molecular characterization of

108 Pages·2002·1.44 MB·English

ISOLATION, IDENTIFICATION, MOLECULAR
CHARACTERIZATION AND DETECTION OF VIRULENCE
ASSOCIATED GENES OF
PASTEURELLA MULTOCIDA OF AVIAN ORIGIN

A
THESIS
SUBMITTED TO THE
ANAND AGRICULTURAL UNIVERSITY
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE
OF
MASTER OF VETERINARY SCIENCE
IN
VETERINARY MICROBIOLOGY
BY
MAYURKUMAR P. BHIMANI
B.V.Sc. & A.H.
(Reg. No.: 04-1687-2011)
DEPARTMENT OF VETERINARY MICROBIOLOGY,
COLLEGE OF VETERINARY SCIENCE & ANIMAL HUSBANDRY
ANAND AGRICULTURAL UNIVERSITY
ANAND - 388 110
2013
DEDICATED TO
My Beloved Parents
and
My Dearest Sister
“ISOLATION, IDENTIFICATION, MOLECULAR CHARACTERIZATION
AND DETECTION OF VIRULENCE ASSOCIATED GENES OF
PASTEURELLA MULTOCIDA OF AVIAN ORIGIN”
Name of Student: Mayurkumar P. Bhimani Major Advisor: Dr. Ashish Roy

DEPARTMENT OF VETERINARY MICROBIOLOGY
COLLEGE OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY
ANAND AGRICULTURAL UNIVERSITY
ANAND-388 001.
ABSTRACT
The present study was undertaken with a view of isolation and identification of
P. multocida from suspected cases of fowl cholera in different avian species.
A total of 312 samples were collected from suspected cases, of which 24
(7.69%) P. multocida isolates (22 from emu and 2 from chicken) were isolated using
blood agar as primary culture medium. These isolates were studied for their
biochemical behaviour, in vitro antibiotic sensitivity pattern, P. multocida species
specific PCR (PM-PCR), molecular characterization by multiplex PCR assay for
capsular typing and detection of various virulence associated genes by PCR.
All the isolates produce non-haemolytic colonies on blood agar and failed to
grow on MacConkey agar. On biochemical behaviour, all the 24 isolates (100%) were
found positive for oxidase, catalase, indole production, nitrate reduction and of
glucose, mannitol, mannose, sucrose, galactose, and sorbitol. All the 24 isolates
(100%) were negative for citrate utilization test and could not ferment dulcitol,
inositol, salicin, xylose, trehalose and lactose. Except two isolates obtained from
poultry, rest all 22 (91.66%) isolates successfully utilized arabinose and maltose.
All 24 (100%) isolates were sensitive to amoxicillin, ampicillin, cefepime,
cefixime, cephotaxime, ciprofloxacin, enrofloxacin, flumequine, gentamicin,
norfloxacin, oxytetracycline, pefloxacin, spectinomycin, streptomycin, sulfadiazine
and tetracycline while all the isolates (100%) were found to be resistance to amikacin,
co-trimoxazole and erythromycin. In addition, all 22 isolates of emu were sensitive to
cephalothin while both isolates of chicken showed resistance to the same.
All the 24 field isolates along with HS vaccine strain (P. multocida P ), known
52
P. multocida capsular type A strain, and five other gram negative bacterial cultures
were tested by PM-PCR, which revealed an amplified product of approximately 465
bp size for all P. multocida isolates, while gram negative bacterial isolates did not.
Later, all the isolates were tested for capsular type by multiplex PCR assay
along with reference strains, which showed an amplified product of 1044 bp size for
all the field isolates and reference strain P. multocida capsular type A. It indicates that
all the field isolates were belonged to capsular type A.
Bone marrow samples which were found to be positive for P. multocida by
cultural and biochemical tests were subjected to direct detection of genomic DNA of
P. multocida from tissue sample by PM-PCR. All tested bone marrow samples showed
amplified product of 465 bp size indicating presence of P. multocida genome.
All the 24 field isolates were subjected to detection of thirteen virulence
associated genes by PCR viz., ptfA, pfhA, nanH, nanB, sodA, sodC, tbpA, hgbA, hgbB,
ompH, oma87 ,toxA, and plpE. It was found that nine genes namely, ptfA, pfhA, nanB,
sodA, sodC, hgbA, ompH and oma87 were present in all the isolates while all the
isolates were negative for the toxA, tbpA and nanH gene. All the 22 (91.66%) emu
isolates were positive for plpE but both chicken isolates were found negative for plpE.
Out of 24 isolates, three emu isolates were found to be negative for hgbB gene.
To conclude, in this study P. multocida infection could be demonstrated in emu
appears to be a very rarely reported indicating a fowl cholera like disease condition
usually associated with severe economic loss to the poultry industry. Both chicken and
emu isolates were of same capsular type A. PCR a rapid, sensitive and effective
method could be employed for the detection of P. multocida among the avian species
studies. Detection of various virulence associated genes has helped characterization of
virulence potential of isolates.
Dr. Ashish Roy
Ph.D.
Professor
Department of Veterinary Microbiology
College of Veterinary Science & A.H.,
Anand Agricultural University
ANAND-388 110
CERTIFICATE
This is to certify that the thesis entitled “Isolation, identification, molecular
characterization and detection of virulence associated genes of Pasteurella
multocida of avian origin” submitted by Mayurkumar P. Bhimani (Reg. No. 04-
1687-2011) in partial fulfillment of the award of the degree of MASTER OF
VETERINARY SCIENCE in the subject of VETERINARY MICROBIOLOGY of
the Anand Agricultural University is a recorded bonafide research work carried out by
his under my guidance and supervision and the thesis has not previously formed the
basis for the award of any degree, diploma or the similar title.
Place : Anand (ASHISH ROY)
Date : /04/2013 Major Advisor
CERTIFICATE
This is to certify that I have no objection for supplying copy of any part of this
thesis at a time through reprographic process, if necessary for rendering service in a
library or documentation centre.
Place : Anand
Date : /04/2013 (Mayurkumar P. Bhimani)
(Ashish Roy)
Major Advisor
ACKNOWLEDGEMENTS
This thesis is the end of my journey of pursuing my M.V.Sc. I have received
immense support and encouragement from numerous people including my well
wishers, my friends, colleagues and institution. At the end of my thesis I would like to
thank all those people who made this study possible; an unforgettable experience for
me, and to all those who contributed in many ways to the success of this work.
I would like to express my deepest sense of Gratitude to my major advisor Dr.
Ashish Roy, Professor, Department of Veterinary Microbiology, for his continuous
advice and encouragement throughout the course of this study.
I am particularly beholden to my Minor Guide Dr. C. J. Dave, Assistant
Professor, Department of Veterinary Pathology, College of Veterinary Science &
Animal Husbandry, Anand for his kind counsel during my study.
I wish to express my sincere gratitude and thanks to members of advisory
committee: Dr. M. K. Jhala, Professor and Head, Department of Veterinary
Microbiology, and Dr. D. N. Rank, Professor and Head, Department of Animal
Genetics and Breeding for their supportive attitude and sustained encouragement
during tenure of my study.
I am also extremely indebted to Dr. B. B. Bhanderi and Dr. R. A. Mathakiya,
Assistant Professor, Department of Veterinary Microbiology for their advices,
constructive criticisms and extensive discussions around my work.
I am very much thankful to Dr. A. M. Thaker, Principal and Dean, College of
Veterinary Science and Animal Husbandry, Anand Campus, Anand for his generous
attitude in providing necessary facilities to carry out the research work.
It’s my fortune to gratefully acknowledge the support of some special
individuals. Words fail me to express my appreciation to Dr. Barvalia for his support,
generous care and the homely feeling at Anand.
I express gratitude to my departmental colleagues for providing a stimulating
and fun filled environment. My thanks go in particular to Dr. Ankita Shah, Dr. Hardik
Patel, Dr. Bharat Patel and Dr. Panna Rabari. My special appreciation goes to Dr.
Ranpariya for his friendship and encouragement.
I also thank my other colleagues, Drs. Butani, Vaghmashi, Nakrani, Dahima,
Savaliya, Suchit, Mukesh, Patil, Brijesh and to undergraduate friends Nikunj, Ketan,
Arun, Prakash, Ritesh and Hitesh for their constant inspiration and for whole hearted
co-operation.
I would also like to extend huge, warm thanks to some special persons
Prashant, Reuben, Abi (Sugar) and Jatin for their love, care and moral support.
I am highly thankful to departmental staff Jayaben, laboratory technician, and
supporting staff Harishbhai and Ravjibhai for their co-operation throughout the course
of study.
Finally, I take this opportunity to express the profound gratitude from my deep
heart to my beloved parents and my siblings for their love and continuous support –
both spiritually and materially. I also would like to thank my nephew and niece for the
laughter gifted to me with their cute and tender voice on the phone.
Lastly, I would like to pay thanks to God almighty who blessed me to achieve
the success.
Place: Anand
Date: /4/2013
(Mayurkumar Bhimani)
CONTENTS
Chapter No. Chapter Page No.
I INTRODUCTION 1 - 4
II REVIEW OF LITERATURE 5 - 24
III MATERIALS AND METHODS 25 - 40
IV RESULTS 41 - 54
V DISCUSSION 55 – 68
69 - 72
VI SUMMARY AND CONCLUSIONS
REFERENCES I - XV
LIST OF TABLES