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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