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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
44
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
122
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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.)
124
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.)
125
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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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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REFERENCES
Achim, C. L., Schrier, R. D., & Wiley, C. A. (1991). Immunopathogenesis of HIV
encephalitis. Brain Pathology, 1(3), 177-184.
Alibardi, L. (1998). Presence of acid phosphatase in the epidermis of the regenerating tail of
the lizard (Podarcis muralis) and its possible role in the process of shedding and
keratinization. Journal of Zoology, 246(4), 379-390.
Allender, M. C., Fry, M. M., Irizarry, A. R., Craig, L., Johnson, A. J., & Jones, M. (2006).
Intracytoplasmic inclusions in circulating leukocytes from an eastern box turtle
(Terrapene carolina carolina) with iridoviral infection. Journal of Wildlife Diseases,
42(3), 677-684.
Allender, M. C., Mitchell, M. A., Phillips, C. A., Gruszynski, K., & Beasley, V. R. (2006).
Hematology, plasma biochemistry, and antibodies to select viruses in wild-caught
eastern massasauga rattlesnakes (Sistrurus catenatus catenatus) from Illinois. Journal
of Wildlife Diseases, 42(1), 107-114.
Appleton, H. (1987). Small Round Viruses: Classification and Role in Food Borne
Infections.
Ariel, E. (2011). Viruses in reptiles. Veterinary Research, 42(1), 100.
Asterita, M. F. (1985). The physiology of stress. New York: Human Sciences.
Axelrod, J., & Reisine, T. D. (1984). Stress hormones: their interaction and regulation.
Science, 224(4648), 452.
Axthelm, M. (1985). Clinicopathologic and virologic observations of a probable viral disease
affecting boid snakes. Annu. Meet. Am. Assoc. Zoo. Vet., Scottsdale, Arizona, 108-
109.
Axthelm, M. (1989). Viral encephalitis of boid snakes.
Baldwin, D., Srivastava, P., & Krummen, L. (1991). Differential actions of corticosterone on
luteinizing hormone and follicle-stimulating hormone biosynthesis and release in
cultured rat anterior pituitary cells: interactions with estradiol. Biology of
Reproduction, 44(6), 1040.
Bechtel, H. B. (1995). Reptile and amphibian variants: colors, patterns, and scales: Malabar,
Fla.: Krieger Pub. Co.
Bellairs Ad’A, B. S. (1985). Autotomy and regeneration in reptiles. In B. F. Gans C (Ed.),
Biology of the Reptilia (Vol. 15). New York: John Wiley &Sons.
Bellairs, A. A., Zoologist, G. B., & Zoologiste, G. B. (1969). The Iife of Reptiles (Vol. 2):
Cambridge Univ Press.
© COP
UPM
142
Bennett, R. A. (1996). Neurology. In R. Mader (Ed.), Reptile Medicine and Surgery (1 ed.,
pp. 141-148). Philadelphia: Saunders.
Bergstrom, K. S. B., Guttman, J. A., Rumi, M., Ma, C., Bouzari, S., Khan, M. A., et al.
(2008). Modulation of intestinal goblet cell function during infection by an attaching
and effacing bacterial pathogen. Infection and Immunity, 76(2), 796-811.
Boorman, G. A. (1990). Pathology of the Fischer Rat: Reference and Atlas: Academic Press.
Boyce, J., Kociba, G., Jacobs, R., & Weiser, M. (1986). Feline leukemia virus-induced
thrombocytopenia and macrothrombocytosis in cats. Veterinary Pathology Online,
23(1), 16.
Bronson, D. L., Saxinger, W., Ritzi, D. M., & Fraley, E. E. (1984). Production of virions with
retrovirus morphology by human embryonal carcinoma cells in vitro. Journal of
General Virology, 65(6), 1043.
Brown, D. R. (2002). Mycoplasmosis and immunity of fish and reptiles. Frontiers in
Bioscience, 7, 1338-1346.
Bustard, H. R., & Maderson, P. (1965). The eating of shed epidermal material in squamate
reptiles. Herpetologica, 21(4), 306-308.
Carlisle nowak, M., Sullivan, N., Carrigan, M., Knight, C., Ryan, C., & Jacobson, E. (1998).
Inclusion body disease in two captive Australian pythons (Morelia spilota variegata
and Morelia spilota spilota). Australian Veterinary Journal, 76(2), 98-100.
Causey, O. R., Shope, R. E., & Bensabath, G. (1966). Marco, Timbo and Chaco, newly
recognized arboviruses from lizards of Brazil. American Journal of Tropical Medicine
and Hygiene 15, 239-243.
Chan, K. T., Papeta, N., Martino, J., Zheng, Z., Frankel, R. Z., Klotman, P. E., et al. (2008).
Accelerated development of collapsing glomerulopathy in mice congenic for the
Hivan1 locus. Kidney Iinternational, 75(4), 366-372.
Chang, L. W., & Jacobson, E. R. (2010). Inclusion body disease, a worldwide infectious
disease of boid snakes: a review. Journal of Exotic Pet Medicine, 19(3), 216-225.
Chia, J., Chia, A., Voeller, M., Lee, T., & Chang, R. (2010). Acute enterovirus infection
followed by myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and viral
persistence. Journal of Clinical Pathology, 63(2), 165-168.
Clark, H. F., & Karzon, D. T. (1972). Iguana virus, a herpes-like virus isolated from cultured
cells of a lizard, Iguana iguana. Infection and Immunity, 5(4), 559.
Cock Buning, T. (1985). Qualitative and quantitative explanation of the forms of heat
sensitive organs in snakes. Acta Biotheoretica, 34(2), 193-205.
© COP
UPM
143
Coffin, J. M., Hughes, S. H., Varmus, H. E., Rosenberg, N., & Jolicoeur, P. (1997).
Retroviral Pathogenesis.
Cualing, H., Bhargava, P., & Sandin, R. L. (2012). Non-Neoplastic Hematopathology and
Infections: Wiley-Blackwell.
Cuconati, A., & White, E. (2002). Viral homologs of BCL-2: role of apoptosis in the
regulation of virus infection. Genes & Development, 16(19), 2465.
Cunningham, D. L., Van Tienhoven, A., & Gvaryahu, G. (1988). Population size, cage area,
and dominance rank effects on productivity and well-being of laying hens. Poultry
Science, 67(3), 399-406.
Cuperus, R., Schäppi, M., Shah, N., Lindley, K., Milla, P., & Smith, V. (2005). Hypertrophic
eosinophilic gastroenteropathy is associated with reduced enterocyte apoptosis.
Histopathology, 46(1), 73-80.
Dauphin-Villemant, X. F. (1986). Nychthemeral variations of plasma corticos Teroids in
captive female lacerate vivipara Jacquin Influence of stress and reproductive state.
General and Comparative Endocrinology, (Vol. 67, pp. 292-302).
Del Rosario, A. D., Bui, H. X., Singh, J., Ginsburg, R., & Ross, J. S. (1994). Intracytoplasmic
eosinophilic hyaline globules in cartilaginous neoplasms: a surgical, pathological,
ultrastructural, and electron probe x-ray microanalytic study. Human Pathology,
25(12), 1283-1289.
Denardo, D. (2006). Anatomy, Physiology and Behaviour. In D. Mader (Ed.), Reptile
Medicine and Surgery (2nd ed., pp. 119-123). St. Louis: Saunders.
DeNardo, D. F., & Licht, P. (1993). Effects of corticosterone on social behavior of male
lizards. Hormones and Behavior, 27, 184-184.
DeNardo, D. F., & Sinervo, B. (1994). Effects of corticosterone on activity and home-range
size of free-ranging male lizards. Hormones and Behavior, 28(1), 53-65.
Denk H, F. W., Eckerstorfer R, Schmid E, Kerjaschki D. (1979). Formation and involution of
Mallory bodies (alcoholic hyaline) in murine and human liver revealed by
immunofluorescence microscopy with antibodies to prekeratin. Proceedings of the
National Academy of Sciences USA, 76, 4112-4116.
Doherty, R., Carley, J., Standfast, H., Dyce, A., Kay, B., & Snowdon, W. (1973). Isolation of
arboviruses from mosquitoes, biting midges, sandflies and vertebrates collected in
Queensland, 1969 and 1970. Transactions of the Royal Society of Tropical Medicine
and Hygiene, 67(4), 536-543.
Domínguez, V., Govezensky, T., Gevorkian, G., & Larralde, C. (2003). Low platelet counts
alone do not cause bleeding in an experimental immune thrombocytopenic purpura in
mice. Haematologica, 88(6), 679-687.
© COP
UPM
144
Dries, D. J. (2008). Histological and Histochemical Methods: Theory and Practice. Shock,
30(4), 481.
Driggers, T. A., F. (1997). Infectious disease. In L.Ackerman (Ed.), The biology, Husbandry
and Health Care of Reptiles (Vol. 3, pp. 593 – 612). Neptune City: TFH Publications.
Ellerman, V., & Bang, O. (1908). Experimental leukemia in chickens. Zentr. Bacteriol.
Parasiterik. Abt, 1, 595-609.
Eustis S. L., B. G. A., Harada T., Popp J. A. (1990). Pathology of the Fischer Rat. Reference
and Atlas San Diego, California: Academic Press.
Evans, E. E. (1963). Comparative Immunology. Antibody Response in Dipsosaurus dorsalis
at Different Temperatures.
Evans, E. E., & Cowles, R. B. (1959). Effect of Temperature on Antibody Synthesis in the
Reptile, Dipsosaurus Dorsalis.
Frank, W. (1984). Non-hemoparositic protozoans. In F. L. F. G. L. Hoff, E. R. Jacobson
(Ed.), Diseases of Amphibians and Reptiles (pp. 259-323). New York: Plenum Press.
Frantzidou, F., Kamaria, F., Dumaidi, K., Skoura, L., Antoniadis, A., & Papa, A. (2008).
Aseptic meningitis and encephalitis because of herpesviruses and enteroviruses in an
immunocompetent adult population. European Journal of Neurology, 15(9), 995-997.
Fredrikson, M., Sundin, O., & Frankenhaeuser, M. (1985). Cortisol excretion during the
defense reaction in humans. Psychosomatic Medicine, 47(4), 313-319.
French, S. (1983). Present understanding of the development of Mallory's body. Archives of
Pathology & Laboratory Medicine, 107(9), 445.
Funk, R. (2006). Biology–Snakes. In D. Mader (Ed.), Reptile Medicine and Surgery (Second
Edition ed., pp. 42-58). Missouri: Saunders Elsevier.
Funk, R. S. (2006). Snakes. In D. Mader (Ed.), Reptile Medicine and Surgery (2nd ed., pp.
42-58). St. Louis: Saunders.
Galabov, A. S. (1981). [28] Induction and characterization of tortoise interferon. Methods in
Enzymology, 78, 196-208.
Galabov, A., & Velichkova, E. H. (1975). Interferon production in tortoise peritoneal cells.
Journal of General Virology, 28(2), 259.
Galabov, A., Savov, Z., & Vassileva, V. (1973). Interferon production in arbovirus-infected
cell cultures of tortoise (Testudo graeca) kidney. Acta Virologica, 17(1), 1.
Garner, M., & Raymond, J. (2004). Methods for diagnosing inclusion body disease in snakes.
Exotic DVM., 6(3), 90-92.
© COP
UPM
145
Girling, S., Raiti, P., & Association, B. S. A. V. (2004). BSAVA Manual of Reptiles: British
Small Animal Veterinary Association.
Grassman, M., & Crews, D. (1989). Ovarian and adrenal function in the parthenogenetic
whiptail lizard Cnemidophorus uniparens in the field and laboratory. General and
Comparative Endocrinology, 76(3), 444-450.
Grassman, M., & Hess, D. L. (1992). Sex differences in adrenal function in the lizard
Cnemidophorus sexlineatus: II. Responses to acute stress in the laboratory. Journal of
Experimental Zoology, 264(2), 183-188.
Greene, H. W., & Fogden, M. (2000). Snakes: the evolution of mystery in nature: University
of California Press.
Hailey, A., & Theophilidis, G. (1987). Cardiac responses to stress and activity in the
armoured legless lizard Ophisaurus apodus: comparison with snake and tortoise.
Comparative Biochemistry and Physiology Part A: Physiology, 88(2), 201-206.
Hanley, C. S., & Hernandez-Divers, S. (2003). Practical Gross Pathology of Reptiles.
Harada, T., Maronpot, R. R., Enomoto, A., Tamano, S., & Ward, J. M. (1996). Changes in
the liver and gallbladder. Pathobiology of the Aging Mouse, 2, 207-241.
Hawkes, M. T., & Vaudry, W. (2005). Nonpolio enterovirus infection in the neonate and
young infant. Paediatrics & Child Health, 10(7), 383.
Heldstab, A., & Bestetti, G. (1984). Virus associated gastrointestinal diseases in snakes. The
Journal of Zoo Animal Medicine, 15(3), 118-128.
Hemsworth, P., Barnett, J., & Hansen, C. (1981). The influence of handling by humans on the
behavior, growth, and corticosteroids in the juvenile female pig. Hormones and
Behavior, 15(4), 396-403.
Herniou, E., Martin, J., Miller, K., Cook, J., Wilkinson, M., & Tristem, M. (1998). Retroviral
diversity and distribution in vertebrates. Journal of Virology, 72(7), 5955.
Herrath, M. G., & Oldstone, M. (1996). Virus-induced autoimmune disease. Current Opinion
in Iimmunology, 8(6), 878-885.
Hicks, J. W. (1998). Cardiac shunting in reptiles: mechanisms, regulation and physiological
functions. Biology of the Reptilia, 19, 425-483.
Holt, P. (1981). Drugs and dosages. Diseases of the Reptilia, 2, 551-584.
Holz, P. (1999). The reptilian renal-portal system: influence on therapy. Zoo and Wild
Animal Medicine Current Therapy, 4, 249-252.
Hoyer, R. F., O'Donnell, R. P., Mason, R. T., & Ovaska, K. (2006). Current distribution and
status of Sharp-tailed Snakes (Contia tenuis) in Oregon. Northwestern Naturalist,
87(3), 195-202.
© COP
UPM
146
Hsu, E. (1998). Mutation, selection, and memory in B lymphocytes of exothermic
vertebrates. Immunological Reviews, 162(1), 25-36.
Huder, J. B., Boni, J., Hatt, J. M., Soldati, G., Lutz, H., & Schupbach, J. (2002). Identification
and characterization of two closely related unclassifiable endogenous retroviruses in
pythons (Python molurus and Python curtus). Journal of Virology, 76(15), 7607.
Jacobson ER, S. D. (2007). Identifying reptile pathogens using electron microscopy. In J. ER
(Ed.), Infectious Diseases and Pathology of Reptiles: A Colour Atlas and Text (pp.
299-349). Boca Raton, FL: CRC Press.
Jacobson, E. (2007). Viruses and viral diseases of reptiles. In J. ER (Ed.), Infectious Diseases
and Pathology of Reptiles: A Color Atlas and Text (pp. 395-460). Boca Raton, FL:
CRC Press.
Jacobson, E. R. (2007). Infectious diseases and pathology of reptiles: color atlas and text:
CRC.
Jacobson, E. R., Gaskin, J., & Mansell, J. (1993). Viral diseases of reptiles. Zoo and Wild
Animal Medicine, Current Therapy, 3, 153-159.
Jacobson, E. R., Orós, J., Tucker, S. J., Pollock, D. P., Kelley, K. L., Munn, R. J., et al.
(2001). Partial characterization of retroviruses from boid snakes with inclusion body
disease. American Journal of Veterinary Research, 62(2), 217-224.
Jacobson, E., Gaskin, J., Page, D., Iverson, W., & Johnson, J. (1981). Illness associated with
paramyxo-like virus infection in a zoologic collection of snakes. Journal of the
American Veterinary Medical Association, 179(11), 1227.
Jensen, K., & Gluud, C. (1994). The Mallory body: morphological, clinical and experimental
studies (Part 1 of a literature survey). Hepatology, 20(4), 1061-1077.
Keeble, A. (2004). Neurology. In P. R. S. J. Girling (Ed.), BSAVA Manual of Reptiles (2nd
ed., pp. 273-288). Quedgeley: BSAVA.
Kenyon, R. H. (1988). Viral strain dependent differences in experimental Argentine
hemorrhagic fever (Junín virus) Infection of Guinea Pigs: DTIC Document.
Keogh, E. (1938). Ectodermal lesions produced by the virus of Rous sarcoma. British Journal
of Experimental Pathology, 19(1), 1.
Khan, A. S., Sears, J. F., Muller, J., Galvin, T. A., & Shahabuddin, M. (1999). Sensitive
assays for isolation and detection of simian foamy retroviruses. Journal of Clinical
Microbiology, 37(8), 2678-2686.
Khan, A. S., Sears, J. F., Muller, J., Galvin, T. A., & Shahabuddin, M. (1999). Sensitive
assays for isolation and detection of simian foamy retroviruses. Journal of Clinical
Microbiology, 37(8), 2678-2686.
© COP
UPM
147
Kiernan, J. (2008). Histological and Histochemical Methods: Theory and Practice (4th ed.).
Bloxham, UK: Scion.
Kim, C. H. (2010). Homeostatic and pathogenic extramedullary hematopoiesis.
Journal of Blood Medicine, 1, 13-19.
Klenk, K., & Komar, N. (2003). Poor replication of West Nile virus (New York 1999 strain)
in three reptilian and one amphibian species. The American Journal of Tropical
Medicine and Hygiene, 69(3), 260-262.
Kluge, A. G. (1993). Aspidites and the phylogeny of pythonine snakes: Australian Museum.
Knowles, N. (2011). Picornaviridae.com. Retrieved 31 July, 2001
Kopito, RR. , (2000). Aggresomes, inclusion bodies and protein aggregation. Trends in Cell
Biology, 10 (12), 524 – 530.
Kowalski, M., Bergeron, L., Dorfman, T., Haseltine, W., & Sodroski, J. (1991). Attenuation
of human immunodeficiency virus type 1 cytopathic effect by a mutation affecting the
transmembrane envelope glycoprotein. Journal of Virology, 65(1), 281.
Krakowka, S. (1987). Immunity, Viral Pathology and Assessment of Immune Dysfunction in
Virology and Toxicology. Toxicologic Pathology, 15(1), 18-26.
Kreger, M. D., & Mench, J. A. (1993). Physiological and behavioral effects of handling and
restraint in the ball python (Python regius) and the blue-tongued skink (Tiliqua
scincoides). Applied Animal Behaviour Science, 38(3-4), 323-336.
Kumar, A., Kushwaha, R., & Singh, U. (2011). Preneoplastic and neoplastic
megakaryocyte/platelet disorders: Three case reports. Indian Journal of Cancer, 48(3),
363.
Kupila, L., Vuorinen, T., Vainionpää, R., Hukkanen, V., Marttila, R., & Kotilainen, P.
(2006). Etiology of aseptic meningitis and encephalitis in an adult population.
Neurology, 66(1), 75-80.
Lance, V. A., & Lauren, D. (1984). Circadian variation in plasma corticosterone in the
American alligator, Alligator mississippiensis, and the effects of ACTH injections.
General and Comparative Endocrinology, 54(1), 1-7.
Le Gall, O., Christian, P., Fauquet, C. M., King, A. M. Q., Knowles, N. J., Nakashima, N., et
al. (2008). Picornavirales, a proposed order of positive-sense single-stranded RNA
viruses with a pseudo-T= 3 virion architecture. Archives of virology, 153(4), 715-727.
Lenihan, D. J., Greenberg, N., & Ten-Ching, L. (1985). Involvement of platelet activating
factor in physiological stress in the lizard, Anolis carolinensis. Comparative
Biochemistry and Physiology Part C: Comparative Pharmacology, 81(1), 81-86.
© COP
UPM
148
Licht, P., Breitenbach, G. L., & Congdon, J. D. (1985). Seasonal cycles in testicular activity,
gonadotropin, and thyroxine in the painted turtle, Chrysemys picta, under natural
conditions. General and Comparative Endocrinology, 59(1), 130-139.
Liévin-Le Moal, V., & Servin, A. L. (2006). The front line of enteric host defense against
unwelcome intrusion of harmful microorganisms: mucins, antimicrobial peptides, and
microbiota. Clinical Microbiology Reviews, 19(2), 315.
Lillywhite, H. (1987). Temperature, energetics, and physiological ecology. Snakes: Ecology
and Evolutionary Biology, 422-477.
Lillywhite, H. B., & smith, L. H. (1981). Haemodynamic responses to haemorrhage in the
snake, Elaphe obsoleta obsoleta. Journal of Experimental Biology, 94(1), 275.
Long, R. E., Knutsen, G., & Robinson, M. (1986). Myeloid hyperplasia in the SENCAR
mouse: differentiation from granulocytic leukemia. Environmental Health
Perspectives, 68, 117.
Love B, L. K. T. ( 2000). The corn snake manual Escondido. In Calif (Ed.), Advanced
Vivarium Systems.
Luna, L. G. (1968). Manual of histologic staining methods of the Armed Forces Institute of
Pathology: New York: McGraw-Hill.
Lunger, P., Hardy Jr, W., & Clark, H. (1974). C-type virus particles in a reptilian tumor.
Journal of the National Cancer Institute, 52(4), 1231.
Lustig, S., Jackson, A. C., Hahn, C. S., Griffin, D. E., Strauss, E. G. and Strauss, J. H. (
1988). Journal of Virology, 62(7), 2329 – 2336.
Mader, D. R., & ScienceDirect. (2006). Reptile Medicine and Surgery.
Maderson, P. (1965). The structure and development of the squamate epidermis. Biology of
the Skin and Hair Growth, 129-153.
Madsen, T., Ujvari, B., Shine, R., & Olsson, M. (2006). Rain, rats and pythons:
Climate driven population dynamics of predators and prey in tropical Australia.
Austral Ecology, 31(1), 30-37.
Manetto, V., Abdul-Karim, F., Perry, G., Tabaton, M., Autilio-Gambetti, L., & Gambetti, P.
(1989). Selective presence of ubiquitin in intracellular inclusions. The American
Journal of Pathology, 134(3), 505.
Manzo C, z. M., Gobbetti A, Di fiori MM, Angelini F. (1994). Is corticosterone involved in
the reproductive process of the male lizard, podarcis sicula. Hormones and Behavior ,
28(2), 117-129.
Marschang, R. E. (2011). Viruses Infecting Reptiles. Viruses, 3(11), 2087-2126.
© COP
UPM
149
Martin, J., Herniou, E., Cook, J., Waugh O'Neill, R., & Tristem, M. (1997). Human
endogenous retrovirus type I-related viruses have an apparently widespread
distribution within vertebrates. Journal of Virology, 71(1), 437.
Martin, J., Kabat, P., Herniou, E., & Tristem, M. (2002). Characterization and complete
nucleotide sequence of an unusual reptilian retrovirus recovered from the order
Crocodylia. Journal of Virology, 76(9), 4651-4654.
Mathews, J., & Vorndam, A. (1982). Interferon-mediated persistent infection of Saint Louis
encephalitis virus in a reptilian cell line. The Journal of General Virology, 61, 177.
Matt, K. S., Moore, M. C., Knapp, R., & Moore, I. T. (1997). Sympathetic mediation of stress
and aggressive competition: plasma catecholamines in free-living male tree lizards.
Physiology & Behavior, 61(5), 639-647.
McFerran, J., Clarke, J., & Connor, T. (1971). The size of some mammalian picornaviruses.
Journal of General Virology, 10(3), 279.
Mitchell, M., & Mader, D. (2006). Salmonella: diagnostic methods for reptiles. Reptile
Medicine and Surgery, 2, 900-905.
Molenaar, G. J. (1992). Anatomy and physiology of infrared sensitivity of snakes. Biology of
the Reptilia, 17, 367-453.
Moore, M. C., Thompson, C. W., & Marler, C. A. (1991). Reciprocal changes in
corticosterone and testosterone levels following acute and chronic handling stress in
the tree lizard, Urosaurus ornatus. General and Comparative Endocrinology, 81(2),
217-226.
Morales, M. H., & Sánchez, E. J. (1996). Changes in vitellogenin expression during
captivity-induced stress in a tropical anole. General and Comparative Endocrinology,
103(2), 209-219.
Munson, L. (2005). Necropsy of wild animals. Wildlife Health Center.
Murray, M. (1996). Pneumonia and normal respiratory function. Reptile Medicine and
Surgery. WB Saunders Co., Philadelphia, Pennsylvania, 396-405.
Musaji, A., Meite, M., Detalle, L., Franquin, S., Cormont, F., Préat, V., et al. (2005).
Enhancement of autoantibody pathogenicity by viral infections in mouse models of
anemia and thrombocytopenia. Autoimmunity Reviews, 4(4), 247-252.
Newman, E. A., & Hartline, P. H. (1982). The infrared vision of snakes. Scientific American,
246(3), 116-127.
Norkin, S. A., Weitzel, R., Campagna-Pinto, D., MacDonald, R. A., & Mallory, G. K. (1960).
“Alcoholic” Hyalin in Human Cirrhosis Histochemical Studies. The American Journal
of Pathology, 37(1), 49.
Norris, D. O. (2007). Vertebrate Endocrinology: Academic Press.
© COP
UPM
150
Nugent, D. (2002). Childhood immune thrombocytopenic purpura. Blood Reviews, 16(1), 27-
29.
Oros, J., Tucker, S., & Jacobson, E. (1998). Inclusion body disease in two captive boas in the
Canary Islands. Veterinary Record, 143(10), 283.
Orrigi, F. C., & (2007). Reptile Immunology. . In E. R. Jacobson (Ed.), In Infectious Diseases
and Pathology of Reptiles (pp. 131-166). New York: Taylor & Francis.
Phair, J., & Palella, F. (2011). Renal disease in HIV-infected individuals. Current Opinion in
HIV and AIDS, 6(4), 285.
Popovic, M., Sarngadharan, M., Read, E., & Gallo, R. C. (1984). Detection, isolation, and
continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS
and pre-AIDS. Science, 224(4648), 497.
Pough, F. H., Andrews, R. M., Cadle, J. E., Crump, M. L., Savitsky, A. H., Wells, K., et al.
(2004). Herpetology: Prentice Hall Upper Saddle River (NJ).
Rand, M. L., & Fraser Wright, J. (1998). Virus-associated idiopathic thrombocytopenic
purpura. Transfusion Science, 19(3), 253-259.
Raymond, J. T., Garner, M. M., Nordhausen, R. W., & Jacobson, E. R. (2001). A disease
resembling inclusion body disease of boid snakes in captive palm vipers (Bothriechis
marchi). Journal of Veterinary Diagnostic Investigation, 13(1), 82.
Reed, L. J., & Muench, H. (1938). A simple method of estimating fifty per cent endpoints.
American Journal of Epidemiology, 27(3), 493-497.
Regenmortel, M. H. V., Fauquet, C. M., Bishop, D. H. L., Carstens, E., Estes, M., Lemon, S.,
et al. (2000). Virus taxonomy: classification and nomenclature of viruses. Seventh
report of the International Committee on Taxonomy of Viruses: Academic Press.
Reid, W., Sadowska, M., Denaro, F., Rao, S., Foulke, J., Hayes, N., et al. (2001). An HIV-1
transgenic rat that develops HIV-related pathology and immunologic dysfunction.
Proceedings of the National Academy of Sciences, 98(16), 9271.
Rhoades, R. E., Tabor-Godwin, J. M., Tsueng, G., & Feuer, R. (2011). Enterovirus infections
of the central nervous system. Virology.
Rivier, C., & Rivest, S. (1991). Effect of stress on the activity of the hypothalamic-pituitary-
gonadal axis: peripheral and central mechanisms. Biology of Reproduction, 45(4),
523-532.
Roingeard, P. (2008). Viral detection by electron microscopy: past, present and future.
Biology of the Cell, 100, 491-501.
Saad, A., & El Deeb, S. (1990). Immunological changes during pregnancy in the viviparous
lizard, Chalcides ocellatus. Veterinary Immunology and Immunopathology, 25(3),
279-286.
© COP
UPM
151
Sandor, T., & Mehdi, A. (1979). Steroids and evolution. Hormones and Evolution
(Barrington EJW, ed). New York: Academic Press, 1-72.
Schaeffer, D. O., & Waters, R. M. (1996). Neuroanatomy and Neurological Diseases of
Reptiles.
Schilliger, L., Selleri, P., & Frye, F. L. (2011). Lymphoblastic lymphoma and leukemic blood
profile in a red-tail boa (Boa constrictor constrictor) with concurrent inclusion body
disease. Journal of Veterinary Diagnostic Investigation, 23(1), 159.
Schultz, C., Temming, P., Bucsky, P., Göpel, W., Strunk, T., & Härtel, C. (2004). Immature
anti inflammatory response in neonates. Clinical & Experimental Immunology,
135(1), 130-136.
Schumacher, I. M., Rostal, D., Yates, R. A., Brown, D. R., Jacobson, E. R., & Klein, P. A.
(1999). Persistence of maternal antibodies against Mycoplasma agassizii in desert
tortoise hatchlings. American Journal of Veterinary Research, 60, 826-831.
Schumacher, J. (2006). Inclusion body disease virus. In D. Mader (Ed.), Reptile Medicine
and Surgery (2nd ed., pp. 836-840). St. Louis: Saunders.
Schumacher, J., Jacobson, E. R., Homer, B. L., & Gaskin, J. M. (1994). Inclusion body
disease in boid snakes. Journal of Zoo and Wildlife Medicine, 511-524.
Selye, H. (1950). The physiology and pathology of exposure to stress.
Semple, J. (2002). Immune pathophysiology of autoimmune thrombocytopenic purpura.
Blood Reviews, 16(1), 9-12.
Sinervo, B., & DeNardo, D. F. (1996). Costs of reproduction in the wild: path analysis of
natural selection and experimental tests of causation. Evolution, 1299-1313.
Sundaram, C., Shankar, S., Thong, W. K., & Pardo-Villamizar, C. A. (2012). Pathology and
Diagnosis of Central Nervous System Infections. Pathology Research International,
2011.
Suttie, A. W. (2006). Histopathology of the spleen. Toxicologic Pathology, 34(5), 466-503.
Swanson, S. K., Mento, S. J., Weeks-Levy, C., Brock, B. D., Kowal, K. J., Wallace, R. E., et
al. (1988). Characterization of Vero cells. Journal of Biological Standardization,
16(4), 311-318.
Thoolen, B., Maronpot, R. R., Harada, T., Nyska, A., Rousseaux, C., Nolte, T., et al. (2010).
Proliferative and nonproliferative lesions of the rat and mouse hepatobiliary system.
Toxicologic Pathology, 38(7 suppl), 5S.
Tokarz, R. R. (1987). Effects of corticosterone treatment on male aggressive behavior in a
lizard (Anolis sagrei). Hormones and Behavior, 21(3), 358-370.
Ujvari, B., & Madsen, T. (2006). Age, parasites, and condition affect humoral immune
response in tropical pythons. Behavioral Ecology, 17(1), 20-24.
© COP
UPM
152
Underwood, G. (1967). A contribution to the classification of snakes: British Museum
(Natural History) London.
Valli, V., & Parry, B. (1993). The hematopoietic system. Pathology of Domestic Animals, 3,
101-265.
Van Regenmortel, M., & Fauquet, C. (2000). Virus taxonomy: classification and
nomenclature of viruses: Seventh Report of the International Committee on
Taxonomy of Viruses.
Vancraeynest, D., Pasmans, F., Martel, A., Chiers, K., Meulemans, G., Mast, J., et al. (2006).
Inclusion body disease in snakes: a review and description of three cases in boa
constrictors in Belgium. Veterinary Record, 158(22), 757.
Varmus, H. E. (1997). Retroviruses: Cold Spring Harbor Laboratory Pr.
Vogt, P. (1997). Historical introduction to the general properties of retroviruses. Retroviruses.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1-25.
Wendling, F., Varlet, P., Charon, M., & Tambourin, P. (1986). MPLV: a retrovirus complex
inducing an acute myeloproliferative leukemic disorder in adult mice. Virology,
149(2), 242-246.
West, G., Garner, M., Raymond, J., Latimer, K. S. & Nordhausen, R. (2001).
Meningoencephalitis in a Bodens python (Morelia boeleni) associated with
paramyxovirus infection. Journal of 'Zoo and Wildlife Medicine, 32, 360-365.
Wever, E. (1978). The reptile ear: its structure and function. Princeton, NJ: Princeton
University Press.
Wilkins, M., Lindley, R., Dourakis, S., & Goldin, R. (1991). Surgical pathology of the liver
in HIV infection. Histopathology, 18(5), 459-464.
Wilson, B. S., & Wingfield, J. C. (1992). Correlation between female reproductive condition
and plasma corticosterone in the lizard Uta stansburiana. Copeia, 691-697.
Wnuk, A. M. (2001). Liver damage in HIV-infected patients. Medical science monitor:
International Medical Journal of Experimental and Clinical Research, 7(4), 729.
Wozniak, E., McBride, J., DeNardo, D., Tarara, R., Wong, V., & Osburn, B. (2000). Isolation
and characterization of an antigenically distinct 68-kd protein from nonviral
intracytoplasmic inclusions in Boa constrictors chronically infected with the inclusion
body disease virus (IBDV: Retroviridae). Veterinary Pathology Online, 37(5), 449.
Wyatt, C. M., Morgello, S., Katz-Malamed, R., Wei, C., Klotman, M. E., Klotman, P. E., et
al. (2008). The spectrum of kidney disease in patients with AIDS in the era of
antiretroviral therapy. Kidney International, 75(4), 428-434.
© COP
UPM
153
Zapata, A., Varas, A., & Torroba, M. (1992). Seasonal variations in the immune system of
lower vertebrates. Immunology Today, 13(4), 142-147.
Ziegel R.F., C. H. F. (1969). Electron microscopic observations on a “C”-type virus in cell
cultures derived from a tumor-bearing viper. Journal of the National Cancer Institute,
43, 1097–1102.
Zimmerman, L., Vogel, L., & Bowden, R. (2010). Understanding the vertebrate immune
system: insights from the reptilian perspective. Journal of Experimental Biology,
213(5), 661.
Zwart, P., Hetzel, U., & Dik, K. (2001). Osteitis deformans and concomitant inclusion body
disease in a boa (boa constrictor). Verhandlungsbericht Erkrankungen der Zootiere,
40, 61-66.