Investigation on Avian leukosis in Broiler parent chickens in Khartoum By Maz Magzob Abd Ellatif ...

87 Pages·2005·0.93 MB·English

Investigation on Avian leukosis in Broiler
parent chickens in Khartoum

By

Maz Magzob Abd Ellatif Mohamed
B.V.Sc. (University of Nyala, 2000)

Supervisor
Dr. Abdelmelik Ibrahim Khalafalla

A thesis submitted to the University of Khartoum in partial fulfillment
of the requirements for the degree of Master of Veterinary Medicine, by
courses and complementary research.

Department of Microbiology
Faculty of Veterinary Medicine
University of Khartoum

October, 2004
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DEDICATION
To my father, mother
brothers and sister
to whom I Love

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Table of contents

DEDICATION…………………………………………………..............................……..I
TABLE OF CONTENTS………………………………………..............................……..II
LIST OF TABLES AND FIGURES ……………………………...................................VII
LIST OF ABBREVIATIONS ……………………………………………………........VIII
AKNOWLEDGEMENT………………………………………………………………….X
ENGLISH ABSTRACT…………………………………………………………............XI
INTRODUCTION ………………………………………………………………………..1
CHAPTER ONE: LITERATURE REVIEW.........………………….............................3
1. Avian oncogenic viruses……………………………………………………………......3
1.1. Taxonomy……….…………………………………………………............................3
1.1.1. Retroviruses ……..…………………………………………………………………3
1.1.2.Herpes virus……...……………………….…………………………………………5
1.2. Morphology….………..…………………………………………………....................5
1.2.1. Retrovirus………..………………………………………………………………….5
1.2.2.Herpes virus……………….……………….……………………………………......5
1.3. Viral genome…….. …………………………………………………………………..6
1.3.1Retrovirus…………………………………………………………………….………6
1.3.2. Herpes virus……………………………………………...........................................7
1.4. Viral stability…...…………………………………………………………….............7
1.4.1. Retrovirus……….………………………………………………………………….7
1.4.2. Herpes virus………..……………………………………………………………….7
1.5. Viral replication………………………………………………………………………8
1.5.1 Retrovirus…………………………………………………….……………...............8
1.5.2. Herpes virus………………………………………………………………...............8
1.6.Epidemiology………………………………………………………………………….9
1.6.1. Distribution…………………………………………………………………………9
1.6.1.1. Retrovirus infection………………………………………………………………9
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1.6.1.2. Herpes virus infection…………………………………………………………...9
1.6.2.Transmission…….………………………………………………………………....10
1.6.2.1. Retrovirus infection….………………………………………………………….10
1.6.2.2. Herpes virus infection……………………………………………………...........10
1.7. Manifestation of the disease………………………………………………………...11
1.7.1. Clinical signs.………………………………………………………………... …...11
1.7.1.1. Retrovirus infection …………………………………………………………….11
1.7.1.1.1. Diseases due to non defective viruses…………………....................................11
1.7.1.1.2. Diseases due to defective viruses …………………….............................. …...12
1.7.1.2. Herpes virus infection .………………………………….....................................13
1.7.2. Pathogenesis ………………………………………………………………………13
1.7.2.1. Retrovirus infection …………………………………………………………….13
1.7.2.2. Herpes virus infection …………………………………………………………..15
1.7.3. Viral susceptibility ………………………………………………………………..17
1.7.3.1. Retrovirus …………………………………………………………………. ……17
1.7.3.2. Herpes virus …………………………………………….....................................17
1.8. Diagnosis…………………………………………………………………….............17
1.8.1. Clinical diagnosis………………………………………………………………….17
1.8.1.1. Retrovirus infection……………………………………………………………..17
1.8.1.2. Herpes virus infection………………………………...........................................18
1.8.2. Histopathology ……………………………………………………………………18
1.8.2.1. Retrovirus infection……………………………………………………………..18
1.8.2.1.1. Lymphoid leukosis…………………………………………………………….19
1.8.2.1.2. Erythroblastosis………………………………………………………………..19
1.8.2.1.3. Myeloblastosis………………………………………………………………...19
1.8.2.1.4. Myelocytomatosis……………………………………………………………..19
1.8.2.1.5. Haemangiomas………………………………………………………………...20
1.8.2.1.6 Renal tumors…………………………………………………………………...20
1.8.2.1.7. Ostopetrosis……………………………………………………………………20
1.8.2.1.8.Connective tissue tumors………………………………………………………20
1.8.2.2. Herpes virus infection …………………………………………………….. ……20
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1.8.3. Viral isolation ……………………………………………………………..............21
1.8.3.1. Cell culture ……………………………………………………………………. .21
1.8.3.1.1. Retrovirus …………………………………………………………………......21
1.8.3.1.2. Herpes virus ………………………………………………………………….21
1.8.3.2. Embryonating egg …………………………………………………………….. 22
1.8.3.2.1. Retrovirus ………………………………………………………………….... 22
1.8.3.2.2. Herpes virus ……………………………………………………………......... 22
1.8.4. Serological diagnosis ………………………………………………………......... 22
1.8.4.1. Retrovirus infection ……………………………………………………….........22
1.8.4.2. Herpes virus infection …………………………………………………………..23
1.8.5. Molecular diagnosis ……………………………………………………………....24
1.8.5.1. Retrovirus infection …………………………………………………………….24
1.8.5.2. Herpes virus infection…………………………………………………………...24
1.9. Management and control ……………………………………………………………25
1.9.1. Retrovirus infection ………………………………………………………………25
1.9.2. Herpes virus infection ……………………………………………………….........26
CHAPTER II: MATERIALS AND METHODS ……………….................................27
2.1. Study area and farm description ……………………………....................................27
2.2. Collection of samples and processing ……………………………………………... 27
2.3. Preparation and sterilization of glass wares ………………….................................. 27
2.4. Preparation and sterilization of plastic wares ……………………………………... 28
2.5. Preparation and sterilization of filters and papers ………………………………… 28
2.6. Histopathology ………………………………………………………………......... 28
2.7. Virus isolation trials……………………………………………….......................... 28
2.7.1. Preparation of samples …………………………………….................................. 28
2.7.2. Fertile eggs …………………………………………………................................ 29
2.7.3. Inoculation on the chorioallantoic membrane …………………………………… 29
2.7.4.Harvest of the chorioallantoic membrane (CAM) ………….................................. 29
2.7.5. Preparation of chicken embryo fibroblast cell culture ……....................................29
2.7.6. Inoculation of chicken embryo fibroblast (CEF) monolayers.................................30
2.8. Polymerase Chain Reaction (PCR) ………………………………………………....31
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2.8.1. DNA extractions ………………………………………………………………….31
2.8.2. Positive controls …………………………………………………………………..31
2.8.2.1. Avian leukosis virus (ALV) ………………………………. ……………………31
2.8.2.2. Reticuloendotheliosis virus (REV) ………………………..................................32
2.8.3. Primer design and PCR conditions………………………………………………..32
2.8.3.1. Reconstitution of primers………………………………………………………..32
2.8.3.2. PCR for Avian leukosis viruses…………………………………………………32
2.8.3.3. PCR for Reticuloendotheliosis virus (REV)…………………………………….34
2.8.4. Analysis of PCR products………………………………………………………....35
2.8.4.1 Gel electrophoresis……………………………………….....................................35
CHAPTERIII: RESULTS………………………………………...................................36
3.1. Morbidity and Mortality Rates………………………………………………………36
3.2. Clinical signs……………………………………………….......................................36
3.3. Post mortem findings ……………………………………………………………….37
3.4. Histopathological findings ………………………………………………………….37
3.5. Viral isolation trials………………………………………………………………….41
3.5.1. Egg inoculation ………………………………………...........................................41
3.5.2. Cell culture………………………………………………………………………...41
3.6. Polymerase Chain Reaction (PCR)………………………………………………….41
3.6.1. PCR with primer pair H5/AD1……………………………………………………41
2.6.2. PCR with primer pair H5/H2…………………………….......................................43
2.6.3. PCR with primer pairH5/H7………………………………………………………44
3.6.3. PCR with primer pair REV-F/REV-R…………………………………………….45
CHAPTER VI: Disscussion……………………………………………………………46
Conclusion and Recommendations……………………………........................................52
References………………………………………………………………………………53
APPENDIXES…………………………………………………………………………...69
1. Preparation of Reagents and Solutions………………………………………………..69
1. 1. Normal Saline (NS)…………………………………………………………………69
1.2. Phosphate buffered saline (PBS)……………………………………………………69
1.3. Preparation of phosphate diluent (PD)………………………………………………70
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1.4. Preparation of Stock Trypsin (2.5%..………………………......................................70
1.5. Preparation of Stock Versa(5%)………………………………………………….....70
1.6. Preparation Trypsin Versin solution………………………………….……………..70
2. Preparation of Media Additives………………………………………………………70
2.1. Preparation of Tryptose phosphate broth (29.5%) ………….....................................70
2.2. Preparation of Lactalbumin hydrolysate (5%) ……………………………………..70
2.3. Preparation of Yeast extracts solution(1%)…………………………………………70
2.4. Preparation of Sodium bicarbonate solution(NaHco3)(7.5%)………………………71
2.5. Bovine serum………………………………………………………………………..71
3. Preparation of antibiotic solutions…………………………………………………….71
4. Mycostatin……………………………………………………………………………..71
5. Preparation and sterilization of media and solutions…………….................................71
5.1. Growth and Maintenance media ……………………………………………………72
6. Preparation of Thioglycolatemedium…………………………………………………72
7. Preparation of Buffers…………………………………………………………………72
7.1. Glycerol (50%)………………………………………………………………………72
7.2. Formaline (10%)…………………………………………………………………….72
8. Preparation of materials for gel electrophoresis………………………………………73
8.1. Agarose 1% …………………………………………………………………………73
8.2. TAE Buffer………………………………………………………………………….73
8.3. Loading dye…………………………………………………………………………73
8.4. 100 bp Ladder (Invitrogen)………………………………………………………….72
8.5. Ethidiumbromide……………………………………………………………………73
ﺔﺣوﺮﻃ ﻻا ﺺﺨﻠﻣ

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List of Tables and Figures

Table1……………………………………………………………………………………34
Table 2……………………………………………….......................................................34
Figure 1………………………………………………......................................................33
Figure 2………………………………………………......................................................36
Figure 3………………………………………………......................................................38
Figure 4………………………………………………......................................................39
Figure 5………………………………………………......................................................40
Figure 6………………………………………………......................................................42
Figure 7…………………………………………………………………………………..43
Figure 8…………………………………………………………………………………..44
Figure 9…………………………………………………………………………………..45

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Abbreviations

ACOLD Arab Company for Livestock Development
AGID Agar Gel Immuno Difusion
ALV Avian Leukosis Virus
ATV Avian Tumor Viruses
C.onc Cellular oncogene
CAM Chorioallantoic membrane
CEF Chick Embryo Fibroblast
CEL Chick Embryo Liver
CIE Counter Immuno Electrophoresis
CK Chick Kidney
CPE Cytopathic effect
DCF Direct Complement Fixation
EAV Endogenous Avian Leukosis Virus
EB Erythroblastosis
ELISA Enzyme Linked Immunosorbent Assay
HTV Herpes Turkey Virus
IFT Immunoflorescent Test
ISH In Situ Hybridization
LL Lymphoid Leukosis
LTR Long Terminal Repeat
MDV Marek׳s Disease Virus
PBS Phosphate Buffer Saline
PCR Polymerase Chain Reaction
PD Phosphate Diluent

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PS Physiological Saline
REV Reticuloendotheliosis Virus
RT-PCR Reverse Transcriptase- Polymerase Chain Reaction
V.onc Viral oncogene

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