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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
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