View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by KhartoumSpace DETECTION OF ORTHOPOXVIRUS ANTIBODIES IN SERA OF SOME DOMESTIC SPECIES IN THE SUDAN BY Siham Tag Elsir Karam Alla Alkidir (B.V.M.2002, University of Khartoum) Athesis submitted to the University of Khartoum in fulfillment of requirement for the degree of Master of Veterinary Medicine by Research Department of Microbiology Faculty of Veterinary Medicine University of Khartoum (June 2008) DEDICATION To my mother, father Husband, daughters, Sisters, brothers And friends With deepest love Acknowledgments Praise to Allah who gave me the ability and health to accomplish, this research work. My sincere thanks and gratitude to my supervisor Prof. Abdelmalik Ibrahim Khalafalla for his guidance, support and encouragement throughout this work. I am also indebted to Dr.Tag Elsir Mohamed, Central Veterinary Research Laboratory in Soba for his help and provision of some essential materials. My deep thanks to my colleague Dr. Afraa Tagelsir for her help and support. I am extremely grateful to the staff of the Department of Microbiology, Faculty of Veterinary Medicine, University of Khartoum, for providing facilities to conduct this work, special thanks to Mr.Sharani Omer for his technical assistance during the laboratory work, And to Miss. Mawahib Awad for her assistance during this study. My gratitude is also extended to all staff of the Department of Preventive Medicine, namely Dr. Kitma Hassan Elmalek, Dr. Abdelwahid Saeed for their support, and Mr. Hussein Abdelraheem and Miss. Zainab Altayeb for their assistance. Also I want to thank Prof. Ahmed Gameel, Department of pathology, for his Help in histopathology. TABLE OF CONTENT DEDECATION.................................................................................... i ACKNOWLEDGMENT..................................................................... ii TABLE OF CONTENT ..................................................................... iv LIST OF FIGUERS.......................................................................... viii LIST OF TABLES ............................................................................. ix LIST OF ABBREVIATIONS ............................................................. x ARABIC ABESTRACT .................................................................... xi ENGLISH ABESTRACT ................................................................. xii INTRODUCTION............................................................................... 1 CHAPTERI: LITERATURE REVIEW .............................................. 3 1.1 Diseases caused by viruses of the genus orthopoxvirus ................ 3 1.2 Camel pox ..................................................................................... 3 1.2.1 Etiology of CP............................................................................ 3 1.2.2 Importance of the disease........................................................... 4 1.2.3 Epidemiology of CP ................................................................... 4 1.2.4 Clinical symptoms...................................................................... 6 1.2.5 Diagnosis of CP.......................................................................... 7 1.3 Cowpox ......................................................................................... 8 1.3.1 Cause of cow pox ....................................................................... 8 1.3.2 Occurrence ................................................................................. 8 1.3.3 Transmission .............................................................................. 8 1.3.4 Clinical & Pathologic Features................................................... 9 1.3.5 Diagnosis.................................................................................... 9 1.3.6 Prevention ................................................................................ 10 1.3.7 Public health significance......................................................... 10 1.4 Poxviridae ................................................................................... 10 1.5 Taxonomic structure of the family .............................................. 10 1.6 Orthopoxvirus ............................................................................ 11 1.7 Classification............................................................................... 12 1.8 Morphology................................................................................. 13 1.9 Physiochemical and physical properties...................................... 14 1.9. 1 Nucleic acid............................................................................. 14 1.9.2 Lipids ....................................................................................... 14 1.10 Diagnosis of Orthopoxvirus ...................................................... 14 1.11 Vaccinia virus............................................................................ 16 1.12 Basic biology............................................................................. 16 1.13 Host resistance........................................................................... 17 1.14 Origin ........................................................................................ 18 1.15 Use as a vaccine ........................................................................ 18 1.16 History....................................................................................... 19 CHAPTER II: MATERIALS AND METHODS .............................. 20 2.1 Virus strain.................................................................................. 20 2.2 Hyper immune serum .................................................................. 20 2.3 Serum samples ............................................................................ 20 2.4 Preparation and sterilization of glassware ................................... 20 2.5 Preparation of solution and cell culture media ............................ 21 2.5.1 Phosphate buffer saline (PBS) and phosphate diluent (PD) ..... 21 2.5.2 Glasgow Minimum Essential Medium (GMEM) ..................... 21 2.5.3 Tryptose phosphate broth (TPB) .............................................. 21 2.5.4 Lactalbumin hydrolysate .......................................................... 21 2.5.5 1%Yeast extract solution.......................................................... 21 2.5.6 7.5%Sodium bicarbonate solution............................................ 21 2.5.7 Thioglycolate medium.............................................................. 22 2.5.8 Trypsin-Versene solution ......................................................... 22 2.5.9 Penicillin-Streptomycin solution .............................................. 22 2.5.10 Fungizon solution................................................................... 22 2.6 Embryonated Chicken Eggs ........................................................ 22 2.6.1 Chorioallantoic Membrane Inoculation.................................... 23 2.6.2 Harvest of Chorioallantoic Membrane ..................................... 23 2.7 Pock lesion histopathology.......................................................... 24 2.8 Preparation of Cell Culture.......................................................... 24 2.9 Virus propagation........................................................................ 25 2.10 Virus titration ............................................................................ 25 2.11 Agar Gel preparation................................................................. 25 2.12 Agar gel immunodiffusion test.................................................. 25 2.12.1 Preparation of agar gel ........................................................... 25 2.12.2 Standardization of AGID test ................................................. 25 2.12.3 Examination of the sera for antibodies against Orthopox viruses using AGID test……………… …. ………………………………...26 CHAPTER III: RESULT .................................................................. 27 3.1Growth of Vaccinia virus on the chorioallantoic membrane ........ 27 3.2 Histopathology of the CAM0 ...................................................... 27 3.3 Growth of vaccinia virus in Cell Culture..................................... 27 3.4 Sero-prevalence of orthopox virus in Sheep, cattle and camel serum samples ............................................................................. 27 CHAPTER IV: DISCUSSION.......................................................... 35 CONCLUSIONS............................................................................... 40 REFERENCES.................................................................................. 41 APPENDICES................................................................................... 52 LIST OF FIGURES Figure 1 Pock lesions produced by Vaccinia virus on CAM of embryonated eggs.................................................................................31 Figure 2 CAM section showing swollen of the cells with pale cytoplasm and some inclusions bodies……………………………………………32 Figure 3 CAM section showing foci of epithelial thickening…….…...33 Figure 4 Vero cell culture infected with Vaccinia Virus showing rounding and plaque formation ………………………………………34 Figure 5 Precipitation line in AGID test for detection of antibodies against orthopox viruses……….………………………………..…….35 LIST OF TABLES Table 1 Testing sera collected from cattle, sheep and