UNIVERSITI PUTRA MALAYSIA

COMPARATIVE IN VITRO AND IN VIVO PATHOGENESIS OF EXPERIMENTALLY INDUCED

INCLUSION BODY DISEASE OF BOIDS

OMAR EMAD IBRAHIM

FPV 2013 25

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UPMCOMPARATIVE IN VITRO AND IN VIVO

PATHOGENESIS OF EXPERIMENTALLY INDUCED INCLUSION BODY DISEASE OF BOIDS

OMAR EMAD IBRAHIM

DOCTOR OF PHILOSOPHY

UNIVERSITI PUTRA MALAYSIA

2013

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UPM COMPARATIVE IN VITRO AND IN VIVO PATHOGENESIS OF EXPERIMENTALLY

INDUCED INCLUSION BODY DISEASE OF BOIDS

By

OMAR EMAD IBRAHIM

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

In Fulfilment of the Requirements for the Degree of Doctor of Philosophy

March 2013

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IN THE NAME OF ALLAH THE MOST GRACIOUS AND MERCIFUL

DEDICATION WITH LOVE AND GRATITUDE TO:

MY PARENTS, WHO MADE IT POSSIBLE;

MY BELOVED WIFE, WHO MADE THIS WRITING ENDURABLE;

AND TO RAND, AHMAD, AL-HAMZA AND AL-FAROUK, WHO MADE IT ALL WORTHWHILE

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

requirement for the degree of Doctor of Philosophy

COMPARATIVE IN VITRO AND IN VIVO PATHOGENESIS OF EXPERIMENTALLY INDUCED INCLUSION BODY DISEASE OF BOIDS

By

OMAR EMAD IBRAHIM

March 2013

Chair: Professor Noordin Mohamed Mustapha, PhD

Faculty: Veterinary Medicine

The inclusion body disease (IBD) is an infectious fatal disease of boid snakes characterised

by behavioral abnormalities, wasting and secondary infections. Microscopically, the disease

is identified by the presence of large eosinophilic cytoplasmic inclusions in multiple tissues

and thus giving rise to the name of the disease.

To date, no exact agent has been conclusively incriminated as the cause of the inclusion body

disease (IBD). A total of forty-eight boa and python snakes suspected of (IBD), (15) boa (33)

python cases were submitted for necropsy to the Department of Veterinary Pathology and

Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia from March 2008

to June 2009 from a recently officiated snake park located 50 kilometres south of UPM.

Using a cell culture and in vivo approach to search for the aetiological agent, identification

and de novo assembled the cytopathic effects and the morphology and size of two viruses

related to small round viruses the size of around 29.5 – 36 nanometer (nm). A continuous

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Vero cell line was established and used to propagate and isolate the viruses in culture. In

total, small round viruses were detected in 20 out of 30 suspected cases of IBD. These viruses

have a typical small round viruses organization but were highly divergent. The result of virus

clarification showed a visible opaque band, of the purified virus at the 30% interface while

the negatively stained particles under electron microscopy showed spherical with icosahedral

symmetry viral particles sized between 29.5 – 36.5 nm. More importantly, the two viral

isolates, CPE, band, size and morphology were similar for both the boa and python. The

presence of small round viruses out of mammals reveals that these viruses infect an

unexpectedly broad range of species and represent a new reservoir of potential human

pathogens.

The findings suggest that IBD is a multisystemic viral infection based on the

histopathological findings in the natural cases of IBD in boa and python. In short, it suggests

that the incriminating virus or agent is definitely not associated with popularly believed

Type-C retrovirus.

Twenty five female BALB/c mice (6 – 8 weeks of age) were used to study the pathogenesis

apart from verifying Koch’s postulate of IBD via inoculation of boa and python isolates in a

murine model. The findings demonstrated the ability of IBD virus from both boa and python

to induce an acute and chronic infection in mice.

In conclusion, this is the first detailed study on isolated IBD virus in an attempt to adapt the

viruses in vitro and assessing their pathogenic potential in a murine model.

Key words: IBD, boa, python, virus, mice

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi

keperluan untuk ijazah Doktor Falsafah

PERBANDINGAN IN VITRO DAN IN VIVO PATOGENESIS DARI UJI KAJI MENDORONG PENYAKIT MEMASUKKAN BADAN DARI BOIDS

Oleh

OMAR EMAD IBRAHIM

Mac 2013

Pengerusi: Profesor Noordin Mohamed Mustapha, PhD

Fakulti: Perubatan Veterinar

Penyakit jasad rangkuman (IBD) merupakan penyakit berjangkit dan merbahaya pada ular

boid yang bercirikan perubahan perilaku, kurus-kering dan jangkitan sekunder.Secara

mikroskopi, penyakit ini disahkan dengan kehadiran rangkuman besar serta bereosinofil pada

sitoplasma pelbagai tisu dan dari sinilah nama penyakit tersebut diambil.

Sehingga kini, tiada agen khusus telah dikenalpasti secara tepat sebagai penyebab penyakit

ini. Masing-masing sebanyak 48 dan 33 karkas dari boa dan ular sawa yang diterima untuk

nekropsi di Jabatan Patologi dan Mikrobiologi Veterinar, Fakulti Perubatan Veterinar,

Universiti Putra Malaysia digunakan dalam kajian ini. Kesemua kes diterima mulai Mac 2008

- Jun 2009 dari sebuah taman ular yang baru dirasmikan terletak 50 km selatan dari UPM.

Dengan menggunakan kultur sel dan pendekatan in vivo dalam mencari etiologi membawa

kepada pengenalpastian dan himpunan de novo kesan sitopati dan morfologi serta kedua-dua

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virus bulat kecil bersaiz 29.5-35 nm yang terlibat. Garisan sel Vero berterusan yang

dikukuhkan telah digunakan untuk menjana dan mengasing virus dalam kultur. Secara

keseluruhan, virus dikesan pada 20 dari 30 kes disyaki IBD. Virus ini mempunyai himpunan

virus kecil bulat tetapi amat berlainan. Hasil dari pengasingan virus menunjukkan garisan

legap dari permukaan 30% virus tertulin manakala zarah yang diwarnakan secara negatif di

bawah mikroskop elektron menunjukkan zarah virus simetri ikosahedron bersaiz antara 29.5-

36.5nm. Lebih penting lagi, kedua-kuda isolat virus, CPE, garisan, saiz dan morfologi adalah

sama bagi boa dan ular sawa. Kehadiran virus bulat kecil ini di luar mamalia menandakan

bahawa virus ini boleh menjangkiti julat sepsis yang luas dan mewakili tabungan potensi

patogen manusia.

Penemuan ini mencadangkan bahawa IBD ialah jangkitan virus multisistem berdasarkan pada

hasil histopatologi pada kes semulajadi IBD pada boa dan ular sawa. Pendekata,it

mencadangkan bahawa virus atau agen terlibat tidak berkait dengan kepercayaan popular

disebabkan oleh retrovirus Jenis C.

Dua puluh lima ekor mencit BALB/c betina berusia 6-8 minggu digunakan untuk kajian

pathogenesis selain dari mengesahkan postulat Koch, dengan inokulat pada isolat boa dan

ular sawa pada model murin. Penemuan menunjukkan keupayan virus IBD daripada boa dan

ular sawa untuk mengaruh jangkitan akut dan kronik pada mencit.

Sebagai rumusan, ini merupakan kajian terperinci pengaisngan virus IBD dalam usaha untuk

menyesuaikan virus in vitro dan menilai potensi kepatogenan pada model murin.

Kata kunci: IBD, boa, ular sawa, virus, mencit

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ACKNOWLEDGEMENTS

At the time of completing this thesis, I would like to take this opportunity to express my

gratefulness to the Almighty ALLAH, lord of all creations, who gives me strength, courage,

inspiration and love to be able to go through all the days of my life and also afforded me

great understanding and wisdom to complete my thesis, all the glory to his name.

I want to pause long enough to shine the spotlight on the people who helped me along the

way, if only for a moment. They deserve it.

First and foremost I offer my sincerest gratitude to my supervisor Professor Dr. Noordin

Mohamed Mustapha for giving me the opportunity to complete my PhD, his help and

guidance throughout my graduate program. He is very understanding, caring, supportive and

a great person to work with. I had a tremendous learning experience as a graduate student

because I was able to study and grow under his mentorship and guidance. His constructive

suggestions, criticisms and provoking have been most valuable. I am forever indebted for his

kindness, patience and motivation.

I wish to express my faithful gratitude and sincere thanks to my committee members, Prof.

Dato’ Dr. Sheikh-Omar Abdul Rahman who filled an important place in my life as a mentor

of spiritual values in this work. I would also like to express my heartfelt thanks and

appreciation to Professor Dr. Zuki Abu Bakar Zakaria for his constructive supports which

were really helpful towards the completion of my study.

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This work would not have been possible without the support and helping hand from the staff

members of the virology laboratory, in particular to Mr. Mohd Kamarudin Awang, Mr. Mohd

Nazri Abd Hamid, and Mr. Shahrudin Uda Ibrahim.

I am also extremely grateful to all the staff in Microscopy Unit and Molecular Biomedicine

of Institute of Bioscience, particular, Mr. Rafizu Zaman Haron, Mrs. Aminah Jusoh, Mrs.

Faridah Akmal, Dr. Tan Sheau Wei and Mrs. Nancy Liew Woan Charn.

I would like to offer my special thanks and I am forever grateful to my best friend, Dr. Amer

Khazaal Salih Al-Azawy for his assistance. Thank you for all the kind help.

My thanks also go to Dr. Aqil Mohammad Daher, Dr. Hamid Al-Tammemi, Dr. Ajwad

Awad, Dr. Karim, Dr. Latif, Dr. Nabeel, Dr. Ibrahim, Dr. Majed Hamed, Dr. Mayada Hasson,

Dr. Nathera Mohamed, Dr. Faruk Bandi, Dr. Saeed Sharif, Dr. Faiz Fawzi, Dr. Hemen

Othman and Dr. Abdul Rahman for their cooperation.

My sincere thanks also to the Malaysian Government and Universiti Putra Malaysia in

particular for supporting me throughout the course of my study.

I wish to express my deepest and heartfelt appreciation to my father Prof. Dr. IMAD and my

greatest supporter my mother. I also would love to thank my beloved wife, my daughter

RAND, and my sons AHMAD, ALHAMZA and ALFAROUK. I am also thankful to my

sister SURA and my brother ABDULLAH also to my uncle Dr. TARIK for being supportive,

helpful throughout my study. Without my family, I would never be able to accomplish this

challenging task.

Finally, many thanks to all who have helped or contributed in one way or other towards the

completion of this study.

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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 were as follows:

Noordin Mohamed Mustapha, PhD Professor

Faculty of Veterinary Medicine

Universiti Putra Malaysia

(Chairman)

Zuki Abu Bakar Zakaria, PhD Professor

Faculty of Veterinary Medicine

Universiti Putra Malaysia

(Member)

Dato’ Sheikh Omar Abdul Rahman, 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 my original work except for quotation and citations which have

been duly acknowledged. I also declare that it has not been previously, and is not

concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other

institution.

_____________________

OMAR EMAD IBRAHIM

Date: 1 March 2013

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TABLE OF CONTENTS

Page DEDICATION iiABSTRACT iii ABSTRAK vACKNOWLEDGEMENTS vi APPROVAL viii DECLARATION xi LIST OF TABLES xxLIST OF FIGURES

CHAPTER

GENERAL INTRODUCTION 1

LITERATURE REVIEW 3

2.1 CLASSIFICATION OF LARGE SNAKES 3

2.2 ANATOMY AND PHYSIOLOGY OF SNAKES 42.2.1 THE INTEGUMENTARY SYSTEM 4

2.2.3 THE MUSCULOSKELETAL SYSTEM 5

2.2.4 THE CARDIOVASCULAR SYSTEM 6

2.2.5 THE RESPIRATORY SYSTEM 7

2.2.6 THE DIGESTIVE SYSTEM 8

2.2.7 THE UROGENITAL SYSTEM 9

2.2.8 THE NERVOUS SYSTEM AND THE SENSES 10

2.3 THE IMMUNE SYSTEM 112.3.1 THE INNATE COMPONENT 13

2.3.2 THE HUMORAL COMPONENT 13

2.3.3 THE CELLULAR COMPONENT 14

2.3.4 NONSPECIFIC HUMORAL FACTORS 15

2.3.5 FACTORS THAT AFFECT THE IMMUNE RESPONSE 16

2.4 STRESSES IN THE CAPTIV REPTILES 172.4.1 THE STRESS REACTION 18

2.4.2 HOW IS THE STRESS REGULATED 18

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2.4.3 EFFECTS OF STRESS ON THE BODY SYSTEMS 19

2.4.4 UNADAPTIVE STRESS RESPONSE 21

2.4.5 STRESS IN CAPITIVITY 21

2.4.6 WHAT IS THE SIGNIFICANCE OF STRESS IN REPTILE MEDICINE 23

2.5 COMMON INFECTIOUS DISEASES OF SNAKES 242.5.1 BACTERIAL DISEASES OF SNAKES 24

2.5.2 Salmonella 24

2.5.3 Aeromonas 25

2.5.4 Pseudomonas 26

2.5.5 Mycobacteria 26

2.5.6 VIRAL DISEASES OF SNAKES 27

2.5.7 Paramyxovirus 27

2.6 HISTORY OF INCLUSION BODY DISEASE 292.6.1 AETIOLOGY OF IBD 29

2.6.2 CLINICAL SIGNS IN BOA NATURALLY INFECTED WITH IBD 31

2.6.3 CLINICAL SIGNS IN PYTHON NATURALLY INFECTED WITH IBD 32

2.6.4 POSTMORTEM FINDINGS 32

2.6.5 HISTOPATHOLOGICAL FINDINGS IN BOA NATURALY INFECTED WITH

32

IBD 32

2.6.6 HISTOPATHOLOGICAL FINDINGS IN PYTHON NATURALLY INFECTED 34

WITH IBD 34

2.6.7 CLINICAL PATHOLOGY 34

2.7 DIGNOSIS OF INCLUSION BODY DISEASE 352.7.1 THE INTRACYTOPLASMIC INCLUSION BODY STRUCTURE 35

2.7.2 DIFFERENTIAL DIAGNOSES OF INCLUSION BODY DISEASE 35

2.8 DISEASE PREVALENCE 36

2.8.1 EPIDEMIOLOGY 37

2.8.2 OCCURENCES IN MALAYSIA 37

2.9 MANAGEMENT OF INCLUSION BODY DISEASE 372.9.1 DISEASE PROGNOSIS 38

2.9.2 DISEASE PREVENTION 38

CHAPTER 3 39

COMPARATIVE PATHOLOGY OF INCLUSION BODY DISEASE IN BOA AND PYTHON 39

3.1 INTRODUCTION 39

3.2 MATERIALS AND METHODS 41

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3.2.1 Animals 41

3.2.2 Case investigations 41

3.2.3 Tissue processing 41

3.3 RESULTS 423.3.1 Case distribution based on species 42

3.3.2 Gross pathological findings of IBD from naturally infected cases of boa 43

3.3.3 Histopathological findings of IBD from naturally infected cases of boa 46

3.3.4 Gross pathological findings of IBD from naturally infected cases of Python 56

3.3.5 Histopathological findings of IBD from naturally infected cases of Python 60

3.4 DISCUSSION 70

CHAPTER 4 77

ISOLATION AND IDENTIFICATION OF THE POSSIBLE AETIOLOGY FOR BOID INCLUSION BODY DISEASE 77

4.1 INTRODUCTION 77

4.2 MATERIALS AND METHODS 784.2.1 Animals 78

4.2.2 Source of virus 78

4.2.3 Tissue homogenisation 78

4.2.4 Virus Isolation in Cell Culture 79

4.2.5 Virus clarification 79

4.2.6 Virus purification by sucrose gradient 80

4.2.7 Electron microscopy 80

4.3 RESULTS 824.3.1 Virus Isolation from boa 82

4.3.2 Virus isolation from python 91

4.4 DISSCUSION 974.4.1 The Cytopathic effects 97

4.4.2 The virus isolates characteristics 99

CHAPTER 5 101

EXPERIMENTAL INFECTION OF BOA AND PYTHON IBD VIRUS ISOLATES INA MOUSE MODEL 101

5.1 INTRODUCTION 101

5.2 MATERIALS AND METHODS 102

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5.2.1 Animals and Management 102

5.2.2 Virus stocks 102

5.2.3 Determination of virus titers 102

5.2.4 Experimental design of virus inoculation in BALB/c mice 103

5.2.5. Haematology 103

5.2.6 Gross Pathology 104

5.2.7 Histopathological Examination 104

5.2.8 Reisolation of virus from experimental mice 105

5.2.9 Tissue homogenates 105

5.2.10 Virus isolation in cell culture 105

5.3 RESULTS 1065.3.1 Clinical observations and gross pathology 106

5.3.2 Histopathology of acute inoculation study with viral isolate from boa 106

5.3.3 Histopathology of chronic inoculation study with viral isolate from boa 111

5.3.4 Histopathology of acute inoculation study with viral isolate from python 116

5.3.5 Histopathology of chronic inoculation study with viral isolate from Python 120

5.3.6 Haematology 126

5.3.7 Virus reisolation from acute boa and python inoculation studies 127

5.4 DISCUSSION 129

CHAPTER 6 138

GENERAL DISCUSSION, CONCLUSION AND RECOMENDATION FOR FUTURE RESEARCH 138

REFERENCES 141

BIODATA OF STUDENT 162

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LIST OF TABLES

Table Page

3-1 the number and their respective percentages of cases based on species 42

4-1 list of ibd positive cases based on cytopathic effects (cpe) on vero cell culture 83

5-1 shows all the haematological changes in acute and chronic (boa and python virus isolations) inoculated in balbc mice 126

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LIST OF FIGURES

3.5 Photomicrograph, kidney of a boa constrictor showing renal tubular epithelial cells containing amphophilic intracytoplasmic inclusions (↓ arrows), with no evidence of inflammatory cells infiltration (H&E stain, X 1000 Magnification. Mag.)

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3.6 Photomicrograph, kidney of a boa constrictor showing renal tubular epithelial cell containing amphophilic intracytoplasmic inclusion (arrow) adjacent to the nucleus and exceeding the nucleus size. No evidence of inflammatory cells infiltration with congestion of renal blood vessel. (H&E stain, X 1000 Mag.).

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3.7 Photomicrograph, kidney of a boa constrictor showing renal tubular epithelial cells containing amphophilic intracytoplasmic inclusions. The inclusions showed variation in size (↓ arrows). Few cells showed clear nuclear changes (↓arrows) (H&E stain, X 1000 Mag.).

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3.8 Photomicrograph, kidney of a boa constrictor showing complete diffuse renal tubular epithelial cells degeneration with multifocal(more than 20) amphophilic intracytoplasmic inclusions (arrows) is adjacent to the degenerated cell’s nucleus (H&E stain, X 400 Mag.).

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3.9 Photomicrograph, kidney of a boa constrictor showing diffuse degeneration of the renal tubular epithelial cells with vacuolation and containing multifocal amphophilic intracytoplasmic inclusions (arrows). No evidence of inflammatory cells infiltration (H&E stain, X 100 Mag.).

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3.10 Photomicrograph, brain of a boa constrictor showing vacuolation and eosinophilic intracytoplasmic inclusion in neurons (arrows) (H&E stain, X100 Mag.).

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3.11 Photomicrograph, liver of a boa constrictor showing Coagulative necrosis (arrow) (H&E stain, X 200 Mag.).

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3.12 Photomicrograph, liver of a boa constrictor showing hepatocytes cytoplasmolysis. No evidence of inflammatory cells infiltration (H&E stain, X 400 Mag.).

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3.13 Photograph, lung of a python harbouring Rhabdias species (arrow). 57

Figurs3.1

Photograph of a boa constrictor showing evidence of twirling

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3.2. Photograph, buccal cavity of a boa showing mild stomatitis with absence of fibrinous organization

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3.3 Photograph, liver of a boa showing congestion 44

3.4 Photograph, boa lung showing a normal pinkish colour and spongy consistency

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3.14 Photograph, python depicting the presence of the mite, Ophionyssus natricisis (arrow)

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3.15 Photograph, buccal cavity of a python showing severe fibrinou stomatitis

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3.16 Photograph, kidney of a python that is swollen showing less clear demarcation of the renal lobes.

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3.17 Photograph, liver of a python. Enlarged, congested, areas of necrosis can also be seen.

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3.18 Photograph, lung of a python that is voluminous, congested and presented with focal areas of haemorrhage.

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3.19 Python. Photomicrograph, brain showing vacuolation and amphophilic intracytoplasmic inclusion in neurons (arrows) (H&E stain, X 1000 Mag.).

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3.20 Python. Photomicrograph, brain showing vacuolation and amphophilic intracytoplasmic inclusion in neurons (arrows) (H&E stain, X 1000 Mag.).

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3.21 Python. Photomicrograph, intestine showing patchy loss of mucosa with enterocytes vacuolation (read arrow) (H&E stain, X 200 Mag.).

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3.22 Python. Photomicrograph, liver showing hepatocytes vacuolation (H&E stain, X 100 Mag.).

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3.23 Python. Photomicrograph, liver showing hepatocytes vacuolation (read arrows) and eosinophilic intracytoplasmic inclusion in the vacuolated hepatocytes (↓ arrows) (H&E stain, X 400 Mag.).

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3.24 Python. Photomicrograph, liver showing hepatocytes vacuolation and eosinophilic intracytoplasmic inclusion in the vacuolated hepatocytes with three histiocytic granulomas formation (arrows) (H&E stain, X100 Mag.).

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3.25 Python. Photomicrograph, liver showing histiocytic granuloma formation (read arrow) with eosinophilic intracytoplasmic inclusion in the hepatocytes (↓ arrow) (H&E stain, X200 Mag.).

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3.26 Python. Photomicrograph, kidney the epithelial renal tubules showing degeneration and Coagulative necrosis (H&E stain, X100 Mag.).

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3.27 Python. Photomicrograph, kidney showing Coagulative necrosis of epithelial renal tubules and glomeruli (arrow) (H&E stain, X100 Mag.).

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4.1 Uninfected African green monkey kidney cells (Vero cell) showing a monolayer, confluent, continuous cell line (X 100 Magnification Mag.)

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4.2 Vero cells Boa virus inoculation after 24 hours post inoculation first passage showing enlarged rounded cells (cellular swellings), (arrow) early CPE (X 100 Mag.)

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4.3 Vero cells Boa virus inoculation after 48 hours post inoculation first passage showing an increased number of enlarged rounded cells (cellular ) (arrows) (X 200 Mag.)

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4.4 Vero cells Boa virus inoculation after 72 hours post inoculation first passage showing aggregates of enlarged rounded cells [cellular swellings and clumping]( arrow) (X 200 Mag.)

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4.5Vero cells first passages, Boa virus inoculation after 5 days post inoculation, note the generalized cell rounding. Many cells are involved in some stage of infection, degenerated and cells detached from the glass surface (X200 Mag.)

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4.6 Vero cells Boa virus inoculation after 24 hours post inoculation second passage showing enlarged rounded cells [cellular swellings] ( arrow) early CPE (X200 Mag.)

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4.7 Vero cells Boa virus inoculation after 48 hours post inoculation second passage showing an increased number of enlarged rounded cells (Cellular swellings) (arrows) (X400 Mag.)

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4.8 Vero cells Boa virus inoculation after 72 hours post inoculation second passage showing diffuse cellular aggregates of enlarged rounded cells [cellular swellings] and clumping (arrow) (X200 Mag.)

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4.9 Photomicrograph of centrifuge tube showing a visible opaque band, of the purified virus at the 30% interface of the sucrose gradient (arrow)

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4.10 Electron micrograph of negatively stained virions of the 4th passage purified from cell culture (Vero cells) (boa virus isolate) showing (small rounded viral particles) spherical with icosahedral symmetry. The size between 29.5 – 36.5 nm (bar: 200 nm)

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4.11 Electron micrograph of negatively stained virions of the 4th passage purified from cell culture (Vero cells) (boa virus isolate) showing (small rounded viral particles) spherical with icosahedral symmetry. The size between 29.5 – 36.5 nm with cluster aggregate (bar: 200 nm)

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4.12 Vero cells Python virus inoculation after 24 hours post inoculation first passage showing enlarged rounded cells [cellular swellings] ( arrow) early CPE (X100 Mag.)

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4.13 Vero cells Python virus inoculation after 48 hours post inoculation first passage showing an increased number of enlarged rounded cells [cellular swellings] (arrows) (X400 Mag.)

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4.14 Vero cells Python virus inoculation after 72 hours post inoculation first passage showing diffuse cellular aggregates of enlarged rounded cells (cellular swellings) and clumping (X200 Mag.)

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4.15 Vero cells Python virus inoculation after 4 days post inoculation first passage note the generalized cell rounding. Many cells are involved in some stage of infection, cellular detachment (X40 Mag.)

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4.16 Vero cells Python virus inoculation after 7 days post inoculation second passage. Note the generalized cell rounding and cellular detachment (X40 Mag.)

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4.17 Photomicrograph of centrifuge tube showed a visible opaque band, of the purified virus at the 30 – 40% interface of the sucrose gradient(arrows).

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4.18 Electron micrograph of negatively stained virions of the 4th passage purified from cell culture (Vero cells) (python virus isolate) showed (small rounded viral particles) spherical with icosahedral symmetry. The size between 29.5 – 36.5 nm (bar: 200 nm)

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4.19 Electron micrograph of negatively stained virions of the 4th passage purified from cell culture (Vero cells) (python virus isolate) showed (small rounded viral particles) spherical with icosahedral symmetry. The size between 29.5 – 36.5 nm (bar: 200 nm)

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5.1 Photomicrograph of the kidney of mouse from Group 1, showing vacuolar degeneration of renal tubular epithelium (arrowheads), with Coagulative necrosis of tubules (arrows) (H & E X40 Magnification Mag.)

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5.2 P Photomicrograph of the lung of mouse from Group 1, (A) showing clear thickening of interalveolar septa with embolic formation in the blood vessels of the alveolar wall (arrows) (H & E X100 Mag.). Stretching and emphysema of alveolar wall are prominent in this section. (B) The lung also showed changes in the bronchiolar mucosa demonstrating increased in the cellularity of the respiratory epithelium (arrows) (H & E X200 Mag.)

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5.3 Photomicrograph of the brain of mouse from Group 1, showing (A) oedema of the cortical parenchyma of the brain tissue (arrow) (H & E X400 Mag.). (B) Acute vascular reaction of cerebral artery (arrow) (H & E X1000 Mag.)

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5.4 Photomicrograph of the liver of mouse from Group 1, showing centrilobular hepatocellular disorientation (arrows) (H & E X400 Mag.). (B) Disorientation of hepatocytes with much more severe degenerative changes and infiltration of Kupffer cells (arrows) (H &E X400Mag.)

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5.5 Photomicrograph of the brain of mouse from Group 2 showing (A) proliferation of astrocytes in the neocortical layer and neuronal necrosis (H & E X400 Mag.). (B) Reactive capillaries and diffuse

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oedema of the molecular layer of cerebellum (H & E X100 Mag.). (C) Focal astrocyte granuloma in the cerebral white mater (H & E X400 Mag.). (D) Gliosis of diffuse pattern in the white matter (H & E X400 Mag.). (E) Non-inflammatory reactive astrogliosis in cerebrum (H & E X400 Mag.)

5.6 Photomicrograph of the lung of mouse from Group 2 showing (A) haemorrhage all over the pulmonary tissue filling air spaces bronchial lumen (arrow) (H & E X40 Mag.). (B) Profuse flooding of erythrocytes with no distinct pathological changes in the pulmonary tissue (arrow) (H & E X100 Mag.)

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5.7 Photomicrograph of the spleen of mouse from Group 2 showing (A) highly reactive germinal center at white pulp and increase cellularity (arrow)( H & E X100 Mag.). (B) Showing red pulp extramedullary haematopoiesis predominantly comprised of darkly staining erythroid elements and a single megakaryocyte (arrow) (H & E X400 Mag.). (C) Focal white pulp hyperplasia (double arrow) (H & E X100 Mag.)

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5.8 Photomicrograph of the liver of mouse from Group 2 showing Kupffer cell hyperplasia predominantly comprised of darkly staining as focal aggregates of Kupffer cells (arrows) are randomly distributed around central veins and in portal areas as well as in the hepatic sinusoids (A, B ,C and D) ( H & E X100 ,X100 ,X400 ,X400 Mag.). Hepatocytes vacuolation (arrowheads) (H & E)

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5.9 Lung of a mouse from Group 3 showing high number of infiltrating inflammatory cells mostly lymphocytes. The intra-alveolar blood vessel wall was congested. Spaces were formed in the pulmonary tissue due to emphysema (arrow) (H & E X200 Mag.)

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5.10 (A) Spleen of a mouse from Group 3 showing exaggerated cellularity in the red pulp areas where the germinal center showed a high number of mononuclear cells intermingled within abundant erythrocytes. Large nucleated foamy–like cells detected easily in thegerminal centers (arrows) (H & E X400 Mag.) (B) Spleen of a mouse from Group 3 showing mild hypercellular reaction and blood vessel engorged with blood (arrow) (H & E X400 Mag.)

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5.11 Liver of a mouse from Group 3 showing A necrotic hepatocytes exhibiting cytoplasmolysis and pyknotic nuclei is seen (arrow). No inclusion bodies of any form were seen either in the nucleus or cytoplasm (H & E X1000 Mag.)

118

5.12 Kidney of a mouse from Group 3 showing vascular lumen distended, dilated and filled with blood with some of the renal tubular epithelium being swollen (arrow) (H & E X200 Mag.)

119

5.13 Small intestine of a mouse from Group 3 showing elongated villus (villous hyperplasia) (double arrows) (H & E X100 Mag.)

119

5.14 Photomicrograph of the lung of a mouse from Group 4 showing thickened interalveolar septa (arrow), broncheactasia and marked emphysema are widespread among the pulmonary tissue with engorged pulmonary blood vessels (H & E X100 Mag.)

121

5.15 (A) Photomicrograph of the spleen of a mouse from Group 4 showing highly reactive germinal center at white pulp and increase cellularity

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(arrow) (H & E X40 Mag.). (B) Showing red pulp extramedullary haematopoiesis predominantly comprised of darkly staining erythroid cells and megakaryocytes (arrows) (H & E X400 Mag.)

5.16 Photomicrograph of a liver of mouse from Group 4 showing (A) hepatocellular vacuolation and Kupffer cell hyperplasia predominantly comprised of darkly staining as focal aggregates of Kupffer cells (arrow) are randomly distributed in portal areas (H & E X200 Mag.). (B) Liver of mouse from Group 4 shows the degenerative changes as hepatocytes exhibiting cytoplasmolysis, no inclusions of any types were evident (H & E X400 Mag.)

123

5.17 Photomicrograph of the kidney of a mouse from Group 4 showing subcapsular haemorrhage (arrow) and prominent degeneration of tubular epithelial cells. Infiltration of mononuclear cell (H & E X200 Mag.)

123

5.18 Photomicrograph of the brain of mouse from Group 4 showing (A) disorganization and degeneration of Purkinje cells (arrow) (H & E X200 Mag.). (B) The cerebral cortex of a mouse showing diffuse gliosis (arrow) (H & E X200 Mag.). (C) The cerebral cortex showing granuloma-like formation composed of oligodendrocytes and microglia (arrow) (H & E X200 Mag). (D) Cerebral cortex of a mouse showing high number of neuronal cells resembling astrocytosis some are arranged in pairs (arrow) (H & E X200 Mag.)

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5.19 Photomicrograph of an intestine of a mouse from Group 4 showing (A) severe hyperplasia of Goblet cells (H &E X 100 Mag.) (B) The lacteal is infiltrated with (mononuclear) chronic inflammatory cells (arrow) (H &E X 200 Mag.)

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5.20 (A) Boa virus isolate reisolated from mice (group 1 acute infection), third passage Vero cell showing CPE typical to primary virus isolation from boa (X100 Mag.). (B) Python virus isolate reisolated from mice (group 3 acute infection), third passage Vero cell showing CPE typical to primary virus isolation from python (X100 Meg.)

128

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CHAPTER 1

GENERAL INTRODUCTION

Inclusion Body Disease (IBD) is the leading cause of death in large non-venomous captive

snakes of the Boidae and Pythonidae family. It is a common disorder characterised by the

formation of eosinophilic intracytoplasmic inclusion bodies in the epithelial cells of major

organs (Schumacher, Jacobson, Homer, & Gaskin, 1994).The intracytoplasmic inclusion

bodies in boa constrictor were mainly disseminated in the visceral organs, however the

inclusions in python most often localised in the brain. Similarly, the development of the

disease in boa constrictor takes much more compared to pythons.

In recent years, there has been an increasing interest in the understanding of the disease

aetiology, pathogenesis, route of transmission, epidemiology and treatment (Vancraeynest et

al., 2006). Chronologically, the past thirty years showed a high incidence of IBD in captive

Burmese python (late seventies extending into the mid eighties), while in the early nineties

more cases were diagnosed in boa constrictors (Schumacher et al., 1994).

Considerable amount of literature on IBD showed the relationship between the C-type

retrovirus like particles albeit never been proven via Koch’s postulate (Schumacher et al.,

1994, Jacobson et al., 2001). Thus, until today the agent, route of entry, cross species

infection and pathogenesis of IBD still remains as an enigma both in boa and python. We

hypothesized that “pathogenesis of inclusion body disease is not associated with type C

retrovirus infection”

As such, issues involve in the aetiology and pathogenesis associated with the agents causing

IBD between python and boa need to be elucidated.

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Therefore the objectives for this study were to:

1. elucidate the pathological changes of IBD in captive boa and python

2. isolate and identify the etiological agents of IBD

3. investigate the in vitro pathogenesis of IBD associated with the agents isolated from

boa and python

4. investigate the in vivo pathogenesis of the disease associated with the virus from boa

and python in mouse model

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