PATHOBIOLOGY AND PREVENTION OF BUFFALOPOX By Muhammad Numan M.Sc. (Hons.) 99-ag-838 Thesis submitted in partial fulfillment of the requirements for the award of the degree of DOCTOR OF PHILOSOPHY IN PATHOLOGY Department of Pathology Faculty of Veterinary Science University of Agriculture Faisalabad Pakistan (2015)DECLARATION Thereby declare that the contents of the thesis “Pathobiology and prevention of buffalopox” are product of my own research and no part has been copied from any published source (except references, standard mathematical or generic models/ equations/ formulae/ protocols ete.). I further declare that this work has not been submitted for award of any other degree. The university may take action if the information provided is found inaccurate at any stage. (ines of any default scholar will be proceeded against as per HEC plagiarism policy) Muhammad Numan Reg. No. 99-ag-838 PhD Scholar, Department of Pathology, Faculty of Veterinary Science, University of Agriculture, Faisalabad.The Controller of Examinations, University of Agriculture, Faisalabad. We, the supervisory committee, certify that the contents and form of thesis submitted by Mr, ‘Muhammad Numan (99-ag-838) have been found satisfactory and recommend that it be processed for evaluation by the External Examiner (s) for the award of degree. SUPERVISORY COMMITTEE: Chairperson (Dr. Farzana Rizvi) Member (Prof. Dr. Muhammad Tariq Javed) Member (Prof. Dr. Ghulam Muhammad)DEDICATED To MY BELOVED MOTHER All that I am, I owe to my angel mother AFFECTIONATE FATHER The greatest gift I ever have from Allah SWEET SONS & WIFE The most beloved friends of mineACKNOWLEDGEMENTS All praises and humble thanks to ALMIGHTY ALLAH, The Creator of the universe, the most Generous, the most Compassionate whose blessings and cxaltations flourished my thoughts to complete the whole task in the form of this write up. My special praises to Holy Prophet Hazrat Muhammad (PBUH) whose eternal teachings would remain the source of inspirations and guidance for the mankind forever. 1 feel privilege to express my heartfelt appreciation and sincerest gratitude to my research supervisor Dr. Farzana Rizvi, Associate Professor, Department of Pathology, University £ Agriculture, Faisalabad for her keen interest, able guidance, valuable suggestions and providing all possible facilities throughout the course of my studies and research work. I do not have words in my command to thank the members of my supervisory committee Prof. Dr. Muhammad Tariq Javed, Department of Pathology and Prof, Dr. Ghulam Muhammad, Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad for their kind behavior, valuable suggestions and technical guidance due to which this work could be accomplished with success. 1 offer my special and sincere thanks to Prof. Dr. Muhammad Zargham Khan, Department of Pathology, University of Agriculture, Faisalabad for his valuable suggestions and guidance, He facilitated not only my research work but his help is far beyond the contents of this thesis, I deem it my utmost pleasure in expressing my gratitude with the profound benedictions to Prof. Dr. Ahrar Khan, Chairman, Department of Pathology, University of Agriculture, Faisalabad for providing me with strategic command at every step. 1 further extend my special thanks to Dr. Fazal Mahmood, Dr. Muhammad Kashif Salemi, Dr. Alhafiza Shafia Tahseen Gul, Dr. Aisha Khatoon for kind cooperation, encouragement and moral support throughout my research work. | would like to thank my friends Dr. Muhammad Shahbaz Yousaf, Assistant Professor, University of Veterinary and Animal Sciences, Lahore, Dr. Mudasser Habib, Senior Scientist, Nuclear Institute for Agriculture and Biology, Faisalabad, and Dr, Zafarul-Ahsan Qureshi, Additional Principal Veterinary Officer, Veterinary Research Institute, Lahore for their nice contribution during my research work. I would like to appreciate the help extended by PhD & MPhil fellows, Laboratory Technicians, Assistants and Attendants of Department of Pathology. It would be very difficult for me to bear the cost of this project, but financial support of Higher Education Commission (HEC), Pakistan (PIN # 117-8560-AV7-107, SAP # 50018244), and study leave grant from Livestock and Dairy Development Department, Punjab for doing PhD made execution of this research possible and very convenient ‘No acknowledgements could ever adequately express my obligations to my Tectionate and adoring ‘beloved parents, wife, sons, brother and sisters whose hands always rose in prayers for me and without whose ‘moral and financial suppor, the present distinction would have merely been a dream. They always acted as a lighthouse for me in the dark oceans of life path, No words can really express the feelings that [have for them. I apologize if I have caused anger or offence to any body and the errors that remain in the manuscript are mined alone, May ALLAH bless all these people with long, happy and peaceful lives (Ameen).TABLE OF CONTENTS Acknowledgements... Table of contents... List of tables.... List of plate: List of figures List of abbreviations Abstract... CHAPTER NO. | TITLE PAGE No. 1 INTRODUCTION 1 2 REVIEW OF LITERATURE 4 3 MATERIALS AND METHODS 36 4 RESULTS 45 5 DISCUSSION 98 6 SUMMARY 108 REFERENCES 1LIST OF TABLES Table No. Title Page No. 41 Number of buffalo farms visited in different areas of Punjab 46 42 Effect of milker’s fever on BPXV infection in buffaloes 47 43 Effect of milker’s skin lesion on BPXV infection in buffaloes. 48 44 | Effect of other species on BPXV infection in buffaloes 49 Effect of introduction of young animals on BPXV infection in 51 buffaloes . 46 Effect of total no. of animals on BPXV infection in buffaloes 52 47 | Effect of BPXV infection on reluctance in milking in buffaloes 53 48 | Effect of disease on milk yield in BPXV infection in buffaloes 54 49 Effect of septic condition in animals on BPXV infection in ° buffaloes 410 Effect of milking from multiple persons on BPXV infection in 37 57 buffaloes 4.11 | Effect of housing on BPXV infection in buffaloes 58 412 Effect of age on BPXV infection in buffaloes 59 4.13 | Effect of type of herd on BPXV infection in buffaloes 60 414 Effect of area of sampling on BPXV infection in buffaloes 61 is _| Fests influencing significantly in BPXV infection including all @ the variables in buffaloes Factors influencing significantly in BPXV infection after 4.16 65 controlling the effect of age in buffaloes‘Non influencing factors in BPXV infection after controlling the 417 67 effect of age in buffaloes ig ENBetofBPXV infection on hematoTogy of buffaloes in 2 years, | 5-7 years and 8-10 years age groups Number of farms showing positive BPXV infection through PCR 4.19 | in different areas of Punjab 9 gag | ASS OF infectivity of Vero cell culture aitemuated BEXV | inoculated intradermally in the skin of rabbits 421 __ Estimation of viral contents of vaccinal material - Calculation of TCIDs» ffom wild type of virus for challenge dose 4.22 94 preparation 423 Protective efficacy of BPX vaccine in rabbits (on lesion basis) 96LIST OF PLATES Plate No. Title Page No. 4.1 | Different stages of pox lesions on teats of BPXV infected buffalo 69 4.2 | Different stages of pox lesions on teats of BPXV infected buffalo | 70 4.3 | Different stages of BPX lesions in milkers nR qq Photograph of PCR resulis indicating positive BPXV seab | samples 45 Photograph of PCR results indicating positive BPXV stab [ 7 samples 4g | Development oF eytopathic effeets in normal and BPXV infected | ” Vero cells ° 4.7 | Gross pathological changes in rabbits after BPXV inoculation 5 4g | Histopathological changes in rabbit skin after infection with 89 ° BPXV o7 49 Preventable fraction of BPXV vaccineLIST OF FIGURES Figure No. Title Page No. Effect of BPXV infection on TLC & DLC in buffalo of 2-4 year | —_ a age group ° wD Effect of BPXV infection on TLC & DLC in buffalo of 5-7 year | age group in Effect of BPXV infection on TLC & DLC in buffalo of 8-10 year | age groupAbbreviation aa AGID BPX BPXV BV CF CFT CMLV CPE CPX CPXV DMS DNA DPI ds eg. FBS GMEM HA hpi br LIST OF ABBREVIATIONS Full name of abbreviations used in thesis Amino acid Agar gel immunodiffusion Buffalopox Buffalopox virus Bovine vaccinia Conversion factor Complement fixation test ‘Camelpox virus Cytopathic effect ‘Cowpox Cowpox virus Dexamethasone Deoxyribonucleic acid Day post infection Double stranded For example Foetal bovine serum Glasgow's minimal essential medium Hemagglutinin Hour post infection Hour That is