has been separated by TLC in liquid fase butanol : acetic acid : water. The 4
compounds show a typical colour reaction with ninhydrin solution.
GAM60
ИМОБИЛИЗАЦИЯ И СВОЙСТВА НА ПЛЕСЕННА
ЦЕЛУЛАЗА ВЪРХУ ПОЛИАМИД
Гинка Делчева, Иван Пищийски, Георги Добрев
В настоящата работа е представена имобилизацията на плесенна целулаза от

ксиланолитичен комплекс, продуциран от Aspergillus niger B03. Като носител е
използван синтетичен полимер полиамид директно или след активиране със
свързващ агент глутаров алдехид и хексаметилендиамин. Определени са
температурен и рН оптимум, температурна и рН стабилност на свободния и
имобилизиран ензим. Изследвани са кинетичните параметри Km и Vmax на двете
форми на ензима.
GAM61
PRIMARY CHARACTERIZATION OF A NEWLY
DISCOVERED BACTERIOCIN-LIKE SUBSTANCE DURACIN
PRODUCED FROM ENTEROCOCCUS DURUM M-3
Svetoslav G. Dimov
Sofia University “St. Kliment Ohridski”, Faculty of Biology, Department of Genet-
ics, e-mail: svetoslav@biofac.uni-sofia.bg
Bacteriocins are ribosomally synthesized bacterial toxins acting on the target cells as
membrane depolarizing and pore forming agents. Many bacteriocins are produced by
lactic acid bacteria isolated from dairy products which inhibit the growth of pathogen
microflora thus preventing spoilage.
Here is described a new bacteriocin-like substance - duracin, produced by Enterococcus
162
durum M-3 isolated from traditional Bulgarian yellow cheese “kashkaval”. The research
was emphasized on the purification of the bacteriocin molecule, determination of the
spectrum of antibacterial activity, and the characteristics of the producer strain. It was
determined that the duracin molecule represents a few kilodaltons peptide which was
produced at maximum at the end of the exponential phase of growth and having relatively
broad range of cytotoxic activity among Gram-positive bacteria.
GAM62
NOVEL BACTERIOCIN FROM ENTEROCOCCUS FAECIUM
3587 – SPECTRUM OF ACTIVITY AND SOME MOLECULAR
CHARACTERISTICS
Viktoriya M. Peeva, Pavlina M. Ivanova and Svetoslav G. Dimov

Sofia University “St. Kliment Ohridski”, Faculty of Biology, Department of Genet-
ics, e-mail: jaki_santan@mail.bg
Many lactic acid bacteria produce bacteriocins – small peptide bacterial toxins inhibiting
species in most cases closely related to the producer thus helping them in the concurrence
for the occupation of the ecological recesses.
In this study we focused our investigation on the following points: 1) characteristics
of the production of the bacteriocin-like substance during growth; 2) determination of the
spectrum of antibacterial activity of the produced bacteriocin; 3) purification of the
bacteriocin; and 4) preliminary characteristics of the bacteriocin molecule.
It was found that the maximum activity was observed at the end of the exponential
phase of growth and the bacteriocin production was not associated with plasmids hence
no plasmids were isolated from the producer strain. The molecule was purified by preparative
PAGE and it was active only against closely related enterococci but not against other
Gram-positive or Gram-negative bacteria.
GAM63
LACTOCOCCUS LACTIS SUBSP. LACTIS HV219 –
A PROBIOTIC?
SD Todorov1, M Botes (nee Brink)1, ST Danova2 and LMT Dicks1

1
Department of Microbiology, University of Stellenbosch, 7600 Stellenbosch, South
Africa
2
Department of Microbial Genetics, The Stephan Angeloff Institute of Microbiology,
Bulgarian Academy of Science, 1113 Sofia, Bulgaria
Probiotic bacteria have to resist a number of intestinal stress conditions, such as bile
163
salts, acids and pancreatic juice to survive passage through the gastro-intestinal tract.
Adhesion to intestinal epithelial cells and production of antimicrobial substances are
additional criteria used to select probiotic strains. In this study, a strain isolated from
vaginal secretions was selected based on antimicrobial peptide (bacteriocin) production,
antibiotic resistance, hydrophobicity, resistance to bile salts and growth at low pH. Growth
was monitored in MRS broth, modified to contain 0.3%, 0.6%, 0.8%, 1.0%, 3.0% and 5.0%
bile, respectively. MRS broth was adjusted to pH 3.0, 4.0, 5.0, 7.0, 9.0, 11.0 and 13.0,
respectively. Lactococcus lactis subsp. lactis HV219 showed good adhesion to human
Caco-2 cell-lines.
Bacteriocin HV219, produced by Lactococcus lactis subsp. lactis HV219, is active
against Escherichia coli, Enterococcus faecalis, Lactobacillus casei, Listeria innocua,
Proteus vulgaris and Pseudomonas aeruginosa. Activity was lost when treated with
proteolytic enzymes, SDS, Triton X-114 and Triton X-100, but not at pH 2.0 to 10.0 or after
20 min at 121 °C. The mode of activity is bacteriolytic, as confirmed by atomic force
microscopy. The strain harbours a plasmid of 3.0 kb. Growth of Lactococcus lactis subsp.
lactis HV219 was not significantly affected in the presence of increasing bile concentrations.
Low hydrophobicity (4.20%) was observed for L. lactis subsp. lactis HV219.
GAM64
EFFECT OF MEDIUM COMPONENTS ON PRODUCTION OF
BACTERIOCINS JW3BZ AND JW6BZ BY LACTOBACILLUS
PLANTARUM ISOLATED FROM BOZA
JW von Mollendorff, SD Todorov and LMT Dicks

Department of Microbiology, Stellenbosch University, 7602 Stellenbosch, South
Africa
Bacteriocins JW3BZ and JW6BZ, produced by Lactobacillus plantarum JW3BZ and

JW6BZ, respectively, inhibits the growth of Lactobacillus sakei, Enterococcus mundtii,
Enterococcus faecalis, Lactococcus lactis subsp. lactis and Lactobacillus casei. The
growth of Streptococcus caprinus was only inhibited by bacteriocin JW6BZ and that of
Listeria innocua LMG 13568 by bacteriocin JW3BZ. Treatment with Proteinase K,
trypsin and pronase inactivated the bacteriocins. No change in activity was observed
in the presence of á-amylase. Maximal activity (25 600 AU/ml) was recorded for
bacteriocin JW3BZ and JW6BZ in MRS broth, adjusted to pH 5.0 to 6.5. Optimal
production of bacteriocin JW3BZ was recorded in the 20.0 g/l glucose and 20.0 g/l maltose.
In the case of bacteriocin JW6BZ, maximal activity was recorded in the presence of 20.0 g/
l glucose and 20.0 g/l mannose, respectively. Modified MRS medium, containing tryptone
and meat extract (1:0.6), yielded activity levels of 25 600 AU/ml for bacteriocins JW3BZ
and 51 200 AU/ml for bacteriocin JW6BZ. Increased concentrations (5.0 g/l) of K2HPO4
doubled bacteriocin JW3BZ activity (51 200 AU/ml). Concentrations of 2.0 g/l to 20.0 g/l
KH2PO4 doubled the activity of bacteriocin JW3BZ. In the case of JW6BZ, concentrations
164
of 2.0 g/l to 20.0 g/l KH2PO4 doubled the bacteriocin activity. A reduction in bacteriocin
activity was recorded when strains JW3BZ and JW6BZ were grown in the presence of
glycerol. The inclusion of selected vitamins in the growth media led to different levels of
bacteriocin JW3BZ and JW6BZ activity. The optimal concentration of tri-ammonium citrate
for production of bacteriocins JW3BZ and JW6BZ was 5.0 g/l. Low activity levels for
both bacteriocins were recorded in BHI broth, M17 broth and skim milk (20.0 g/l).
GAM65
FACTORS AFFECTING THE ADSORPTION OF
BACTERIOCIN ST194BZ TO LACTOBACILLUS SAKEI AND
ENTEROCOCCUS FAECIUM
SD Todorov1, M Meincken2 and LMT Dicks1

1
Department of Microbiology and 2Department for Forest and Wood Science, SPM
unit, University of Stellenbosch, 7600 Stellenbosch, South Africa
Lactobacillus plantarum ST194BZ, isolated from boza, a cereal based fermented

beverage from Bulgaria, produces two bacteriocins, viz. ST194BZ(a) of 3.3 kDa and
ST194BZ(b) of 14.0 kDa. A combination of the two bacteriocins inhibits the growth of
Lactobacillus casei, Lactobacillus sakei, Lactobacillus delbrueckii subsp. bulgaricus,
Enterococcus faecalis, Escherichia coli, Enterobacter cloacae and Pseudomonas
aeruginosa. A maximum total bacteriocin activity of 12 800 AU/ml was recorded in MRS
broth. Bacteriocins ST194BZ(a) and ST194BZ(b) withstand 20 min at 121oC and incubation
at pH 2-10, but is inactivated when treated with proteolytic enzymes such as pepsin,
papain, á-chymotrypsin, trypsin and Proteinase K.
Images obtained by atomic force microscopy showed clear signs of membrane damage
of Lactobacillus sakei, accompanied by the leakage of DNA and â-galactosidase.
Adsorption of the bacteriocin to cells increased when the cells were treated with buffers
at pH values above neutral. An increase in bacteriocin ST194BZ adsorption to cells of
Enterococcus faecium and L. sakei was observed with an increase in incubation temperature.
Treatment of the latter two species with various inorganic salts and solvents gave different
results regarding the adsorption of bacteriocin ST194BZ. In general, pre-treatment of the
two sensitive cells with Triton X-100, Triton X-114 and chloroform increased the adsorption
of bacteriocin ST194BZ. Treatment of E. faecium and L. sakei with Na-EDTA and SDS led
to a decrease in the adsorption of bacteriocin ST194BZ. Variable results were recorded
with inorganic salts.
165
GAM66
DEFORMATION OF BACTERIAL CELLS AS A RESULT OF
BACTERIOCINS PRODUCED BY LACTIC ACID BACTERIA
M Meincken2, SD Todorov1, JW von Mollendorff1 and LMT Dicks1

1
Department of Microbiology and 2Department for Forest and Wood Science, SPM
unit, University of Stellenbosch, 7600 Stellenbosch, South Africa
The interaction between bacteriocins and target cells was studied using different
methods, such as atomic force microscopy (AFM), cell lysis, and extracellular leakage
of DNA and â-galactosidase. Most methods used to detect bacteriocin-cell interactions
are based on the observation of indirect reactions or cellular changes. AFM reveals the
direct effect of a bacteriocin on a sensitive cell. This includes deformation of the cell
surface, vesiculation and leakage of the cytoplasm.
AFM images were obtained in air and with tapping mode on a Multimode AFM from
Veeco. A silicon non-contact cantilever from Nanosensors (Neuchatel, Switzerland) with
a resonance frequency of 160 kHz and a spring constant of approximately 50 N/m was
used. The results clearly showed changes in cell morphology, such as collapse of the
apical ends or the cell center, signs of cytoplasm leakage and vesiculation. Differences
observed among the bacteriocins suggests different mode of actions, such as the barrel
stave model and the toriodal model, which describes the formation of pores in the cell
membrane or the carpet model, which leads to a vesiculation of the outer cell membrane.
GAM67
PARTIAL CHARACTERIZATION OF A BACTERIOCIN PRO-
DUCED BY LACTOBACILLUS CURVATUS ISOLATED
FROM CERVELAT SALAMI
A van den Worm, SD Todorov and LMT Dicks

Department of Microbiology, Stellenbosch University, Stellenbosch 7602, South
Africa
A bacteriocin-producing strain of Lactobacillus curvatus (AW42CS) was isolated

from Cervelat salami collected at a local food store (Stellenbosch, South Africa). Bacteriocin
AW42CS inhibited the growth of Gram-positive bacteria (Streptococcus caprinus ATCC
700066, Streptococcus spp. TL2W and TL2R, Lactobacillus sakei DMS 20017, Listeria
innocua LMG 13568, Listeria ivanovii N29, Listeria monocytogenes N22 and N26,
Lactococcus lactis subsp. lactis HV219, and Enterococcus faecalis HKLHS, E90, E88).
The bacteriocin is approximately 2.5 kDa in size as determined by tricine-SDS-PAGE and is
produced at 1600 AU/ml after 21 h of growth in MRS broth. Activity was lost after
treatment with proteolytic enzymes and when heated for 20 min at 121 ÚC. Treatment
166
with á-amylase, lipase, SDS, Tween 20, Tween 80, urea and EDTA, or incubation at
pH 2 to 12 did not inactivate bacteriocin AW42CS. Cells of L. sakei DSM 20017, L
innocua LMG 13568 and E faecalis treated with the bacteriocin did not lyse, suggesting
a bacteriostatic mode of action. The peptide did not adsorb to producer cells. Bacteriocin
AW42CS was partially purified by precipitation with 60% ammonium sulphate and
separation in a SepPakC18 column.
Treated cells of L. sakei DSM 20017 collapsed after contact with bacteriocins JW11BZ
and JW15BZ produced by L. fermentum JW11BZ and JW15BZ. Leakage was observed
after treatment of exponentially growing cells of L. sakei DSM 20017 with bacteriocins
AMA-K and JW6BZ, and L. innocua LMG 13568 treated with bacteriocin ST8KF.
Vesiculation was observed for cells of L. sakei DSM 20017 treated with plantaricin 423 and
bacteriocin ST23LD produced by L. plantarum
GAM68
SANIONINS: ANTIINFLAMMATORY AND ANTIBACTERIAL
AGENTS WITH WEAK CYTOTOXICITY FROM THE
ANTARCTIC MOSS SANIONIA GEORGICO-UNCINATA
Veneta Ivanovaa, Klaus-Juergen Dornbergerb, Albert Haertlb, Ute Moellmannb,

Hans-Martin Dahseb,
Mariana Kolarovaa, Krasja Aleksievaa, Nesho Chipevc
a
26 Acad.Georgy Bonchev St., 1113 Sofia, Bulgaria
b
Leibniz Institute for Natural Product Research and Infection Biology-Hans-Knoell-
Institute, Beutenbergstrasse 11a, D-07745 Jena, Germany
c
Central Laboratory of General Ecology, Bulgarian Academy of Sciences, 2
Gagarin St., 1113 Sofia, Bulgaria
Sanionins A (1) and B (2) were isolated from the moss Sanionia georgico-uncinata,
collected on the Antarctic Livingston Island. The compounds 1 and 2 were purified by
solvent extraction, silica gel column chromatography and preparative HPLC consecutively.
The structures of the both compounds were elucidated by 1 and 2D NMR experiments and
mass spectrometric investigations. Both substances sanionin A (1) and sanionin B (2) for
the first time were isolated from the antarctic moss Sanionia georgico-uncinata and can
be classified into cinnamoyl bibenzyls.
These compounds showed activity against important Gram-positive pathogens like
mycobacteria, multiresistant staphylococci and vancomycin resistant enterococci. This
activity is combined with antiinflammatoric activity and low cytotoxicity. The sanionin A
(trans-isomer) was with better biological activities that sanionin B (cis-isomer).
167
GAM69
MALONYL,4-5-DIHYDRONIPHIMYCIN: NEW POLYOL
MACROLIDE ANTIBIOTIC, PRODUCED BY
STREPTOMYCES HYGROSCOPICUS
Veneta Ivanova, Mariana Kolarova and Krasja Aleksieva

Acad. G. Bonchev-Str. 26, 1113 Sofia, Bulgaria
Malonyl,4-5-dihydroniphimycin was isolated as a new antibiotic from the mycelium of

a Streptomyces hygroscopicus. The chemical constitution was elucidated from the physico-
chemical properties, NMR techniques and mass spectrometry to be a 36-membered macrolide
related to non-polyenic antibiotics. Malonyl,4-5-dihydroniphimycin is a new antibiotic
having a 36-membered ring, a two malonate and a monomethylguanidino groups. The
position of the two malonyl residues is at C-23 and C-19 and displayed activity against
Gram-positive bacteria and filamentous fungi.
GAM70
DIPHENYLETHER AND MACROTRIOLIDES OCCURRING
IN A FUNGAL ISOLATE FROM THE ANTARCTIC LICHEN
NEUROPOGON
Veneta Ivanova1, Udo Graefe2, Brigitte Schlegel2, Mariana Kolarova1, Krasja

Aleksieva1
1
Acad. G.Bonchev Str. 26, 1113 Sofia, Bulgaria
2
Hans-Knoll-Institute of Natural Products Research, Beutenbergstrasse, 11a, D-
07745 Jena, Germany
The fungus Tritirachium sp. 0317 was isolated from the Antarctic lichen Neuropogon
sp. Fermentation of this strain, extraction of the culture broth and preparative separation
of produced compounds furnished 4-carboxy-5,5’-dihydroxy-3,3’-dimethyldiphenylether
(1), macrosphelide A (2) and macrosphelide J (3). The structures were elucidated on the
basis of MS and NMR measurements and display structural features such as diphenylether
and lactone groups, which were found too in the depsidones as typical products of this
lichen. Macrosphelides A and J displayed interesting activities as cell adhesion inhibitors
and moderately cytotoxic agents.
168
GAM71
MICROBIAERATIN, A NEW NATURAL INDOLE ALKALOID
FROM A MICROBISPORA AERATA STRAIN, ISOLATED
FROM LIVINGSTON ISLAND, ANTARCTICA
Veneta Ivanova1, Udo Grдfe2, Hans-Martin Dahse2, Mariana Kolarova1, Krasja

Aleksieva1, Hartmut Laatsch3
1
Acad. G. Bonchev-str. 26, 1113 Sofia, Bulgaria
2
Leibniz Institute for Natural Product Research and Infection Biology-Hans-Knoell-
Institute, Beutenbergstrasse 11a, D-07745 Jena, Germany
3
Department of Organic and Biomolecular Chemistry, University of Gцttingen,
Tammannstrasse 2, D-37077 Gцttingen, Germany
A new sulphur-containing natural alkaloid named microbiaeratin (1a) was isolated

together with the known microbiaeratinin (2) from the culture filtrate of Microbispora
aerata strain. The organism was isolated from penguin excrements, collected on the
Antarctic Livingston Island. The structure was elucidated by one- and two-dimensional
NMR experiments and mass spectrometric investigations. The structure of 1a was finally
elucidated as N-3-β-indolylethyl-15-α-acetylamide-ethylthiazole-12-carboxamide. A low
antiproliferative and cytotoxic effects of 1a was determined with L-929 mouse fibroblast
cells, K-562 human leukemia cells (GI50 >50 µg/ml) and HeLa human cervix carcinoma (CC50
>50 µg/ml). Microbiaeratin (1a) did not show antimicrobial activity.
GAM72
AN INVESTIGATION ON THE OPPORTUNITIES FOR IN-
CREASING STREPTOMYCES AMBOFACIENS BIOSYN-
THETIC ACTIVITY THROUGH INDUCED MUTAGENESIS
Nataliya Hristova, Venelin Baloutzov, Stanislava Karafizova*

University of Sofia “St. Kliment Ohridski”,
Faculty of Biology, Department of Biotechnology,
8 Dragan Tzankov blvd., Sofia 1164
*postgraduate student – MS programme
The present research is related to studying the possibilities for an increase in

Streptomyces ambofaciens ATCC 15154 (a spiramycin producer) biosynthetic activity.
Once having determined the optimal regime of mutagen treating by statistical analysis
of experimental data, the strain has been subjected to both the single and combined action
of N-methyl – N’-nitro – N-nitroso- guanidine and ã-rays.
The biosynthetic potency of the isolated active varieties has been defined by the agar
diffusion method, after the varieties were grown through submerged cultivation.
169
GAM73
ПЕРМЕАБИЛИЗИРАНЕ НА ДРОЖДЕВИ КЛЕТКИ ЗА
ПРОДУКЦИЯ НА Α -КЕТО КИСЕЛИНИ
Донка Д. Костова, Анна В. Куюмджиева

Софийски университет „Св. Климент Охридски”
Катедра по „Обща и промишлена микробиология”
1164 София, бул. „Драган Цанков” №8
Получаването на високо активни дрождеви клетки за продукция на α-кето

киселини, чрез трансформация на D-амино киселини, е ефективна алтернатива на
използването на чисти ензимни препарати. Селекционираният термотолерантен,
лактозоусвояващ щам Kluyveromyces marxianus 903 притежава висока специфична
D-аминооксидазна активност (60 U/g белтък), която е повишена деветкратно (545
U/g белтък) чрез подбор на хранителна среда и условия за ефективна индукция на
ензима.
С помощта на химични (третиране с катионния детергент цетилтриметил
амониев бромид - СТАВ) и физични (лиофилизация) подходи за пермеабилизиране,
D-аминооксидазната активност на цели клетки от селекционирания щам е
повишена многократно. Изследвани са детайлно условията за пермеабилизация
при химично третиране (концентрация на СТАВ, продължителност на
третирането, температура, рН) и различни режими на лиофилизация за постигане
на най-добро пермеабилизиране на клетките. Установено е, че третиране на
клетките с 0,2% СТАВ в 100 mM калиево-фосфатен буфер за 20 минути при 22ЪС,
увеличава 43 пъти D-аминооксидазна активност на клетките, а лиофилизирането
- 26 пъти.
GAM74
ИЗСЛЕДВАНЕ НА КИСЛОРОДНИЯ МАСООБМЕН
ВЪРХУ БИОСИНТЕЗА НА ЕКЗОПОЛИЗАХАРИД P -45
А.В. Атанасова, К. Павлова, Г.Щ. Атанасова, А.И. Тончев, В.В. Лосев, В.Г.
Назаров
От особенно значение при получаването на екзополизахарида Р-45 е

осигуряване на контролируем синтез на полимер и получаване на продукт с
възпроизводими показатели. Важен параметър при синтеза на Р–45 представляват
стойностите на скоростта на консумирания кислород и скоростта на отделения
въглероден диоксид по време на процеса. Представените експериментални данни
показват, че степента на използване на кислорода може да послужи като критерий
определящ добива на екзополизахарида в крайния продукт. Анализът на кислород
и въглероден диоксид се осъществява с помоща на газ- анализатор Tandem на
170
фирмата Adaptive Biosistems. На базата на този вид анализ е определен
респираторния коефициент на микробната култура. Тенденцията на изменение
на този физиологичен параметър показва специфичното ниво на синтез на
екзополизахарида. Това е един от възможните механизми за промяна на степента
на полимризация, състава и качествата на синтезирания екзополизахарид.
GAM75
МАЩАБЕН ПРЕХОД ЧРЕЗ ИЗПОЛЗВАНЕ НА
КИСЛОРОДНИЯ МАСООБМЕН ПРИ БИОСИНТЕЗА НА
АМИНОСИСЕЛИНИ
А.В.Атанасова, А.И.Тончев, В.В.Лосев, С.Б.Петков, В.Г.Назаров
Един от основните проблеми при получаването на биопродукти чрез

дълбочинно култивиране се явяват лимитиращите масообменни процеси. Важен
критичен параметър при култивирането им представлява скоростта на
консумиране на кислород и скоростта на отделяне на въглероден диоксид по
време на процеса. Добивът на много първични метаболити, получени по този
начин е предопределен от кинетиката на растежа на клетките, която обикновенно
предхожда началото на биосинтеза на продукта.
Изследвана е зависимостта между степента на трансформацията на субстрат
и количественото изменение на интензивността на кислородния масообмен в
биореактора. Анализирането на съдържанието на кислород и въглероден диоксид
в изходящите ферментационни газова се осъществява с помоща на газов
анализатор Tаndem на фирма Adaptive Biosistems. Тези on-line пресметнати
параметри се корелират емпирично със степента на трансформацията на субстрата
в активна субстанция. Получените експериментални резултати показват
изменението на стойностите на респираторния коефициент в различните фази на
процеса. Експериментите са проведени на 20L и на 3m3 биореактори. Сравнените
показатели илюстрират създаването на модел на биосинтетичния процес, на
базата на който се придобива допълнителна информация за мащабиране на
процеса.
GAM76
КОЛИЧЕСТВЕНО ОПРЕДЕЛЯНЕ НА ГЕНТАМИЦИН В
ПРОБИ ОТ КРЪВНА ПЛАЗМА ЧРЕЗ
МИКРОБИОЛОГИЧЕН МЕТОД
П. Янкова, Ан. Варсанова
171
Настоящия метод е разработен с цел количествено определяне съдържанието
на гентамицин в проби от кръвна плазма, получена от телета третирани i.m.
(интрамускулно) с гентамицин сулфат инжекционен разтвор еднократно в доза
4mg/kg маса.Същността на метода се основава на дифузия на антибиотика в
агарова среда, инокулирана с подходящ тест микроорганизъм. Имайки предвид
възможността в някой от интервалите за вземане на кръвни проби съдържанието
на гентамицин да варира под 100 ng/ml, важно бе да се определи такъв тест
микроорганизъм и условия за провеждане на анализа, чрез които да се постигне
максимална чувствителност на метода. За целта са тествани три
микроорганизъма: B. subtillis, B. pumilus и B. mycoides в различни концентрации и
на различни среди,като най-подходящ се оказа B. mycoides. Установена е
границата на количествено определяне на гентамицин – 60 ng/ml и границата на
откриване на гентамицин - 20 ng/ml. Определен е срока на годност на изходния и
работните сравнителни разтвори на стандартното вещество, съответно 20 дни и
2 дни, съхранявани при температура 4-80С.
Разработеният метод е валидиран по отношение на линейност, прецизност и
точност.
GAM77
DESIGNING A WHOLE-CELL BIOTRANSFORMATION
DOUBLE-PHASE OXIDATION SYSTEM WITH
STREPTOMYCES ROSEOCHROMOGENES
Boris Marinov , D. Koleva, I. Kostova
Whole cells of Streptomyces roseochromogenes, contains a cytohrome P450 which,

in conjunction with two indigenous electron transfer proteins, roseoredoxin and
roseoredoxin reductase, hydroxylates exogenous mevastatin to pravastatin. In intact cells
the natural electron donor is NADH. Trying to design a whole-cell biotransformation
double-phase oxidation system with Streptomyces roseochromogenes was used a known
hydroxylation of mevastatin to pravastatin. In case to design organic : aqueous system
was overcome through the implementation of mevastatin lacton which is low water soluble.
The prolonged productivity of such a system depends on cell viability, since viable cells
are naturally able to regenerate the co-factor.
Cyclohexane, n-Decane, n-Hexadecane, Glycerin-tributyrat, Glycerin-1-monooleat, Soya
bean oil were the solvents that allowed cell activity and viability. The most adequate
phase ratio was 1:4 but it was possible to be used phase ratio 1:1. For all other tested
organic solvents was found to be toxic, causing cell death at any concentration level.
172
GAM78
БИОФИЗИЧНИ ХАРАКТЕРИСТИКИ НА БАКТЕРИАЛНИ
БИОСЪРФАКТАНТИ
Емилия Стоименова, Eвгения Василева-Тонкова*, Михаела Иванова,

Христина Петкова**, Албена Йорданова***, Анна Сотирова*, Данка
Гълъбова*, Здравко Лалчев
Биологически Факултет, СУ “Св. Климент Охридски”, 1164, София
* Институт по Микробиология, БАН, 1113, София
**Химически факултет, СУ “Св. Климент Охридски”, 1164, София
***Институт по Биофизика, БАН, 1113, София
Биосърфактантите са разнообразни по химичен състав повърхностно активни

вещества - нискомолекулни гликолипиди и липопептиди, както и високомолекулни
липопротеини и други. Особено актуална група биосърфактанти са
гликолипидите, към които принадлежи класа на рамнолипидите, продуцирани от
различни бактерии, най – вече от различни видове на рода Pseudomonas.
Напоследък замърсяването на околната среда с органични вещества се явява
сериозен екологичен проблем и все по-често се налага използването на безвредни
и сигурни биологични методи за тяхното отстраняване. Основните организми,
които разграждат въглеводородите по естествен начин и така спомагат за
пречистването и поддържането на баланса на екосистемите са микроорганизмите.
Въглеводород-разграждащите микроорганизми могат да продуцират
биосърфактанти, които понижават повърхностното и междуфазовото напрежение
и улесняват емулгирането на хидрофобните субстрати, при което значитено се
увеличава достъпността им за разграждане.
Целта на настоящото изследване е да се охарактеризират и сравнят
бактериални биосърфактанти, секретирани от щам на Pseudomonas fluorescens
при култивиране върху различни органични субстрати. Направен е
физикохимичен анализ на повърхностните свойства на биосърфактантите, като е
проследена промяната на повърхностното напрежение на културалната течност
при различно рН и различна солева концентрация на NaCl. Микроорганизмите са
култивирани в минерални среди с различни въглероден източници-хексадекан,
керосин, маслинено олио и др. с цел намиране на подходящо практическо
приложение на новоизолираните биосърфактанти.
173
GAM79
КОМБИНИРАНЕ НА МЕТОДИ ЗА ВИДОВА
ИДЕНТИФИКАЦИЯ НА LACTOBACILLUS DELBRUECKII
SSP. BULGARICUS
Савова Т., Уршев З., Спасова М., Петрова И., Ишлимова Д., Алексиева П.
ЕЛБИ Булгарикум ЕАД, София, ул. „Малашевска” 12А
Е-mail: savova.t@lbbulgaricum.bg
Поради необходимостта от бърза идентификация на културите Lactobacillus

delbrueckii ssp. bulgaricus (L. bulgaricus), съхранявани в LBB колекцията (EЛБИ
Булгарикум ЕАД), беше възприета полифазна система за видова идентификация,
включваща три достъпни и икономически обосновани метода. Установяването
на биохимичните характеристики на изследваните култури по отношение на
усвояването на различни въглехидратни източници с помощта на API-50CHL (API,
Bio-Merieux) комбинирахме с определянето на оптичната форма на образуваната
млечна киселина с ензимен кит (Roche) и анализ на EcoRI рестриктния профил на
16S рДНК (ARDRA). В зависимост от получените резултати, при анализа на 155
култури L. bulgaricus, изолирани от домашно приготвено кисело мляко в периода
1968-1972 г., както и на щамове, получени от единични колониии при
допълнителното пречистване на културите, се оформиха 6 групи.
Първата група определихме категорично като L. bulgaricus, като и трите
избрани от нас метода дадоха съвпадение с типовия щам L. bulgaricus ATCC 11842.
Втората група, съгласно данните от ARDRA анализа, беше определена като L.
bulgaricus, но профилът на захарите, получен с API тест кита, показа 53.6-87.1%
идентичност с L. lactis. Допълнителните изследвания на щамовете от тази група с
помощта на макрорестрикционнен анализ (пулсова електрофореза) показаха
голямо генетично сродство между отделните щамове, от които най-добре проучена
е културата LBB.B144. По-ранни изследвания с анализ на тоталните клетъчни
белтъци с денатурираща ПА гел-електрофореза категорично определиха щам
LBB.B144 като L. bulgaricus (Ж. П. Димитров, непубликувани резултати). С тези
допълнителни данни, приехме работната хипотеза, че втората група лактобацили
са от вида L. bulgaricus, които обаче допълнително усвояват N-ацетилглюкозамин
и трехалоза, което води до подвеждащи резултати с API-теста.
В културите от третата група установихме едновременното присъствие на
щамове с фенотипната характеристика от първите две групи и приехме, че е
необходимо допълнително пречистване на съответните изходни култури.
Четвъртата група лактобацили генотипно определихме като L. bulgaricus, но при
тях не се наблюдаваше развитие при API-теста. Тъй като същите култури се
развиват добре на MRS-бульон, предположихме, че индикаторът бромкрезол
пурпур има инхибиращо въздействие. Смяната на индикатора с хлорфенол ред не
промени резултата. В останалите две групи установихме съществени отклонения
в съдържанието на D(-) млечна киселина. Изолираните единични колонии от тези
174
групи съгласно ARDRA анализа отнесохме към L. bulgaricus и L. helveticus.
В заключение, подбраните от нас три идентификационни метода дадоха
достатъчно надеждни резултати при потвърждаване на принадлежността на
изследваните култури към вида L. bulgaricus. В случая с щамовете, генетично
близки до LBB.B144, беше необходимо използуването на данни и от други анализи.
Единичните случаи на присъствие на L. helveticus бяха категорично отчетени от
възприетата идентификационна методика.
GAM80
СЕЛЕКЦИОНИРАНЕ НА ЩАМОВЕ STR.THERMOPHILUS
ЗА УСЪВЪРШЕНСТВАНЕ НА СТАРТЕРНИ КУЛТУРИ ЗА
ДИРЕКТНО ПРИЛОЖЕНИЕ
Пашова-Балтова К., Нинова Н., Уршев З., Михайлова М..

ЕЛБИ Булгарикум ЕАД, София ул.”Малашевска 12А”
Е-mail: baltova.k@lbbulgaricum.bg
Като основен производител на стартерни култури, ЕЛБИ Булгарикум ЕАД

търси нови възможности за оптимизиране параметрите на симбиотичните закваски,
наложили се от години в производството на българските традиционни продукти,
но използувани досега главно за получаване на производствени закваски в
предприятията.
Съвременното развитие на млечния отрасъл все повече налага използването
на закваски за директно приложение./DVS/. Те изместват от приложение
производствените закваски, утвърдили се в продължение на дълги години при
производството на българско кисело мляко и бяло саламурено сирене.
При разработването на закваски за директно приложение, се вземат предвид
много по-строги изисквания към лиофилизираната култура, най-вече по отношение
на нейната активност, а също и по достигане на подходящи технологични
параметри –киселинообразуване по време на технологичния процес, ниско
посткиселинообразуване по време на съхранение, осигуряване на добра
консистенция на продукта.
Приложението на DVS формите налага и нови изисквания към технологичните
качества на щамовете. Една възможна стратегия за усъвършенствуване на
закваските за DVS приложение е обогатяването им със селекционирани щамове
Str.thermophilusл.
Нови 150 щама Str.thermophilus бяха изолирани от домашно приготвени
кисели млека от различни райони на страната и детайлно охарактеризирани. В
технологичната характеристика на проучените щамове са включени важни
технологични параметри –динамика на киселинообразуване до 16 час и 48 час,
при различни температури на инкубиране-430С, 370С, 340С, натрупване на
биомаса, осмотичен толеранс към висока концентрация на въглехидрати,
175
кинематичен и роторен вискозитет, посткиселинообразуване при различни
температури.
Като култури, които в значителна степен могат да допринесат за подобряване
свойствата на DVS закваските, от натрупаните база данни бяха селекционирани
4 щама с къса лаг фаза на развитие, както и 2 структуроподобряващи щама.
Получените нови комбинации стартерни култури, обогатени с подбраните
щамове Str.thermophilus, приложени като DVS показаха по-добри параметри
относно време на коагулация, структура и консистенция на готовия продукт,
посткиселинообразуване.
GAM81
ОЦЕНКА НА ПРОТЕОЛИТИЧНАТА АКТИВНОСТ НА
КУЛТУРИ LACTOBACILLUS DELBRUECKII SSP.
BULGARICUS
Уршев З., Фачикова Н., Пашова-Балтова К., Петрова И.

ЕЛБИ Булгарикум ЕАД, София, ул. „Малашевска” 12А
Е-mail: zoltan.urshev@lbbulgaricum.bg
Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) е широкоизползувана

млечнокисела бактерия при производството на кисело мляко и бяло саламурено
сирене. Общо 153 култури L. bulgaricus съхранявани в LBB Колекцията на ЕЛБИ
Булгарикум ЕАД бяха охарактеризирани по протеолитичната им активност (ПА)
в мляко при култивиране в продължение на 16 часа при две температури по метод
препоръчан от IDF Стандарт 149А:1997. По този метод ПА се дефинира като
количеството образувани Фолин-позитивни аминокиселини и пептиди по време
на ферментационния процес, а резултатите се представят като тирозинов
еквивалент (mg% Tyr).
Разпределението на стойностите на ПА при инкубиране при 42оС и 37оС
показа изключителна нехомогенност на културите от този бактериален вид по
този показател (от 0.3 до 10.7 mg% Tyr). Получените стойности (средно 6.4 ± 2.1
mg%Tyr за 42оС и 6.2 ± 2.1 mg% Tyr за 37оС, n = 153) по правило бяха значително
по-високи от стойностите, установени за щамове Streptococcus thermophilus (0.8 ±
0.8 mg% Tyr, n = 62) и по-ниски от тези, определени за щамове Lactobacillus helveticus
(10.1 ± 2,5 mg% Tyr, n = 16). За изследваните култури L. bulgaricus беше установена
слаба статистически значима положителна корелация между изменението на
активната киселинност (“pH) и стойностите на ПА, определени в 16 час и при
двете температури (корелационен коефициент r = 0.34, p<10-4 за 42оС и r = 0.40,
p<10-6 за 37оС). При по-ниската температура на инкубиране във ферментиралите
с културите L. bulgaricus млека “pH беше статистически достоверно по-малко от
това в млеката ферментирани при 42оС (средна разлика от 0.24 ± 0.25 pH единици),
докато разликата в ПА на културите, определена на 16 час съответно при двете
176
температури, не беше статистически достоверна.
Варирането на температурата на инкубиране (37оС-42оС) влияе много силно
върху темпа на киселинообразуване на културите L. bulgaricus, но не и върху
общото количество натрупани продукти на протеолизата, като едно възможно
обяснение е наличието на широк температурен интервал на оптимално действие
на протеолитичните ензими на L. bulgaricus. Значителната вариация на ПА на
изследваните култури е добра предпоставка за селекцията на щамове L. bulgaricus
с ниска/висока ПА в зависимост от приложението на съответните стартерни
култури.
GAM82
АКТИМИКРОБНА АКТИВНОСТ НА МЛЕЧНО КИСЕЛИ
БАКТЕРИИ,
ИЗОЛИРАНИ ОТ БЪЛГАРСКИ РЪЖЕНИ КИСЕЛИ ТЕСТА
Любомира Йочева1, Гергана Добрева2, Радка Василева1 и Стефка Антонова-

Николова2
1
– Институт по криобиология и хранителни технологии
2
– Катедра “Обща и промишлена микробиология”, СУ “Св. Кл. Охридски”
Млечно киселите бактерии са сред най-изучаваните микроорганизми, поради

доказаната си способност да синтезират различни биологично активни вещества
– бактериоцини, биологично активни пептиди, екзополизахариди.
От друга страна, в резултат на метаболитната си активност, някои от тях
разграждат съдържащи се в млякото или в брашното белтъци с алергенни
свойства, с което повишават хранителната им стойност.
Разнообразните възможности за приложение на млечно киселите бактерии
насочва изследователите към проучване и на други екологични ниши, освен
млечно киселите продукти, с цел получаване на изолати с желани биологични
свойства.
При проучване на микрофлората на кисели теста, получени чрез спонтанна
ферментация на българско ръжено брашно от различни реколти, са изолирани 88
щама млечно кисели бактерии, на които са изучени различни метаболитни
активности.
Културалните, морфологичните и физиологичните свойства на изолатите ги
отнасят към родовете Lactobacillus и Pediococcus. При изследване антимикробната
им активност спрямо патогенни или причиняващи развала на хляба тест
микроорганизми беше установено, че най-голям е броят на млечно киселите
бактерии, потискащи в различна степен развитието на Bacillus cereus,
предизвикващ картофена болест по хляба, и Staphylococcus aureus. Един от
изолатите показва слаба активност спрямо Listeria sp. Не бяха установени
щамове, инхибиращи тест-микроорганизма Escherichia coli.
177
Изследването е финансирано с договор 47/2006г от Фонд “Научни
изследвания” към СУ “Св. Кл. Охридски”.
GAM83
MILK-CLOTTING ENZYMES FROM SUBMERGE CULTI-
VATED BASIDIOMYCETES
T. Dmitrieva, I. Feist, M. Shamtsyan

St. Petersburg State Technological Institute (Technical University), Moskovsky
Milk clotting enzymes from higher basidiomycetes are a promising source to substitute
rennin in cheese making. The aim of our studies was a screening of the producers of milk
clotting enzymes among various species of higher basidiomycetes.
The quality of cheese significantly depends from enzyme preparations used for milk
clotting. For this purposes animal enzymes extracted from rennet are usually used. Although
various microbial coagulants are developed as inexpensive substitutes for animal enzymes,
but they does not found wide utilization in the countries of developed cheese making,
were are used mainly for the production of cheddar type cheeses.
The fruit bodies, submerged mycelium, grown on various media, as well as the culture
broth of several higher basidiomycetes were studied for milk clotting activity. Mushrooms,
which fruit bodies, mycelium or culture broth posses high milk clotting and low proteolytic
activities were selected. Growth media composition was optimized to achieve higher
enzymatic activity.
Due of separation of enzymes the ratio of milk clotting and general proteolytic activities
in the preparations highly increased.
Milk clotting enzymes were purified and characterized.
GAM84
INTENSIFICATION OF YEAST BIOMASS ACCUMULATION
AND ETHANOL FERMENTATION PROCESSES
I. Koltyga, T. Dmitriyeva, M. Shamtsyan

St. Petersburg State Institute of Technology (Technical University), Russia, St.
Petersburg, 198013, Moskovsky prospect, 26 E-mail: shamtsyan@yahoo.com
К факторам, вызывающим замедление брожения, относят: условия

брожения, ис-ходный состав сусла (содержание сбра-живаемых сахаров,
возможный недо-статок витаминов и азотистых веществ), наличие в среде
компонентов, отрицательно влияющих на дрожжи (тяжелые металлы, остатки
178
пе-стицидов и т. д.), технологические при-емы при переработке сырья и
полупро-дуктов, накопление в сбраживаемой среде продуктов метаболизма
(этиловый спирт, углекислый газ, жирные кис-лоты) и др.
Один из путей интенсификации про-изводства в пивоварении, виноделии,
спиртовой и безалкогольной промыш-ленности — применение сорбирующих
материалов на различных технологичес-ких стадиях. Однако, из всего разнообразия
применяемых сорбирующих материалов лишь мень-шая часть пригодна для
использования на стадии брожения, еще меньше спо-собны сорбировать наиболее
токсичные для дрожжей компоненты сусла — жир-ные кислоты с короткой цепью,
токсичные вещества (микотоксины). Боль-шинство сорбентов, обладающих
спо-собностью удерживать жирные кисло-ты, вместе с токсичными компонентами
выводят из среды вещества, определяю-щие сортовые характеристики готового
продукта — вкус и аромат. Наилучшим средством для решения проблем
недоброда были бы сами дрожжевые клетки или те их составляющие, которые
сорбируют жирные кислоты.
В наших исследованиях изучалась сорбционная способность клеточных стенок

высших грибов (хитинглюканового комплекса, глюканов). Установлено что
исследуемые вещества интенсификацировали выделения спирта в процессе
брожения. путем сорбции компонентов оказывающие негативное воздействие на
этот процесс.
Полученные результаты могут быть использованы доля решения различных
задач в пивоварении, виноделии, спиртовой и безалкогольной промышленности.
GAM85
БАЛИСТИЧНА ДЕЗИНТЕГРАЦИЯ НА БИОМАСА ОТ
LACTOBACILLUS DELBRUECKII SUBSP. BULGARICUS
BTCC 50
Диляна Николова, Валентин Савов, Яна Евстатиева, Светла Илиева, Пенчо

Далев, Атанас Атев
Биологически факултет, Софийски Университет „Св. Климент Oхридски”,
катедра Биотехнология
Пробиотиците имат редица доказани клинични и други полезни ефекти,

благодарение на които могат да се използват както с превантивна цел, така и
като активни агенти с терапевтични свойства. Изследванията върху
пробиотичните свойства на млечнокиселите бактерии, показват участието на
компонентите на бактериалната клетъчна стена в реализирането на тези полезни
свойства. За изолирането на тези компоненти най-често се прилага дезинтеграция
на клетките с последващо фракциониране.
Балистичната дезинтеграция на микробни клетки като физичен метод се влияе
179
от редица параметри - тип и количество биомаса; интензивност на разбъркване;
времетраене; количество балистичен товар и др.
В настоящата работа са проведени експерименти, свързани с оптимизиране
условията за балистична дезинтеграция на биомаса от щам Lactobacillus
delbrueckii ssp bulgaricus BTCC 50. Проучено е влиянието на възрастта на
културата млечникисели бактерии, съотношението между количеството биомаса
и балистичен товар, времетраене и други параметри на процеса дезинтеграция.
На базата на осъществената експериметална работа са установени оптималните
стойности на основни параметри на процеса на дезинтеграция на клетки от щам
Lactobacillus delbrueckii ssp bulgaricus BTCC 50 и е постигнат ефект над 17%.
Благодарности: Настоящата експериментална работа е осъществена с
финансовата подкрепа на Фонд Научни Изследвания при МОН по проект № ВУ-
Б-7/05.
GAM86
HYBRIDIZATION OF LACTOBACILLUS PLANTARUM
226-15 AND LACTOBACILLUS CASEI SUBSP. CASEI C
A. Slavchev, I. Murgov, Z. Denkova
A successful hybridization of protoplasts of Lactobacillus casei subsp. casei C and

Lactobacillus plantarum 226-15 was accomplished.
It was shown, that as a result of the hybridization was obtained a heterogenic population
and amongst its clones were selected strains with high reproductive ability, resistance to
the conditions in the stomach and the intestines, non-sliming, etc.
The recombinant clones Lactobacillus LPC 6T, Lactobacillus LPC 40 and Lactobacillus
LPC 52, which were distinguished for accelerated coagulation of milk, improved acid-
forming and flavour-forming capabilities were selected.
GAM87
INVESTIGATION OF THE INHIBITORY EFFECT OF LACTIC
ACID BACTERIA ON THE CELLS OF ESCHERICHIA COLI
NBIMCC 8739
P. Nedelcheva, Z. Denkova, R. Nikolova
The inhibitory effect of growing cultures of Lactobacillus plantarum 226-15,

Lactobacillus casei subsp. casei C, Lactococcus lactic subsp. lactis l4 and Pediococcus
cerevisiae 2P on the cells of Escherichia coli NBIMCC 8739 was investigated during their
mixed cultivation in skimmed milk at temperatures 23±1°С and 37±1°С.
180
It was established, that during the first 6 h the viable cell counts of both the lactic acid
bacteria and Escherichia coli NBIMCC 8739 increased. During the next 12-24 h the growth
of Escherichia coli NBIMCC 8739 gradually slowed up and held up. This effect was more
distinct during the mixed cultivation of lactic acid bacteria and E. coli at 37°С.
GAM88
THE EFFECT OF DNA REPLICATION ON THE REPAIR OF
DAMAGED
PLASMIDS IN SACCHAROMYCES CEREVISIAE
Miglena Koprinarova, Anastas Gospodinov, George Russev
One of the mechanisms of repair of DNA and maintenance of genome integrity is the
homologous recombination. For homologous recombination repair there should be an
undamaged sequence used as a template. The obvious time period for searching and
finding such an undamaged sequence is the S-phase of cell cycle during which the
eukaryotic DNA is doubled. The close bond between DNA replication and repair is logical
but not firmly established.
The subject of our work was in vivo examination of the repair of plasmids damaged
with trioxalen using yeast Saccharomyces cerevisiae as a host system. Trioxalen causes
interstrand cross-links in the molecule of DNA for the repair of which homologous
recombination and NER (Nucleotide excision repair) is required in yeast.
We transformed previously damaged centromere plasmid (YCp50) in temperature-
sensitive degron (td) mutant strain (YJT18) Saccharomyces cerevisiae. That was a heat-
inducible Cdc45 degron mutant that promotes rapid degradation of Cdc45p at the restrictive
temperature of 37°C. The Cdc45p plays a central role in both initiation and elongation
phases of chromosomal DNA replication. We used a wild-type strain with the same
background as a control. At certain intervals we took aliquotes of yeast culture, isolated
DNA and examined the rate of repair of previously damaged plasmids by Competitive
PCR.
The results showed 50% decrease in the repair capacity of the temperature-sensitive
degron mutant strain in comparison with the wild-type strain indicating that the process
of homologous recombination repair might be coupled to DNA replication in the yeast
strains Saccharomyces cerevisiae.
GAM89
AGEING IN BREWING YEAST
Tamara Ginova, Silvia Mileva

Institute of cryobiology and food technology, Sofia
181
Yeast ageing include changes that occur during cell lifespan leading to senescence
and death. Brewing yeast Saccharomycs cerevisiae ageing refers to some distinct
physiological states : stationary phase, stored yeast, serially used yeast population,
individual age of the cell. Yeast ageing is accompanied by several modifications –
morphological, metabolic, physiological. Cells in extended stationary phase are referred
as aged cultures because the population are composed of individual cells with young and
aged phenotype. Aged cells are alive and in different rate metabolically active, but not
reproductive. Survival in stationary phase during fermentation depends on environmental
stresses. Stored yeast that are exposed to some stress factors (nutrient starvation, low
temperature) and yeast that are used for several successive fermentations are also studied.
We report an extensive changes in cell size, surface wrinkles, granular appearance and
increases of generation time that characterized aged phenotype in age-heterogeneous
population. Mortality in stored yeast increases with diminished of cell glycogen content
Impact of ageing on flocculation properties and fermentation performance of the yeast is
discussed.
GAM90
GROWTH INHIBITORY PROPERTIES OF CHALCONES
AGAINST VARIOUS YEAST SPECIES
K. L. Lahtchev1, D. I. Batovska2, St. Parushev2, V. Bankova2

1
Acad. G. Bonchev Str. Bl. 26, Sofia 1113, Bulgaria
2
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy
of Sciences, Acad. G. Bonchev Str. Bl. 9, Sofia 1113, Bulgaria
Chalcones are natural or synthetic flavonoid compounds with a common structural skeleton
of 1,3-diphenyl-2-propen-1-one. They possess various biological properties including antifungal
activity. For deeper insight of its antifungal mechanisms we first synthesized a series of 19
chalcones with diverse combinations of substituents in ring B and further examined their
structure-activity relationships. The chalcones were tested for their growth inhibitory activity
against 32 strains belonging to baker yeast Saccharomyces cerevisiae (13 strains),
methylotrophic yeast Hansenula polymorpha (16 strains) and lactic Klyveromyces lactis (3
strains). All the strains employed were with defined ploidy and carried specific mutations in
either the biosynthetic pathways or defence systems. The MICs determination revealed that
the most active chalcones were DB1, DB9, DB12, DB24 and DB48 while the rest of the
compounds exhibited antifungal activity at very high concentrations only. The strains
belonging to Kl. lactis appeared to be the most sensitive among the species tested. S.
cerevisiae strains were more resistant to the parent chalcone DB1 then H. polymorpha, but
the opposite was true for DB24. The MICs depended on both the chalcone concentrations
and the strain cell density. At high chalcone concentrations and low cell density a strong
killing effect was observed. Clear differences were observed between the sensitivities/
182
resistances of the strains belonging to the same species. These differences depended on the
strain genotypes and presence of specific mutations. While no effect was evident for the
mutations controlling purine, pyrimidine and some amino acids biosynthetic pathways we
found that H. polymorpha strains carrying mutation gsh1 (impairment of glutathione
synthesis) conferred increased sensitivities to some of the chalcones. This result suggests
that glutathione is important defence against chalcone action.
GAM91
ELECTRO-OPTICAL METHOD AS A NEW APPROACH OF
INVESTIGATION AND DISCRIMINATION OF TWO STRAINS
ESHERICHIA COLI
A. Gurova-Chausheva1, E.Velichkova1, R. Aleksandrova2, S. Danova2, S. P. Stoylov1

1
Institute of Physics Chemistry, Bulgarian Academy of Science, Bulgaria, Sofia
1113, Akad. G. Bonchev Str. Bl.11
2
of Science, 26, Acad.G. Bontchev Str. , 1113 Sofia, Bulgaria
In present study two physico-chemical methods (electro-optics and

microelectrophoresis) were used to investigate Gram negative bacteria Esherichia coli.
The used electro-optic method is based on light scattering by biological particles under
the influence of electrical field, which orients them depending on the intensity and
frequency of the electric field, surface electrical, optical and geometrical properties of
cells. The target strains HB 101 and XL-1-blue (the last one was obtained from another
strain treated by Ca2+, undergone electroporation and so it has become capable to accept
an alien DNA) were comparatively investigated .Our purpose was to evaluate the
discriminative power of the above mentioned methods. Special attention was paid on the
recording of the detectable and reproducible changes.
Considerable differences in the surface electrical, optical and geometrical properties of
tested strain’s cells were established. Thus the differences in characteristics of so called
electro-optical pulses, their amplitudes and relaxation times, were large and significant
and probably may be used for correct further identification and discrimination. Moreover
the electro-optical pulse in question can be masured in seconds, because the speed is one
of the advantages of the electro-optic method. In conclusion the applied electro-optical
method might be considered as a new, promising approach to the investigation and
identification of different microbiological objects from Gram negative taxons.
183
GAM92
APPLICATION OF CO-POLYMER MICROPARTICLES FOR
IMMOBILIZATION OF TRYPSIN
Todor Ivanov1, Michail Kamburov1, Viara Ivanova2*, Jordan Hristov3

1
Department of Biotechnology and 3Department of Chemical Engineering, Univer-
sity of Chemical Technology and Metallurgy, 8 Kl.Ohridsky Blvd., 1576, Sofia,
Bulgaria
2
Department of Organic Chemistry and Microbiology, University of Food Technolo-
gies, 26 Maritza Blvd., 4002, Plovdiv, Bulgaria
Co-polymers were synthesized on the basis of acrylonitrile and maleinic anhydride.

From these co-polymers were formed spherical particles by extrusion. Fine and coarse
powder supports were obtained by grinding of the dry co-polymers. Trypsin was
immobilized on the three supports. From initial mixtures, containing the monomers in
proportion 72% AN - 28% MA and 68% AN – 32% MA, were obtained two co-polymers
PANMA1 and PANMA2, containing 2.4 % and 3.7% maleinic acid, respectively. The polymer
yields were between 70% and 90%. After the polymerization step, the co-polymers were
filtered, washed with water and methanol and dried at 50 ºC. Finally, the particles were
fragmentized and sieved by standard sieves and were separated in three main groups –
particles with size from 100 to 200 ìm, from 200 to 500 ìm and from 500 up to 1000
ìm. The IR-spectrum of the polymers showed sharp peaks at 2200 cm-1, specific for the
nitrile íCa”N group, at 1700 cm-1 - specific for the carbonyl group íC=O, and for the specific
absorption for íOH, in the region of 3000 cm-1, specific for the carboxylic group (3200 – 3300
cm-1). Particles with size between 100 and 200 ìm were more suitable for trypsin
immobilization. Up to 34.0 mg trypsin/g wet support were immobilized on these
particles. The bounded enzyme quantity was two times lower on supports with size > of
500 ìm and only 4 mg/g on extruded spherical particles with diameter between 500
and 1000 ìm.
184
GAM93
IMMOBILIZATION OF TRYPSIN ON CO-POLYMERS OF
ACRYLONITRILE AND MALEINIC ANHYDRIDE
Todor Ivanov1, Michail Kamburov1, Viara Ivanova2*, Jordan Hristov3

1
Department of Biotechnology and 3Department of Chemical Engineering, Univer-
sity of Chemical Technology and Metallurgy, 8 Kl.Ohridsky Blvd., 1576, Sofia,
Bulgaria
2
Department of Organic Chemistry and Microbiology, University of Food Technolo-
gies, 26 Maritza Blvd., 4002, Plovdiv, Bulgaria
Abstract. Optimization of conditions for covalent coupling of trypsin on co-polymers

of acrylonitrile and maleinic anhydride was achieved by variation of pH and trypsin initial
concentration. The maximal immobilized amounts of enzyme (as protein per g support) on
co-polymers PANMA1 and PANMA2 at pH 4.0 were about 50.0 and 34.0 mg/g wet support,
respectively. The relative amidase activity (substrate BAPNA) represented 17 and 19% of
the corresponding enzyme activity in native state. Optimum protein concentration in the
immobilization solutions was determined to be about 12.5 mg/ml, and at this concentration
47.0 and 28.0 mg trypsin/g wet support were bounded on these two polymers. At pH 7.5,
the maximal relative activities of immobilized enzyme on these co-polymers reached 23 and
14%, but at lower bounded protein quantities - 4.0 and 8.0 mg coupled protein/g wet
support. The maximal bounded protein quantities were 14.0 and 27.0 mg trypsin/g wet
support. Trypsin activity was also determined using macromolecular substrates, such as
casein. Immobilized on PANMA1 trypsin with 17% relative amidase activity retained only
5% of its proteolytic activity. The immobilized trypsin preparations were tested for
application in affinity chromatography. About 40% of the coupled enzyme was capable to
bind specifically aprotinine.
185
VETERINARY MICROBIOLOGY
VM1
DEVELOPMENT AND APPLICATION OF RABBIT
POLYCLONAL MONOSPECIFIC AFFINITY PURIFIED ANTI-
BODY AGAINST SYNTHETIC SEQUENCE FROM PEPTIDE
P32 TO DETECT SHEEP POX VIRUS BY IMMUNOHIS-
TOCHEMICAL POLYMER STAINING TECHNIQUE ON
FROZEN TISSUE SECTIONS
Bumbarov V., Eligulashvili R., Yadin H.

Kimron Veterinary Institute, Department of Virology, Beit Dagan, Israel
Conventional laboratory diagnosis of Sheep pox disease is based on transmission

electron microscopy inspections, virus isolation in cell culture and confirmation by virus
neutralization. However virus isolation is a lengthy procedure up to few weeks.
A new Rabbit Polyclonal Affinity Purified Monospecific antibody against synthetic
sequence from peptide P32 of Sheep pox virus was elaborated and developed for rapid
diagnosis of Sheep pox infection.
In this report is discussed advantage at usage of Polymer Staining Techniques of
Immunohistochemistry on cryostat tissue sections using Rabbit Polyclonal Affinity Purified
Monospecific Antibody against synthetic sequence from peptide P32 of Sheep pox virus
as a primary antibody. The interpretation of results is very simple. All of procedure is up
to 2 hours.
VM2
НАХОДКИ И СЕРОТИПИЗИРАНЕ НА LISTERIA
MONOCYTOGENES В МЕСО ОТ ПИЛЕТА БРОЙЛЕРИ
Румен Караколев
Регионален диагностичен ветеринарномедицински институт - Велико Търново
За петгодишен период (2000 – 2005 г.) са изследвани 464 проби от пилета

бройлери - 224 от охладени и 240 от замразени пилета. Общо положителни за L.
monocytogenes са 65 материала или 14,0 % от изследваните. От охладени пилета
са получени 42 изолата L. monocytogenes (18,75 %), а от замразени пилета – 23
изолата от същия вид (9,58 %). След извършеното серотипизиране, 57 изолата са
отнесени към серогрупа І и 8 – към серогрупа ІІ. От изследваните пилета са
получени и 9 изолата L. innocua.
186
VM3
ОПТИМИЗИРАНЕ НА ПВР-ДГГЕ ЗА ДИРЕКТНО
ДОКАЗВАНЕ И МОЛЕКУЛЯРНО ТИПИРАНЕ НА
CAMPYLOBACTER JEJUNI И CAMPYLOBACTER COLI В
ЦЕКАЛНИ ПРОБИ ОТ ПТИЦИ
Х. Найденски
Институт по микробиология “Стефан Ангелов” - БАН
Методът на ПВР-ДГГЕ (полимеразноверижна реакция - дегенеративна

градиентна гел електрофореза) е оптимизиран и успешно приложен за директно
доказване и типиране на Campylobacter jejuni и Campylobacter coli в цекални проби
от бройлери без предварително набогатяване.
27 щама Campylobacter jejuni и 1 щам Campylobacter coli бяха изследвани
посредстом ПВР-ДГГЕ, използвайки праймерите WM12/WM22, произхождащи
от 3’ края на flaA гена и 3’ края на интергенния участък, разделящ тандемно
ориентираните гени flaA и flaB. Получените ДНК фрагменти бяха разделени в 13
групи, което е пряко доказателство за значителното разнообразие от щамове
сред популацията от вида Campylobacter jejuni. Освен това, посредством ПВР-
ДГГЕ бяха доказани бактерии от вида Campylobacter jejuni в изкуствено
контаминирани цекални проби, както и бактерии от видовете Campylobacter jejuni
и Campylobacter coli в естествено контаминирани проби. Оптимизираният
протокол на ПВР-ДГГЕ позволява надеждна и бърза детекция и идентификация
на Campylobacter jejuni и Campylobacter coli в цекални проби от птици - бройлери
и носачки, с граница на позитивиране от 1 х 104 кое/мл.
VM4
COMPARATIVE RESEARCHING OF PH IN SOME MUSELS
FROM SACRIFIED ANIMALS
A.Kuzelov; O. Kirovska Cigulevska

MIK Sv. Nikole; Sveti Nikole, Macedonia e-mail; kuzelovaco@yahoo.co.uk
JZU ZZZ – Skopje; 1000 Skopje, Macedonia, e-mail; kicok38@hotmail.com
Some of the most important factor for identifying meat quality is pH; witch is about 7
in the very first moment from sacrificing animals. That very first moment is known as
moment for starting physical, biochemical and structural changes in the meat structures.
Intensity of those changes depends of animal condition before sacrificing, genetically
code, stress exposure of animals and muscle structure post mortem. The main process
after sacrificing animals is relieving of glucose and milk acid in the muscles. That is the
reason for decreasing of pH factor and getting a PSE meat.
187
In the read meat muscles that glucose relieving process has a low intensity, which
consequence is low pH. Both of the processes are decreasing for the meat quality.
Our researching for PSE and DFD meat in some special kind of pork legs (yorkshire and
landrace). Researching was improved in muscle M. quadriceps femoris and M.
semitendineus after 30 minutes and 24 hours from sacrificing of female animals. These
researching show us that in 13% from male animals had PSE meat and 12% from female had
to. DFD meat had only 2% from the male animals.
PSE meat (low quality meat) had bigger percentage that DFD meat in exploring dad
animals.
VM5
ЕКОЛОГИЧЕСКИ АЛТЕРНАТИВИ НА
АНТИБИОТИЧНАТА ПРОФИЛАКТИКА И ТЕРАПИЯ
Христо Арнаудов1, Румен Караколев2, Пенка Калчева3

1
ВТУ “Св.Св. Кирил и Методий” - Велико Търново,
2
РДВИ – Велико Търново,
3
СД „СИНАПС & ЕСКЛ” – Велико Търново
След триумфа на антибиотиците и сулфонамидите от средата на 20-ти век, в

световната литература напоследък се появи тенденция на повишено внимание
къв естествените лечебни източници и преди всичко към билките.
В настоящата статия се прави преглед на предлагани от различни фирми
лечебни и профилактични продукти на билкова основа, за ветеринарната
медицина. Прави се оценка на тяхната ефикасност и безвредност. Изтъква се, че
използвайки традиционните познания върху дрогите и съвременните научни
постижения /ултразвукова техника, биотехнологии и пр./ могат да се получат
ефикасни и безвредни конкуренти на антибиотиците от етерични растителни масла,
полигликани, алкалоиди и др.
Авторите дават пример с получен от тях продукт от етерично масло от
Origanum vulgaris и микробиялният полигликан Xanthan gum. Препаратът е
ефикасен срещу десетки патогенни микроорганизми, като същевременно не
индуцира появата на антибиотикова резистентност.
В заключение се препоръчва да се извърши цялостен скрининг на богатата
българска флора, който ще подпомогне създаването на широка гама от
медикаменти, биостимулатори, нутритиви и пр., като алтернатива на антибиотици,
сулфонамиди и химически стимулатори.
188
VM6
ЛЕКАРСТВЕНА УСТОЙЧИВОСТ НА БАКТЕРИИТЕ ОТ
РОД MORAXELLA, ИЗОЛИРАНИ ОТ ЗАЙЦИ С
ПНЕВМОНИЯ
С. Иванова1, Н. Коруджийски1, Т. Гълъбинова2 Б. Митов1

1
Национален Диагностичен Научноизследователски Ветеринарно Медицински
Институт- гр. София,
2
Институт за Контрол на Ветеринарни Продукти – гр. София
По методите на микробиологията са изследвани бели дробове от 174 домашни

зайци с пневмония. Изолирани са и са определени таксономично 37 щамове от
група Moraxella.
Обсъжда се значението на тези щамове за пневмонията и тяхната лекарствена
чувствителност във връзка с терапията.
Провеждат се опити по метода на двукратните серийни разреждания в
геометрична прогресия.
Установява се, че щамовете Moraxella са устойчиви на В- лактами,
тетрациклинии някои аминогликозиди., но са чувствителни спрямо
флуорохинолони, което е особено важно за профилактиката и терапията на
пневмониите.
VM7
ДЕЗИНФЕКЦИЯ НА ОБЕКТИ В ПЧЕЛАРСТВОТО,
КОНТАМИНИРАНИ С PAENIBACILLUS ALVE И
ASCOSPHAERA APIS
Калинка Гургулова*, Даринка Илиева*, Йордан христов*, Стоил Караджов*,

Емил Илиев**
*
Национален диагностичен научноизследователски ветеринарномедицински
институт, София
**
Национална ветеринарномедицинска служба, София
Изпитана е биоцидната активност на Vircon S® срещу 3 щама на Paenibacillus

alvei – причинител на европейския гнилец и срещу 3 щама на Ascosphaera apis –
причинител на аскосферозата, като ефикасността е доказана с неутрализиращ
разтвор. Бактерицидното и фунгицидното действия бяха установени по метода
на количествено разреждане на суспензиите и чрез контаминирани тест-носители.
Естериментите бяха извършени в лабораторни условия с различни концентрации
и експозиции.
189
Установено е, че 0,5 % Virkon S® показва гермицидна активност срещу P.
alvei за 30 min и 1% Virkon S® е ефективен срещу A. apis за 15 min.
VM8
МИКРОБИОЛОГИЧНИ ПРОУЧВАНИЯ ПРИ
ПСЕВДОТУБЕРКУЛОЗАТА ПО ОВЦЕТЕ И КОЗИТЕ
Н. Коруджийски, С. Иванова, Б. Гюров, Д. Тодоров, Цв. Дикова

Институт – гр. София
Проучванията са проведени при дедуктивни условия с 45 овце и 15 кози със

съмнение за псевдотуберкулоза.
Постмортално при овцете или при проведения Stemping out са взети
материали от абсцедирани, казеозни лимфни възли и вътрешни органи.
При козите от живи животни са изследвани, абсцедирали долночелюстни и
ингвинални лимфни възли и форункули от млечната жлеза.
Получените микробиологични резултати показват, че псевдотуберкулозата
при овцете и козите е полибактериална инфекция при която се изолират
предимно представители на род Corynebacterium / C. pyogenes, C.
pseudotuberculosis, C.bovis /, род Streptococcus / групи С и Д / и най- често
Streptococcus група А.
VM9
СРАВНИТЕЛНИ ПРОУЧВАНИЯ ВЪРХУ МЕТОДИТЕ ЗА
ДОКАЗВАНЕ НА MYCOBACTERIUM BOVIS ПРИ
ЖИВОТНИ
Магдалена Боновска, Ст. Милашки, А. Абасс, Т. Савова, Е. Гюрова

Централен Научноизследователски Ветеринарномедицински Институт, София
Използваните рутинни диагностични методи (патологоанатомичен,

бактериологичен и биологичен), са сравнени с въведения в лаборатория
“Туберкулоза” към НДНИВМИ молекулярно биологичен метод (PCR). За целта
в продължение на 4 години са изследвани тъканни суспенсии от лимфни възли
(111 броя проби), бял дроб (32), черен дроб (19), сърце (10), млечни проби (10),
носни секрети (10) и кръв от реагирали съмнително и положително на туберкулин
крави от различни стопанства в страната.
От използваните методи най-ниска е чувствителността на микроскопското
изследване (МИ) -средно 86%, по-висока на бактериологичното (БАК) - 94,8%,
190
на патоанатомичното (ПА) - 97% и на PCR - 98,94%. Най-висока е чувствителността
на биологичния метод (БИО) - 100%, проведен с морски свинчета.
Средният процент на съвпадение между отделните стандартни методи и PCR е
съответно: PCR/МИ –89,46%, PCR/БАК – 96,52%, PCR/ПА – 98,33%% и PCR/БИО
– 100%.
Получените резултати показват, че по чувствителност PCR метода съвпада
100% с биологичния метод и е по-чувствителен от останалите стандартни методи.
Въвеждането на PCR може да отмени използването на биопроби. Като се вземе
предвид и бързината на реакцията, убедително се доказва възможността за
въвеждане на метода като дъпълнителен диагностичен тест, който да подобри и
ускори поставянето на диагноза туберкулоза.
VM10
INFLUENCE OF KARBOFURAN ON CAPABILITY OF
FUSARIUM MONILIFORME TO PRODUCE FUMONISINS
UPON MAIZE
Lidiya Borisova, Yuliana Tasheva, Velichka Vrabcheva

Nacional Diagnostic and Research Veterinary Medical Institute “ Prof. dr George
Pavlov “ – Sofia
It,s well known that mouldy of Fusarium species / Fusarium moniliforme /, producing
fumonisins, are contaminators of maize in our country / Popova, T., 1984; Borisova, L.,
2004 /. In fact that carbamate insecticide Karbofuran is widely used for treatment of maize
against storehouse,s pests, it will be interesting to investigate the influence of this
insecticide upon fumonisins,s production in storage of grain.
The aim of our work was to investigate the influence of Karbofuran, especially the time
of adding of insecticide to contaminated substrate, on production of fumonisins.
The results show that effects of Karbofuran in dose 8 g/kg maize /concentracion used
in treated seed-corn/, as inhibitor of biosynthesis of fumonisins, reduse with increasing
age of culture; on the 10 –th day there is no difference between control /non-treated/ and
treated culture; contents of fumonisins on the 3-th week after contamination with
F.moniliforme both the samples are 12 ppm. The contents of fumonisins in maize, in which
Karbofuran was adding together with contamination of substrate is 4,12 ppm. The pesticide
is most active inhibitor of production of mycotoxins as the Karbofuran is added 5 days
before contamination of substrate with F.moniliforme – the content of fumonisins is 0,76
ppm.
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VM11
DETERMINATION OF PSEUDOMONAS
AERUGINOSA IN BULL AND BOAR SEMEN
BY REACTION CO-AGGLUTINATION
Georgi Vasilev
Regional Research Institute of Veterinary Medicine, Stara Zagora, Bulgaria
Investigations were carried out on 118 samples of bull semen and 94 samples of boar
semen aimed at determination of P. aeruginosa by reaction co-agglutination. All samples
with P. aeruginosa, determined by the conventional microbiological methods, manifested
positive co-agglutination at 3 – 8-min intervals. All the control samples and all the samples
free from pseudomonas microorganisms did not react. These results would encourage
further investigations in search of other reliable methods for improvement of semen control.
Key words: Pseudomonas aeruginosa, Co-agglutination, immunization, semen.
VM12
EXPERIMENTS OF INDUCTION OF TOXIGENOUS
MUTANTS OF CLOSTRIDIUM PERFRINGENS TYPE C
E. Iliev*, D. Ilieva**, M. Petkov*, N. Korudgiysky**

*National Veterinary Service, Sofia
**National Diagnostic and Research Veterinary Institut “Prof. Dr. G.Pavlov, Sofia
Comparative studies are carried out to select Clostridium perfingens type C cultivated
on modified agar of Zeissler in six variants with different content of defibrinated blood of
rams. As a result from the experiments the level of toxicity is studied on 4 generations
daughter colonies of strain M. The isolated colonies of Cl.perfringens type C stabilized on
liquid media with the highest blood content keep their toxicity for more than 75 days.
VM13
ПРОМЕНИ В ПАТОГЕННИЯ ПОТЕНЦИАЛ НА YERSINIA
ENTEROCOLITICA ПРИ СЪХРАНЕНИЕ НА
КОНТАМИНИРАНО СВИНСКО МЕСО
М. Илиев, Х. Найденски
Патогенните серотипове на Yersinia enterocolitica и заболяванията, асоциирани
192
с тях имат нарастващ социален и икономически ефект върху обществото. За
реализиране на техният патогенен потенциал са отговорни редица хромозомни и
плазмидно детерминирани фактори на вирулентност.
Настоящето изследване има за цел да определи настъпващите промени в
патогенния потенциал на Y. enterocolitica при различни температури и интервали
на съхранение на контаминирано свинско месо, чрез мониторинг на
преживяемостта и плазмидното носителство сред бактериалните клетки.
Разработена е селективна хранителна среда за мониторинг на фенотипната
експресия на плазмида на вирулентността (pYV), както и ПВР за генотипен
мониторинг. Изследвани са промените в плазмидното носителство на 6 щама Y.
enterocolitica от серотиповете O:3, O:8 и O:9.
Получените резултати разкриват изразена серотипна хетерогенност в
преживяемостта и плазмидната дисоциация. Регистриран е спад в
преживяемостта, детерминирана от дозата на инокулума, продължителността и
температурният режим на съхранение на месото. При съхранение на месото на
+4oC тя е по-ясно изразена при щамовете от силно патогенния серотип O:8, в
сравнение с щамовете от по-слабо патогенните серотипове O:3 и O:9. При силно
патогенните серотипове се наблюдава по-слабо изразена плазмидна дисоциация
(до 32%), за разлика от по-слабо патогенните серотипове при които загубата на
pYV достига до 54% за O:9 и 63% за O:3. Подобен ефект се наблюдава при
съхранение на месото при %20 oC. Паралелно провежданият мониторинг върху
преживяемостта и плазмидната дисоциация посредством ПВР потвърждава
фенотипните изследвания и позволява бърза и надеждна детекция и диференциация
на pYV+ и pYV- бактериални клетки от популацията. След оптимизиране, ПВР се
позитивира в диапазона от 5х101 до 9х101 КОЕ/мл.
VM14
ДОКАЗВАНЕ НА ПАТОГЕННИ СЕРОТИПОВЕ YERSINIA
ENTEROCOLITICA
В КОНТАМИНИРАНО ПРЯСНО МЛЯКО
М. Илиев, Х. Найденски
Консумацията на контаминирани хранителни продукти представлява основен

път за иницииране на инфекция с Yersinia enterocolitica. Патогенните био/
серотипове на вида са хетерогенна група, чийто патогенен потенциал е
комплексно детерминиран от хромозомални и плазмидно локализирани фактори.
Тяхното наличие и комбинирана детекция е важно условие за епидемиологичната
характеристика на контаминиращите щамове Y. enterocolitica.
Независимо от големият брой на регистрираните случаи на хранителни
инфекции след консумация на мляко, контаминирано с Y. enterocolitica, значително
193
по-често се изолират непатогенни щамове в сравнение с патогенните. За оценка
на млякото и млечните продукти като потенциални рискови храни, е необходимо
разработването на адекватна ПВР за детекция на Y. enterocolitica в изкуствено
контаминирани проби. Оптимизирането на реакцията включва избор на праймери
и адекватна обработка на пробите, за отстраняването на възможните инхибитори
на ПВР и съкращаване на времето за анализ. Като основни таргетни участъци от
генома са подбрани гените ail (хромозомно локализиран) и vir F (плазмидно
локализиран), разграничаващи патогенните щамове от серотиповете О:3, О:8 и
О:9 на Y. enterocolitica от непатогенните, както и от други непатогенни видове на
род Yersinia.
Изследвани са ефективността и детекционния лимит на ПВР в проби от прясно
мляко, изкуствено контаминирани с 6 плазмид-положителни (pYV+) щама Y.
enterocolitica от серотиповете О:3, О:8 и О:9, съхранявани на +4oC за 24 часа и 72
часа. Паралелно е провеждан фенотипен мониторинг на преживяемостта на
йерсиниите и плазмидното носителство на изолираните колонии чрез използването
на модифицирана хранителна среда. Оптимизиран е методът за обработка на
пробите и е постигнато значително намаляване на времето, необходимо за
осъществяване на генетичния анализ. ПВР реакцията се позитивира в диапазона
от 1,1х101 до 1,5х101 КОЕ/мл при всички pYV+ щамове, което утвърждава
използваната схема за директна детекция на патогенни щамове Y. enterocolitica в
мляко.
VM15
ВЗИСКАТЕЛНИ БАКТЕРИИ В ЕТИОЛОГИЯТА НА
ПУЕРПЕРАЛНИТЕ ЕНДОМЕТРИТИ ПРИ КРАВИТЕ И
ВЪЗМОЖНОСТИТЕ ЗА ТЕРАПИЯ
Никола Коруджийски
Институт, София
Проведени са проучвания при крави с клиника на акутен катарално - гноен

ендометрит, при който от маточните изтечения не се изолират бактерии - по
рутинните микробиологични методи.
В нативни натривки от изтеченията на същите крави се установява присъствие
на бактерии.
Култивирането на посевки от изтеченията върху специализирани хранителни
среди, в анаеробни условия в среда от 10% СО2 , позволява да се изолират щамове
Bacteroides fragilis, Bacteroides melaninogenicus, Bacteroides oralis, при които са
възможни успешни терапевтични третирания с флуорохинолони и някои
аминогликозиди.
194
VM16
ЛЕКАРСТВЕНА УСТОЙЧИВОСТ НА БАКТЕРИИТЕ ОТ
РОД ALCALIGENES, ИЗОЛИРАНИ ОТ ЗАЙЦИ С
ИНТЕСТИНАЛНИ РАЗСТРОЙСТВА
Т. Гълъбинова1, Б. Митов2, Н. Коруджийски2, С. Иванова2, Л. Ангелов2

Институт за Контрол на Ветеринарни Продукти – гр. София1
Институт- гр. София2
Постмортално е изследвано чревно съдържание и вътрешни органи от

домашни зайци с акутен ентероколит, при което са изолирани 45 щама
монокултури на Alcaligenes faecalis.
Щамовете са проучени за чувствителност на химиотерапевтични средства по
метода на двукратните сериини разреждания върху твърди хранителни среди.
Прави се заключение, че бактериите от вида Alcaligenes faecalis имат значение
за ентеритите при домашните зайци и се отличават с висока и широкоспектърна
лекарствена устойчивост, което е от интерес за профилактиката и терапията.
VM17
НЕОПЛАЗИИ ПРИ ЕСЕТРОВИ РИБИ
Ваня Чикова, Вера Коларова, Ангел Цеков*

Национален научноизследователски ветеринарномедицински институт – София;
* Институт по рибарство и аквакултури - Варна
Значителното намаляване на естествените популации от есетрови риби ги

превръща в атрактивен обект за аквакултура .
Тумори при риби от този вид са сравнително рядко срещани. Изследванията
обхващат материал от 12 есетрови риби от вида Acipenser guldenstadti от 2
рибовъдни ферми в страната. Описана е наблюдаваната клинична картина и са
представени резултатите от патологоанатомично и патохистологично изследване
на туморовидни образувания, разположени в основата на хрилните дъги.
Установени са също макроскопски и микроскопски промени в черен дроб,
далак и сърце.
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VM18
СЛУЧАЙ НА АЕРОМОНОЗА ПРИ ВЕСЛОНОС
Ваня Чикова, Таня Хубенова*, Вера Коларова, Райна Атанасова*, Ангел

Зайков*
Национален научноизследователски ветеринарномедицински институт - София
* Институт по рибарство и аквакултури - Варна
При опитите за интродуциране на веслонос /Polyodon spathula / като нов вид

за аквакултура в страната е установена висока смъртност и е наблюдавана
характерна клинична и патологоанатомична картина.При микробиологичните
изследвания за доказване и идентифициране на етиологичния агент - Aeromonas
hydrophila са използвани стандартни микробиологични техники и експресна
система за бактериална идентификация Мerck MCN6.
PRESENTATIONS OF FIRMS
ZEU-IMMUNOTEC
MICROBIOLOGICAL TESTS FOR SCREENING OF ANTIBI-

OTIC AND SULFONAMIDE RESIDUES IN FOOD
Dr. Elena Domнnguez- ZEU-INMUNOTEC, Maria de Luna 11, Zaragoza, Spain
Veterinary medicines are commonly used for prevention or treatment proposes by

producers of animal and animal products across the world. Residues of these drugs can
carry over into foods such as fresh meat, meat or dairy products, fish and honey.
Veterinary residues pose a hazard to consumers by causing allergic reactions and
developing bacterial resistance. They also have an inhibitory effect on starter cultures,
used in fermented products, causing financial losses in the food industry. In order to
prevent from residues going to the food chain strict EU regulations are in force.
Food testing is strongly advised to ensure anti-microbiological residues are below the
Maximum Limits of Residues (MLR) set in the European legislation.
Microbiological methods, based on the bacteria growth inhibition, are used for screening
of a broad range of antibiotic and sulfonamide residues. These methods are available as
commercial kits allowing food producers and farmers to run their own self-control. Studies
to check the performance of these test kits show good sensitivity to comply with the
European MRLs. They are also very easy to use and require minimum laboratory equipment.
More than 90 samples can be screened in about 3 to 4 hours on site or in the lab.
196
HYGIENA INTERNATIONAL LTD
HYGIENE MONITORING IN SUPPORT OF FOOD SAFETY -

A REVIEW OF METHODS AND TRENDS
Ing. Frans Martens, Hygiena International Ltd., United Kingdom
Good Hygienic Practices are an essential to ensure food safety. They are required by
law under national and international Food Hygiene Regulations and are frequently
considered as pre-requisites to food safety systems based on Hazard Analysis such as
HACCP. The presentation will discuss the availability of simple, user friendly and cost
effective rapid methods for checking sanitation programs and day to day hygiene
monitoring. These methods give immediate feedback to the quality of the cleaning and
have proven to be a powerful tool in food processing industries.
197
INFECTIOUS IMMUNOLOGY
II1
QUALITY OF ANALYSIS
IN CLINICAL MICROBIOLOGY
I. Smirnov, Moskow, Russia
II2
IMPROVED POOLED IGG PREVENTS DEATH
IN EXPERIMENTAL SEPSIS
Tchavdar Vassilev, Jordan Dimitrov and Nina Ivanovska

Stephan Angeloff Institute of Microbiology, 1113 Sofia, Bulgaria
Sepsis and septic shock are one of the major causes for death worldwide. Despite the
high numbers of patients involved, these medical conditions rarely make headlines. The
life-threatening symptoms of septic shock are the result of a generalised, uncontrolled
inflammatory reaction of the body to an invading microorganism (bacterium, virus or a
fungus). The high mortality in some emerging diseases (e.g. of avian flu) is also due to a
dysregulated inflammatory response. Currently available therapeutic strategies in sepsis
are not satisfactory as far as efficacy is concerned. A better understanding of the molecular
mechanisms that are implicated in the pathogenesis of sepsis has provided us with
indications on the design of new class of immunomodulatory agents to combat the
generalized inflammatory reaction in sepsis. The use of therapeutic immunoglobulin (Ig)
preparations represents one such approach in the prevention and in the treatment of
sepsis and septic shock. Immunoglobulin preparations exert anti-inflammatory activity,
mediated by their interactions with complement components, inhibitory receptors on
immune cells, down-regulation of T-, B-cell and dendritic cell functions and effect on
cytokine networks. These anti-inflammatory activities do not result in immunosuppression
unlike high doses of corticosteroids. However, Ig preparations in their present form have
not been fully successful in preventing sepsis-related complications. Therefore, there is
an urgent need for improving the currently available therapeutic strategies to counter
these problems. Conception and designing of next generation Ig preparations is a critical
necessity.
We have recently developed a technology to produce improved pooled therapeutic
human immunoglobulin G with strongly enhanced anti-inflammatory activity. It is based
on the exposure of IgG to prooxidative ferrous ions or to reactive oxygen species. This
198
treatment results in enhanced IgG paratope flexibility and hydrophobicity, leading to
expansion of the spectrum of recognized antigens, regulation of cell proliferation and
protection in experimental sepsis. The injection of a single dose (30 mg/kg) of Fe(II) ion-
exposed pooled human therapeutic IgG, but not of native IgG, resulted in the prevention
of death in an experimental model of sepsis. A beneficial effect was also seen when using
a dose of 150 mg/kg, but not of 6 mg/kg).
II3
ЕФЕКТ НА YOPK ПРОТЕИНА НА YERSINIA PSEUDOTU-
BERCULOSIS ВЪРХУ ИНВАЗИН-МЕДИИРАНОТО
ПОГЛЪЩАНЕ ОТ HELA КЛЕТКИ
Е. Иванова1, Х. Найденски1, А. Веселинова1, F. Deleuil2, H. Wolf-Watz 2

1
Институт по микробиология “Стефан Ангелов” - БАН, София, България
2
Университет Умеа, Швеция
Патогенните бактерии от род Yesinia (Y. pestis, Y. enterocolitica и Y.

pseudotuberculosis) притежават тип III секреционен апарат, който експортира
ефекторни протеин, т.н. Yops (Yersinia outer proteins), кодирани от плазмида на
вирулентността (pYV). Част от тези протеини се транслоцират в еукариотната
клетка, което води до възпрепятстване на фагоцитозата и подтискaне на
възпалителния отговор. Един от тези протеини – YopK, е необходим на Y.
pseudotuberculosis да предизвиква системна инфекция, а нивото на експресия на
YopK влияе върху нивата на транслоцираните Yops в гостоприемниковата клетка.
Целта на настоящето изследване е, да се определи ефекта на YopK върху
инвазин-медиираното поглъщане на Y. pseudotuberculosis от Hela клетки и дали
експресията на YopK протеина от трансфектирани с yopk гена Hela клетки могат
да променят ефекта на хипертранслокация на други Yops при ÄyopK мутантeн
щам. Получените резултати показват, че YopK протеинът не участва в процеса
на блокиране на инвазин-медиираната фагоцитоза. Наличието му в еукариотните
клетки преди инфекция, не може да индуцира сигнален път в клетката, отговорен
за контрола върху количеството транслоцирани Yops при ÄyopK мутантния щам,
нито да блокира Yop транслокацията при дивия щам.
199
II4
RAPID IMMUNOHISTOCHEMISTRY METHOD FOR DE-
TECTION OF TSE PRION PROTEIN ON FROZEN BRAIN
TISSUE SECTIONS
Lubashevsky E., Yadin H., Perl S., Adry N., Gorochov A., Bumbarov V.
Prion diseases are manifested as genetic, infectious or sporadic , lethal

neurodegenerative disordes involving alterations of the prion protein (PrPc). PrPc is
constitutively expressed in normal adult brain and is sensitive to proteinase K digestion,
while the altered PrP-scrapie (PrPsc) conformation is resistant to proteases. Abnormal
PrPsc is found at high levels in the brains of animals affected by scrapie in sheep, BSE in
cattle and Creutzfelt-Jacob disease in humans.
Procedures that detect PrPsc, such as Western immunoblotting, ELISA technology
and immunohistochemistry (IHC) techniques, ara based on the ability to identify infected
animals subclinically by detecting small quantities of PrPsc. The current IHC method –
gold as standard – used to supplement or replace histophathology as the method of
choice for diagnosing diseases ,to obtain reliable results.To maximize sensitivity of IHC
required brain is usually preserved in the fixative paraformaldehyde, lysine, periodic acid
and hydrated autoclaving before immunostaining. These procedures need a relatively
long time (5 days) and sometimes led to artifacts that affect tissue morphology. The aim of
the study is the development of raped efficient HIS method for mass screening prion
protein.
In proposed IHC PrPsc detection is carried out in frozen tissue brain preparations.
II5
“RESPIVAX” MODULATES THE EXPRESSION OF CD86 ON
DIFFERENT CIRCULATING APC SUBSETS
D.Stankulova1, A. Mihova1, H. Taskov1, Vl. Maximov2, M. Nikolova1

1
Central Laboratory of Immunology, National Center of Infectious and Parasitic
Diseases, Sofia, Bulgaria
2
Specialized Hospital for active treatment of Pulmonary Diseases “St Sofia’, Sofia,
Bulgaria
Background: The oral immunomodulator “Respivax” (BulBio-NCIPD) has a well

established stimulatory effect on cellular immune responses. However, the underlying
mechanisms have not been studied in detail. The efficient activation of naïve T lymphocytes
requires the delivery of second signal by the antigen-presenting cell (APC), and CD86
(B7.2) is one of the best characterized co-stimulatory molecules on human APC. Aim: To
study the effect of “Respivax” treatment on CD86 expression by different subsets of
200
circulating human APC. Material and methods: Fifteen patients with recurrent non-specific
respiratory infections were subjected to a standard course of “Respivax” treatment.
Heparinized whole blood samples were collected at baseline, day 20 and day 80. Multicolor
flow cytometry (FACSCanto, B-D) was used to study CD86 expression on circulating
dendritic cells (DC, lin-HLA-DR+), monocytes (Mo, CD14+), and B Ly (CD19+). According
to the mean fluorescence intensity (MFI) of CD86 expressionCD86low and CD86high DC were
distinguished. The effect on B Ly was assessed after 24h in vitro stimulation of whole
blood with LPS. Results: “Respivax” treatment increased the CD86+ DC population from
68.7% to 76.2%, (p<0.01) and promoted its maturation resulting in the increase of CD86high
subset as compared to baseline (a mean of 52% vs. 43%, p<0.05). This increase was more
significant in a subgroup of patients with initially decreased CD86high DC level (a mean of
56.8% vs.39%, p<0.01), and was already detectable at day20. Further on, CD86 MFI was
significantly increased on Mo from patients with initially lower intensity of CD86 expression
(1318 vs. 1071, p<0.01), already at day 20. At baseline 38.2% of the LPS -stimulated B Ly
expressed CD86 vs. 44.6% at day 20 (p<0.02), and 48.2% at day 80 (p<0.01). Conclusion:
These results suggest that one of the mechanisms for stimulation of T cell immune responses
by “Respivax” is the increased co-stimulatory potential of circulating APC, and hence -
the possibility for activation of naïve T-lymphocytes.
II6
КОЖНИЯТ ТУБЕРКУЛИНОВ ТЕСТ В ОЦЕНКАТА НА
ПОСТВАКСИНАЛНАТА БЦЖ АЛЕРГИЯ . КАЧЕСТВА НА
БЪЛГАРСКИЯ PPD ТУБЕРКУЛИН
Е. Сапунджиева, Е. Йорданова, БУЛ-БИО, НЦЗПБ, СОФИЯ
Кожната туберкулинова чувствителност, определена чрез теста на Mantoux

се използва много години за диагностика при туберкулозата, както и за оценка
на постваксиналната алергия. Видът и размера на кожната реакция зависи от
много фактори, в това число: вида на щама и дозата на ваксината, възрастта на
ваксинирания, начина на хранене, годините изминали след ваксинация, честотата
на кожния туберкулинов тест.
За стандартност при оценката на кожния тест от първостепенна важност е
използвания туберкулин и техниката при прилагане и отчитане на резултата.
Представен е анализ на данните от стабилността на българския PPD Tuberculin,
при изследвания проведени съгласно Европейската фармакопея.
Препаратът при съхраняване на тъмно в хладилник при температура 20 – 80С,
запазва своите химико-физични и имунобиологични свойства в продължение на
повече от 3 години.
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II7
ПОЛОВИНВЕКОВНА ИМУНОПРОФИЛАКТИКА НА
ТЕТАНУС С БЪЛГАРСКА ВАКСИНА
Е. Сапунджиева*, И. Тодорова*, Е. Йорданова*, Й. Христова*, М.

Демирева**, Р. Мълчанова**
*БУЛ БИО-НЦЗПБ, София;
** Столична хигиенно-епидемиологична инспекция (СХЕИ)
Тетанусът е остро спорадично ранево инфекциозно заболяване, протичащо с

висок леталитет. И до днес няма специфично лечение, като прилаганата терапия
се свежда до опит за туширане на характерната неврологична симптоматика.
Единственото средство е специфична имунопрофилактика с тетаничен
токсоид. В България тя е въведена през 1959 г.
В резултат на постепенното до пълно обхващане на населението,
заболяемостта от 4.8%000 през 1945 г. е сведена до единични случаи при
неваксинирани лица. През 2003 и 2004 г. заболяемостта е 0%000. Изследван е
имунологичния статус на повече от 6 000 лица от различни възрасти (0 до 90
години). Резултатите показват, че 96% от българското население е защитено от
тетанус (титър на тетанични антитела ≥ 0.01 IU/ ml). Интерпретират се различията
в степента на защита в отделните възрастови групи. Нередовното обхващане с
реимунизации води до почти 50% незащитеност при лица в активна трудова
възраст. Последното крие потенциален риск от заболяване при нараняване.
II8
MONITORING OF CYTOTOXIC CD8 T LYMPHOCYTES IN
HIV-1+ PATIENTS SUBJECTED TO HAART
M. Muhtarova, M. Nikolova, S. Magaev, H. Taskov

Central Laboratory of Immunology, National Center of Infectious and Parasitic
Diseases
Background: One of the goals of highly active antiretroviral therapy (HAART) is

restoration of the differentiation and functional potential of CD8+ cytotoxic T cells (CTL)
in HIV patients. We have previously reported that the expression of CTL receptor CD160
is increased in HIV patients and may predict a good therapy response.
Aim: To study the changes in the co-expression of the cytotoxic mediators granzyme
B (GzmB) and perforin (Per) in HIV-1 patients subjected to HAART, in relation to the CD8
T cell differentiation/functional subsets defined by CD160/CD27/CD28 co-expression.
202
Material and methods: Heparinized whole blood from 9 treatment-naive HIV(+) patients
was analyzed at baseline and after 6 months of HAART in comparison with 11 age- and
sex-matched non-treated HIV+ patients and 10 HIV(-) healthy controls. GzmB/Per
intracellular co-expression and CD27/CD28/CD160 membrane co-expression on CD8+ T
cells were studied by multi-color flow cytometry (FACSCanto, B-D).
Results: At baseline, HIV+ patients were characterized with increased percentage of
intermediate (CD27+CD28-), late (CD27-CD28-) and CD160+ CTL as compared to HIV-
controls. Although the percentage of functional GzmB+Per+ CTL was increased in HIV+
patients (10.4 vs. 3.8, M-W p<0.05), immature GzmB+Per- CTL predominated
disproportionately (39.9 vs. 5.8, M-W p<0.001). At second examination, untreated patients
had no significant changes in any of the studied parameters. However, HAART induced a
significant increase of the GzmB+Per+ (functional) CTL subset (22.5 vs.10.4, Wilcoxon,
p<0.05), Importantly, GzmB+Per+ CTL correlated with the subset of late CTL, expressing
CD160 molecule (CD27-CD160+, Spearman R=0.7, p<0.02).
Conclusion: Expression of CD160 on CD8+ T cells in chronic HIV infection is important
for monitoring the restoration of cytotoxic function in the course of HAART.
II9
PROTECTION AGAINST WHLOOPING COUGH IN
CHILDREN BETWEEN 0-6 YEARS OLD
R. Alexiev1, K. Hadjiisky1, S. Malchanova2, V. Demireva2 and Pl. Nenkov1

1
BB - NCIPD Ltd. - Sofia, Bulgaria; 2 Hygiene Epidemiological Inspection - Sofia,
Bulgaria
The specific immunoprophylaxis of humans with pertussis, as a component of combine

bacterial vaccine leads to production of specific antibodies that is indicator of the whooping
cough prevention. For evolution of immunization procedures and the vaccine itself,
antibody levels against pertussis are useful to show the immune status of the population.
An enzyme-linked immunosorbent assay was used for measuring immunoglobolin G
pertussis antibodies in human sera. The ELISA involves the blinding of bacterial cells to
polystyrene tubes. Results of the direct ELISA test are highly reproducible. It is believed
that a pertussis antibody level of 1:81 provides protection against disease. The titters of
antibody from 1:161 to 1:320 showed full protection of people against whooping cough
and titters over 1:321 are used as a criteria of disease or used as a criteria of passed
disease. The results of estimation of immune status of population in Bulgaria were based
on the same criteria.
The assay was done in plastic plates coated with inactivated bacterial cells. For
investigation 279 human sera were tested in age group between 0 – 6. The tested sera
showed that 54% of persons have full protection against whooping cough and in 42% of
children the obtained titters showed basic immunity. In 4% of patients the titter of antibody
is more than 1:321, which titter is used as a criteria of disease of human sera. All of sera
203
have good level of protection against whooping cough and non-protected patients were
not found.
Present results indicate a good protection against pertussis in Bulgaria in children till
6 years old. This fact is a result of specific immunoprophylaxis by pertussis vaccine, as a
component of DIFTETKOK (DTP) combined bacterial vaccine, produced by BB – NCIPD,
Ltd.
II10
INVESTIGATION ON THE IMMUNE STATUS OF
THE POPULATION AGAINST DIPHTHERIA DURING THE
PERIOD 2001 – 2005
R. Alexiev1, K. Hadjiiski1, S. Malchanova2, V. Demireva2 and Pl. Nenkov1

1
BB - National Centre of Infectious and Parasitic Diseases - Sofia, Bulgaria
2
Hygiene Epidemiological Inspection - Sofia, Bulgaria
The specific immunoprophylaxis of humans with diphtheria toxoid, as a component of

combine bacterial vaccines leads to production of specific antibodies that have main role
in diphtheria prevention. An enzyme-linked immunosorbent assay was used for measuring
immunoglobolin G diphtheria toxin antibodies in human sera. The assay was done in
plastic plates coated with purified diphtheria toxoid. 4711 human sera in different age
groups were tested for investigation. The estimations showed that 67.8% of tested sera
have full protection against diphtheria, 4.4% have basic immunity and in 27.8% lack
protection against disease.
The comparison of different groups of age showed that the best protection against
diphtheria have in children and teenagers younger than 15 years old. In these groups of
age the sera without antibodies against diphtheria are significant. Aging of people leads
to reduction of the percent of protected persons and to increasing the percent of non-
protected people. Despite this, the percent of protected population in group of age between
16 to 65 years old is better than in some European countries and in the USA. The patients
over 65 years old had very low levels of antibodies against diphtheria and in about 83%
lack protection against diphtheria.
Present results indicate a good protection against diphtheria in Bulgaria. This fact is a
result of specific immunoprophylaxis by diphtheria toxoid, as a component of combined
bacterial vaccines, produced by BB – NCIPD, Ltd.
204
II11
DETERMINATION OF MINIMAL SENSITIZING DOZE OF
BCG VACCINE (SUBSTRAIN SOFIA SL222) IN GUINEA PIG
T. Stefanova1, M. Chouchkova1 , S.Nikolaeva2

1
BCG Laboratory, BB-NCIPD Ltd., Sofia
2
Laboratory of Imminomorphology; NCIPD, Sofia
During the investigation of the allergenic potency of BCG strains it is advisable to

vaccinate with decreasing doses of vaccine in order to estimate the lowest dose that
induces tuberculin sensitivity, rather than to compare the effect of large doses of BCG.
The impact of the BCG vaccine dose on the development of cell-mediated and protective
immunity in the guinea pig model has been intensively discussed in different studies
during the last few years.
Although the biological test does not reveal the mathematical correlation of dose-
effect, it is important to look for the determination of the minimal sensitizing dose for every
BCG vaccine. In this study twelve groups of six guinea pigs each were vaccinated with
three different doses (0.12 ng, 1.2 ng and 12 ng) of freeze-dried vaccine (Batch No 42,
NCP=2.698x106/ml). Tuberculin tests were performed in different groups at the 30th, 60th,
90th and 120th day after BCG injection. The negative tuberculin reactions converted to
positive between 60th and 90th day. The dose of 1.2 ng elicited the largest tuberculin
reactions in the animals. This dose contained only about 64-65 culturable particles and
could be regarded as the smallest effective sensitizing dose of the BCG vaccine produced
from Working Seed Lot 7-93. The histological examinations demonstrated that a very low
inoculum (0.12 ng or 6 viable cells) is sufficient to induce a specific inflammation after BCG
vaccination.
II12
ЛИЧЕН ОПИТ ПО ОТНОШЕНИЕ НА ДИАГНОСТИКАТА И
ДИФЕРЕНЦИАЛНА ДИАГНОСТИКА НА ПТИЧИЯ ГРИП
В.Люпке1, В. Йонкова2, В. Люцканова2

1
ВУ “Земеделски колеж”- Велико Търново;
2
МУ – Варна, катедра Микробиология
Територията на Р.България се намира под трасета и сборни места на редица

видове прелетни птици, които могат да бъдат преносители на вируса на птичия
грип. Съществува реална опасност и риск от заразяването на хора и животни при
по-тесен контакт с фецес, секрети и трупен материал.
Диференциалнодиагностично трябва да се изключват псевдочума по птиците,
различни видове инфлуенцавируси и Хламидии.
В настоящето съобщение са представени прилаганите от нас лабораторно-
205
диагностични методи за доказване и диференциране на миксовируси и някои други
причинители на респираторни заболявания. Предлага се създаване на
специализирани за тази цел лаборатории.
II13
CHLAMYDIA TRACHOMATIS (CT) ANTIBODIES IN SERUM
AND GENITAL FLUIDS IN INFERTILE COUPLES
V. Yonkova1, V. Lyoutzkanova1, V. Savouleva2, Y. Yonkov3

1
Dept. of Microbiology and Virology, Medical University – Varna;
2
Dept. of Obstetrics and Gynecology, Medical University – Varna;
3
Dept. of Anatomy, Histology and Embryology, Medical University – Varna
Chlamydia Trachomatis (CT) infections of the genital tract of women and men, although
a major cause of infertility, are often asymptomatic and undetected. The majority of couples
who are infertile have no history of a sexually transmitted disease or pelvic inflammatory
disorders. Since many infertile women now seek in vitro fertilization that is a problem of
priority to detect or to exclude the relation between an unsuspected CT infection and
infertility. The most useful marker of that condition is the local antiCT sIgA, which is
assayed to look for persistent antigenicity, potential subsequent disorders and partuer’s
contamination.
By using conventional methods, materials (cervical mucus – 63, prostatic fluid – 38,
semen – 55, sera – 100) of 55 men and 63 women were tested. All samples were free from
blood. The positives results to CT infection we detected are as follows: 38,1 % (21) of men
and 47,6 % (30) of women. Differences in the prevalence of CT infection between the
various outcome groups did not reach statistical significance (P = 121). Antichlamydial
IgA was present in the cervices of 25,2 % of the women with endometrioses, 18,1 % of
women with tubal factor infertility, and 14,3 % of women whose infertility was due to poor
sperm quality. In conclusion, women with evidence of a current or recent CT genital tract
infection had a poorer outcome in their in vitro fertilization cycles than did women with no
evidence of this infection.
206
II14
IMMUNOBLOT ANALYSIS OF ANTIBODY RESPONSE
TO PLASMID ENCODED RELEASED PROTEINS OF
YERSINIA ENTEROCOLITICA IN PATIENTS
WITH REACTIVE ARTHRITIS
Elica Golkocheva1, Hristo Najdenski1 and Rumen Stoilov2

1
Department of Pathogenic Bacteria, The Stephan Angeloff Institute of Microbiol-
ogy, Bulgarian Academy of Sciences, Sofia; 2 Clinic of Rheumatology, The Medical
University, Sofia
Yersinia enterocolitica has been identified as causative organism of reactive arthritis

in humans. Antibodies developed against the Yersinia outer membrane proteins /Yops/,
in cases of chronic enteritis and reactive arthritis, usually persist at high levels for longer
time periods. Immunoblot analysis of anti-Yersinia antibody response of blood sera from
patients with reactive and rheumatoid arthritis was carried out. The assay was applied by
using plasmid encoded Yops as antigen. Seven strong bonds of the molecular weights: 26
kDa - YopE, 33 kDa - YopN, 36 kDa - YopD, 41 kDa - V-ag, 43 kDa - YopB, 46 kDa - YopM and
51 kDa - YopH were visualised. In sera from patients with reactive arthritis IgG antibodies
were detected against all the seven Yops. IgA antibodies were established against YopM,
YopB, YopD, YopN and YopE. Sera from the patients with other rheumatic diseases were
tested for the presence of anti-Yersinia antibodies too. These sera were negative for the
presence of anti-Yersinia IgA antibodies, but two of them were positive for IgG against
Yop D. Antibodies from the classes IgA and IgG were not detected in the serum samples
from healthy people.
II15
ATTENUATION AND PRESERVED IMMUNOGENIC
POTENTIAL OF YERSINIA PSEUDOTUBERCULOSIS
MUTANT STRAINS EVIDENCED IN ORAL PIG MODEL
H. Najdenski1, E. Golkocheva1, E. Ivanova1, V. Kussovski1, A. Vesselinova1, S.

Garbom2, H. Wolf-Watz2
1
Sofia, Bulgaria;
2
University of Umea, Umea, Sweden
Experimental oral infection of pigs with a wild type Yersinia pseudotuberculosis strain
pIB102, serotype O:3 and two mutant isogenic strains – pIB155,”yopK and pIB44,”ypkA
has been carried out. Clinical findings, microbiological and immunological parameters
were examined in dynamics from day 7 to day 60 post infection (p.i.).
207
All types of infections ran asymptomatically, without hyperthermia, loss of appetite,
etc. Experiments on the blood parameters demonstrated a transient leukocytosis with
lymphocytosis and monocytosis better expressed after pIB155"yopK infection. Even
though pig is usually known as a reservoir of yersiniae, bacterial colonization was found
in tonsils and mesenterial lymph nodes on days 7 and 14 p.i. with wild type strain, and
only in tonsils on day 14 p.i. with both mutant strains. The augmented sensitivity of
mutants to the bactericidal effect of leukocytes and blood sera are the characteristic
features of attenuation in their pathogenicity, in comparison with the wild strain.
Comparative in vitro experiments on the immune response and immunostimulating capacity
of Y. pseudotuberculosis mutant strains verify their preserved immunogenic potential,
predominantly in case of pIB155"yopK. Immunomorphological rearrangements like a
hyperplasia and strong activation of the lymphoid tissue of Peyer’s patches, mesenterial
lymph nodes, spleen and peribronchial lymph tissue of pigs challenged with both mutant
strains were proved. The results obtained give the reason to claim that the genetically
constructed yopK null mutant strain is significantly attenuated but still immunogenic and
has the potential for a live vaccine carrier strain.
II16
HEMOCYANINS AS IMMUNOSTIMULATORS
Reneta Toshkova 1, Emilia Ivanova2, Maria-Dorothea Nastke3, Lyudmila Velkova4,

Stefan Stevanovic3, Rumyana Hristova4, Alexandar Dolashki4, Maria Gardeva4,
Ivan Dimitrov4, Wolfgang Voelter5, Pavlina Dolashka-Angelova4
1
Institute of Experimental Pathology and Parasitology, BAS, Bulgaria;
2
Institute of Microbiology, BAS;
3
Department of Immunology, Institute for Cell Biology, University of Tьebingen,
Germany;
4
Abteilung fьr Physikalische Biochemie des Physiologisch-chemischen Instituts der
Universitдt Tьbingen, Germany;
5
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Bulgaria
Hemocyanin from the giant keyhole limpet Megathura crenulata has been a subject of
biomedical interest because of its remarkable immunostimulatory properties in experimental
animals and man. Molluscan Helix vulgaris (HvH) and Rapana venosa (RvH), and
arthropodan Carcinus aestuarii (CaH) hemocyanins have been studied in order to evaluate
their potential biochemical and medical applications.
It was established that the serum IL-2 production was better expressed in animals
immunized by HvH and CaH then with the native molecule of KLH. Increased IL-2
production in supernatants of in vitro cultivated lymphocytes was observed in animals
immunized with native CaH and KLH. Spleen cells from the mice immunized with other
hemocyanins showed negligible stimulation. It was found that CaH causes increased
208
specific and non-specific proliferation of spleen lymphocytes and Th1 associated cytokin
production in BDF1 mice.
The effect of the molluscan Rapana venosa hemocyanin on the antibody-dependent
cell cytotoxicity (ADCC) and mitogen responsibility of spleen lymphocytes from hamsters
with transplanted myeloid Graffi tumors was demonstrated. After treatment of animals
with KLH or RvH the spleen lymphocyte ADCC decreased during tumor progression,
while ADCC of spleen lymphocytes against own tumor cells increased about twofold in
comparison to that of lymphocytes from untreated tumor bearing hamsters (TBH). The
lymphocytes isolated from normal animals without treatment showed two times lower
cytotoxic activity compared to those from RvH- or KLH- treated controls. RvH induced 3-
5% higher ADCC compared to KLH in all combinations of sera and lymphocytes. We
suggest that this action is caused by stimulation of the Th-1 and T-cytotoxic lymphocyte
population as well as unspecific lymphoproliferative properties of Hc preparations.
209
PARAZITOLOGY
P1
EMERGING AND REIMURGING PARASITIC DISEASES IN
BULGARIA
Kurdova-Mintcheva R., D. Jordanova, T. Marinova, M. Ivanova, N. Tsvetkova, I.

Marinova, R. Harizanov
The impact of emerging and reemerging parasitic diseases in Bulgaria and their health
influence have increased in recent years. Among them, toxoplasmosis, cryptosporidiosis,
pneumocystosis, visceral leishmaniosis, blastocystosis, cystic echinococcosis and
trichinellosis are the leading diseases.
Seroprevalence of toxoplasmosis in the population during the past 10 years was 31.21%-
47.7% with 40-50 cases of acute primary infection detected per year. Cases of
cryptosporidiosis and blastocystosis among travelers, immunocompromised persons and
patients with intestinal disorders were recorded. Indigenous visceral leishmaniasis is also
on rise with endemicity in the southern part of the country. All protozoan infections
mentioned above are often associated with AIDS.
Regarding human cystic echinococcosis, since 1993 there has been a marked rise in
morbidity - 8.47 per 100 000 population in 1996 and 8.32 per 100 000 population in 2002,
with an average of 6.37 per 100 000 population for the last decade. In some regions (Sliven,
Burgas, Chaskovo, Pazardjick, etc.), the morbidity is much higher (15-27 37 per 100 000
population) than the country average. In Bulgaria, trichinellosis outbreaks and sporadic
cases as well as an increased morbidity among the human population has been on the rise,
too. During 1991-2005, the number of outbreaks registered was 139.
The situation regarding emerging and reemerging parasitic diseases in the country
clearly demands joint organized measures for their surveillance and control.
P2
EPIDEMIOLOGICAL ASPECTS OF HUMAN
TRICHINELLOSIS IN BULGARIA (2001 – 2005)
M. Ivanova1, R.Kurdova1, D.Jordanova1, N.Tsvetcova1

National Center of Infectious and Parasitic Diseases, NCIPD, Sofia
Human trichinellosis is an important zoonosis, which is annually registered in Bulgaria.

Among all ages and population groups, every year trichinellosis outbreaks and sporadic
cases are reported. Worldwide different encapsulated and non-encapsulated Trichinella
210
species and genotypes have been described. Some differences have been observed in
the signs, symptoms and clinical course of the disease, caused by various Trichinella
species.
The epidemiology of infection greatly varies depending on Trichinella species. Recent
investigations discovered that in Bulgaria besides T.spiralis, cases of human trichinellosis
are also associated with T.britovi. During 2001 – 2005 period, altogether 49 outbreaks and
73 sporadic cases of human trichinellosis have been officially registered in the country.
For this period 965 people were reported as consumers of infected meat products and 813
(84,25 %) of them developed asymptomatic or clinical form of trichinellosis.
Basic epidemiological aspects of the disease, peculiarities in geographical distribution
of the registered cases and tendencies, which were observed over the past years, regarding
also the source of infection and Trichinella genotypes involved, were revealed in the
study.
P3
STUDY ON THE DIAGNOSTIC VALUE OF SPECIFIC IGE
ANTIBODIES IN HUMAN ECHINOCOCCOSIS
I.Marinova, G.Nikolov, A.Mihova, R.Kurdova, B.Petrunov
Cystic echinococcosis is a widespread parasitic zoonosis, caused by the cestode

Echinococcus granulosus. In human, as intermediate host, develops the larval stage of
the parasite and different antigens (cyclophilline, EgEF-1) lead to the formation of specific
IgE antibodies. Although the contemporary immunologic methods detect them only in 42
– 52 % of the patients, the specificity is very high – 95 – 100 %.
In the present study150 sera – 75 sera from patients with proved echinococcosis and
75 sera from people without echinococcosis (blood donors, patients with nonparasitic
diseases and samples with false positive results for IgG in IHA or ELISA) were examined.
Automatic system UniCap, based on ELISA, was used for detection of IgE antibodies.
The level of the specific antibodies was determined quantitatively and the results were
arranged in 7 patients groups. The examination of the sera from patients with echinococcosis
revealed IgE antibodies from negative to extremely high levels. Specific IgE antibodies
were not detected in the samples from people without echinococcosis – specificity of the
reaction 100 %.
These data prove the high specificity of IgE antibodies, which makes them very useful
for confirmation of the diagnosis in patients with doubtful results in the routinely applied
serological tests.
211
P4
DETECTION OF CROSS-REACTIVE BANDS IN CYSTIC
FLUID BY WESTERN BLOT ANALYSIS
I. Marinova, I. Rainova, A. Tchernov, R. Kurdova

Natiоnal Centre of Infectious and Parasitic Diseases, Sofia
Presence of cross-reactive interactions among different helminthic antigens can lead

to missinterpretation of positive results in serological tests. Detection of antibodies against
particular parasitic proteins by Western blot (WB) is а way to overcome this issue. The
aim of the present investigations was to examine by WB with echinococcus antigens,
sera from patients with parasitic diseases in order to reveal common bands indicating
cross-reactivity. Sera from patients with amoebiasis, cysticercosis, taeniarhynchosis,
trichinellosis, alveococcosis, toxocarosis and a pool of human sera from healthy individuals.
Electroforesis of cystic fluid was performed in 15% separating gel according to Laemmli
and electrophoretic transfer of proteins from polyacrylamide gels to PVDF membrane was
made according to Towbin. All samples revealed bands with high molecular weight. Sera
from patients with alveococcosis, cysticercosis and toxocarosis showed false positive
results by reacting with the bands with diagnostic value – 12 kDa and 24-26 kDa. Samples
from the other parasitoses did not react with the bands with diagnostic value 8, 12 and 24-
26 kDa, and were interpreted as negative results.
P5
APPLICATION OF IGG АVIDITY FOR DIAGNOSIS OF
ACUTE TOXOCAROSIS
I.Rainova
National Center of Infectious and Parasitic Diseases (NCIPD), Sofia
Toxocarosis is a parasitic disease in humans, caused by larvae of dog ascarid Toxocara

canis. The infection is generally diagnosed by demonstration of specific immunoglobulins
to Toxocara excretory/secretory (E/S) antigens in sera of infected patients. The test that
has been clinically useful is the ELISA reaction with E/S antigens. This diagnostic method
detects the antibodies for months or even years after infection and this is the reason why
the discrimination between chronic and recent infection is very difficult. That is why we
use assay with measuring IgG avidity to distinguish acute from chronic Toxocara infection.
Sera samples tested in ELISA with E/S antigen and positive for toxocarosis were treated
with urea and then tested in ELISA again. The index of avidity was calculated as the ratio
of IgG values in sera treated with urea and the value of IgG in non-treated sera, multiplied
by 100. An index up to 40 is considered as low avidity which means freshly acquired
infection (36 to 40 borderline) and more than 40 is high avidity (chronic infection). From
1184 patient sera, tested in ELISA, 129 were positive for toxocarosis and most of them
212
showed high avidity. It means that predominantly patients are in the chronic stage of
infection at the time of examination.
P6
АНТИТЕЛА СРЕЩУ TOXOPLASMA GONDII В ЧОВЕШКИ
IG ПРЕПАРАТИ
И. Райнова1, Ю. Начева2, А. Чернов1

1
Национален център по заразни и паразитни болести (НЦЗПБ) – София
2
Бул Био НЦЗПБ –ЕООД – София
Три партиди човешки Ig препарати, произведени в Бул Био НЦЗПБ - ЕООД

бяха изследвани в реакциа пасива хемаглутинация (РПХА) и ELISA за наличие
на антитела срещу Toxoplasma gondii., като във всички изпитвани препарати бяха
установени антитела срещу паразита.
Получените резултати могат да бъдат обяснени с високия процент
серопозитивност за токсоплазмоза сред населението и трябва да се отчита
възможността за наличие на антитела срещу T.gondii при пациенти, лекувани с
човешки Ig препарати.
P7
IDENTIFICATION OF FREE-LIVING AMOEBAE BY PCR.
N. Tsvetkova, R. Kurdova
National center of infectious and parasitic diseases, Sofia
Free-living amoebae (FLA) group include different genera, such as Acanthamoebae,

Naegleria, Hartmannella, Vahlkampfia, Vannella etc., wide spread in the nature. Some species
of the genera Acanthamoebae, Naegleria and Hartmannella have been implicated in human
pathology.
The aim of the present study was to apply PCR tests for detection and identification of
FLA in water samples.
Materials and methods. Different types of water habitats, including rivers, tap, well,
spring, mineral water, a lake and wastewater treatment plant were examined for the presence
of cysts of FLA. DNA was isolated using DNAsy Tissue kit (Quigen). Culturing on 1.5%
nonnutrient agar plates and 3 PCR tests were carried out. Reference amoebae strains
representatives of the genera Acanthamoebae, Naegleria and Hartmannella were included
in the study. Separation through 1.5 % agarose gel and visualization under UV light
illumination of the amplification products was performed.
Results. Seven types of water habitats, including 41 sources with 126 samples were
213
examined for the presence of cysts of FLA. Forty-six of the samples tested were found
positive on 1,5% nonnutrient agar culture plates and 42 – PCR positive. Acanthamoebae
spp. cysts were detected in 18/16 samples (culture/PCR); Hartmannella spp. cysts – in 17/
17 samples and both Acanthamoebae spp. + Hartmannella spp. cysts in 11/10 samples.
Conclusion. Amoebae of the genera Acanthamoebae and Hartmannella were detected
with high frequency in water samples from sources examined. Further, tests for
pathogenicity have to be done aiming at study the potential of the amoebae to produce
diseases in animals and humans.
P8
VISCERAL LEISHMANIASIS IN BULGARIA
R.Harizanov, G. Filipov, D. Yordanova, R. Kurdova

Visceral leishmaniasis is a protozoal infection spreading over large territories of the

globe. According to the WHO data, more than 350 millions of people in 88 countries are
leaving at risk and more than 12 millions are already infected. Annually among 100 and 150
thousands new cases are registered, and many others remain undiagnosed.
Foci of visceral leishmaniasis exist in every border Bulgarian country. In Turkey for
1982 – 1991 period, over 500 clinical cases of human leishmaniasis were registered, and
parasitological and serological surveys confirmed presence of the disease in dogs. In
Greece endemic regions are situated in the south and peninsula parts of the country
mainly, and canine visceral leishmaniasis estimates to 22 %. Endemic regions exist also in
Serbia and Montenegro, while in Macedonia sporadic cases are annually reported.
In the past a number of authors reported in their publications for the presence of
indigenous visceral leishmaniasis in Bulgaria, while the last described cases dated from
1952.
At the end of eighties of the twentieth century, indigenous cases of visceral
leishmaniasis reappeared, and their transmission occurred in the country. As in the past,
nowadays the infection has the peculiarities of the Mediterranean clinico-epidemiological
variant and infected persons are not only children, but adults also. Domestic and wild
animals from the Canidae family are considered to be the reservoir and source of infection,
and the main vectors for disease transmission are species of the genus Phlebotomus.
During the 17 years observation period (1988 – 2005), 95 cases of visceral leishmaniasis
have been registered - 55 cases (57,89%) in children between 0 to 18 years of age and 40
cases (42,11 %) in persons over 18 years.
214
P9
ECHINOCOCCOSIS DISTRIBUTION AMONG CHILDREN
AND ADOLESCENTS IN BULGARIA (1990 – 2005)
D. Yordanova, R. Kurdova
Echinococcosis /hydatidosis/ is a serious health, social and economical problem for

our country. During the past 15 years a sharp increase in the basic epidemiological indices
(morbidity, mortality rate and lethality rate) has been observed.
This study was made on the basis of official notification data received for operated
patients and those with PAIR, from the surgical units and clinics.
Since 1990 to 2005 year period a constant tendency of elevation in echinococcosis
morbidity rate among the whole country population was established. Morbidity dynamics
in the age group of 0 – 19 years ranges between 0,53 %000 to 10,54 %000. Rise in the relevant
proportion of echinococcosis cases in the young-adolescent age group was observed.
This index is extremely important for the purposes of investigation of the parasitic
transmission and intensity of disease transmission in the country.
Territorial distribution of echinococcosis cases in the country is uneven. The most
affected territories are those of Plovdiv, Burgas and Sliven regions.
Organ localization of the hydatid cysts in children and adolescents most frequently is
in the lungs, liver, both lungs and liver and other organ localizations (kidneys, spleen,
brain, subcutaneous tissues).
The high echinococcosis morbidity index and the high relative proportion of
echinococcosis cases among children in the country, in comparison to other Mediterranean
Balkan countries, led to elaboration of a national programme for control of this parasitosis.
P10
CLINICAL FORMS AND CHEMOTHERAPY
OF TRICHINOSIS
D. Vuchev1, K. Anichina2, K. Eneva1, V. Blagoeva1, A. Russinova3, M.

Darakchieva4, G. Stancheva3
1
Medical University, Plovdiv, Department of Infectious Diseases, Epidemiology,
Parasitology and Tropical Medicine;
2
Bulgarian Patent Department, Sofia;
3
Center for Disease Control, Plovdiv;
4
Center for Disease Control, Smolian
In the last two decades the number of registered trichinosis cases has been on the
rise. This fact made effective etiological therapy with benzimidazoles (thiabendazole,
mebendazole and albendazole) particularly important. This study is aimed at outlining a
215
treatment strategy for trichinosis with benzimidazoles and its clinical forms (asymptomatic,
mild, moderate and severe) and stages (intestinal and muscular) as well as for its
chemoprophylaxis. Experimental-laboratory and clinical data on disease outbreaks are
presented. Subsequent follow-up examinations are necessary in the convalescence period.
Key words: trichinosis, clinical, chemotherapy, benzimidazoles
P11
EPIDEMIOLOGICAL FEATURES OF TRICHINOSIS IN CEN-
TRAL SOUTHERN BULGARIA (PLOVDIV, PAZARDJIK AND
SMOLIAN REGIONS)
D. Vuchev1, K. Eneva1, V. Blagoeva1, A. Russinova2, M. Darakchieva3, G.

Stancheva2, N. Paliiska4
1
Medical University, Plovdiv, Department of Infectious Diseases, Epidemiology,
Parasitology and Tropical Medicine;
2
Center for Disease Control, Plovdiv;
3
Center for Disease Control, Smolian;
4
Center for Disease Control, Pazardjik
A surge in the number of human trichinosis cases has been noticed in the last decades.
The aim of this study is to analyze morbidity rate and clinical features of trichinosis in
Plovdiv, Pazardjik and Smolian regions (Central Sothern Bulgaria) over a 10-year period.
These regions are among the most severely affected and disease outbreaks as well as
sporadic cases are registered annually. Trichinosis distribution and morbidity rate dynamics
in the different administrative and landscape zones are presented. Sources of the disease
( boars and domestic pigs ) and the ratio of rural to urban population affected are analyzed.
Analysis is based on epidemiological surveys, conducted by the Centers for Disease
Control in Plovdiv, Smolian and Pazardjik as well as on clinical and laboratory observations.
Main factors contributing to trichinosis management are: strict epidemiological and
veterinary control, adequate clinical, laboratory and parasitological diagnosis, health
promotion and education of population, especially in the regions where temporary
synantropic foci of infection exist.
Key words: trichinosis, epidemiology, distribution, prevention
216
PLANT AND SOIL MICROBIOLOGY
PSM1
SPREAD AND DETECTION OF PHYTOPLASMA DISEASES
Dimitrijka Sakalieva
Bulgaria, 4000 Plovdiv, 12 Mendeleev str., Agricultural University
e-mail: d_sakalieva@au-plovdiv.bg
Phytoplasmas are associated with several diseases in flower crops, vegetables and
weed, especially from Aster yellows group (16 Sr I). Sometimes they were detected in
mixed infection with phytoplasmas belonging to other ribosomal groups, especially in
woody plants. Phytoplasmas of this group were often identified also in insects.
When cloned phytoplasma DNA probes were produced a large increase was noticed:
their use shows clear evidence that phytoplasma differentiation was possible on the basis
of their DNA sequences. Polymerase chain reaction with primers from sequences of
randomly cloned phytoplasma DNA, from 16S rDNA, from ribosomal protein gene and
others opened the ‘modern era’ of phytoplasmology.
The use of molecular technologies has provided tools to differentiate phytoplasmas
and preliminary results indicate that mixed phytoplasma infection are not uncommon,
especially in woody host plants or insects. These findings have proven that the concept
that each disease is caused by one type of phytoplasma is not generally applicable. In
fact, phytoplasmas infecting flower crops or vegetables, result in belonging to different
subgroups in the 16 Sr I group.
PSM2
MANIFESTATION OF TOLERANCE TO SHARKA
(PLUM POX) VIRUS OF PLUM CULTIVARS IMPORTED
IN BULGARIA
Antoniy Stoev *, Pencho Iliev **

* Plant Protection Institute, Kostinbrod 2230, Bulgaria
** Experimental Station of Plum Growing, Dryanovo, Bulgaria
Several plum cultivars were determined as tolerant to sharka virus (PPV) under natural
conditions. This mode of reaction gives opportunity for an enrichment of plum production
variety.
217
PSM3
PROPOSALS FOR IMPROVEMENT OF THE VARIETY
TESTING OF WHEAT AND BARLEY ACCORDINGLY THEIR
REACTION TO THE MOST IMPORTANT IN BULGARIA
VIRUSES
Nonka Bakardjieva, Antoniy Stoev

Plant Protection Institute, Kostinbrod 2230, Bulgaria
The report substantiates the necessity of an addition of the variety testing system so
that the reaction of the cereal plants to barley yellow dwarf virus (BYDV) and wheat dwarf
virus (WDV) to be evaluated. The tasks specified as main for the improvement already
mentioned are creation of infectious experimental plots and maintenance of viruliferous
vectors.
PSM4
MORPHOLOGIC AND CULTURAL VARIATION IN
PHOMOPSIS FOENICULI
R. Rodeva1, J. Gabler2
1
Institute of Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
2
Institute for Resistance Research and Pathogen Diagnostics, Federal Centre for
Breeding Research on Cultivated Plants, 06449 Aschersleben, Germany
Phomopsis foeniculi Du Man. et Vegh. causes umbel browning and stem necroses on
fennel (Foeniculum vulgare Mill.). The fungus was isolated from naturally infected umbels
and stems.
Twelve isolates were studied for variation of their morphological and cultural characters
on five nutrient media each variant represented by five replications. The experiments were
carried out twice. The isolates showed great variability. They differed in colony colour
and linear growth, the quantity of pycnidia and ability to produce teleomorph in vitro. The
best growth of isolates was observed on V-8 agar and oat agar. Some of isolates scarcely
produced pycnidia in vitro. The most abundant were pycnidia on potato dextrose and oat
agar. They were black, superficial, scattered or aggregated in the colony center. Two types
of conidia were formed in the pycnidia: á- and â-conidia. Alpha conidia were less
abundant than beta conidia, oblong to fusiform, straight to slightly curved, 2-3 guttulate,
5-11 x 2-4 ìm. Beta conidia were filiform, curved or sometimes hamate, eguttulate, 15-
30 x 1-2.5 ìm.
The most suitable media for the sexual production were malt - yeast agar and oat agar.
Ascomata developed as black patches on both nutrient media but only two isolates
succeeded to produce mature perithecia with ripe ascospores. The perithecia were globose
218
to subglobose with long necks, often clustered and contained numerous asci with a
conspicuous refractive apical ring and 8 biseriate ascospores. The ascospores were
unicellular, colourless, ellipsoidal, guttulate, papillate, with rounded ends, 10–15 x 3-4.5
ìm.
PSM5
IN VITRO AND IN PLANTA INTERACTIONS BETWEEN
THREE SCLEROTIAL PLANT PATHOGENS
R. Rodeva, R. Pandeva
Institute of Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
An investigation was undertaken to assess in vitro and in planta interactions between

Botrytis cinerea Pers. Fr., Sclerotinia sclerotiorum (Lib.) de Bary and Verticillium dahliae
Kleb. The three fungi form sclerotia and are among the more devastating plant pathogens.
The ability to survive in adverse conditions and remain viable for long periods in the
absence of hosts makes them difficult to control. They are soil inhabiting, have common
host plants, similar requirements for resources and other niche overlaps. Their interactions
are potentially important determinant of spatial distributions and functioning of their
populations.
Strains obtained from two naturally infected host plants: pepper (Capsicum annuum
L.) and caraway (Carum carvi L.), were included in the study. In vitro tests were made in
dual cultures. Plugs of mycelial inoculum with 5 mm diameter were removed from the
growing margin of the colony and were placed 3.5 cm apart on agar plates in 9-cm petri
dishes, one pairing per dish and incubated in the dark at room temperature. Strains were
paired in all combinations - with itself and with each of the remaining ones. All treatments
were replicated three times. Mycelial compatibility was macro- and microscopically recorded
4 and 14 days after inoculation. Pairings were scored as compatible when the two strains
merged to form one colony with no distinct interaction zone and as incompatible when the
two strains failed to grow together and a thin mycelial-free space remained between them
or when a dark interaction line was observed at the zone of contact. Most pairings showed
incompatible mycelial interactions. All pairings of a strain with itself were compatible. In
planta tests were made by inoculations of pairings on the stem of both host plants at
distance of 5 cm between the points of inoculation. All strains used in this study were in
some way affected by the presence of another fungus.
219
PSM6
БИОЛОГИЧНИ СВОЙСТВА И ЛИОФИЛИЗАЦИЯ НА
ИЗОЛАТИ НА ВИРУСА НА ХЛОРОТИЧНИТЕ ЛИСТНИ
ПЕТНА ПО ЯБЪЛКАТА (ACLSV) ОТ РАЗЛИЧНИ ОВОЩНИ
ВИДОВЕ В БЪЛГАРИЯ
Aнелия Борисова1, Анжела Йорданова2

1
Институт по земеделие, Кюстендил 2500, България;
2
Националната банка за промишлени микроорганизми и клетъчни култури,
София 1113, п.к. 239, България
Проучени са биологичните свойства на пет български изолата на Аpple

chlorotic leaf spot virus (ACLSV), произхождащи от ябълки, череши и праскови,
идентифицирани чрез ELISA.
Изолатите на вируса са пренесени от естествено инфектираните дървета върху
пет вида дървесни индикатори чрез методa на двойното окулиране. Всички изолати
предизвикват сходни, слабо разграничаващи се симптоми, характерни за
съответните индикаторни дървета - Cydonia oblonga (C7/1), Мalus sylvestris R
12740-7А и Malus platycarpa. По листата на Prunus tomentosa и Аronia melanocarpa
не се наблюдават признаци на вирусна инфекция.
Пет тревисти вида, чувствителни към ACLSV, са инокулирани механично със
сок от инфектирани венчелистчета и листа на отделните изолати. При четири от
използваните индикатори се наблюдават локални симптоми, а Chenopodium quinoa
реагира с локални лезии и системна инфекция.
Проведено е вакуумно-сублимационно сушене на листа, венчелистчета и
флоем от заразени дървета ябълка, череша и праскова. Инфекциозността е
тестирана на Ch. quinoa. Установено, е че вирусът се запазва най-добре в
лиофилизирани венчелистчета.
PSM7
EFFECT OF INOCULATION WITH AZOSPIRILLUM AND
AM FUNGI ON THE PLANT BIOMASS OF WHITE THISTLE
(SYLIBIUM MARIANUM L.)
E. Jonova, N. Kaloyanova
“N. Poushkarov” Institute of Soil Science
A pot experiment with meadow-cinnamonic soil (luvisol) was carried out to study the
effect of single and double inoculation with Azospirillum brasilense sp. 36/24 and Glomus
mossae on the growth and development of white thistle (Sylibium marianum L.). The
experiment was carried out without fertilization, with two types of soil fertilization
220
(N50P100K100, N100P200K100) and with leaf fertilization using Bioleaf.
The plant shoots increase more with the mycorisal fungi when there is no fertilization.
The effect of double inoculation on the root mass is noticeably higher.
The N50P100K100 variant showed the opposite - the inoculation decreases the yield from
the plant shoots. In this case the root mass increases only with the application of
Azospirillum bacteria whereas in the other variants it decreases. In comparison with the
lower doses, the N100P200K100 variant decreases the weight of the plant shoots in the control,
while the mixed inoculation increases it. The root mass increases only with single
Azospirillum inoculation.
Our experiment with leaf fertilization didn’t show any particular effect as a result from
the inoculations.
PSM8
ЕФЕКТ ОТ ИНОКУЛАЦИЯТА С МЕСТНИ ЩАМОВЕ
ФОСФАТРАЗЛАГАЩИ БАКТЕРИИ ВЪРХУ РАЙГРАС
Костадинка Недялкова
Институт по почвознание “Н.Пушкаров”, София, ул. “Шосе Банкя” 7
Инокулацията с полезни почвени микроорганизми с цел подобряване

храненето на културите и повишаване на продукцията е предмет на многобройни
изследвания през последните десетилетия.
В настоящата работа се изследва влиянието на инокулацията с
фосфатразлагащи бактерии, изолирани от наши почви, върху продукцията на
биомаса и усвояването на азот, фосфор и калий от райграс. Опитът е изведен във
вегетационна къща. Почвата е наторена с N, P, K във всички варианти. Извършена
е инокулация с четири бактериални щама, притежаващи фосфатразлагаща
активност. Проследен е ефекта върху количеството суха биомаса (зелена маса и
корени) и съдържание на азот, фосфор и калий при три откоса на зелената маса.
В резултат на бактериалните инокулации количеството на зелената маса при
I откос се увеличава незначително (3-5%). Чувствително повишение на биомасата
(15%) е установено при II откос при инокулация с щам 67. При III откос
количеството на зелената маса намалява и ефект от бактериалните инокулации
не се наблюдава. Под влияние на инокулациите се увеличава съдържанието на
фосфор и калий в корените.
221
PSM9
TAXONOMICAL IDENTIFICATION OF ARSENIC RESIS-
TANT AND ARSENIC TRANSFORMING
SULFATE - REDUCING BACTERIA
Krasimira S. Krumova, Veneta I. Groudeva

Department of General and Industrial Microbiology, Lab. ”Geomicrobiology”,
1164 Sofia, Bulgaria; e-mail: krumova@biofac.uni-sofia.bg
Abstract: Twenty eight bacterial strains, which are able to transform arsenic compounds,
were isolated and characterized in the laboratory of ”Geomicrobiology” at the Department
of “General and Industrial Microbiology”, SU “St. Kliment Ohridski”. It was determined,
that 11 of these isolates are able to efficiently oxidize the high mobile and high toxic
arsenite [As (III)] to less mobile and less toxic arsenate [As (V)]. The others 17 strains are
able to reduce arsenate [As (V)] to arsenite [As (III)]. Оn the basis of the determination
via classical methods, the isolated bacteria were related to different genera within the
big group of sulfate-reducing bacteria. The confirmation of the taxonomical belonging
of these strains was done on basis of the amplification of their DNA with SRB specific
primers for each genus.
Key words: Sulfate-reducing bacteria, arsenite oxidizing and arsenate reducing bacteria;
PSM10
ВЛИЯНИЕ НА ПРОТЕИНОВ ХИДРОЛИЗАТ ОТ
КЕРАТИНОВИ ОТПАДЪЦИ ВЪРХУ ПОЧВЕНАТА
МИКРОФЛОРА
Мая Нусторова, 1 Адриана Гущерова2, Диана Брайкова 2, Евгения Василева2

1
Лесотехнически университет –София
2
БАН-Институт по микробиология
Кератин-съдържащите отпадни материали / вълна, четина, рога, перушина и

др. / от редица индустриални производства са потенциален източник на ценни
продукти, богати на протеини и аминокиселини с възможности за широко
практическо приложение. Перспективен метод за използване на кератиновите
отпадъци е трансформирането им в евтини и екологично безопасни протеинови
хидролизати.
Изследвана е възможността за използване на протеинов хидролизат от
кератинови отпадъци като почвен подобрител чрез активиране на почвената
микрофлора. Препаратът е изготвен на базата на алкална хидролиза на
животински отпадни продукти. Той съдържа 75 – 80% водонеразтворими
222
съединения – пептиди, аминокиселини, соли, липиди, въглехидрати, пигменти,
калиеви йони. Изследването е извършено в лабораторни условия – 8-месечен
съдов опит с три варианта на торене. Използвана е почва – чернозем-смолница,
засята с райграс /lolium perenne/.В динамика са проучвани почвени,
микробиологични и растителни показатели. Данните сочат трайна тенденция за
повишаване на почвената биогенност, растежа и натрупването на растителна
биомаса и при трите варианта на торене. Определена е оптималната концентрация
на въздействие на препарата.
PSM11
ИЗСЛЕДВАНИЯ ВЪРХУ ТОЛЕРАНТНИ КЪМ
ЗАСУШАВАНЕ ГЕНОТИПОВЕ СОЯ. І. ОТЗИВЧИВОСТ
КЪМ ИНОКУЛАЦИЯ С BRADYRHIZOBIUM JAPONICUM
Анна Маркова, Радка Алтимирска, Йорданка Киркова, Георги Стоименов

Институт по почвознание “Н.Пушкаров”, София
При селекция на соята е необходимо да се имат предвид не само стопанските

й качества, но и нейната способност да фиксира атмосферен азот.. В този аспект
изследването на отзивчивостта на тази култура към инокулация със специфичните
за нея азотфиксиращи бактерии от вида Bradyrhizobium japonicum е от особено
важно значение.
При условията на съдов и полски опити с излужена ливадно-канелена почва
от с.Цалапица,Пловдивско са изпитани вирулентността и ефективността на
Bradyrhizobium japonicum при сухоустойчиви и отзивчиви на напояване сортове и
линии соя. Установена е различна отзивчивост на соята към инокулация в
зависимост от генотипа и почвената влажност.
PSM12
ИЗСЛЕДВАНИЯ ВЪРХУ ТОЛЕРАНТНИ КЪМ
ЗАСУШАВАНЕ ГЕНОТИПОВЕ СОЯ ІІ.
МИКРОБИОЛОГИЧНА АКТИВНОСТ В РИЗОСФЕРАТА
НА СОЯ
Радка Алтимирска, Анна Маркова, Христо Стойков, Красимир Чачев

Засушаването като важен абиотичен фактор е от особено значение както по

отношение продуктивността на соята, така и по отношение на микробиологичната
223
активност в почвата. Освен симбиотичните азотфиксиращи бактерии съществено
значение за храненето на соевите растения има и ризосферната им микрофлора.
В условията на съдов и полски опити върху излужена ливадно-канелена почва/
с.Цалапица, Пловдивско/, бяха изследвани основните физиологични групи
микроорганизми, участващи в трансформацията на хранителните вещества в
ризосферата на различни сухоустойчиви и отзивчиви на напояване сортове и
линии соя. Установените изменения в микробиологичната активност, както и
тези в добива варират в зависимост от генотипа на соята и изпитваната влажност.
PSM13
ВЛИЯНИЕ НА ХЕРБИЦИДА РИЛЕЙ ВЪРХУ ОСНОВНИ
СВОЙСТВА НА BRADYRHIZOBIUM JAPONICUM
Радка Донкова – Институт по почвознание “Н.Пушкаров”, София
В условията на съдов опит с две почви (излужена смолница, Божурище и

алувиално-ливадна, Цалапица) и соя сорт “Даниела” е проучено влиянието на
хербицида рилей върху основни свойства на Br. japonicum. Хербицидът е изпитан
в две концентрации – препоръчваната за употреба в практиката и двукратно
увеличена – съответно 200 и 400 ml/dka за алувиално-ливадната почва и 280 и
560 ml/dka за излужената смолница.
Установено е отрицателно влияние на рилей върху вирулентността,
азотфиксиращата активност и ефективност на Br. japonicum. Наблюдаваният ефект
е по-силно проявен при вариантите с високата концентрация на хербицида, при
които е отчетено двукратно намаление на вирулентността, 30% по-малко
фиксиран азот и окооло 20% по-нисък добив. Хербицидното действие показва
зависимост от почвените свойства. То е по-силно проявено при алувиално-
ливадната почва.
PSM14
МИКРОБИОЛОГИЧНА ХАРАКТЕРИСТИКА НА ПОЧВИ В
РАЙОНА НА КЦМ- ПЛОВДИВ
Радка Донкова и Николай Динев

Институт по почвознание „Н.Пушкаров”, София
Функциониращите в страната комбинати за цветни метали генерират проблема

за замърсяването на почвите с тежки метали и металоиди. Акумулирането на тези
замърсители в почвата би могло да окаже влияе върху състоянието и
функционирането на почвените микробиални съобщества. което да промени
224
устойчивостта на почвената екосистема като цяло. Целта на настоящето
изследване е да се установи съдържанието на тежки метали в района на КЦМ
Пловдив и влиянието им върху почвената микрофлора. Определени са
съдържанието на тежки метали в почвата, количеството на основни групи почвени
микроорганизми и общата биологична активност на проби , взети на различно
отстояние от комбината по мониторингови точки. Установено е, че основни
замърсители за района са Cd, Pb и Zn, чиито концентрации в някои от изследваните
точки превишават пределно-допустимите с няколко порядъка. Наблюдава се
промяна в съотношението между отделните групи почвени микроорганизми и
намаляване на общата биологична активност, което е указание за стесняване
спектъра на микробиологичната дейност и понижаване функционалната
активност на микроорганизмите.
PSM15
ВЛИЯНИЕ НА КАДМИЯ ВЪРХУ
МИКРОБИОЛОГИЧНАТА АКТИВНОСТ НА
КАРБОНАТЕН ЧЕРНОЗЕМ
Галина Петкова, Радка Донкова

Тежките метали могат да предизвикат сериозни нарушения в биоценозите,

почвеното плодородие и здравето на човека поради своята токсичност и
способността им да се акумулират трайно в околната среда. От особено значение
е влиянието им върху почвената микрофлора, от дейността на която зависи
функционирането на почвата като жива система и поддържане на биологичната
й продуктивност.
В моделен опит в динамика е проучено влиянието на нарастващи
концентрации кадмий върху основни групи почвени микроорганизми и общата
биологична активност на карбонатен чернозем. Установено е, че кадмия има
отрицателно действие върху някои от проучваните групи микроорганизми при
концентрации по-ниски от ПДК. Най-чувствителни са микроорганизмите
свързани с азотната трансформация на органичното вещество. Сравнително най-
устойчиви са актиномицетите. Въз основа на получените данни е направена
оценка на възможността за използване на проучваните показатели като
индикатори за замърсяване на почвата с кадмий.
225
ACTUAL PROBLEMS OF THE BIOSCIENCE
BS1
SCIENCE FUNDING IN BULGARIA
Albena Vutsova, Ministry of Education and Science, Sofia
It is evident that science as a whole has a significant role to play in support of a society
which has placed knowledge creation at the heart of its vision. What is more, science is of
primary importance for the development of economy and for strengthening its structure
(especially for the countries in transition). It is not by chance that we use the term
“knowledge based economy”. The challenges bared by this term are building on the well-
documented public interest in science, using all forms of communication to foster dialogue
and interest, maintaining international competitiveness in high technology areas and, in
addition, providing scientific and technological competence as a precondition for global
competitiveness and the key to successful partnerships.
To address these challenges, one of the most important prerequisites for the
development of a knowledge-based society is the funding of science. Scientific research
funding is closely related to the adoption and implementation of scientific, technological
and innovation policy – three different spheres of activity, however, incorporated within a
dynamic framework.
Bearing in mind that scientific and technological policy and scientific research funding are
interrelated, we should look at the sources of funding and the way funds are allocated.
Development of a stable, vital and competitive research system in Bulgaria,, contributing
to the creation of knowledge-based economy depends on several factors, namely (but not
exhaustively) :
- General increase of state expenditures for research and development
- Mix of financial instruments – public funds, private sources and European funds
- Allocation of funds on a competitive basis
226
BS2
БИОИНФОРМАТИЧНИ РЕСУРСИ В
МИКРОБИОЛОГИЯТА:
ПРЕДИЗВИКАТЕЛСТВА И ПЕРСПЕКТИВИ
Димитър Василев, Агро Био Институт, София
През последните няколко десетилетия напредъкът на молекулярната биология

и наличното модерно оборудване за научни изследвания позволиха секвенирането
на големи части от генома на множество видове организми. В действителност до
сега са секвенирани напълно геномите на няколко вида бактерии и на няколко
еукариоти (напр. хлебните дрожди). Човешкият геном също беше изцяло
секвениран в рамките на мащабен международен проект. Популярни бази-данни
със секвенции като GenBank и EMBL, ресурси за търсене и анализ на молекулярна
информация (EMBOSS,), обектно ориентирани бази данни и софтуер за обработка
се създават с нарастваща скорост за съхраняване, анализ, визуализиране на
събраната информация. Този огромен приток на информация е необходимо да
бъде грижливо съхраняван, организиран и индексиран. За тази цел
информационните технологии и техните приложения в биологията създадоха една
нова, бързоразвиваща се научна област, наречена биоинформатика.
Най-елементарните задачи на биоинформатиката засягат създаването и
поддържането на бази-данни с биологична информация. Последователности от
нуклеинови киселини, получени след секвенирането им, както и протеинови
последователности, получени от тях, представляват болшинството от
информацията на тези бази данни. Докато съхраняването и организацията на
милиардите нуклеотиди е далече от обичайната дейност, изграждането на базата
данни и създаването на интерфейс, чрез който изследователите ще могат
едновременно да имат достъп до информацията и да подават нови данни, е само
началото.
Най-неотложната задача на биоинформатиката включва анализа на
секвенираната информация. Този процес се осъществява от т.нар. изчислителна
биология, която включва: предсказване и откриване на гените в ДНК
последователностите в различните организми; изработване на методи за
предсказване на триизмерната структура и/или функция на новооткритите
протеини и структурни РНК последователности; групиране на протеиновите
секвенции в семейства от свързани секвенции и разработване на протеинови
модели; подреждане на сходни протеини и създаване на “филогенетични дървета”
за изследване на еволюционните връзки.
Бързото навлизане и приложение на биоинформатиката в
микробиологията създава предпоставки за все по-мащабно и динамично развитие
на огромни международни проекти засягащи особено вирусологията,
изследването на не-малък брой социално значими заболявания: СПИН, хепатит,
in silico моделирането и разработването на различни видове лекарства.
227
FIRST AUTHOR INDEX - ABSTRACT NUMBER
Abashev Y. P. VP32 Dimitrova D. Bl. GAM50, GAM51, GAM52

Abrashev R. GAM49 Dimov Sv. G. GAM61
Ackermann J- U. GAM19 Dmitrieva T. GAM83
Aleksandrova R. GAM21 Dobrev G. GAM16
Aleksiev I. V22 Donkova R. PSM13, PSM14
Alexiev R. II9, II10 Dubyago N. MM35
Alexieva Z. GAM26 Dundarov S. V8
Altimirska R. PSM12
Andonov A.P. V13 Evstatieva Y. GAM58
Angelov P. MM10
Angelova M. GAM11 Filipov Ch. VP38
Argirova R. V14
Arnaudov H. VM5 Galabov A. S. Tb1, V2
Atanassova M. GAM54 Galabova D. GAM10
Atanassova А. V. GAM74, GAM75 Galabinova T. VM16
Gavazova R. V20
Bakalova St. GAM41 Georgiev D. GAM57
Bakardjieva N. PSM3 Georgieva I. L. VP31
Beshkov D. V24 Georgieva J.H. GAM42
Bikumandla A. GAM56 Gerginova M. GAM28, GAM29
Blazheva D. MM5 Ginova T. GAM89
Bojkova K. MM16 Gladniska T. MM24
Boneva B. VP39 Gocheva Y. GAM48
Bonovska M. Tb8, VM9 Golkocheva E. II14
Borisov K. V19 Gotev N. MM25, MM26
Borisova A. PSM6 Gotseva A. V29
Borissova P. VP37 Gouliamova D. E. GAM3
Bostandjieva R. VP41 Grigorova V. MM14, MM15
Brankova N. MM21 Groudeva Tz. GAM4
Bumbarov V. VM1 Groudeva V. GAM7, GAM8, GAM30
Gulluce M. MM17
Chankova D. MM30 Gurgulova K. VM7
ChikovaV.VP43,VM15, VM16,VM17,VM18 Gurova-Chausheva A. GAM91
Chouchkova M. Tb2
Christova D. GAM36 Hadjiolova T. V16
Christova I. MM23 Harizanov R. P8
Hristova N. GAM72
Decheva A. MM22
Delcheva D. GAM 60 Ianis M. GAM35
Derekova A. GAM44 Ignatova Tz. GAM22
Dimitonova S. MM28 Iliev I. GAM15.
228
Iliev M. VM13, VM14 Mileva M. V12
Ilieva D. VP42 Minchev P. Tb5
Iovcheva L. GAM82 Mitov I. MM12
Ivanov T. GAM92, GAM93 Mladenov K. MM27
Ivanova E. II3 Mladenova Z. V15
Ivanova I. GAM9 Muhtarova M. II8
Ivanova L. VP40 Murgov I. MM19
Ivanova K. MM36
Ivanova M. P2 Najdenski H. VM3, II15
Ivanova S. VM6 Naumova S. GAM45
Ivanova V. GAM68, GAM69, GAM70, GAM71 Nedelcheva P. GAM87, MM18
Nedjalkova K. PSM8
Kabaivanova L. GAM32 Nenkov I. V28
Kalcheva H. GAM46 Nenova R. MM4
Kalfin E. MM7 Nikolov L. GAM17, GAM18
Kalvachev Z. V26 Nikolova D. GAM85
Kantardjiev T. Tb6, MM11 Nikolaeva-Glomb L. V4
Karakolev R. VM2 Nustorova M. PSM10
Koltyga I. GAM84
Koprinarova M. GAM88 Orozova P. MM29
Korsun N. VP34
Korudjiisky N. VM8, VM15 Pashova-Baltova K. GAM80
Kostova D. GAM73 Pavlova S. V17
Kouzmanov A. MM31 Peeva V. M. GAM62
Krumov N. GAM40 Pencheva V. MM9
Krumova E. V12, GAM47 Peneva M. V21
Krumova Kr. GAM5, PSM9 Petkova G. PSM15
Kurdova-Mintcheva R. P1 Petrova P. GAM14
Kuzelov A. GAM20, VM4 Petrova V. GAM12
Kuzmin V. V3 Popov A. MM32
Lahtchev K. GAM90 Rainova I. P5, P6

Lozitsky V. V4 Raleva S. V23
Lubashevsky E. II4 Ratkov A. GAM23
Lupke V. II12. Remichkova M. V10
Lyutskanova D. GAM59 Rodeva R. PSM4, PSM5
Rukanova D. MM13
Manasiev Y. GAM27 Rusev V. VP36
Margesin R. GAM6
Marinov B. GAM77 Safarikova M. GAM53
Marinova I. P3, P4 Sakalieva D. PSM1
Markova A. PSM11. Sapungieva E. II6, II7
Meincken M. GAM66 Savova T. GAM79
Mekouchinov K. V30 Serbezov V. MM8
229
Serkedjieva J. V9 Topalova Y. GAM2
Shilev S. GAM39 Toshkova R. II16
Shishkov S. VP33 Tosi S. GAM1
Simeonova L. V7 Tsekova K. GAM37
Slavchev A. GAM86 Tsvetkova N. P7
Slavov S. V27 Tumbarski J. D. V6
Slavova-Azmanova N. GAM13
Smirnov I. II1 Urshev Z. GAM81
Sotirova A. GAM33
Spiridonova V. MM34 Valcheva V. Tb7
Sredkova M. MM1 Van den Worm A. GAM67
Stankulova D. II5 Vasilev G. VM11
Stefanova D. Tb4 Vassilev D. BS2
Stefanova T. II11 Vassilev T. II2
Stefanova V. GAM55 Vassileva-Pencheva R. V5
Stoev A. PSM2 Vatcheva R. MM33
Stoilov R. MM3 Velcheva D. V18
Stoilova I. GAM31. Vilhelmova N. VP35
Stoimenova E. GAM78 von Mollendorff J.W. GAM64
Stoyancheva G. MM2 Vuchev D. P10, P11
Stoycheva T. GAM25 Vutsova A. BS1
Teoharov P. V25 Wild F. V1

Terziyska A. GAM43
Terziiski D. MM20 Yankova Z. Tb1
Todorov K. GAM24 Yankova P. GAM76
Todorov S.D. GAM63, GAM65 Yonkova V. II13
Todorova T. GAM38 Yonova E. PSM7
Tomova I. MM6 Yordanova D. P9
230
231
ELEVENTH CONGRESS
OF THE BULGARIAN MICROBIOLOGISTS
St. Constantine, Varna, October 5-7, 2006
232
ELEVENTH CONGRESS
OF THE BULGARIAN MICROBIOLOGISTS
St. Constantine, Varna, October 5-7, 2006
233

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PROGRAM AND ABSTRACTS

International House of Scientists

St. Constantine, Varna

October 5-7, 2006

Congress October 5, 2006 October 6, 2006 October 7, 2006

Posters Session I Session II

Congress Organizers ……………………………………………..

Union of Scientists in Bulgaria –

Bulgarian Society for Microbiology

Bulgarian Society of Medical Microbiology

The Stephan Angeloff Institute of Microbiology,

President: Prof. Angel S. GALABOV, DSc,

Local organizing committee:

Liliana HARALAMPIEVA, MS, Assist. Prof. Ralitca NEDKOVA, MS

The Stefan Angeloff Institute of Microbiology,

БИОКОМ Tрендафилов ЕООД

представлявана за България от фирма КРОСПОЙНТ ЕООД

Medical Technics Engineering Ltd.

General and Applied Microbiology (GAM)

Medical Microbiology (MM)

Veterinary Microbiology (VM)

Infectious Immunology (II)

Plant and Soil Microbiology (PSM)

Actual Problems of the BioScience (BS)

Thursday, October 05, 19:00

Friday, October 06, 20:00

Registration: Wednesday, October 04 16:00 – 19:00

Chairpersons: Angel S. Galabov

9:00 Welcoming Addresses

President of the Organizing Committee

Memorial Session Dr. Stamen Grigorov

9:20 Tb1 Vitae of Dr. Stamen Grigorov

14:00 V1 Henipaviruses, a new family of paramyxoviruses,

14:30 V2 Structural basis for antiviral drug-resistance of a

15:00 V3 The hierarchical QSAR technology for effective

16:00 V4 Christo Russeff Memorial Lecture

16:20 V5 Rational treatment course schedule effective

16:50 V7 In vivo effective anti-flu combination of antivirals

17:05 V8 Must monesine preparative be used for prophylaxis of

17:20 V9 In vitro anti-influenza virus effect of a protease inhibitor

17:35 V10 Anti-herpes activities of Pseudomonas sp. S-17

17:50 V11 Antioxidant effects of plant polyphenols quercetin and

18:05 V12 A plant polyphenol extract reduces oxidative stress in

18:20 Session end

Hall 2: Medical Microbiology

Chairpersons: G. Terziiski, Sofia

14:00 MM1 Polyphase identification analysis of clinical

14:30 MM2 Study of vaginal lactobacilli from Bulgarian

14:50 MM3 Frequency of the triggering infection at patients

16:00 MM4 Microbiological investigation of imported

16:15 MM5 Characteristics of the cultivation of

16:30 PCR-DIAPOPS method for detection of

17:00 MM6 Contamination of hotels from Bulgarian Black

17:15 MM7 Normal flora is found in human blood

17:30 MM8 Q-fever – an insufficiently known and

17:45 MM9 Clinical aspects of lung mycotic infections

18:15 Session end

Hall 3: Veterinary Microbiology

Chairpersons: I. Ivanov, Sofia

14:00 Microbiological methods for detection of

14:20 VM1 Development and application of rabbit polyclonal

14:40 VM2 Findings and serotyping of Listeria

15:00 VM3 Optimization of PCR-DGGE for direct detection