UNIVERSITI PUTRA MALAYSIA

BOVINE MUCOSAL IMMUNE RESPONSE TO INTRANASAL EXPOSURE WITH LIVE PASTEURELLA MULTOCIDA B:2

KHIN MYAT NWE

FPV 2009 7

BOVINE MUCOSAL IMMUNE RESPONSE TO INTRANASAL EXPOSURE WITH LIVE

PASTEURELLA MULTOCIDA B:2

KHIN MYAT NWE

DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA

2009

BOVINE MUCOSAL IMMUNE RESPONSE TO INTRANASAL EXPOSURE WITH LIVE PASTEURELLA MULTOCIDA B:2

By

KHIN MYAT NWE

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

July 2009

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy

BOVINE MUCOSAL IMMUNE RESPONSE TO INTRANASAL EXPOSURE WITH LIVE PASTEURELLA MULTOCIDA B:2

By

KHIN MYAT NWE

July 2009

Chairman : Professor Mohd Zamri-Saad, PhD Faculty : Veterinary Medicine

Haemorrhagic septicaemia (HS) is an infectious disease of cattle and buffalo caused by

Pasteurella multocida B:2. It is endemic in most parts of tropical Asia, Africa and

India, causing high mortality in livestock and is considered as one of the most

economically important livestock diseases in Southeast Asia. Vaccination has been used

to control the disease and the oil adjuvant vaccine is most often used although alum

precipitate and broth vaccines are also available. Although the available injection

vaccines are effective in providing protection, the low vaccination coverage, particularly

among cattle and buffaloes kept extensively, is one of the main reasons that lead to

disease outbreaks, particularly among those extensively kept, semi-wild cattle and

buffaloes.

ii

Therefore, the disease remains a significant obstacle to sustainable agriculture in the

region and attempts should be made to increase the vaccination coverage. The

development of live attenuated vaccine that can be administered intranasally may be an

answer. This study reports on the use of an attenuated P. multocida B:2, that has been

created by manipulating the gdhA genes, as a component of live haemorrhagic

septicaemia vaccine. It is as an alternative way to protect the animals from

haemorrhagic septicaemia and in the same time to increase vaccination coverage.

The respiratory tract contains an important component of the mucosal immune system,

and the first line of immunological defense since it is exposed continuously to inhaled

antigens. Intranasal exposures to live wild-type and gdhA derivative of P. multocida B:2

not only successfully stimulated the mucosal immunity of the respiratory tract, but also

the systemic immunity. This was evidenced by the increased in the size of BALT, the

numbers of lymphocytes, the levels of IgA in the lung lavage fluid and the levels of IgG

in the serum of exposed calves. However, the nasal associated lymphoid tissues

(NALTs) were successfully stimulated only following intranasal exposures to the wild-

type P. multocida B:2.

Following intranasal exposures of calves to live gdhA derivative of P. multocida B:2 at

2-week interval and challenged by wild-type P. multocida B:2 four weeks later, the

exposed calves were not only able to prevent establishment of infection by wild-type P.

multocida B:2 but also produced similar effects in the susceptible commingled calves.

When immuno-suppression was created in calves by subcutaneous injections of

iii

dexamethasone for 3 consecutive days, and immediately followed by the intranasal

exposures to the gdhA derivative of P. multocida B:2, both mucosal and systemic

immunities of the immuno-suppressed calves failed to be stimulated. This was obvious

when the serum IgA and IgG levels of the exposed, stressed calves were similar to that

of unexposed calves. On the other hand, the unstressed calves showed significantly

(p<0.05) higher serum IgA and IgG levels following intranasal exposures to the gdhA

derivative of P. multocida B:2. Similarly, the IgA and IgG levels in the lung lavage

fluid of calves treated with dexamethaosne were significantly (p<0.05) lower than those

without dexamethasone treatments. It was concluded that dexamethasone reduced the

availability of immune cells, thus reducing immune responses.

iv

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan ijazah Doktor Falsafah Veterinar

TINDAKBALAS KEIMUNAN MUKOSA LEMBU TERHADAP PASTEURELLA MULTOCIDA B:2 HIDUP MENERUSI PENDEDAHAN

INTRANASAL

Oleh

KHIN MYAT NWE

Julai 2009

Pengerusi : Professor Dr. Mohd Zamri Saad, PhD

Fakulti : Fakulti Perubatan Veterinar

Sampar berdarah merupakan penyakit berjangkit yang menyerang lembu dan kerbau

disebabkan oleh bakteria Pasteurella multocida B:2. Penyakit ini didapati endemik di

kebanyakan bahagian tropika Asia, Afrika dan India, di mana menyebabkan kematian

yang tinggi terhadap haiwan-haiwan ternakan dan merupakan salah satu penyakit yang

menyebabkan kerugian besar daripada segi ekonomi di Asia Tenggara. Pemvaksinan

telah digunakan untuk mengawal penyakit ini dan vaksin minyak adjuvan digunakan

dengan lebih meluas berbanding vaksin ‘alum precipitate’ dan ‘broth’. Walaupun

pemvaksinan melalui suntikan telah terbukti berkesan dalam memberi perlindungan,

namun kesulitan dalam memberi vaksinasi terutama pada lembu dan kerbau yang

diternak secara separa liar telah menyebabkan berlakunya wabak penyakit.

v

Oleh yang demikian, penyakit ini telah menjadi halangan besar kepada sektor pertanian

di Asia Tenggara dan usaha harus dilakukan untuk mempertingkatkan lagi perlindungan

melalui vaksinasi. Penghasilan vaksin hidup yang dilemahkan dan diberi melalui

intranasal mungkin menjadi jawapan kepada permasalahan yang dihadapi kini.

Saluran pernafasan adalah komponen penting dalam sistem keimunan mukosa, dan

merupakan pertahanan paling hadapan dalam sistem keimunan benda hidup disebabkan

oleh pendedahan berterusan kepada antigen yang disedut dari udara. Pendedahan

intranasal terhadap P. multocida B:2 liar hidup dan gdhA diterbitkan dari P. multocida

B:2 iaitu vaksin yang dilemahkan bukan sahaja telah berjaya merangsang keimunan

mukosa di saluran pernafasan, malah turut merangsang penghasilan keimunan sistemik.

Ini dibuktikan melalui peningkatan dalam saiz BALT, jumlah limfosit, paras IgA dalam

cairan peparu dan paras IgG di dalam serum lembu yang telah divaksinkan.

Walaubagaimanapun, NALT hanya berjaya dirangsang melalui pendedahan intranasal

terhadap P. multocida B:2 liar sahaja.

Berikutan pendedahan lembu-lembu kepada gdhA hidup yang diterbitkan dari P.

multocida B:2 melalui vaksinasi intranasal pada selang dua minggu dan diberi cabaran

keupayaan menggunakan P. multocida B:2 liar dua minggu kemudiannya, menunjukkan

bahawa lembu-lembu tersebut bukan sahaja berupaya melawan jangkitan dari P.

multocida liar malah turut menghasilkan kesan yang sama terhadap lembu-lembu yang

bercampur dengan lembu yang telah diberi vaksin. Seterusnya, lembu-lembu tersebut

diberi immunosupressi menerusi suntikan intra-muskular untuk tiga hari berturut-turut,

vi

dan disusuli dengan pendedahan intranasal terhadap gdhA yang diterbitkan dari P.

multocida B:2. Keputusan menunjukkan lembu-lembu yang diberi immunosuppresi

gagal merangsang penghasilan keimunan mukosa dan sistemik. Perkara ini jelas sekali

dilihat apabila paras antibodi serum IgA dan IgG lembu-lembu yang telah diberi

tekanan secara immunosepressi dan didedahkan dengan vaksin yang dilemahkan adalah

sama sepertimana lembu-lembu yang tidak diberi rawatan. Dengan kata lain, lembu-

lembu yang tidak diberi tekanan dan didedahkan dengan gdhA yang diterbitkan dari P.

multocida B:2 menunjukkan signifikan (p<0.05) lebih tinggi dalam paras serum IgA

dan IgG. Perkara yang sama dapat dilihat melalui paras serum IgA dan IgG dalam

cairan peparu lembu-lembu yang dirawat dengan dexamethasone, dimana secara

signifikan (p<0.05) lebih rendah berbanding dengan yang tidak diberi rawatan

dexamethasone. Kesimpulannya, dexamethasone telah mengurangkan keupayaan sel-sel

keimunan dan seterusnya mengurangkan tindakbalas keimunan.

vii

ACKNOWLEDGEMENTS

I would like to express my heartiest appreciation especially to my main supervisor,

Professor Dr. Mohd Zamri Saad for his immeasurable and encouraging guidance

throughout the course of research and in dictation of this thesis. Similarly, sincere thanks

are also due to my co-supervisors, Associate Professor Dr. Noordin Mohamed Mustapha

and Associate Professor Dr. Mohd Effendy Abdul Wahid for their patience, advices and

helpful discussion with me throughout this journey.

I would also like to thank the following people who contributed their efforts in making

this research a success:

Dr. Md Sabri Mohd Yusoff and all HS team members, Mr. Mohd Jamil Samad, Mrs.

Jamilah, Mrs. Latifah, Ms. Darul and all Histopathology laboratory members for their

help, understanding, encouragement and support.

Mr Ariff Ahmad, Mr. Raziman and Mr. Kamal for their assistance and invaluable time

spent and staff of Faculty of Veterinary Medicine and who contributed directly or

indirectly in sharing their knowledge, skill and assistance throughout the course of my

study.

I would like to express my gratitude to all colleagues and friends who have helped and

supported me throughout these years.

viii

I obliged all my success to my parents, Mr. Win Maung and Mrs. Nu Nu Yee and my

brothers and sisters, for their blessing and love, for giving strength and continuous

encouragement throughout my upbringing which has led me to where I am now.

A special thank to my late father who initiated and piloted me to overcome all

challenges and catch successes throughout my whole life.

My heartfelt and special thanks to my husband, Mr. Nay Aung, my elder son Aung Pyae

and my younger son Hein Seck for their love, patience, sacrifices, understanding and

special encouragement.

Thank you all.

ix

This thesis was submitted to the Senate of 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) Noordin Mohamed Mustapha, PhD Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Mohd Effendy Abdul Wahid, PhD Associate Professor Centre for Marine Biotechnology, Universiti Malaysia Terengganu (Member) HASANAH MOHD. GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: 11 September 2009

x

DECLARATION I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not currently, submitted for any other degree at Universiti Putra Malaysia or at any other institution. KHIN MYAT NWE Date: 2 October 2009

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TABLE OF CONTENTS Page ABSTRACT ii ABSTRAK v ACKNOWLEDGEMENTS viii DECLARATION xii LIST OF TABLES xvi LIST OF PLATES xvii LIST OF FIGURES xix LIST OF ABBREVIATIONS xxi CHAPTER 1 INTRODUCTION

1.1

2 LITERATURE REVIEW 2.1 Pasteurella multocida 2.2 Haemorrhagic septicaemia 2.3 Live-attenuated Pasteurella multocida B:2 2.4 Nasal-associated lymphoid tissues (NALT)

2.1 2.1 2.2 2.4 2.6

2.5 Bronchus-associated lymphoid tissue (BALT) 2.6 Mucosal and humoral immunities in the respiratory tract 2.7 The effects of stress on immunity

2.9 2.13 2.19

3 GENERAL MATERIALS AND METHODS 3.1 Animals

3.1 3.1

3.2 Inocula 3.3 Preparation of the inocula 3.4 Experimental procedure

3.2 3.2 3.3

3.5 Clinical observations 3.6 Histological examinations

3.4 3.4

3.7 Determination of antibody levels 3.5 3.8 Sample processing for bacterial isolation 3.9 Statistical analysis

3.6 3.7

xii

4 BOVINE RESPIRATORY IMMUNE RESPONSES FOLLOWING INTRANASAL EXPOSURES TO Pasteurella multocida B:2

4.1

4.1 Introduction 4.1 4.2 Materials and Methods

4.3

4.2.1 Animals 4.3 4.2.2 Inocula 4.4 4.2.3 Experimental procedure 4.4 4.2.4 Post-mortem examination and sampling lung lavage 4.2.5 Determination of antibody levels

4.5 4.6

4.2.6 Determination of BALT and NALT responses 4.6 4.2.7 Statistical analysis 4.7 4.3 Results 4.3.1 Clinical and pathological responses

4.7 4.7

4.3.2 Responses by BALT 4.7 4.3.3 Responses by NALT 4.12 4.3.4 Serum antibody responses 4.14 4.3.4.1 The IgG levels 4.3.4.2 The IgA levels 4.3.5 Antibody levels in the lung lavage fluid 4.3.5.1 The IgG levels 4.3.5.2 The IgA levels 4.4 Discussion 5. INTRANASAL EXPOSURES TO LIVE ATTENUATED gdhA DERIVATIVE Pasteurella multocida B:2 TO PREVENT INFECTION BY WILD TYPE Pasteurella multocida B:2

4.14 4.15 4.16 4.16 4.17 4.18 5.1

5.1 Introduction 5.1

5.2 Materials and Methods 5.3 5.2.1 Animals 5.3 5.2.2 Inocula 5.3 5.2.3 Experimental procedure 5.4 5.2.4 Sampling 5.5 5.2.5 Sample processing 5.5 5.2.6 Statistical analysis

5.7

xiii

5.3 Results 5.7 5.3.1 Clinical and pathological observations 5.7

5.3.2 Bacterial isolation 5.8 5.3.3 Serum IgG level 5.14 5.3.4 Lung lavage IgA level

5.15

5.4 Discussion

5.16

6 THE EFFECT OF DEXAMETHASONE ON HUMORAL IMMUNE RESPONSES OF CALVES FOLLOWING INTRANASAL EXPOSURES OF LIVE ATTENUATED gdhA DERIVATIVE Pasteurella multocida B:2

6.1

6.1 Introduction

6.1

6.2 Materials and Methods 6.2 6.2.1 Animals 6.2 6.2.2 Inocula 6.3 6.2.3 Experimental procedure 6.3 6.2.4 Determination of antibody levels 6.4 6.2.5 Statistical analysis

6.5

6.3 Results 6.5 6.3.1 Serum IgA levels 6.5 6.3.2 Serum IgG levels 6.7 6.3.3 IgA levels in the Lung lavage fluid 6.8 6.3.4 IgG levels in the Lung lavage fluid

6.9

6.4 Discussion

6.10

7 GENERAL DISCUSSION

7.1

REFERENCES R.1 APPENDICES A.1 BIODATA OF STUDENT B.1

xiv

LIST OF TABLES

Table Page 3.1 Lung lesion scoring protocol 3.3 3.2 The scoring system to determine clinical signs as described by 3.5 Hodgson et al. (2005). 4.1 The average number and type of bronchus associated lymphoid tissue

(BALT) in calves exposed intranasal to live Pasteurella multocida B: 2 4.8

4.2 Average size of BALT and the number of lymphocytes in calves exposed intranasal to live Pasteurella multocida B:2 4.9

4.3 Average size of NALT and the number of lymphocytes in calves 4.12 exposed intranasal to live Pasteurella multocida B:2

xv

LIST OF PLATES

Plate Page 4.1 The bronchus-associated lymphoid tissue (BALT) of a calf

exposed intranasally to the live wild-type Pasteurella multocida B:2 showing large nodular type BALT containing numerous lymphocyte cells.

4.10

4.2 The aggregate type of bronchus-associated lymphoid tissue (BALT) of a calf exposed intranasally to the live wild-type Pasteurella multocida B:2 showing a large BALT containing numerous lymphocyte cells.

4.10

4.3 The aggregate type of bronchus-associated lymphoid tissue (BALT) of a calf exposed intranasally to the live gdhA derivative of Pasteurella multocida B:2 showing a large BALT containing numerous lymphocyte cells.

4.11

4.4 The nodular type of bronchus-associated lymphoid tissue (BALT) of a calf exposed intranasally to the live gdhA derivative of Pasteurella multocida B:2 showing a large BALT containing numerous lymphocyte cells.

4.11

4.5 The aggregate type of nasal-associated lymphoid tissue (NALT) of a calf exposed intranasally to the live wild-type Pasteurella multocida B:2 showing a large NALT containing numerous lymphocyte cells.

4.13

4.6 The nodular type of nasal-associated lymphoid tissue (NALT) of a calf exposed intranasally to the live wild-type Pasteurella multocida B:2 showing two large NALT containing numerous lymphocyte cells and lymph nodules.

4.13

4.7 The nodular type of nasal-associated lymphoid tissue (NALT) of a calf exposed intranasally to the live wild-type Pasteurella multocida B:2 showing a large NALT containing numerous lymphocyte cells.

4.14

xvi

5.1 Visceral organs of a calf of Group 1 reveals no significant lesion 5.9 after intranasal challenge exposure to the live gdhA derivative of Pasteurella multocida B:2 5.2 Lungs of a calf exposed intranasal to the live gdhA derivative of 5.9 Pasteurella multocida B:2 showing a normal pink appearance 5.3 Lung of a control calf of Group 3 that was not exposed to the 5.10 live gdhA derivative Pasteurella multocida B:2 but challenge exposed intra-tracheal to the wild-type Pasteurella multocida B:2 showing patches of haemorrhages and dark red acute pneumonia 5.4 Lung section of a control calf of Group 3 showed haemorrhages 5.10 into the alveolar spaces after intra-tracheal challenge with wild -type Pasteurella multocida B:2. 5.5 Lung section of a control calf of Group 3 showing mild 5.11 Haemorrhages and neutrophils after challenged by live wild-type Pasteurella multocida B:2. 5.6 Lung section of a control calf of Group 3 showing thickened inter 5.11 -alveolar septa due to congestion of capillaries and oedema . 5.7 A bronchial area of lung section of a calf showing relatively 5.12 normal lung section without lesion after intranasal exposures to live gdhA derivative of Pasteurella multocida B:2 and challenged by the wild-type Pasteurella multocida B:2. 5.8 Lung section of a control calf of Group 3 challenged intra-tracheal 5.12 with live wild-type Pasteurell multocida B:2 showing evidence of thrombosis with endothelial degeneration in certain area of the blood vessel.

xvii

LIST OF FIGURES Figure Page

4.1 Serum IgG levels following intranasal exposures to Pasteurella multocida B:2.

4.15

4.2 Serum IgA levels following intranasal exposures to Pasteurella multocida B:2.

4.16

4.3 The IgG levels in the lung lavage fluid of calves following intranasal exposures to Pasteurella multocida B:2.

4.17

4.4 The IgA levels in the lung lavage fluid of calves following intranasal exposures to Pasteurella multocida B:2.

4.18

5.1 Photograph of a multiplex PCR Pasteurella multocida B:2 results on the samples from the control calves of Group 3.

5.13

5.2 The serum Ig G levels following intranasal exposures to live gdhA derivative of Pasteurella multocida B:2.

5.14

5.3 The lung lavage IgA level of the different groups of calves following intranasal exposures to live gdhA derivative of Pasteurella multocida B:2.

5.15

6.1 The serum IgA levels in groups of calves exposed to live gdhA derivative of Pasteurella multocida B:2 with or without dexamethasone treatment.

6.6

6.2 The serum IgG levels in groups of calves exposed to gdhA derivative of Pasteurella multocida B:2 with or without dexamethasone treatment.

6.8

xviii

6.3 The IgA level in the lung lavage fluid of calves exposed to live gdhA derivative of Pasteurella multocida B:2 with or without dexamethasone treatment.

6.9

6.4 The IgG level in the lung lavage fluid of calves exposed to live gdhA derivative of Pasteurella multocida B:2 with or without dexamethasone treatment.

6.10

xix

LIST OF ABBREVIATIONS

BALT - bronchus-associated lymphoid tissues

B.H.I - brain-heart infusion broth

oC - degrees centigrade

cfu - colony forming unit

dH2O - distilled water

DEX - dexamethasone

EDTA - ethylenediamine tetraacetate

g - gram

gdhA - glutamate dehydrogenase

GALT - gut associated lymphoid tissue

HS - Haemorrhagic Septicemia

H & E - Hematoxylin and Eosin

IgA - Immunoglobulin A

IgG - Immunoglobulin G

Incontact - Incontact group

I/N - Intranasal administration group

kg - kilogram

L - litre

M - Molar

mg - milligram

mL - milliliter

mM - milliMolar

xx

xxi

NALT - nasal associated lymphoid tissue

PBS - Phosphate Buffered Saline

PCR - polymerase chain reaction

pH - isoelectric point

% - percent/ pencentage

RT - room temperature

rpm - revolution per minute

s - second

TBE - tris-boric-EDTA

Taq - Thermus aquaticus

μl - microlitre

μg - microgram

μM - microMolar

CHAPTER 1

INTRODUCTION

The Gram-negative bacterium, Pasteurella multocida is the etiological agent of

hemorrhagic septicemia in cattle, fowl cholera in birds and atrophic rhinitis in pigs

(Mannheim, 1984). Pasteurella multocida strains have been categorized into 5

serogroups, namely serogroups A, B, D, E, and F based on the antigenicity of their

capsule (Carter, 1967; Rimler, 1987). They are further categorized into 16 serotypes;

namely serotypes 1 to 16 based on the characteristic of their lipopolysaccharide

(LPS) antigens (Heddleston, 1972). The capsular serogroups are generally related to

disease predilection, with hemorrhagic septicemia strains belonging to serogroups B

or E (Verma and Jaiswal, 1998).

Hemorrhagic septicemia is endemic in most parts of tropical Asia, Africa and India,

causing high mortality in livestock (Bain et al., 1982). It is considered to be the most

economically important disease of cattle and buffaloes in Southeast Asia and causes

significant economic losses in India and Africa (Bain et al., 1982; Verma and

Jaiswal, 1998). Cattle and buffaloes are the most common hosts, but pigs, sheep,

goats, deer and camels are also susceptible to the infection (Dawkins et al, 1991;

Blackall et al., 2000). Vaccination with killed vaccines is practiced in areas where the

disease is endemic and has reduced the incidence of disease, but the duration of

immunity is short while the vaccination coverage is low, leading to disease outbreaks

(Bain et al., 1982; Verma and Jaiswal, 1998).

The respiratory tract of mammals has a mucosal cell lining, which is constantly

exposed to environmental materials including antigens. Therefore, the respiratory

tract must either mount an immune response or maintain immunological tolerance to

prevent establishment of infection (Holt, 1993; Debertin et al., 2003). Since

Bienenstock et al. (1973a) called attention to the presence of sub-epithelial lymphoid

tissue of the respiratory tract known as the bronchus-associated lymphoid tissue

(BALT), the morphology and function of BALT in the respiratory tract of laboratory

animals and man has been studied by many investigators (Bienenstock, 1985;

Anderson et al., 1986; Mair et al., 1987). Large aggregates of lymphoid follicles,

such as those seen in the intestine as Peyer’s patches, are also found in the upper

airways, especially at bifurcations of the bronchial tree. All of these lymphoid tissues

are involved in the immune response in the lung against inhaled antigens.

The specific immune effector lymphocytes are believed to originate either from

precursors present in sub-epithelial lymphoid tissue scattered throughout the

respiratory tract or from a precursor in the regional and systemic lymphoid tissues.

Thus, following intranasal exposure to the antigen, there will be stimulation of the

mucosal immunity of the respiratory tract, and the bronchus-associated lymphoid

tissues (BALTs) are expected to show morphological changes while a high level of

secretory immunoglobulin is expected in the lung lavage fluid and serum. This study

describes the morphological changes in BALT and the nasal associated lymphoid

tissues (NALT), the antibody levels in the lung lavage fluid and serum following

stimulation of the mucosal immunity of respiratory tract by intranasal exposures to

live Pasteurella multocida B:2.

1.2