بسم االله الرحمن الرحيم

Molecular Characterization of Pasteurella multocida Vaccine

Strains

By:

Hajir Badawi Mohammed Ahmed

B.V.M Khartoum University (2006)

Supervisor:

Dr. Awad A. Ibrahim

A dissertation submitted to the University of Khartoum in partial

fulfillment of the requirements for the degree of M. Sc. in

Microbiology

Department of Microbiology,

Faculty of Veterinary Medicine,

University of Khartoum

June, 2010

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Dedication

To my mother

Father

Brother, sister and friends

With great love

 

Acknowledgments

First and foremost, I would like to thank my

Merciful Allah, the most beneficent for giving me

strength and health to accomplish this work.

Then I would like to deeply thank my supervisor Dr.

Awad A. Ibrahim for his advice, continuous

encouragement and patience throughout the period

of this work.

My gratitude is also extended to prof. Mawia M.

Mukhtar and for Dr. Manal Gamal El-dein, Institute

of Endemic Disease.

My thanks extend to members of Department of

Microbiology Faculty of Veterinary Medicine for

unlimited assistant and for staff of Central

Laboratory Soba.

I am grateful to my family for their continuous

support and standing beside me all times.

My thanks also extended to all whom I didn’t

mention by name and to the forbearance of my

friends, and colleagues who helped me.

Finally I am indebted to all those who helped me so

much to make this work a success.

 

Abstract

The present study was carried out to study the national haemorrhagic

septicaemia vaccine strains at their molecular level. The vaccine is

bivalent contain Pasteurella multocida serotype E:2 and B:2. The

vaccine strains were obtained from Central Veterinary Research

Laboratory, Department of Biological Product, Soba. Culture

characteristic and colonial morphology was ascertained in common

laboratory media. The bacterial strains were characterized by using

sodium-dodecyl sulphate polyacrylamide gel electrophoresis SDS-

PAGE, western blotting and by using PCR for capsular serotyping.

Firstly, the two strains were characterized by SDS-PAGE technique.

The bacteria were cultured in liquid media, and then the bacterial

whole cell lysates were prepared. SDS-PAGE was carried out for both

strains and the proteins bands were stained with coomassie brilliant

blue. Then the molecular weights of the proteins bands were

determined; they were 175 kDa, 165 kDa, 150 kDa, 123 kDa, 102kDa,

90 kDa, 85 kDa, 70 kDa, 64 kDa, 60 kDa, 51 kDa, 42 kDa, 37 kDa, 22

kDa and16 kDa. The protein profiles of both vaccine strains were

similar.

P. multocida strains protein profiles were investigated by

immunoblotting using specific hyperimmune sera prepared in rabbits.

Immunostaining with enzyme conjugate was done after transfer of

proteins in nitrocellulose acetate paper. Eight proteins in whole cell

lysate, of approximately 175 kDa, 102 kDa, 90 kDa, 85 kDa, 70 kDa,

42kDa, 37 kDa, and 16 kDa, were recognized by the sera.

 

The PCR assay was performed for strain B and E of P. multocida by

using primers that amplify capsule gene. Two extraction methods for

DNA were used. There were the Boiling method and the kits method

in which a lysis buffer is used. The two methods of DNA extraction

gave good DNA yield. However the kit method was better in this

respect than the boiling method. Primers of strain E gave an

amplification product of 511 bp for vaccine strain E. In contrast

primers of strain B did not amplify strain B DNA vaccine strain but

amplified strain B field strain which were obtained from Department

of Microbiology Faculty of Veterinary Medicine, University of

Khartoum stock culture and the amplicon was 760 bp.

 

المستخلص

اللقاح . القومي لمرض التسمم الدموي للقاح المستوى الجزيئي دراسةل بحثال اأجري هذ .للبكتريا الباسترولا مالتوسيدا B:2, E:2 ثنائي العترة يتكون من النوعين المصلليين

من مركزالمعامل والأبحاث البيطرية المركزية بسوبا قسم المنتجات اللقاح جلبت عترات لوحظت الخصائص المزرعية و أشكال المستعمرات في الأوساط المزرعية . يةالبيولوجالترحيل الكهربائي بالصوديوم دوديصايل ثم درست عترات البكتريا باستعمال . العامة

لتحديد النوع سلفيت بولي اكريلميد جل و الوسترن بلوت و تفاعل البلمرة التسلسليحيل الكهربائي بالصوديوم دوديصايل سلفيت أجري اختبار التر أولا. المصلي للمحفظةحطام الخلايا حضر محلول زرعت البكتريا في وسط سائل ثم. بولي اكريلميد جل

أجري اختبار الترحيل الكهربائي بالصوديوم دوديصايل سلفيت بولي اكريلميد . البكتيريةق ثم حدد جل للنوعيين المصليين وتم صبغ البروتين بصبغة كوماسي البريلينت الأزر

70 ،85 ،90، 102، 123، 150 ،165، 175ي الوزن الجزيئي للبروتينات وكانت كالأتوكانت بروتينات عترتي اللقاح ,نكيلو دالتو 16 و 22 ،37، 42، 51 ،60 ،64 ،

. متشابهه

النوعي باستعمال المصل وذلك أيضا تم تعريف نوعي الباسترولا باختبار الوسترن بلوتبعد نقل تم عمل التصبيغ المناعي بالإنزيم المرتبط .لكلا العترتين رانبالمحضر في الأ

من محلول الخلايا تم اتثمانية بروتين. النايتروسيليلوز خلات البروتينات في ورق، و 37، 42، 70، 85، 90، 102، 175التعرف عليها بالمصل وأوزانها الجزيئية

.كيلو دالتون 16

. جين المحفظة كثارلاالبلمرة التسلسلي للنوعين باستعمال بادئات أجري اختبار تفاعل التجارية Kitال تم استخلاص الحمض النووي منزوع الأكسجين بالغليان وباستعمالحيث

تم الحصول على حمض نووي بكلا الطريقتين ولكن. استخدم فيها الدارئ المحللالتي و .كانت الأفضل Kitطريقة ال

في انتاج اللقاح خدمالمست E للعترةاكثار الحمض النووي E العترة استطاعت بادئاتلم تستطع اكثار الحمض التي Bالعترةبادئات بعكس. زوج قاعدي 560ئ بوزن جزي

الذي Bللعترة في انتاج اللقاح ولكن تم اكثار النوع الحقلي خدمالمست B للعترةالنووي دقيقة كلية الطب البيطري جامعة الخرطومجلب من مخزون البكتريا من قسم الأحياء ال

. زوج قاعدي 711بوزن جزيئي

 

Table of content Title page Dedication……………………………………………………… i Acknowledgement……………………………………………. ii Abstract……………………………………………………….. iii Arabic abstract………………………………………………… v Table of contents……………………………………………… vi List of figures………………………………………………….. ix List of abbreviations…………………………………………... x Introduction……………………………………………………. 1 Chapter One: Literature Review: 4 1.1 Hemorrhagic septicemia………………………………….. 4 1.2 Etiology…………………………………………………..... 4 1.3 Taxonomy…………………………………………………. 5 1.4 Morphological, biochemical and cultural characteristics... 5 1.5 Phenotypic and genotypic characterization……………….. 5 1.6 Epidemiology……………………………………………… 6 1.6.1Transmission……………………………………………… 7 1.6.2 Mortality and morbidity…………………………………. 7 1.6.3 Host range……………………………………………….. 7 1.6.4 Geographic distribution…………………………………. 8 1.7 Antigenic structure and serotyping………………………... 9 1.7.1 Designation of serotypes………………………………… 11 1.8 Pathogenesis……………………………………………… 12 1.8.1 Lipopolysaccharide (LPS)……………………………… 12 1.8.2Capsule………………………………………………….. 13 1.8.2 Enzyme…………………………………………………. 13 1.8.3 Toxins…………………………………………………… 14 1.8.4 Bacteriocins…………………………………………….. 14 1.9 Immunity to Pasteurella multocida………………………. 15 1.10 Common antigens………………………………………... 16 1.11 Diagnosis………………………………………………… 17 1.11.1 Clinical signs………………………………………….. 18 1.11.2 Gross lesions………………………………………….. 18 1.11.3 Collection of rewarding specimen …………………….. 19 1.11.4 Morphology and staining……………………………… 19 1.11.5 Growth characteristics and colony morphology………. 19 1.11.6 Biochemical properties………………………………... 20 1.11.7 Serological test and serotyping methods……………… 21 1.11.8 Molecular methods……………………………………. 22 1.11.8.1 Polymerase chain reaction technique……………… 22

 

1.11.8.2 SDS-PAGE………………………………………….. 24 1.11.8.2.1 SDS PAGE of whole cell lysate …………………. 25 1.11.8.2.2 SDS PAGE of outer membrane protein (OMPs) … 26 1.11.8.3 Western blotting…………………………………….. 26 1.11.8.3Transfer method……………………………………… 27 1.11.8.3.1Wet transfer………………………………………… 27 1.11.8.3.2 Semi-dry transfer………………………………….. 28 1.11.8.4 Characterization of P. multocida antigen by immunoblotting……………………………………………….

28

1.12 Prevention and control………………………………….. 29 1.12.1Vaccination……………………………………………... 29 1.12.1.1 Inactivated vaccines…………………………………. 30 1.12.1.2 Live attenuated vaccines…………………………….. 30 Chapter two: Materials and Methods: 32 2.1 Pasteurella multocida …………………………………….. 32 2.2. Laboratory animals………………………………………. 32 2.2.1 Rabbits………………………………………………….. 32 2.2.2 Rats……………………………………………………… 32 2.3 Glass ware…..…………………………………………….. 32 2.4 Plastic ware.……………………………………………… 32 2.5 Pasteurella multocida strains…………………………….. 32 2.6 Hyperimmune serum production…………………………. 33 2.6.1 Immunization schedule…………………………………. 33 2.6.2 Detection of antibody…………………………………… 34 2.6.2.1 Agar gel immunodiffusion test……………………….. 34 2.7 Whole cell lysate preparation……………………………... 34 2.7.1 SDS PAGE……………………………………………... 35 2.7.1.1 Preparation of glass plates…………………………… 35 2.7.1.2 Separating gel…………………………………………. 35 2.7.1.3 Stacking gel…………………………………………… 35 2.7.1.4 Sample loading………………………………………... 36 2.7.1.5 Staining and destaining of gel………………………… 36 2.7.2 Western blotting………………………………………… 36 2.7.2.1Enzyme antibody conjugate…………………………… 36 2.7.2.2 Protein transferring……………………………………. 37 2.7.2.3 Staining of nitrocellulose membrane…………………. 37 2.8 PCR method………………………………………………. 36 2.8.1 PCR primers……………………………………………. 36 2.8.2 DNA extraction………………………………………… 37 2.8.2.1 Boiling extraction…………………………………….. 37 2.8.2.2 Kit extraction…………………………………………. 37

 

2.8.3 DNA amplification…………………………………….. 39 2.8.3.1 PCR product detection……………………………….. 39 2.8.3.2 Electrophoresis of PCR product……………………… 39 Chapter Three: Result 40 3.1 Growth characteristics…………………………………….. 40 3.2 Morphological characteristics…………………………….. 40 3.3Agar gel immuno diffusion test…………………………… 40 3.4 SDS PAGE……………………………………………….. 40 3.5 Western blotting………………………………………….. 41 3.6 PCR………………………………………………………. 41 Chapter Four: Discussion 48 Conclusions……………………………………………………. 52 Recommendations…………………………………………….. 53 References……………………………………………………. 54 Appendices…………………………………………………… 67

 

List of figure

Fig. Page

1 Growth of P. multocida in blood agar greyish colonies 42

2 P. multocida bipolarity stained with methylene blue 43

3 AGID test, continuous precipitation line between sera against strain E and strain B and E 44

4 Whole cell lysate protein profile of P. multocida vaccine strains by SDS-PAGE 45

5 Immunogenic bands of P. multocida detected with hyperimmune serum and developed by enzyme conjugate 46

6 PCR of P. multocida strain E and B field and vaccine strains 47

 

List of Abbreviations

AGID  Agar gel immuno diffusion test  APV  Alum-precipitated vaccine  APS  Ammonium persulphate  BA  Blood agar  BHI  Brain heart infusin  CSY  Casein-sucrose-yeast  CVRL  Central Veterinary Research Laboratories CIEP  Counter immuno electrophoresis test  dNTP  Deoxynucleotide triphosphate  DNA  Deoxyribose nucleoic acid  D.W  Distilled water ELISA  Enzyme linked immuno sorbent assay HS  Haemorrahgic septicemia  IHT  Indirect haemagglutination test IROMPs  Iron-regulated outer membrane proteins KDa  Kilo dalton LPS  Lipopolysaccharide  MCA  Maconkey agar  MW  Molecular weight  OAV  Oil-adjuvated vaccine  OMPs  Outer membrane proteins  PBS  Phosphate buffered saline PCR  Polymerase chain reaction  RIP  Radioimmunoprecipitation RNA  Ribonucleic acid  SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel

electrophoresis  SCE  Sonicated cell extract  S.C  Subcutaneously TEMED  Tetramethylenediamine TBE  Tris boric EDTA 

WCL  Whole cell lysate 

 

Introduction

Haemorrhagic septicaemia (HS) is a major disease of cattle and

buffaloes characterized by an acute, highly fatal septicaemia with high

morbidity and mortality (Bain et al., 1982; Carter and De Alwis, 1989;

Mustafa et al, 1978). The disease has been recorded in wild mammals

in several Asian and European countries (Carigan et al, 1991).

Outbreaks mostly occur during change in the climatic conditions, as

high humidity and high temperatures.

The disease is caused by P. multocida, a Gram-negative coccobacillus

residing mostly as commensal bacteria in the upper respiratory tract of

animals. The Asian serotype B:2 and the African serotype E:2 (Carter

and Heddleston system) (Carter, 1955; Heddleston et al., 1972)

corresponding to 6:B and 6:E (Namioka-Carter system) (Namioka and

Bruner, 1963), are mainly responsible for the disease.

Haemorrhagic septicaemia occurs in Africa, Asia, Central, South

America and Europe. The disease was also reported in Sudan and

serotypes E and B were isolated (Shigidi and Mustafa, 1979). The

clinical manifestations of the typical disease caused by B:2 or E:2

strains include a rise in temperature, respiratory distress with nasal

discharge, and frothing from the mouth, followed by recumbency and

death. In the recent past, HS has been identified as a secondary

complication in cattle and buffalos following outbreaks of foot and

mouth disease (FMD) (De Alwis, 1992; Carter and De Alwis, 1989).

 

Vaccination is considered an effective mean of controlling this

disease. Local isolates are usually used for vaccine preparation. In

haemorrhagic septicaemia, capsular antigen, LPS or LPS-protein

complex, and outer membrane proteins, including the iron-regulated

outer membrane proteins are effective immunogens for serogroups B

and E (Carter and De Alwis, 1989). However, no published report is

written for immunogenic proteins of P. multocida that cause

haemorrahgic septicaemia in Sudan. Vaccinations with inactivated

whole-cell preparations posses the problem that they do not provide

long-lasting immunity (Verma and Jaiswal, 1998; De Alwis, 1999).

The key antigens of P. multocida B:2 that evoke protective immunity

to HS in cattle have been defined and P. multocida B:2 outer-

membrane proteins (OMPs) have been implicated as protective

antigens (Vasfri and Mittal, 1997; Srivastava, 1998). For other

serotypes of P. multocida, OMPs are recognized as important

immunogens. Several OMPs are immunogens and the antibodies

produced against these OMPs demonstrate a strong protective action,

such antigens may be used as a component of subunit vaccines

(Rimier, 2001; Gatto et al., 2002; Prado et al., 2005).

P.multocida is characterized serologically by identification of

capsular antigens by using passive haemagglutination test and by

detection somatic antigens using gel diffusion tests. Serotyping of

P.multocida is currently only undertaken by regional reference

laboratories (Carter, 1955). Five serogroups A, B, D, E and F are

currently distinguished in the Carter system. A limitation of the

capsule typing is the difficulty in inducing antibodies to specific

antigens. Most workers found it relatively easy to induce antibodies

 

against B and E serogroup specific antigens, but not the other

serogroup specific antigens. In many instances a non-encapsulated

strain is found to be unserotypeable (Rimier and Rhoades, 1989). A

multiplex Polymerase Chain Reaction was introduced as a rapid

alternative to capsular serotyping system (Townsend et al., 2001).

using this technique, however only the capsular serotying information

could be ascertained; this are helping in serotyping instead of the

conventional method of serotyping.

Objective of the study:

- To determine the protein profiles of Pasteurella multocida by

SDS PAGE.

- To detect immunogenic proteins of Pasteurella multocida

strains used in vaccine production by immunoblotting.

- To determine the efficacy of PCR using capsule gene primers in

compared with serotyping in the study of P. multocida.

 

CHAPTER ONE

LITERATURE REVIEW

1.1 Haemorrhagic septicaemia

Haemorrhagic septicemia is an acute, highly fatal septicaemic disease of

cattle and buffaloes, characterized by fatal septicaemia with high

morbidity and mortality. The symptoms progress rapidly from dullness

and fever to death within hours and recovery is rare (Carter, 1967).

1.2 Etiology

Haemorrhagic septicaemia is caused by specific serotypes within the

bacterial species of P. multocida; more frequently by two specific

serotypes of P. multocida, serotype B:2 and E:2 in Asia and Africa,

respectively (Carter, 1955; Heddleston et al., 1972). These serotypes are

corresponding to the newer 6:B and 6:E classification i.e Namioka-

Carter system (Namioka and Bruner, 1963). In this respect a few

countries such as Egypt and Sudan, have recorded both serotypes (Farid

et al., 1980; Shigidi and Mustafa, 1979).

P. multocida is a Gram-negative bacterial pathogen which is the

causative agent of a range of diseases in animals, including fowl cholera

in avian species (Carter, 1972), haemorrhagic septicaemia in ungulates

(De Alwis, 1984), atrophic rhinitis in swine (White et al., 1993), and

snuffles in Rabbit (Manning, 1984). This bacterium also causes infection

in humans; primarily through dog and cat bites (Talan, 1999).

 

1.3 Taxonomy

The classification of Pasteurella multocida is:

Kingdom:Bacteria.

Phylum:Proteobacteria.

Class:GammaProteobacteria.

Order:Pasteurellales.

Family: Pasteurellaceae.

The family Pasteurellaceae contains genera Actinobacillus,

Haemophilus, Lonepinella, Mannheimia and Phocoenobacter. This

taxonomy is based on outer membrane proteins (OMPs) for iron

acquisitions have roles in infection and pathogenesis. Characterization of

cell surface proteins of members of the Pasteurellaceae family including

Haemophilus, Actinobacillus, Pasteurella, and the Mannheimia genera of

organisms has highlighted several redundant iron acquisition receptors

for transferrin, siderophores, and heme/heme-containing protein (Chung

et al., 2008). More taxanomy was done on the basis of the 16r RNA

sequencing and phylogentic analysis (Dewhirst et al., 1992).

1.4 Morphological, biochemical and cultural characteristic

P. multocida is a small, Gram negative coccobacillary rod, with bipolar

staining characteristics from tissues, nonmotile, non-spore forming and

non-haemolytic, aerobic to facultative anaerobic and produce indole and

ferment carbohydrates with slight gas production (Quinn et al., 1994).

1.5 Phenotypic and genotypic characterization

Phenotypic characterization is based on the bacterium biochemical

reaction where, dulcitol and sorbitol fermentation method is argued.

There were significant variations in the phenotypic properties of P.

 

multocida they have been reported (Heddleston, 1976). Mutters, et al.

(1985) reclassified genotypically as members of the genus Pasteurella on

the basis of DNA-DNA hybridization studies. Three clusters of P.

multocida showing 84 to 100, 91 to 100, and 89 to 100% DNA

reassociation between strains. There were subsequently classified as P.

multocida subsp. multocida, P. multocida subsp. gallicida, and P.

multocida subsp. septica. Representatives of the existing capsular types

were found to be closely related on the basis of DNA-DNA hybridization

(Pohl, 1981), despite the diversity of disease manifestations and hosts.

Further, the study of Kuhnert, et al (2000) also showed that variant

phenotypes of P. multocida shared at least 98.5% 16S rRNA sequence

similarity with the recognized subspecies of this species.

1.6 Epidemiology

Haemorrhagic septicaemia (HS) is an endemic disease in most countries

of Asia and sub-Saharan Africa. Within the Asian Region, countries can

be classified into three categories, on the basis of incidence and

distribution of the disease. These are respectively countries where the

disease is endemic or sporadic, clinically suspected but not confirmed, or

free. Economic losses due to HS are confined to losses in animal

industry. Only a few attempts have been made to estimate economic

losses (Dutta et al., 1990).

Organism causing HS does not survive outside the animal body to any

significant degree to be a source of infection. Moist conditions prolong

its survival. Thus the disease tends to spread more during the wet season.

Also movements of animals, work stress in work animals, levels of low

nutrition etc. all of which favour the precipitation of outbreaks

(Benkirane and De Alwis, 2002).

 

1.6.1Transmission

Infection occurs by inhalation or ingestion of P. multocida bacteria.

Higher incidence of HS is associated with moist, humid conditions, high

animals population density, and extensive free grazing system of

management, where large herds graze freely in common pastures

(Benkirane and De Alwis, 2002).

1.6.2 Mortality and morbidity

In situations where occasional sporadic outbreaks occur in some regions

within endemic countries, mortality may be very high unlike in endemic

areas where regular, seasonal outbreaks occur, where losses in each

outbreak are low and confined to young animals. The phenomenon of

naturally acquired immunity resulting from the so-called non-fatal

infection largely controls the mortality and morbidity patterns

(Benkirane and De Alwis, 2002).

1.6.3 Host range

Cattle and water buffaloes are the principal hosts of hemorrhagic

septicemia (De Alwis, 1984) and it is widely considered that buffaloes

are the more susceptible. Outbreaks of hemorrhagic septicemia have

been reported in sheep and swine where it is not a frequent or

significant disease. Cases have been reported in deer, elephants and

yaks. There is as yet no evidence of a reservoir of infection outside the

principal hosts; there are cattle, water buffaloes, and bison (Heddleston

and Gallagher, 1969). The disease was reported in camels in Sudan in

Blue Nile Province and the causative agent was identified to be

serotype B:6 (Hassan and Mustafa, 1985).

 

1.6.4 Geographic distribution

Occurrences of the disease in certain parts of the world like Southeast

Asia, where favourable conditions often coincide, are the area of

highest incidence. The disease occurs in the Middle East and Africa

where the environmental circumstances and predisposing conditions are

not as clearly defined as in Southeast Asia. In Asia, the disease is

frequently associated with the rainy season and poor physical condition

(De Alwis, 1984)

Haemorrhagic septicaemia was recognized in Japan as a specific

disease of cattle caused by particular strains of pasteurella as early as

1923. Since 1926, the disease has been controlled, and the last recorded

case in cattle in Japan occurred In 1952.The B:2 serotype has been

recovered from haemorrhagic septicaemia in countries of Southern

Europe, the Middle East, and South East Asia, including China

(Anonymous, 1991). This same serotype has been reported from Egypt

and the Sudan. The E:2 serotype has been recovered from hemorrhagic

septicemia occurring in Egypt , Sudan, the Republic of south Africa,

and several other African countries. There is no report of either

serotype being recovered from Australia, New Zealand, and countries

of South and Central America. There is no evidence that the disease has

spread from carrier bison in the western United States to neighboring

cattle. Given the conditions in which hemorrhagic Septicemia occurs in

endemic areas where primitive husbandry practices, low country plains,

and well-defined dry and wet seasons, it seems unlikely that the disease

will reach epidemic proportions in the United States (De Alwis, 1984).

The disease was reported in Sudan in Blue Nile, Kassala Nothern

Kordofan, and Upper Nile Province. The serotypes B:6 and E:6 were

 

isolated and identified from cases of Haemorrahagic septicaemia in cattle

by Shigidi and Mustafa, (1979).

1.7 Antigenic structure and serotyping

Early attempts at serological classification of P. multocida date back to

the 1920. Agglutination absorption test has identified Groups I, II, III

and IV (Cornelius, 1929), whereas Yusef, (1935) used precipitation test

to identify Groups I, II, III and IV. Rosenbach and Merchant, (1939)

used agglutination fermentation identified Groups I, II and III. Little and

Lyon, (1943) used slide agglutination and identified Types 1, 2 and 3.

However, Roberts, (1947) developed a system of serological

classification based on passive protection tests in mice. He used antisera

prepared in rabbits to protect mice against challenge with a wide range

of strains. On the basis of mouse protection, he was able to identify four

types, which he designated types I, II, III and IV. This was the first

classification to meet some degree of acceptance. Since all HS strains

fell into Roberts's type I, this designation became fairly well established.

Lately, Hudson, (1954) added a fifth serotype.

Carter used a precipitation test (Carter, 1952) and subsequently, an

indirect haemagglutination test (Carter, 1955) was able to identify four

serological types. These were based on agglutination of human type O

erythrocytes coated with crude extracts of outer cell components from

the bacterial cultures. These crude capsular extracts supernatants were

prepared by heating suspensions of the bacteria at 56°C for 30 minutes

and removing the cells by centrifugation. He designated these four

capsular types A, B, C and D (Carter1952, 1955). The strains that caused

HS were grouped into Carter type B. Subsequently, he found that the

 

strains that caused HS in Africa did not fall strictly into any of these

groups, though they were related to type B, and they were included in a

separate group designated type E (Carter, 1961). Later, he found that

type C was not a consistent type and it was deleted (Carter, 1963). This

method of identifying serotypes has become established as the Carter

indirect haemagglutination test (IHA).

Three decades later, Rimier and Rhoades, (1987) isolated a consistent

type from turkeys which did not fit into any existing serogroups, this was

designated serogroup F. Since fresh human type O erythrocytes may not

always be available in a laboratory, the IHA test has been modified by

various workers for practical convenience. Carter and Rappay, (1962)

used formalinised human type O cells, which could be stored in a

laboratory for long periods. More recently, Sawada et al. (1982) used

glutaraldehyde-fixed sheep erythrocytes. The test has now been modified

for the detection of antibodies as well, using erythrocytes coated with

cell extracts from known reference cultures. Wijewardana et al. (1986)

used fresh sheep erythrocytes, and adopted the test both for identification

of serotype and for antibody detection. Namioka and Murata, (1961 a)

described a simplified and rapid method of identifying the capsular types

using a slide agglutination test in which fresh cultures are agglutinated

with hyperimmune rabbit sera. Namioka and Murata, (1964) and

Namioka and Bruner, (1963) developed what is described as a somatic

typing test, based on releasing core (somatic) bacterial components by

agglutinating acid (HCI)-treated cells with rabbit antiserum. Using this

method, 11 somatic types were identified. Type-specific antiserum was

produced by a complicated system of absorptions. Another drawback to

this system is that some cultures undergo auto agglutination after the

 

HCI treatment and therefore are rendered untypeable. Heddleston et al.

(1972) developed an agar gel precipitation test also for somatic typing.

In this test, the antigen used was the supernatant of culture suspensions

heated at 100°C for one hour. The antiserum was prepared in chicken.

Using this method, 16 different somatic types were recognized. This test

was originally used to type avian strains from fowl cholera but is now

extended to strains from all host species.

1.7.1Designation of serotypes

Currently, the most acceptable and widely used serotype designation

system is a combination of Carter capsular typing and Heddleston

somatic typing. Using this method, the Asian and African HS serotypes

are designated B:2 and E:2 and a non-HS type B strain of Australian

origin as B:3,4. This strain was originally isolated from a bovine wound

but has subsequently been associated with occasional HS-like

septicaemic disease in cattle in North America and deer in the United

Kindom . Since there are only two of Namioka's types (6 and 11) among

the capsular type B strains, and only one (6) among the capsular type E

strains, a combination of capsular and Namioka typing is also used

occasionally (i.e. 6:B and 6:E for the Asian and African strains). Under

this system, the avirulent Australian strain is designated 11:B. Since both

systems are used in the literature.

In the Carter-Heddleston system, the capsular type is expressed first,

followed by the somatic type. In the Namioka-Carter system, expression

is made in the reverse order. Broadly, two typing systems are adopted.

One is the capsular typing by Carter’s IHA test (Carter, 1955) or by

AGID tests (Anon, 1981; Wijewardena, 1982). The other is somatic

 

typing by the method Of Namioka and Murata (Namioka, 1978;

Namioka and Murata, 1961b) and by the method of (Heddleston, et al.

1972). It is generally agreed that designation of serotypes should be

based on a somatic– capsular combination.

1.8 Pathogenesis

Upon entry of the Pasteurella organism into the animal, it is believed that

the initial site of multiplication is the tonsillar region. The outcome of

this infection depends on an interaction between the virulence of the

organism and its rate of multiplication in vivo, and the specific immune

mechanisms and nonspecific resistance factors of the host animal. Thus,

the dose of infection is a vital factor and if the organism overcomes the

host's defence mechanisms, clinical disease will result. If the defence

mechanisms dominate over the organism, this is described as an arrested

infection and the animal becomes an immune carrier. Such animals

possess solid immunity, and the presence of large numbers of such

immune animals following an outbreak of disease contributes to 'herd

immunity' (De Alwis et al., 1986).

1.8.1Lipopolysaccaride (LPS)

The LPS of P. multocida are similar to those of other gram-negative

bacteria. They constitute the endotoxins of the organism, and are the

basis of somatic typing. LPS are largely responsible for the toxicity in

the HS causing serogroup B:2, and play an important role in the

pathogenesis of the disease (Rebers et al., 1967). Purified LPS extracts

have been shown to have antiphagocytic activity in vitro by using

phagocytic uptake assays in an ovine mammary neutrophil system and

[3H] labeled type B strain of P. multocida. Muniandy et al. (1993) found

 

that capsular polysaccharide extracts known to contain 20%

lipopolysaccharides (LPS), potassium thiocyante extracts and Westphal

type LPS extracts inhibited phagocytosis. These workers also found that

when encapsulated cells and de-encapsulated cells were used, the

percentage of de-encapsulated cells phagocytosed was significantly

higher than when encapsulated cells of P. multocida were used. These

observations indicated that HS-causing strains of P. multocida appeared

to possess a factor in their capsule that inhibited the ability of phagocytes

to engulf and destroy invading bacterial cells. It is well established that

the endotoxins of gram negative bacteria consist predominantly of LPS.

The toxic effects of the LPS of P. multocida associated with HS have

been demonstrated, where it produced experimental HS in calves and

pigs by different routes using type B strains. Also administered

endotoxin prepared from this strain to a calf induced symptoms and

lesions resembled those of experimental infection (Rebers et al., 1967).

1.8.2Capsule

Dissociation of colonies is associated with reduction or loss of virulence

and also with loss of antigenicity. Well capsulated cultures make good

vaccines; for this reason, vaccine seed cultures are passaged in

laboratory animals or even in natural host species periodically. However,

the relationship between the capsule and virulence is not absolute. There

are capsulated variant cultures that are of low virulence or are avirulent,

while non capsulated strains may be virulent (Wijewardana et al., 1986).

 

1.8.2 Enzyme

P. multocida has been found to produce a number of enzymes.

Neuraminidase is produced by members of serogroups A, B, D and E

(Rimier and Rhoades, 1989). Its activity is found to be highest in strains

of serogroup A and D. Activity of neuraminidase of type E was inhibited

by homologous antiserum only, while those of types B and D were

inhibited by antisera against serogroups A, B, D and E. The production

of hyaluronidase and chondroitinase by serotype B:2 associated with HS

is well documented (Carter and Chengappa, 1980). Hyaluronidases are

enzymes that are normally associated with invasive mechanisms in

bacteria, helminths and snake venoms. Type B strains, bearing other

somatic antigens, such as the B: 3, 4 cattle and deer strains, fail to

produce hyaluronidase. Whilst it may be concluded that hyaluronidase

production is a character exclusively restricted to serotype B: 2 strains

that cause HS. De Alwis et al. (1995) described a type B:2 mutant that

was of low virulence to mice and rabbits and a virulent to cattle and

buffaloes, yet produced hyaluronidase. No clear relationship has been

established between the ability to produce hyaluronidase or any other

enzyme and virulence.

1.8.3 Toxins

Serogroups A and D have been found to produce protein toxins, more

toxigenic strains being present in serogroup D. These toxins are directly

involved in the pathogenesis of disease, as in naturally occurring

atrophic rhinitis in swine. No correlation has been found between toxin

production and somatic types. Toxins of serogroups A and D are similar,

if not identical, and antiserum produced against one neutralizes the other

(Rimier and Rhoades, 1989). True exotoxins are not produced by strains

 

of the B group associated with HS. Toxic effect (endotoxic shock) can be

produced by injection of culture supernatants (which contain free

endotoxins) or endotoxin preparations. With the exception of a few

serogroups A and D strains that produce protein toxins, proteins of P

multocida are nontoxic.

1.8.4 Bacteriocins

Bacteriocins are bacteriocidal proteins produced by many species of

bacteria and which are active against members of their own species or

closely related species. Production of bacteriocins is believed to be

determined by a genetic element. Bacteriocins activity has been

demonstrated in bovine and avian strains of P. multocida (Rimier and

Rhoades, 1989). Thirty-three bovine and bison strains belonging to

serotypes A, B and D were tested for bacteriocins activity Chengappa

and Carter, (1977); 14 were found to produce bacteriocins. Seventeen

strains were susceptible to their bacteriocins. The role of bacteriocins in

the pathogenesis of disease has not been investigated.

1.9 Immunity to Pasteurella multocida

A protective immune response comprises humoral immunity or cellular

immunity, or both, is effective to eliminate or reduce the load of

organism. Humoral immunity or antibody mediated immunity is a main

type of immunity against P.multocida specially that LPS of P.multocida

stimulates antibody production (Wijewardana and Sutherland, 1990).

Proteins are believed to be important immunogens and play a vital role

in the protective mechanism. The association of outer membrane

proteins (OMPs) with protective immunity has been widely investigated.

 

Muniandy and Mukkur, (1993) observed that the immunogenicity of

certain LPS preparations was due to the presence of OMPs.

Serological relationships exist between LPS of serogroups B and E

(Mosier, 1993). Electrophoretic analysis of purified LPS preparations

has also established relationships between B and E and some type A

strains (Rimier, 1990). This is not surprising, since all strains of both

Asian and African origin possess the Namioka somatic antigen type 6

and Heddleston type 2, although in the two serotyping procedures the

LPS components used are different (De Alwis, 1987). Although crude

LPS preparations are associated with immunity, it has been shown that

highly purified LPS are nonimmunogenic to mice and rabbits (Muniandy

et al., 1993).

The OMPs of gram-negative bacteria such as porin and OmpA have been

considered effective vaccine candidates. Two major OMPs of

P.multocida are related to the families of porin (protein H; OmpH) and

heat modifiable (OmpA). Based on the electrophoretic migration of

OmpH, different OMP patterns were identified among capsular serotype

strains of P. multocida, representing various host species and geographic

origins, while the electrophoretic mobility of OmpA of P. multocida

varied slightly among different strains OmpH possessed both specific

and cross-reacting epitopes which are abundantly expressed on the

bacterial surface. OmpA possessed cross-reacting epitopes which are not

exposed on the cell surface, as shown by immunoelectron microscopy

cited by Vasfi and Mittal, (1997).

1.10 common antigens

P. multocida shares common antigens with other closely related gram-

negative bacteria. Antigenic relationships with Yersinia

 

paratuberculosis, Mannheimia haemolytica, Haemophilus canis,

Haemophilus influenza, Actinobacillus lignieresi and Escherichia coli

have been reported (Bain, 1963; Prince and Smith, 1966). Cross-

protection has been detected in a study of 11 isolates of P. multocida

from cases of HS, bovine pneumonia and fowl cholera were showed to

belong various serotypes (Rimier, 1996). A serotype A:5 strain and a

fowl cholera strain were found to protect against a number of other

strains, irrespective of the disease caused. This protection was attributed

to antigen components of molecular weight 20-120 kDa.

Homogeneity in protein profiles among 14 strains associated with HS

was detected. Strains of Asian and North American origin (B:2)

displayed a major protein band of molecular mass 32 kDa. On other

hand, strains of African origin (E:2), gave a similar band at 37 kDa.

Other bands at 27, 45 and 47 kDa were shared by all strains, irrespective

of serotype. Using monoclonal antibodies and an immunoblotting

technique Ramdani and Adler, (1993) identified protein fractions of 29

and 36 kDa in the cytoplasmic and periplasmic fractions and 42 kDa in

the membrane fraction.

1.11 Diagnosis

A clinical, provisional diagnosis of HS is based on a combination of clinical

signs, gross pathological lesions and a consideration of relevant epidemiolo-

gical parameters and other similar diseases prevalent in the locality. A

variety of diagnostic techniques have been developed over the years for HS.

These include Blood smear, culture and biological tests for isolation of the

causative agent as using biochemical, serological tests and molecular

methods such as PCR, (Benkirane and De Alwis, 2002).

 

1.11.1 Clinical Signs:

The majority of cases in cattle and buffalo are acute or peracute with

death occurring from 6 to 24 hours after the first recognized signs. In a

few outbreaks, animals may survive for as long as 72 hours. Dullness,

reluctance to move, and elevated temperature are the first signs.

Following these signs, salivation and nasal discharge appear, and

edematous swellings are seen in the pharyngeal region and then spread to

the ventral cervical region and brisket. Visible mucous membranes are

congested, and respiratory distress is soon followed by collapse and

death. Recovery, particularly in buffaloes, is rare. Chronic

manifestations of hemorrhagic septicemia do not appear to occur (De

Alwis, 1992).

1.11.2 Gross Lesions

Widely distributed hemorrhages, edema, and general hyperemia are the

most obvious tissue changes observed in infected animals. In almost all

cases, there is an edematous swelling of the head, neck, and brisket

region. Incision of the edematous swellings reveals a coagulated

serofibrinous mass with straw colored or blood-stained fluid. This edema

distends tissue spaces. There are subserosal petechial hemorrhages

throughout the animal, and blood-tinged fluid is frequently found in the

thoracic and abdominal cavities. Petechiae may be found scattered

throughout some tissues and lymph nodes, particularly the pharyngeal

and cervical nodes, which are also swollen and often hemorrhagic (OIE

Manual, 2008).

 

1.11.3Collection of rewarding specimen

The septicaemia in HS occurs at the terminal stage of the disease.

Therefore, blood samples taken from sick animals before death may not

always contain P. multocida organisms. A blood sample or swab

collected from the heart is satisfactory if it is taken within a few hours of

death. . If there is no facility for postmortem examination, blood can be

collected from the jugular vein by incision or aspiration (Wijewardana et

al., 1986).

1.11.4 Morphology and staining

This organism is short rod or coccobacillus, 0.2-0.4 by 0.6-2.5 mm in

size. Repeated laboratory subcultures of old cultures or cultures have

grown under unfavorable conditions tend to be pleomorphic and longer

rods and filamentous forms appear. In tissues exudates and recently

isolated cultures, the organism shows the typical coccobacillary forms. It

is a gram-negative organism and in fresh cultures and animal tissues,

gives typical bipolar staining, particularly with Leishman or methylene

blue stain (Wijewardana et al., 1986).

1.11.5 Growth characteristics and colony morphology

P. multocida grows in most common laboratory media such as nutrient

agar but the growth is very poor. Special media such as dextrose-starch

agar and casein-sucrose-yeast (CSY) medium support an abundant

growth. Blood agar and CSY agar with 5% blood (bovine, sheep) are

convenient media for routine laboratory culture (Wijewardana, et al

1986). The optimum growth temperature is 35-37°C. In enriched media

at 37°C; colonies 1-3 mm in diameter are produced after 18-24 hours

culture. The organism shows different types of colonies, which are

 

related to the capsular type. Colonies of types B and E vary in size,

depending on the degree of capsulation. They will range from larger

greyish colonies, when freshly isolated or when grown in media

containing blood serum, to smaller colonies that give a yellowish-green

or bluish green iridescence when viewed in transmitted light. Rough

colonies may be produced by old cultures. These are the smallest

colonies of all forms, and are noniridescent in oblique light. Production

of rough colonies is the result of loss of capsular material or loss of LPS.

Passage of rough cultures in natural host animals or laboratory animals

or subculture in media containing animal tissues, causes reversion to the

capsulated, iridescent colony forms. Dissociation also occurs during

storage of stock cultures either in stock culture media or in lyophilised

form. In such instances, an animal passage should be carried out upon

reconstitution of the stock culture (Wijewardana et al., 1986).

1.11.6 Biochemical properties

Many biochemical methods have been used to study P. multocida. These

include: catalase, indole, oxidase and sugars fermentation tests. Shigidi

and Mustafa, (1979) tested 42 strains of P.multocida isolated from

different outbreaks of HS and from healthy cattle in various parts of

Sudan. The isolates did not cause haemolysis on blood agar (BA) and

failed to grow on MacConkey agar (MCA). All strains produced indole,

catalase, oxidase and reduced nitrate to nitrite. All the strains fermented

xylose, glucose, fructose, galactose, mannose, sucrose and sorbitol with

acid production. None of the strains fermented rhamnose, lactose,

trehalose, raffinose, dulcitol or salicin. The results were variable with

some of the carbohydrates; 16 strains fermented arabinose, four

fermented maltose and nine fermented mannitol. None of the strains

 

changed litmus milk, utilized citrate, liquefied nutrient gelatin or

produced urease.

1.11.7 Serological test and serotyping methods

Serological tests for detecting antibodies are not normally used for

diagnosis. The indirect haemagglutination test (IHA) can be used for this

purpose. High titers detected by the IHA test are indicative of recent

exposure to HS. As HS is a disease that occurs mainly in animals reared

under unsophisticated husbandry conditions, where disease-reporting

systems are also poor, there is often considerable delay in notification of

outbreaks. When notification is made in such situations, high IHA titers

from 1/160 up to 1/1280 or higher among in-contact animals surviving in

affected herds, are indicative of recent exposure to HS (OIE Manual,

2008).

Several serotyping tests are used for the identification of the HS-causing

serotypes of P. multocida. These consist of rapid slide agglutination

(Namioka & Murata, 1961 a) indirect haemagglutination (IHA) test for

capsular typing (Carter, 1955) and an agglutination test using

hydrochloric-acid-treated cells for somatic typing (Namioka and Murata,

1961b). The agar gel immunodiffusion (AGID) test (Heddleston et al,

1972; Wijewardena, 1982; Anon, 1981) and the counter immune

electrophoresis test (CIEP) are also used for this purpose (Carter and

Chengappa, 1981)

 

1.11.8 Molecular methods

Molecular methods such as PCR, ribotyping or restriction

endonuclease analysis have an epidemiological significance because

they enable strain differentiation within serotypes and hence some

epidemiological inferences, for investigations extending beyond

routine diagnosis. (Benkirane & De Alwis, 2002).

1.11.8.1 Polymerase chain reaction technique

The polymerase chain reaction (PCR) is a technique developed in 1984

by Kary Mullis, (Mullis, 1990). widely used in molecular biology,

microbiology, genetics, diagnostics, clinical laboratories, forensic

science, environmental science, hereditary studies, paternity testing,

and many other applications. The name, polymerase chain reaction,

comes from the DNA polymerase used to amplify a piece of DNA by

in vitro enzymatic replication. The original molecule or molecules of

DNA are replicated by the DNA polymerase enzyme, thus doubling

the number of DNA molecules. Then each of these molecules is

replicated in a second "cycle" of replication, resulting in four times the

number of the original molecules. Again, each of these molecules is

replicated in a third cycle of replication. This process is known as a

"chain reaction" in which the original DNA template is exponentially

amplified. With PCR it is possible to amplify a single piece of DNA,

or a very small number of pieces of DNA, over many cycles,

generating millions of copies of the original DNA molecule. PCR has

been extensively modified to perform a wide array of genetic

manipulations, diagnostic tests, and for many other uses (Saiki et al.,

1985; Saiki et al., 1988).

 

PCR technology can be applied for rapid, sensitive and specific detection

of P. multocida. The rapidity and high specificity of two of the P.

multocida-specific assays provide optimal efficiency without the need

for additional hybridization. Although the use of hybridization can

confirm specificity, this approach is usually possible only in specialized

laboratories. The P. multocida-specific PCR identify all subspecies of P.

multocida (OIE, 2008).

Nucleic acid based differentiation of closely related P.multocida vaccinal

strains was performed after morphological and biochemical

characterization. HS-specific and species-specific PCR analysis of P.

multocida vaccinal strains was demonstrated to be useful in

distinguishing hemorrhagic septicemia-causing type B strains. The PCR

assay performed for species specific P. multocida by using primer pair

KMT1T7 and KMTISP6 resulted in amplification of all the strains.

Another PCR analysis carried out for HS causing strain conformation by

using primer pairs KTT72 and KTSP61 showed that only H.S. causing

strains were amplified. It was also observed that PCR amplification

performed directly on bacterial colonies or cultures was an extremely

rapid, sensitive method of P. multocida identification (Townsend et al.,

1998).

Recently a multiplex PCR was introduced as a rapid alternative to

capsular serotyping system. Comparative analysis of the five capsular

biosynthetic regions confirmed a genetic basis for the serological

differences observed between strains. By using these genetic differences,

a rational, DNA-based typing system for P. multocida was developed

(Townsend et al., 2001). Notably, the PCR-based system was not

affected by the geographical distribution of isolates. For example,

 

isolates classified as serogroup A by conventional serotyping from

Australia, Vietnam, and the United States have all produced the

appropriate amplicon with the serogroup A cap-specific primers.

However by this technique only the capsular serotyping information

could be ascertained (Townsend et al., 2001). Gautam et al., (2004)

introduced a PCR technique specific for P.multocida serogroup A.

1.11.8.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SDS-PAGE

SDS PAGE is a technique used in biochemistry, genetics and molecular

biology to separate proteins according to their electrophoretic mobility;

a function of the length of polypeptide chain or molecular weight as

well as degree of protein folding, posttranslational modifications and

other factors. The solution of proteins to be analyzed is first mixed with

sodium dodecyl sulphate (SDS), an anionic detergent which denatures

secondary and non–disulfide–linked tertiary structures, and applies a

negative charge to each protein in proportion to its mass. Without SDS,

different proteins with similar molecular weights would migrate

differently due to differences in folding, as differences in folding

patterns would cause some proteins to better fit through the gel matrix

than others. Adding SDS solves this problem, as it linearizes the proteins

so that they may be separated strictly by molecular weight primary

structure, or number and size of amino acids. The SDS binds to the

protein in a ratio of approximately 1.4 g SDS per 1.0 g protein although

binding ratios can vary from 1.1-2.2 g SDS/g protein, giving an

approximately uniform mass: charge ratio for most proteins, so that the

distance of migration through the gel can be assumed to be directly

related to only the size of the protein. A tracking dye may be added to

 

the protein solution to allow the experimenter to track the progress of the

protein solution through the gel during the electrophoretic run (Laemmli,

1970).

The protein analysis of P. multocida organism is usually performed by

(SDS-PAGE) technique. It is important that strains of P. multocida used

for the production of vaccine be antigenically similar and

immunologically homologous to the strains of organisms prevalent in the

field (Sridevi et al., 1999).

1.11.8.2.1 SDS PAGE of whole cell lysate

Johnson et al. (1991) examined a wide range of P. multocida strains of

different serogroups by electrophoretic techniques. They found a high

degree of homogeneity in protein profiles among 14 strains associated

with HS. Strains of Asian and North American origin (B:2) displayed a

major protein band of molecular mass 32 kDa like strains of African

origin (E:2). Further, it also gave a similar band at 37 kDa. Other bands

at 27, 45 and 47 kDa were shared by all strains, irrespective of their

serotype.

HS related P. multocida isolates, collected from different localities of

Pakistan, were characterized on the basis of whole cell proteins by (SDS-

PAGE) technique. He found no quantitative difference was observed

among different isolates (Nawaz, 2006).

1.11.8.2.2 SDS PAGE of Outer Membrane Protein (OMPs)

The analysis of total membrane proteins by (SDS-PAGE), in cells of

serotype B:2 strains grown under iron-replete and iron-restricted

conditions (Veken et al. 1994;Veken et al. 1996), revealed different

 

specific protein components that were expressed by the same strain,

depending on the culture conditions . A variety of protein components of

various molecular weights have also been isolated from the Indian

vaccine strain P52, by various extraction methods including sonication

and precipitation with ammonium sulfate gel. Their immunogenic merits

have been tested in rabbits and mice (Pati et al. 1996; Srivastava, 1996).

P.multocida strains isolated from adult cattle with HS and calves with

typical bronchopneumonia, to determine electrophoretic profiles of

OMPs and compare reference strains of the serotypes B:2 and A:3, using

SDS-PAGE. The electrophoretic profiles of field isolates of P. multocida

sampled from adult cattle and calves, and the reference strains, serotypes

B:2 and A:3, exhibited 9 to 14 bands with different molecular weights,

ranging from 18 kDa to 115 kDa (Jablonska & Opacka, 2006).

1.11.8.3 Western Blotting

The Western blot, i.e. protein immunoblot is an analytical technique used to

detect specific proteins in a given sample of microbial homogenate or

extract. It uses gel electrophoresis to separate native or denatured proteins by

the length of the polypeptide (denaturing conditions) or by the 3-D structure

of the protein (native/ non-denaturing conditions). The proteins are then

transferred from the gel to nitrocellulose membrane, where they are probed

detected using antibodies specific to the target protein (Towbin et al. 1979;

Renart et al. 1979). This method is used in the fields of molecular biology,

biochemistry, immunogenetics and other molecular biology disciplines.

The method originated from the laboratory of George Stark at Stanford. The

name western blot was given to the technique by W. Neal (Burnette, 1981)

and is a play on the name Southern blot, a technique for DNA detection

 

developed earlier by Edwin M. Southern. Detection of RNA is termed

northern blotting and the detection of post-translational modification of

protein is termed Eastern blotting.

1.11.8.3Transfer method

Transfer can be done in wet or semi-dry conditions. Semi-dry transfer is

generally faster but wet transfer is a less prone to failure due to drying of the

membrane and is especially recommended for large proteins, >100 kda

(Hames and Rickwood, 1998).

1.11.8.3.1Wet transfer

The gel and membrane are sandwiched between sponge and paper

(sponge/paper/gel/membrane/paper/sponge) and all are clamped tightly

together ensuring no air bubbles have formed between the gel and

membrane. The sandwich is submerged in transfer buffer to which an

electrical field is applied. The negatively charged proteins travel towards the

positively-charged electrode, but the membrane stops them, binds them, and

prevents them from continuing on. A standard buffer for wet transfer is the

same as the 1X Tris-glycine buffer used for the migration/running buffer

without SDS but with the addition of methanol to a final concentration of

20%. For proteins larger than 80 kDa, it is recommended that SDS is

included at a final concentration of 0.1%. 

1.11.8.3.2 Semi-dry transfer

A sandwich of paper/gel/membrane/paper wetted in transfer buffer is

placed directly between positive and negative electrodes (cathode and

anode respectively). As for wet transfer, it is important that the

membrane is closest to the positive electrode and the gel closest to the

negative electrode. The proportion of Tris and glycine in the transfer

 

buffer is not necessarily the same as for wet transfer (Hames and

Rickwood, 1998).

1.11.8.4Characterization of P. multocida antigen by immunoblotting

Immunoblotting was done for serotype B:2. As a step for identification

of individual antigens that may protect against HS, proteins present in a

sonicated cell extract (SCE) and outer-membrane protein (OMP)

preparation of a wild-type P. multocida serotype B:2 were investigated

by immunoblotting with sera from calves that had been protected against

challenge with a virulent strain of P. multocida B:2 by vaccination with a

aroA derivative live-attenuated strain B. Five proteins in SCE, of

approximately 50, 37,30, 26 and 16 kDa, were recognised by the sera. In

an OMP preparation, two bands, at 37 and 50 kDa, were recognised as

strongly immunogenic (Ataei et al., 2009). Using monoclonal antibodies

and an immunoblotting technique Ramdani & Adler, (1993) identified

protein fractions of 29 and 36 kDa in the cytoplasmic and periplasmic

fractions and 42 kDa in the membrane fraction.

Western blot was used to confirm that immunogens of P. multocida in

rabbits, compared with the radioimmunoprecipitation procedure (RIP)

for identification of outer membrane immunogens and also for

identification of antibody-accessible proteins on the cell surface is

important in the selection of vaccine candidates, indicating that the two

systems were similar in detecting P. multocida outer membrane

immunogens (Lu et al., 1988).

Immunoblotting and ELISA was used to evaluate antigenic complexes

immunogenic properties of outer membrane proteins (OMPs) and iron-

 

regulated outer membrane proteins (IROMPs) prepared from strain of

serotype B:2,5 for the immunization of calves. The occurrence of

antibodies against specific outer membrane proteins as detected by

immunoblotting and ELISA in the sera of immunized cattle suggest a

beneficial immunogenicity of the vaccines (Kedrak & Opacka, 2003).

1.12 Prevention and control

In endemic areas the only practical ways to protect animals are by an

organized program of vaccination and maintenance of animals in as good

a condition as possible. Avoiding crowding, especially during wet

conditions will also reduce the incidence of disease. Animals that are

exposed to P. multocida serotypes 6:B and 6:E and survived are

considered solidly immune (OIE Mannual, 2008).

1.12.1Vaccination

Peracute nature of disease, febrile condition of animals and development

of resistance against antibiotics usually result in therapeutic failure.

Therefore, an effective control of disease could only be achieved by

vaccination. The three types of vaccines used against HS are bacterins,

alum-precipitated vaccine (APV) and oil-adjuvanted vaccine (OAV). To

provide sufficient immunity with bacterins, repeated vaccination is

required. Administration of dense bacterins can give rise to shock

reactions, which are less frequent with the APV and almost nonexistent

with the OAV.

 

1.12.1.1 Inactivated vaccines

Vaccination is routinely practiced in endemic areas where three

preparations are used; dense bacterins combined with either alum

adjuvant or oil adjuvant, and formalin-inactivated bacterins injected

subcutaneously (s.c.). There preparations give some protection against

HS, but they provide only short-term immunity (Chandrasekaran et al.,

1994). The high viscosity of oil-adjuvant vaccines makes them

unpopular among field users. The oil adjuvant bacterin is thought to

provide protection for up to one year and the alum bacterin for 4–6

months. Non- the –less, there is a disadvantage, that maternal antibody

interferes with vaccine efficacy in calves (Shah and De Graaf, 1997;

Verma and Jaiswal, 1997, 1998; Sawada et al, 1985)

1.12.1.2 Live attenuated vaccines

A live HS vaccine prepared using an avirulent P. multocida strain B:3,4

deer strain has been used for control of the disease in cattle and

buffaloes over 6 months of age. It is administered by intranasal aerosol

application; a natural route of entry into the host. This allows targeting

the immunostimulatory factors at the same sites of the immune system

that occur in the natural infection. For live strains to be used as

vaccines, the mode of attenuation should be well defined. The vaccine

has been recommended by the Food and Agriculture Organization of

the United Nations (FAO) as a safe and potent vaccine for use in Asian

countries. However, there is no report of its use in other countries and

killed vaccines are the only preparations in use by the countries affected

with HS (OIE, 2008; Myint et al. 2005; Myint and Carter, 1989).

 

CHAPTER TWO

MATERIAL AND METHOD

2.1 Pasteurella multocida

Lyophilized vaccine strains B and E were obtained from Biological

Department of the Central Veterinary Research Laboratories (CVRL),

Soba, Sudan. Lyophilized field strains B and E were obtained from the

Department of Microbiology, Faculty of Veterinary Medicine, University

of Khartoum.

2.2 Laboratory animals

2.2.1 Rabbits:

Four healthy local breed of rabbits were purchased from the market. The

animals were kept under close observation and were fed well for ten

days.

2.2.2 Rats:

Two healthy Rats were obtained from the Department of Microbiology

Faculty of Veterinary Medicine.

2.3 Glass wares:

Petri dishes, flasks, beakers, bottles and sonicator tubes were sterilized

by oven at 160 °c for 2 hours.

2.4 Plastic wares:

Eppendorf tubes, plain tube and rubbers were sterilized by autoclave at

121°c for 15 min.

2.5 Pasteurella multocida strains:

Each of the lyophilized B and E P. multocida vaccine was reconstituted

in nutrient broth (Appendix, І) and was incubated at 37°c for 24 hours.

 

Point five milliliter of each strain culture was revived in a rat by giving

subcutaneous injection. The rats died in less than 24 hours and were

dissected aseptically. Heart blood, heart and liver were collected and

inoculated into blood agar (Appendix, І) and incubated at 37°C for 24

hours. The growth was observed and colonial morphology was checked.

Blood smears stained with methylene blue were prepared to check the

bipolarity (Carter, 1985).

2.6 Hyperimmune serum production

Hyperimmune serum was prepared against specific strains in rabbits.

Vaccine strains B and E were cultured in brain heart infusin broth (BHI)

(Appendix, І) for 24 hours in shaking water bath. About fifty ml of

culture for each strain were centrifuged at 4000 rpm at 4°C for 15 min

(Megafuge, Germany). The pellet was washed 3 times with phosphate

buffer saline (PBS) pH 7.2 (Appendix, П), and the final pellets were

suspended in 5 ml of 0.3% formalin saline and were left overnight to kill

the bacteria .The suspensions were centrifuged at 4000 rpm at 4°C for

15 min and the pellets were suspended in 10 ml normal saline. The

turbidity of the cell suspension was adjusted to that of McFarland's tube

No. 3 which corresponds to108 cfu/ml.

2.6.1 Immunization schedule

Rabbits were inoculated intravenously in ear vein at 3 day intervals with

0.2, 0.5, 1.0, 1.5 and 2.0 ml of the killed bacteria suspension. Proof

bleeding was done and the sera were detected for presence of antibody

by Agar gel immunodiffusion test. The rabbits were inoculated

subcutaneously 1 week after the last injection with 0.5 ml of a similar

suspension as a booster dose to raise antibody titer. The animals were

 

bled 10 days later. The serum was separated and stored at –20°C as

described by OIE Manual, (2008).

2.6.2 Detection of antibody

Agar gel immunodiffusion (AGID) test was done to detect the presence

of antibodies in serum.

2.6.2.1 Agar gel immunodiffusion test

The gel was prepared by dissolving 1gram of agarose (SIGMA) powder

was dissolved in 100 ml of 0.1M PBS (Appendix, П) and was melted in

microwave. A volume of 0.5ml of phenol was added as bacteriostatic

agent. The agar solution was poured in 60 mm petri dishes and left to

cool and solidify. Wells were cut with gel puncture. Thirty micro litter of

whole cell lysate (WCL) bacterial antigen was placed in one well and in

other well 30µl of the serum was dropped. The gel incubated in humidity

chamber for 24-48 hours. The test was read against the illuminator

chamber.

2.7 Whole cell lysate preparation

Each P. multocida vaccine strain was cultured in 250 ml BHI broth and

incubated at 37°C overnight in shaking water bath. Purity of each culture

was checked. The cultures were centrifuged at 4000 rpm for 15 minutes

at 4°C in 15 ml falcon tubes and pellets were collected and washed 3X

with PBS. The final pellets were redissolved in distilled water vortexed

and sonicated in sonicator (MSE-England) for 30 seconds stroke and 30

seconds cooling upto10 cycles, the amplitude was kept 18 rpm in cycles

(Srivastava, 1998). Cell debris and unbroken cells were removed by

centrifugation at 3000 rpm for 15 min and the supernatants were kept at -

20°c until used.

 

2.7.1 SDS PAGE

The SDS PAGE of whole cell lysate (WCL) vaccine strains were carried

out according to Lammeli method (Lammeli, 1979) using electrophoresis

apparatus (Bio Rad, Germany) with 12% separating gel and 5% stacking.

Polyacrylamide gel was prepared and polymerized by the addition of

tetramethylenediamine (TEMED) (Sigma).

2.7.1.1 Preparation of glass plates

Firstly, the glasses were cleaned up with alcohol and the bottom of the

electrophoresis glasses were sealed with 2% agarose and were left to

solidify.

2.7.1.2 Separating gel

The seperating gel containing 4.2 ml of 30% acrylamide+bisacrylamide,

in 1.5 M Tris-HCl,100 µl of 10 %SDS, 3.2 ml D.W and 100 µl 99%

ammonium persulphate (APS) (Sigma). Fourteen microliter of TEMED

were added immediately before pouring the separating gel and leveled

by methanol. Methanol was discarded and distilled water was added and

the glass plates were covered with wet tissue and kept for overnight at

4°C for better resolution.

2.7.1.3 Stacking gel

The stacking gel was prepared by adding 1.3 ml of 30%

acrylamide+bisacrylamide, in 0.5M Tris-HCl, 100 µl of 10 %SDS, 2 ml

D.W and 100 µl of APS. Ten microliter of TEMED was added and

immediately poured off the stacking gel and the comb was placed, the

gel was left to solidify, then the comb was removed.

 

2.7.1.4 Sample loading

Ten microliter of sample were mixed with 10 µl of sample buffer

(Appendex, III) containing bromophenol blue and boiled for 5 min. Then

10 µl of heated sample was loaded into each well and also 5 µl of protein

marker 10-175kDa (gene direx, USA) was loaded along with the sample.

The electrophoresis apparatus was filled with running buffer (Appendix,

III). Then the apparatus was connected with constant voltage (100v) for

2hours or until the bromophenol blue reached the bottom of the slides.

2.7.1.5 Staining and destaining of gel

The gels were placed into container with a staining solution (Appendix,

III) containing Coomassie brilliant blue dissolved in methanol. Gels

were left in staining solution for overnight under slow shaking. The gels

were destained in destaining solution (Appendix, III) containing

methanol until the bands become visible.

2.7.2 Western blotting

2.7.2.1 Enzyme antibody conjugate:

The conjugate antibody was antiwhole IgG molecule, goat anti rabbit

IgG conjugated with horse radish peroxidase enzyme purchased from

Adcam Company, Malaysia

2.7.2.2 Protein transferring

Proteins were electrophoretically transferred to nitrocellulose membrane

of average pore size 0.45 Mm (Schleicher and Schueul, Gerrmany). The

sponge pads were wetted with transfer buffer and were put in the transfer

apparatus, place one piece of wet filter paper, the nitrocellulose

membrane, the gel, another piece of wet filter paper were placed in

respective order. Bubbles were squeezed out by adding transfer buffer

 

(Appendix, IV) at every step and by rolling with a pipette after each

addition. The apparatus was filled with transfer buffer and the lid was

closed and was connected to the power supply and run at 100 volts for 1

hour.

2.7.2.3 Immuno peroxidase staining of nitrocellulose membrane

The nitrocellulose membranes were put on a container and were covered

with 2% gelatin as blocking buffer (Appendix, IV). Then the membranes

were incubated on the shaker with low speed at room temperature for 1

hour. The blocking buffer was discarded and the membranes were

covered with primary antibody diluted at 1:200 in serum diluent

(Appendix, IV) and incubated on a shaker at room temperature for 1

hour. The membranes were washed with washing buffer (Appendix, IV)

three times (5 minutes for each wash at high speed). Then the

membranes were covered with secondary antibody (antirabbit horse,

raddish peroxidase- conjugated) (Appendix, IV) diluted 1:1000 and

incubated for 2hours. This was followed by washing the membranes

three times like in the first wash. Finally the substrate 4-chloro α naphtol

(Appendix, IV) was added and incubated for 15 min. Distilled water was

added to stop the reaction. The membranes were air dried and preserved

in dark until photographed (Towbin, 1979).

2.8 PCR method

2.8.1 PCR Primers

Primers for each strain were obtained as freeze dried oligonucleotide

(Vivantis, Malaysia) with sequences that amplify capsule gene with

specific molecular weight (MW) band according to strain.

Strain B

 

CAPB-FWD 5’-CAT-TTA-TCC-AAG-CTC-CAC-C-3’ MW 5668

CAPB-REV 5’-GCC-CGA-GAG-TTT-CAA-TCC-3’ MW 5460

Strain E

CAPE-FWD 5’TCC-GCA-GAA-AAT-TAT-TGA-CTC-3’ MW 6389

CAPE-REV 5’-GCT-TGC-TGC-TTG-ATT-TTG-TC-3’ MW 6096

2.8.2 DNA extraction

Two methods of DNA extraction were used. The first method of DNA

extraction was by boiling method and the second method by the kit

(PUREGENE ,Gentra System, Minneapolis, USA).

2.8.2.1 Boiling extraction

A pure colony of P. multocida was inoculated into 5 ml of BHI broth and

incubated at 37°C for 24 h. One point five milliliters of this broth culture

was transferred into an eppendorf tube and centrifuged at 3000 x g for 10

min. The pellet was washed twice in PBS and the final pellet was

resuspended in 100 µl of sterile deionized distilled water. The mixture

was boiled for 30 min and immediately chilled on ice for 30 min. The

sample was then thawed and centrifuged at 3000 x g for 5 min. The

supernatant was stored at -20°C for further use as DNA template as

described by Antony et al. ( 2007).

2.8.2.2 Kit extraction:

DNA was extracted by using DNA isolation kit. Five hundred microlittre

of overnight bacterial BHI culture were centrifuged at 15.000xg in 1.5ml

micro tube for 5 second to pellet cells and carefully the supernatant were

removed. Then 300µl of cell lysis solution was added to the cells pellet,

 

and the mixture was inspirated and expirated by micropipette until cells

were suspended and incubated at 80°C for 5 min, followed by the

addition of 1.5µl RNase a solution to the cell lysate. The samples were

then mixed by inverting the tube 25 times and the tubes were incubated

at 37°C for 15 minutes, cooled to room temperature and 100µl protein

precipitation solution was added to the cell lysate. The tubes were

vortexed at high speed for 20 seconds and then centrifuged at 15.000xg

for 3 minutes. Then 300µl of 100% Isopropanol were added to the

supernatant fluid into a clean 1.5 microfuge tube, mixed by inverting

gently 50 times, and centrifuging at 15.000xg for 1 minute to pellet the

DNA. The supernatant was poured off and the tubes were drained

briefly, after that DNA was washed with 300µl of 75% ethanol,

centrifuged at15.000xg for 1 minute and the ethanol was poured off. The

tubes were then allowed to dry for 10 minutes. 20µl DNA hydration

solution was added and DNA was dissolved by incubating for 1 hour at

65°C and then stored at -20°C until used.

2.8.3 DNA amplification:

DNA amplification was done in PCR tube, 50 µl reaction mixture was

prepared. One microlitres of template DNA was added to a reaction

mixture containing 3.2 µM each of primer, 200 µM of each dNTP, 1 X

Taq buffer with 1.5 M MgCl2 and 2 units of Taq DNA polymerase. The

amplification reaction was carried out in an automated thermal cycler

(Biometra, Germany) according to the following programme, an initial

denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at

94°C for 30 second, annealing at 56°C for 30 second, extension at 72°C

for 30 second and a final extension at 72°C for 5 min.

 

2.8.3.1 PCR product detection:

Detection was done by electrophoresis to determine molecular weight of

bands.

2.8.3.2 Electrophoresis of PCR product

The product was analyzed by 2% agarose gel with 0.05% ethidium

bromide in 1x tris boric EDTA (TBE) running buffer (Appendix, V)

prepared by adding 2g agarose to100 ml of TBE buffer and melted in

microwave then 1.5 µl of ethidium bromide were added and poured in

electrophoresis apparatus, the comb was placed until solidification of

gel. Then comb was removed and samples were loaded. Three

microlittre PCR product was mixed with 3µl loading dye (Appendix, V)

and was loaded into gel wells. Standard molecular size marker low range

DNA ruler with fragments size 100 bp was also loaded and used as DNA

molecular size marker to ascertain the size of the amplified PCR product.

 

CHAPTER THREE

RESULT

3.1 Growth characteristic

P. multocida, namely B:6 and E: 6 vaccine strains were showed luxuriant

growth on blood agar having translucent grayish colonies varies in size

and there was no blood hemolysis (Fig,1). In liquid media showed

homogenous growth. The growth was better in BHI broth than nutrient

broth.

3.2 Morphological characteristic

P. multocida when stained with Gram stain showed Gram negative short

rod or coccobacilli. Blood smears from injected rats showed bipolar

staining bacteria (Fig,2).

3.3Agar gel immune diffusion test

Sera were collected from immunized rabbits and tested for presences of

antibody .The gel showed presence of continuous precipitation line

between strain B and strain E against the sera of strain E or B and other

line which might be polysaccharide (Fig,3).

3.4 SDS PAGE

For the analysis of proteins of P. multocida organism, SDS-PAGE was

done according to Lammeli method (Lammeli, 1979). The gel obtained

after analysis was stained with Coomassie brilliant blue dye. By this

technique different polypeptides bands of the organism’s proteins were

observed. The protein profiles of P. multocida whole cell lysate prepared

for each strain, there were 15 protein bands of molecular weight 175

 

kDa, 165 kDa, 150 kDa,123 kDa, 102 kDa, 90 kDa, 85 kDa, 70 kDa, 64

kDa,60 kDa, 51 kDa,42 kDa,37 kDa, 22 kDa and16 kDa (Fig,4). Some

of these bands are major protein with molecular weight 102 kDa, 85 kda,

70 kDa, 62 kDa, 42 kDa, and 37 kDa. Polysaccharides were observed as

a thin a band near the start of the gel.

3.5 Western blotting

Immunoblotting using antiserum against P. multocida strain B prepared

in rabbits and using nitrocellulose membrane containing bands

transferred from gel according to Towbin (1979) method, revealed eight

protein bands of molecular weight 175 kDa, 102 kDa, 90 kDa, 85 kDa,

70 kDa, 42 kDa, 37 kDa, and 16 kDa (Fig,5).

3.6 PCR

The two methods used for extraction of DNA gave good yield for DNA,

but the Kits method was the best in yield.

The strains of P. multocida E and B when subjected to amplification

using the primer pairsgave an amplification of 511 bp and 760 bp for

strain E and strain B respectively when subjected to gel electrophoresis.

Primers of strain E amplified vaccine strain B DNA. Field strains B and

E were specifically amplified. Vaccine strain E gave DNA amplification

product (band) but vaccine strain B did not give an amplification product

(band) when strain B primers were used as depicted in Fig. 6.

 

Fig .1 Growth of P. multocida in blood agar showed grayish colonies.

 

Fig. 2 Pasteurella multocida bipolar staining with methylene blue

(Blue dots)

 

Fig.3 AGID test, continuous precipitation line between antisera against B and E and strain E of P. multocida.

E

Anti E

Anti B

 

 

Fig .4Whole cell lysate proteins profile of P. multocida vaccine strains by SDS-PAGE

Lane 1 protein marker, lane2-5 strain E, lane 6-10 strain B, lane 11 protein marker.

P= polysaccharide.

1        2         3            4         5           6          7            8          9          10               11 175 

130 

95 

70 

62 

 

51 

 

42 

 

29 

P

 

Fig.5 Immunogenic bands of P. multocida detected with hyper immune serum and developed by enzyme labeled antibody.

M= protein marker

 

 

 

42 

 

 

 

95 

 

175 

 

M 

 

Fig.6 PCR of P. multocida strain E and B field and vaccine strains

Lane M =protein marker, lanes 1-4 boiling extraction, lanes 5-8 kits

extraction

1and 6 = vaccine strain B, 2 and 5= field strain B, 3 and 7= vaccine

strain E, 4 and 8 field strain E, 9=vaccine strain B with primer E, 10

vaccine strain B with primer E

          M     1    2    3     4    5    6   7     8    9    10  

500 bp 

1000bp

 

CHAPTER FOUR

DISSCUTION

P. multocida is an aerobic, chemo-organotrophic organism that has

different serotypes that cause disease in animal and poultry. Serotypes

B and E cause hemorrhagic septicaemia in cattle and buffaloes. In all

countries where pasteurellosis occurs vaccinations are considered as

an effective means of controlling this disease and local isolates are

usually used for vaccine preparation. Bacterins were used for

immunization, in addition to precipitated alum or aluminium

hydroxide gel vaccines and dense bacterin. Further several authors

immunized cattle with live vaccines. In order to improve the

immunogenicity of vaccines, the causative organism has been

fractionated and various cell surface components have been studied

(Carter and De Alwis, 1989; Chandrasekaran et al., 1994).

In these study cultural characteristics of two P. multocida vaccine

strains was studied on blood agar. The optimum growth temperature

was 37°C after 18-24 hours incubation culture gave translucent

grayish colonies that varies in size 1 to 3 mm were seen and there was

no blood hemolysis. In liquid media incubated in shaker there was

homogenous growth. P.multocida is Gram negative short rod that

gave bipolar staining from tissue and these confirmed results had been

reported by (Shigidi and Mustafa, 1979; Arawwawela et al., 1981;

Wijewardana et al., 1986).

The techniques in molecular biology have significantly increased

understanding of the epidemiology of Pasteurella diseases. SDS-

PAGE has shown to establish the unique properties of the bacterial

 

proteins (Johnson et al., 1991). In this study the proteins profile of P.

multocida organism was studied by SDS-PAGE and clear proteins

bands were obtained following staining of the gel with Coomassie

brilliant blue dye. By this technique, different polypeptides of the

organism’s proteins were observed. The protein profiles of P.

multocida whole cell lysate revealed 15 bands on SDS-PAGE analysis

some of these bands are of high molecular weight include175 kDa,

165 kdDa, 150 kDa, 123 kDa, 102 kDa protein band. Type of the

medium used and the method of protein extraction, as the growth on

BHI medium enhanced the production of high molecular weight (Jain

et al., 2005) which is substantiated by the correlate observation in this

study. Jain et al., (2005) who observed expression of protein of

molecular weight 102 kDa along with other major OMP bands in

capsular type B buffalo isolates. Further they also examined four

reference strains of serotype B: 2 to determine their protein profiles

and compared them with field isolates. Their results revealed that

fractions of molecular weight of 25 to 55, 75, 80, 86 and 90 kDa were

most frequently observed among all isolates and reference strains.

In this study bands with molecular weight 90 kDa ,85 kDa, 70 kDa, 64

kDa,60 kDa, 51 kDa,42 kDa,37 kDa, 22 kDa and16 kDa. Some of

these bands were found by other authors. Johnson et al. (1991)

determined the proteins profile of the capsular serotype B and E

strains isolated from animals with hemorrhagic septicemia and placed

the isolates in two distinct groups on the basis of the molecular masses

(32 to 37 kDa) of the major proteins. They reported that polypeptide

of 37 KDa was the most immunogenic of all the isolates and this band

was found in African strain 37 kda. This finding is in agreement with

our present result.

 

Detected band, with hyperimmune serum using immunoblotting were

with molecular weights 175 kDa, 102 kDa, 90 kDa, 85 kDa, 70 kDa,

42 kDa, 37 kDa and 16 kDa correspond to those bands obtained by

Pati et al. (1996) who reported proteins of 44, 37 and 30 kDa

determined that they were the major immunogens when use subunit

vaccines comprising OMPs from P. multocida serotype B:2 in

immunizing buffalo calves. There for the present study provided

further wider OMPs are protective and could be used in vaccines

against haemorrhagic septicaemia.

The identification and sequence analysis of the biosynthetic locus of

the capsule of an organism can lead to a greater understanding of its

capsular polysaccharide composition and can provide a genetic basis

for the serological differences observed between strains. Genetic

analysis of the serogroup B biosynthetic locus revealed only three

gene products with similarity to proteins known to be involved in

polysaccharide biosynthesis, while six gene products had no similarity

to known proteins. However, the structure of the type B capsule

remains unknown (Boyce et al., 2000).

Determination of the nucleotide sequence of the serogroup E

biosynthesis region provided little information about the capsular

polysaccharide composition. Region 2 of serogroup E contains nine

genes, two of which showed similarity to genes involved in

polysaccharide biosynthesis. These two genes have homologs in the P.

multocida B:2 cap locus, indicating that N-acetyl-D-

mannosaminuronic acid is a component of both the serogroup B and

the serogroup E capsules; the remaining seven genes, five have

 

homologs in the B:2 cap locus but still have no known function, one

encodes a putative glycosyltransferase, and the other is unique to

serogroup E. In our study DNA bands were obtained by serotype E

and B using primer E cap locus and there were no bands using primers

strain B cap locus but there was band in field strain B. This finding

according to Townsend et al., (2001) is indicative the bivalent vaccine

contain only strain E.

 

Conclusions

- Protein profile of P. multocida is similar in two vaccine strains.

- Some P. multocida proteins as obtained by ultrasonicaition

followed by SDS PAGE were reported by others.

- The study further provided that whole cell lysate, include OMPs

of P. multocida were immunogenic and could be used in

subunit vaccines against haemorrhagic septicaemia.

- Bivalent haemorrhagic septicaemia vaccine when subjected to

PCR gave amplification product for strain E only.

- PCR can be used for capsular serotyping of P. multocida.

 

Recommendations

- Research must be done to determine if there is a difference

between vaccine strains and field strains in protein profile.

- Further studies are required to confirm the immunogenicity of

these proteins prior to use them as antigen in subunit vaccine.

- Research should be done in subunit vaccine and how to use

them by mucosal routes to induce local immunity.

- Strain B of national vaccine should be recharacterized.

 

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Appendices

Appendix І

Bacteriological media:

Nutrient Broth:

Meat (beef) extracts 10g

Peptone 10g

NaCl 5g

Distilled water 1000 ml

Blood agar:

Defibrinated blood 50ml

Nutrient agar 950 ml

Nutrient Broth was gelled by addition of 2% agar. Sterilized by

autoclaving at 121°C for 15 min. Then the blood was added at 40°C

Brain heart infusion broth (BHI):

BHI 37g

D.W 1000ml

The media were sterilized by autoclaving at 121°c for 15 min.

 

Appendix П

Buffers:

Normal saline:

NaCl 8.5g

D.W 1000ml

The buffer was sterilized by autoclaving at 121°C for 15 min.

Phosphate buffer saline:

NaCl 8g

NaH2PO4 1.15g

KH2PO4 0.2g

KCl 0.2g

D.W 1000 ml

The pH was optimized to 7.2.

0.1M phosphate buffer (PB), pH 7.8:-

Sodium phosphate (NaH2PO4.H2O) 52.4g

Deionized water to final volume 3.8L

The pH was adjusted to 7.8

 

Appendix III

SDS PAGE Buffers:

Running buffer:

Tris-OH 3.02g

Glycine 14.4g

SDS 1g

800 ml of D.W was added pH was adjusted to 8.3 then completed to

1000ml.

Sample buffer:

Tris- OH 1ml

Glycerol 0.8ml

SDS 10% 1.6ml

2-mercapto-ethanol 0.4ml

Bromophenol blue 0.05% 0.2ml

D.W 4ml

Staining buffer:

GAA 5.75ml

Methanol 28.5ml

D.W 62.5ml

Coomassie brilliant blue 0.157-0.2g

De-staining solution:

GAA 10ml

Methanol 50ml

D.W 100ml

 

Appendix IV

Western blotting buffers: Transfer Buffer: glycine 14.4g Tris base 3.02g Methanol 100ml D.W 900ml pH was Adjust to 8.3. Blocking buffer: Gelatin 2g PBS 100ml Gelatin was added to PBS and heated in water bath until dissolve Serum diluents: Gelatin 1g PBS 100ml Tween20 50 µl Gelatin was added to PBS and heated in water bath until dissolved. Then Tween20 was added. Preparation of primary antibody: Hyperimmune sera 125 µl Serum diluent 25ml Preparation of conjugate: Anti rabbit conjugate 25 µl Serum diluents 25ml Washing buffer: PBS 250ml Tween20 125 µl Preparation of substrate: 4. Chloro α. naphthol 0.002g Methanol 5ml PBS 25ml H2O2 100 µl

 

Appendix V:

PCR reagent: Tris boric EDTA Buffer (TBE):

Tris base 108 g

Boric acid 55g

0.5M EDTA (pH8.0) 40 ml

Stock 10x TBE

Loading dye:

Bromophenol blue (11%) 10 µl

Glycerol 40 µl

DDW 50 µl