UNIVERSITI PUTRA MALAYSIA

ABUBAKAR SALISU MUHAMMAD

FPV 2012 19

RESPONSE OF GASTROINTESTINAL TRACT TO Pasteurella multocida SEROTYPE B:2 INFECTION IN BUFFALOES(Bubalus bubalis Linnaeus)

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RESPONSE OF GASTROINTESTINAL TRACT TO Pasteurella multocida SEROTYPE B:2 INFECTION IN BUFFALOES

(Bubalus bubalis Linnaeus)

BY

ABUBAKAR SALISU MUHAMMAD

Thesis Submitted to the School of Graduate Studies, Universiti

Putra Malaysia, in Fulfilment of the Requirement for the Degree

of Doctor of philosophy

November, 2012

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in

fulfilment of the requirement for degree of Doctor of Philosophy

RESPONSE OF GASTROINTESTINAL TRACT TO Pasteurella multocida SEROTYPE B:2 INFECTION IN BUFFALOES

(Bubalus bubalis Linnaeus)

By

ABUBAKAR SALISU MUHAMMAD

November, 2012

Chairman: Professor Mohd Zamri-Saad, DVM, PhD

Faculty: Veterinary Medicine

Pasteurella multocida B:2, which causes haemorrhagic septicaemia (HS) of

ruminants, is believed to enter the host via the respiratory tract. Among the

consequences of the respiratory route of infection are septicaemia, increased

permeability of blood vessels and presence of the organism in several

organs. However, the respiratory tract may not be the only portal of entry

and route of spread of P. multocida B:2. Circumstantial evidence had

suggested the involvement of gastrointestinal tract in the pathogenesis of HS

in ruminants. Nevertheless, the pathogenesis and pathology of the disease

following oral infection has not been well documented since previous

reports on the disease were limited to incidental observations. The response

of gastrointestinal tract following oral exposure to P. multocida B:2 was

studied and compared its severity with intratracheal exposure. The safety,

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antibody pattern and mucosal immune response in the gastrointestinal and

respiratory tracts following oral or intranasal exposure to gdhA (glutamate

dehydrogenase) derivative P. multocida B:2 in buffalo calves were also

investigated.

The clinical signs observed in this studies includes; dullness, depression,

recumbency, pyrexia, dyspnoea, congested mucous membranes, nasal

discharge, lacrimation and salivation following single oral exposure to P.

multocida B:2, however mean clinical score were significantly higher in

intratracheally exposed group, inaddition diarrhea was noted in group of

calves exposed orally to P. multocida B:2 twice, 2 weeks apart. The

pathological alterations as the result of oral or intratracheal exposure to P.

multocida B:2 included generalized lymphadenopathy, acute ulcerative

rhinitis, acute fibrinous pneumonia, pleurisy, hydropericardium,

hydroperitoneum, haemorrhagic encephalitis, acute enteritis, colitis and

necrotizing and haemorrhagic typhilitis and proctitis, however lesions

scoring revealed higher scores in gastrointestinal following orally exposure,

while respiratory tract showed higher scores in intratracheally exposed

group. Following oral or intratracheal exposure, P. multocida B:2 was

isolated from the intestinal segments of the calves that developed severe

clinical HS and they had to be sacrificed at 48 h post-exposure. Similarly,

the ultrastructural changes in the infected calves were typical of acute

cellular injury, with degeneration of endothelium and vascular walls of the

gastrointestinal and respiratory tracts. There were deciliation in the

respiratory tract, and microvilli degeneration and disruption in the

gastrointestinal tract. Scanning electron microscopy revealed P. multocida

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B:2 presence on the surfaces of oesophagus, nasal mucosa and trachea of

calves in both oral- and intratracheal-exposed group. The lesions were

confirmed through the immunoperoxidase technique, to be associated with

the inoculated P. multocida B:2,. P. multocida B:2 antigen was detected not

only in the bacterial clusters in the gastric pits, intestinal epithelia and

capillaries, Brünner’s glands and crypt of Lieberkühn but also seen to

interact with infiltrating neutrophils, macrophages and erythrocytes in

congested vessels and in haemorrhages.

Concurrently the mucosal-associated lymphoid tissue (MALT) response in

the gastrointestinal and respiratory tracts of buffalo calves following oral

exposure to live wild-type P. multocida B:2 was also evaluated. In calves

exposed to both oral and intranasal P. multocida B:2, lymphoid nodules

with increased sized and lymphocyte number were detected. With the

confirmation of bronchus-associated lymphoid tissue (BALT) and gut-

associated lymphoid tissue (GALT) involvement in P. multocida B:2

infection, an experiment was conducted to evaluate the safety, antibody

pattern, and responses of BALT and GALT to exposure to gdhA derivative

P. multocida B:2. Large dose oral and intranasal gdhA derivative P.

multocida B:2 exposures elicited sustained and significantly higher serum

IgG and IgA responses. Similarly, the IgG and IgA levels in bronchioaveolar

fluid and intestinal washings were higher and the BALT and GALT

responses were significant.

This study showed that P. multocida B:2 were present in various segments

and tissues of the gastrointestinal tract following oral or intratracheal

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exposure. Therefore, it can be concluded that P. multocida B:2 infection can

be transmitted via the gastrointestinal tract. Both oral and intranasal routes

of administration of gdhA derivative Pasteurella multocida B:2 elicited high

serum antibody response although mucosal IgA and IgG responses were

variable. Thus, oral and intranasal infections with large doses of live

attenuated P. multocida B:2 were safer than with wild-type P. multocida

B:2. Both of these routes can be considered as potential alternative route for

vaccine administration against HS in buffalo calves.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia

sebagai memenuhi keperluan ijazah Doktor Falsafah Veterinar

GERAK BALAS SALUR GASTROUSUS TERHADAP JANGKITAN

Pasteruella multocida SEROTYPE B:2 PADA KERBAU

(Bubalus bubalis Linnaeus)

Oleh

ABUBAKAR SALISU MUHAMMAD

November, 2012

Pengerusi: Professor Mohd Zamri-Saad, DVM, PhD

Fakulti: Perubatan Veterinar

Pasteurella multocida B:2, penyebab sampar berdarah (HS) pada ruminan,

dipercayai memasuki perumah melalui salur pernafasan. Antara akibat

jangkitan melalui jalan pernafasan ini ialah septisemia, peningkatan

kebolehtelapan salur darah dan penumpuan organisma dalam beberapa

organ. Bagaimanapun salur pernafasan mungkin bukan satu-satunya haluan

masuk dan cara sebaran P. multocida B:2. Bukti tidak langsung

menyarankan ada penglibatan salur gastrousus dalam patogenesis HS

dalam ruminan. Sungguhpun begitu, patogenesis dan patologi penyakit

berikutan jangkitan oral belum pernah didokumenkan sepenuhnya kerana

laporan sebelum ini hanya terhad kepada pemerhatian secara kebetulan.

Kami mengkaji gerak balas salur gastrousus berikutan pendedahan oral

kepada P. multocida B:2 dan membandingkan keterukannya dengan

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pendedahan intratrakea. Keselamatan, pola antibodi dan gerak balas dalam

salur gastrousus dan pernafasan berikutan pendedahan oral dan intranasum

kepada gdhA (glutamate dehydrogenase) terbitan P. multocida B:2 dalam

anak kerbau juga diselidik.

Peranan jangkitan melalui saluran pernafasan sudah terbukti secara ujikaji.

Kajian kini menghuraikan persembahan klinikal, termasuk kesuraman,

kemuraman, terbaring, pireksia, dispnea, kesesakan membran mucus,

lelehan hidung, pelakrimaan dan pengliuran berikutan pendedahan oral dan

cirit-birit dalam kumpulan anak kerbau yang terdedah kepada P. multocida

B:2 sebanyak dua kali dalam jarak 2 minggu antara satu. Perubahan

patologi akibat daripada jangkitan ini termasuk limfadenopati menyeluruh,

rinitis ulseratif akut, pneumonia berfibrin akut, pleurisi, hidroperikardium,

hidroperitoneum, ensefalitis hemoraj, enteritis akut, kolitis dan tifilitis

nekrosis dan hemoraj dan proktitis. Berikutan pendedahan oral atau

intratrakea, P. multocida B:2 telah dipencil daripada segmen usus anak

lembu yang menunjukkan HS klinikal teruk dan terpaksa disembelih pada

jam 48 pasca-pendedahan. Begitu juga dengan perubahan ultrastrukur pada

anak kerbau terjangkit, yang tipikal untuk kecederaan sel akut, dengan

degenerasi endotelium dan dinding salur darah pada salur gastrousus dan

pernafasan. Pada salur pernafasan berlaku penyahsiliuman dan pada salur

usus pula berlaku degenerasi dan kerosakan mikrovilus. Mikroskopi

electron imbasan menunjukkan kewujudan P. multocida B:2 pada

permukaan esofagus, mukosa nasum dan trakea pada anak kerbau dalam

kedua-dua kumpulan terdedah oral dan intratrakea. Lesi ini disahkan

melalui teknik imunoperoksidas adalah berkaitan dengan P. multocida B:2

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yang diinokulat. Antigen P. multocida B:2 dikesan bukan sahaja pada

gugusan bakteria dalam pit gaster, epitelium dan rerambut usus, kelenjar

Brünner dan kripta Lieberkühn tetapi juga dapat dilihat bertindak balas

dengan neutrofil, makrofaj dan eritrosit yang menyusup ke dalam salur

tersesak.

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ACKNOWLEDGEMENTS

All praises are due to ALLAH, lord of the world for the abundant privileges

too numerous to mention and the strength to undergo a training of the mind

(Ph.D program).

I wish to sincerely acknowledge the advisory and supervisory guidance of

the chair of my committee, Prof. Dr. Mohd Zamri-Saad for his unique style

and good research direction, and to Prof. Dr. Jasni Sabri and Prof. Dr. Md

Zuki Abu Bakar @ Zakaria for their understanding and constructive

criticism right from the conception, through execution to completion of the

research. You all remain accessible at all times in the course of my studies; I

will forever remain indebted to you.

I would use this opportunity to acknowledge the technical assistance of the

following people; who assisted in animal handling and post-mortem, Mr.

Ariff Ahmad, Mr. Noraziman Sulaiman, Dr. Shahirudin Shamsudin, Mr.

Ghazali Md Yusoff, Mr. Mohd Najib Yahya, Mr. Apparau Somanaidu; In

histopathology, Puan (Mrs) Jamilah Jahari, Puan (Mrs) Latifah Mohd

Hanan, En. (Mr.) Mohd Jamil Samad; In electron microscopy, many staff of

the microscopy unit at the Institute of Bioscience, particularly Puan (Mrs)

Irmazian Abd Shukor, Mrs. Aminah Jusoh, Mrs. Farrah Deba Jamiauddin,

Mrs. Faridah Akmal Ismail, Mr. Rafiuz Zaman Haroun, Mr Abdu Shukor of

the Veterinary Research Institute, Ipoh, Perak and Prof. Dr. Rasedee

Abdullah for technical assistance with abstract translation to Malay.

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The support and advice of my past and present laboratory mates were very

useful throughout the journey (Post-docs. Dr. Hani, Dr. Benjamin, and Drs

Didik, Shahrom, Sriyanto, Aan, Amal, Rafidah, Annas, Adza and Shaqinah;

and Firdaus, Atya, Wawa, Wani). Discussions with other students and

faculty members help in improving the outcome of the project and thesis.

Drs Faruk Bande, Muhtar Anka, Ibrahim Anka had at various times assisted

in animals restraints, blood sampling and/or offering useful advice

occasionally which in turn improve the outcome of work.

To my parents from whom, I learnt hard work and being independent. They

have continued to support my course with untiring love. With you around, I

feel stable emotionally, psychologically and financially throughout the

journey. To my siblings for the consistent calls and concern all through, you

all continue to inspired me and keep my spirit high all along my Ph.D

Journey.

To my wife for unparallel sacrifice shown for abandoning her medical

practice in Nigeria to make sure we raise our two most cherish divine gifts

together (Maryam and Hafsat). This concern and many more commitments

showed, re-kindled and boost my spirit and determination to succeed.

Lastly, I will especially once more express my profound gratitude to Prof.

Dr. Mohd Zamri-Saad (My Supervisory committee Chair) for the offer of

Special Graduate Research Allowance and funding the entire Ph.D project.

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This is my Journey and the people behind the scene, Thank you all

ALHAMDULILLAH.

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I certify that a Thesis Examination Committee has met on 21st November,

2012 to conduct the final examination of Abubakar Salisu Muhammad on

his thesis entitle “Response of Gastrointestinal tract to Pasteurella

multocida Serotype B:2 infection in Buffaloes (Bubalus bubalis

Linnaeus)” in accordance with the Universities and University Colleges Act

1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15

March 1998. The Committee recommends that the student be awarded the

Doctor of Philosophy.

Members of the Thesis Examination Committee were as follows:

Abdul Rahman bin Omar, PhD Professor Institute of Bioscience Universiti Putra Malaysia (Chairman)

Mohd Hair bin Bejo, PhD

Professor

Faculty of Veterinary Medicine

Universiti Putra Malaysia

(Internal Examiner)

Md Sabri bin Mohd Yusoff, PhD

Senior Lecturer

Faculty of Veterinary Medicine

Universiti Putra Malaysia

(Internal Examiner)

Patricia E. Shewen, PhD

Professor

Department of Pathobiology

University of Guelph, Canada

(External Examiner)

SEOW HENG FONG, PhD Professor and Deputy Dean School of Graduates Studies Universiti Putra Malaysia

Date:

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This thesis was submitted to the Senate of the Universiti Putra Malaysia and

has been accepted as fulfilment of the requirement for the degree of Doctor

of Philosophy. The Members of the Supervisory Committee are as follows:

Mohd Zamri-Saad, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman) Jasni Sabri, PhD Associate Professor Faculty of Veterinary Medicine Universiti Malaysia Kelantan (Member) Md Zuki Abu Bakar @ Zakaria, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) ___________________ BUJANG BIN KIM HUAT, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is based on my original work except for quotations

and citations, which have been duly acknowledged. I also declare that it has

not been previously or concurrently submitted for any other degree at

Universiti Putra Malaysia or other institution.

ABUBAKAR SALISU MUHAMMAD

Date: 21 November 2012

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TABLE OF CONTENTS

Page ABSTRACT ii ABSTRAK vi ACKNOWLEDGEMENT ix APPROVAL xii DECLARATION xiv LIST OF TABLES xix LIST OF FIGURES xxi LIST OF ABBREVIATION xxviii

CHAPTER

1 INTRODUCTION 1 1.1 Research hypotheses 4 1.2 Objectives of the study

5

2 2 LITERATURE REVIEW 6 2.1 Pasteurella multocida 6 2.1.1 Pasteurella multocida infections 7 2.1.2 Serology and identification of Pasteurella

multocida 8

2.1.3 Composition of Pasteurella multocida capsule 9 2.1.4 Adhesins and their role in pathogenesis 10 2.1.5 Membrane-associated enzymes and pathogenic

mechanism of Pasteurella multocida

11

2.2 Haemorrhagic septicaemia 14

2.2.1 Aetiology of haemorrhagic septicaemia 14 2.2.2 Species susceptibility to haemorrhagic

septicaemia 14

2.2.3 Factors influencing the transmission and clinical presentation

16

2.2.4 Occurrence and role of carrier animals in haemorrhagic septicaemia

17

2.2.5 Pathology of haemorrhagic septicaemia

18

2.3 Mucosal immune system in animals 21

2.3.1 Development of mucosal immunity in animals 21 2.3.2 Conjuctiva-associated lymphoid tissue (CALT) 24 2.3.3 Nasal-associated lymphoid tissue (NALT) 24 2.3.4 Larynx-associated lymphoid tissue (LTALT) 25 2.3.5 Bronchus-associated lymphoid tissue (BALT) 26 2.3.6 Gut-associated lymphoid tissues (GALT)

27

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2.4 Dynamics of mucosal immunity 31

2.4.1 The role of M-cell in mucosal immune response 34 2.4.2 Lymphocyte responses and regulation 35 2.4.3 Migration of mucosal lymphocytes

37

2.5 Haemorrhagic septicaemia vaccines and vaccination 38

2.6 Benefits and limitations of mucosal immunization in disease control

40

3 CLINICO-PATHOLOGICAL CHANGES IN BUFFALO CALVES FOLLOWING ORAL EXPOSURE TO Pasteurella multocida B:2

43

3.1 Introduction

43

3.2 Materials and Methods 44

3.2.1 Animals 44 3.2.2 Inocula preparation 45

3.2.3 Experimental procedure 46 3.2.4 Post-mortem examination 47 3.2.5 Bacterial isolation 48 3.2.6 Statistical analysis

49

3.3 Results 50

3.3.1 Clinical observations 50 3.3.2 Gross and histopathology 52 3.3.3 Bacterial isolation

62

3.4 Discussion 63

4 ULTRASTRUCTURAL CHANGES AND BACTERIAL LOCALIZATION IN BUFFALO CALVES FOLLOWING ORAL EXPOSURE TO Pasteurella multocida B:2

65

4.1 Introduction

65

4.2 Materials and Methods 66 4.2.1 Animals 66 4.2.2 Inocula preparation 66 4.2.3 Experimental design 66 4.2.4 Bacteriological examination 67 4.2.5 Immunohistochemistry (IHC) for P. multocida

B:2 67

4.2.6 Electron microscopic examination

68

4.3 Results 70

4.3.1 Clinical observations 70

4.3.2 Bacterial isolation 70

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4.3.3 Immunohistochemical detection of Pasteurella multocida B:2

72

4.3.4 Ultrastructural observations

76

4.4 Discussion 85

5 IMMUNOPEROXIDASE FOR EVALUATIONS OF LESIONS IN THE GASTROINTESTINAL TRACT OF BUFFALO CALVES ORALLY EXPOSED TO Pasteurella multocida B:2

88

5.1 Introduction

88

5.2 Materials and Methods 89 5.2.1 Animals 89 5.2.2 Inocula preparation 90 5.2.3 Experimental procedure 90 5.2.4 Bacterial isolation 91 5.2.5 Post-mortem and immunoperoxidase

examination 91

5.2.6 Statistical analysis

93

5.3 Results 93

5.3.1 Clinical Observations 93

5.3.2 Bacterial Isolation 93 5.3.3 Pathology and immuno-detection

94

5.4 Discussion 101

6 THE RESPONSES BY GUT-ASSOCIATED AND BRONCHUS-ASSOCIATED LYMPHOID TISSUES OF BUFFALO CALVES FOLLOWING ORAL EXPOSURE TO Pasteurella multocida B: 2

104

6.1 Introduction

104

6.2 Materials and Methods 105 6.2.1 Animals 105 6.2.2 Inoculum preparation 106 6.2.3 Experimental procedure 106 6.2.4 Tissue examination 107 6.2.5 Statistical analysis

107

6.3 Results 107

6.3.1 Bronchus-associated lymphoid tissue 108 6.3.2 Gut-associated lymphoid tissue 109 6.4 Discussion 113

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7 SAFETY, ANTIBODY PATTERN AND RESPONSES BY BRONCHUS-ASSOCIATED AND GUT-ASSOCIATED LYMPHOID TISSUES OF BUFFALO CALVES FOLLOWING ORAL ADMINISTRATION OF LIVE ATTENUATED Pasteurella multocidA B:2

115

7.1 Introduction

115

7.2 Materials and Methods 117 7.2.1 Animals 117 7.2.2 Inocula preparation 117 7.2.3 Experimental design 118 7.2.4 Clinical observations 119 7.2.5 Bacterial examination 119 7.2.6 Serological examination 120 7.2.7 Post-mortem examination 121 7.2.8 Tissue examination 121 7.2.9 Statistical analysis

121

7.3 Results 122 7.3.1 Clinical observations 122 7.3.2 Serological response 123 7.3.3 Bronchus-associated and gut-associated

lymphoid tissues responses

124

7.4 Discussion

137

8 GENERAL DISCUSSION, CONCLUSIONS AND SUGGESTIONS FOR FUTURE RESEARCH

140

REFERENCES 153 APPENDICES 168 BIODATA OF STUDENT 187 LIST OF PUBLICATIONS 188