TThheessiiss RReeff.. NNoo.. ________________________________ DDDEEETTTEEERRREEEMMMIIINNNAAATTTIIIOOONNN OOOFFF OOOPPPTTTIIIMMMAAALLL TTTIIIMMMEEE OOOFFF VVVAAACCCCCCIIINNNAAATTTIIIOOONNN AAAGGGAAAIIINNNSSSTTT IIINNNFFFEEECCCTTTIIIOOOUUUSSS BBBUUURRRSSSAAALLL DDDIIISSSEEEAAASSSEEE (((GGGUUUMMMBBBOOORRROOO))) AAANNNDDD MMMOOOLLLEEECCCUUULLLAAARRR DDDIIIAAAGGGNNNOOOSSSIIISSS OOFF CCLLIINNIICCAALL CCAASSEESS IINN CCEENNTTRRAALL EETTHHIIOOPPIIAA MVSc Thesis BY SAMSON TEREFE KASSA ADDIS ABABA UUUNNNIIIVVVEEERRRSSSIIITTTYYY,,, CCCOOOLLLLLLEEEGGGEEE OOOFFF VVVEEETTTEEERRRIIINNNAAARRRYYY MMMEEEDDDIIICCCIIINNNEEE AAANNNDDD AAAGGGRRRIIICCCUUULLLTTTUUURRREEE,,, DDDEEEPPPAAARRRTTTMMMEEENNNTTT OOOFFF CCCLLLIIINNNIIICCCAAALLL SSSTTTUUUDDDIIIEEESSS,,, MMMVVVSSSccc PPPRRROOOGGGRRRAAAMMM IIINNN VVEETTEERRIINNAARRYY EEPPIIDDEEMMIIOOLLOOGGYY JUNE, 2017 BBIISSHHOOFFTTUU,, EETTHHIIOOPPIIAA DDDEEETTTEEERRREEEMMMIIINNNAAATTTIIIOOONNN OOOFFF OOOPPPTTTIIIMMMAAALLL TTTIIIMMMEEE OOOFFF VVVAAACCCCCCIIINNNAAATTTIIIOOONNN AAAGGGAAAIIINNNSSSTTT IIINNNFFFEEECCCTTTIIIOOOUUUSSS BBUURRSSAALL DDIISSEEAASSEE ((GGUUMMBBOORROO)) AANNDD MMOOLLEECCUULLAARR DDIIAAGGNNOOSSIISS OOFF CCLLIINNIICCAALL CCAASSEESS IINN CCEENNTTRRAALL EETTHHIIOOPPIIAA AAA ttthhheeesssiiisss sssuuubbbmmmiiitttttteeeddd tttooo ttthhheee CCCooolllllleeegggeee ooofff VVVeeettteeerrriiinnnaaarrryyy MMMeeedddiiiccciiinnneee aaannnddd AAAgggrrriiicccuuullltttuuurrreee ooofff AAAddddddiiisss AAAbbbaaabbbaaa UUUnnniiivvveeerrrsssiiitttyyy iiinnn pppaaarrrtttiiiaaalll fffuuulllfffiiillllllmmmeeennnttt ooofff ttthhheee rrreeeqqquuuiiirrreeemmmeeennntttsss fffooorrr MMMaaasssttteeerrr'''sss DDDeeegggrrreeeeee in Veterinary Epidemiology By Samson Terefe Kassa June, 2017 BBiisshhooffttuu,, EEtthhiiooppiiaa Addis Ababa University College of Veterinary Medicine and Agriculture Department of Veterinary Epidemiology __________________________________________________________________________ As members of the Examining Board of the final MVSc open defense, we certify that we have read and evaluated the thesis prepared by Samson Terefe Kassa titled: ‘DETEREMINATION OF OPTIMAL TIME OF VACCINATION AGAINST INFECTIOUS BURSAL DISEASE (GUMBORO) AND MOLECULAR DIAGNOSIS OF CLINICAL CASES IN CENTRAL ETHIOPIA’, and recommend that it be accepted as fulfilling the thesis requirement for the Master's Degree in Veterinary Epidemiology. _______________ _______________ Chairperson Signature Date _______________ ________________ External Examiner Signature Date _______________ _______________ Internal Examiner Signature Date Dr. Teshale Sori (Assoc.Prof.) _______________ _______________ Major advisor Signature Date Dr. Fufa Abunna (Assoc.Prof.) _______________ _______________ Co-advisor Signature Date I hereby certify that I have read the revised version of this thesis prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. Dr. Teshale Sori (Assoc.Prof.) _______________ _______________ Major advisor Signature Date Dr. Fufa Abunna (Assoc.Prof.) _______________ _______________ Co-advisor and Department chairperson Signature Date BIOGRAPHICAL SKETCH I, the author of this MVSc Thesis, was born in December 1986 in Nazareth, Ethiopia. I attended Elementary, Junior Secondary school at Merti Junior school and my High School Education at Arbegnoch Senior Secondary School. After a successful completion of my High School Education, I had joined Addis Ababa University and graduated with DVM degree in 2011. Soon after graduation, I was employed by the Agricultural Technical and Vocational Educational Training (ATVET) Department of the Ministry of Agriculture and Rural Development to teach at Agarfa ATVET College as Instructor. Then after, I was employed by Elfora agro-industries as a Poultry Health Manager. I was then employed by Ethiopian Institute of Agricultural Research as a Veterinary Researcher where I am still working. After 2015, I have joined the school of graduate studies of College of Veterinary Medicine and Agriculture of Addis Ababa University to pursue Master's Degree in Veterinary Epidemiology. STATEMENT OF THE AUTHOR I first, declare that this dissertation is my bonafide work and that all sources of materials used for this dissertation have been duly acknowledged. This dissertation has been submitted to the requirements for MVSc degree at Addis Ababa University, College of Veterinary Medicine and Agriculture and is deposited at the University’s Library to be made available to borrowers under rules of the Library. I solemnly declare that this dissertation is not submitted to any other institution anywhere for the award of any academic degree, diploma, or certificate. Brief quotations from this dissertation are allowable without special permission provided that accurate acknowledgment of source is made. Requests for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the School of Graduate Studies when in his judgment the proposed use of the material is in the interest of scholarship. In all other instances, however, permission must be obtained from the author. Name: Samson Terefe Kassa Signature: ______________ College of Veterinary Medicine and Agriculture Date of Submission: ____________________ TABLE OF CONTENTS PAGES ACKNOWLEDGEMENTS .................................................................................................... iii LIST OF ABBREVIATIONS ................................................................................................ iii LIST OF TABLES .................................................................................................................. vi LIST OF FIGURES ............................................................................................................... vii LIST OF ANNEXS ................................................................................................................ viii ABSTRACT ............................................................................................................................. ix 1. INTRODUCTION ................................................................................................................ 1 2. LITERATURE REVIEW .................................................................................................... 5 2.1. History of IBD ................................................................................................................ 5 2.2. Disease Definition ........................................................................................................... 6 2.3. Etiology ........................................................................................................................... 7 2.3.1. Taxonomy of the Virus .............................................................................................. 8 2.3.2. Morphology of the virus, structural protein and functions of IBDV proteins .......... 9 2.4. Epidemiology of the virus .............................................................................................. 9 2.4.1. Host range................................................................................................................. 9 2.4.2. Physico-chemical nature of the virus...................................................................... 10 2.4.3. Route excretion and transmission of the virus ........................................................ 10 2.4.4. Molecular epidemiology and field evolution .......................................................... 11 2.4.5. Antigenic variation of IBDV strains ....................................................................... 12 2.4.6. IBDV reassortment, recombination and reversion to virulence ............................. 13 2.5. Immune response of IBD ............................................................................................. 14 2.5.1. Innate immunity ...................................................................................................... 14 2.5.2. Humoral immunity and cellular immunity .............................................................. 15 2.5.3. Immunosuppression mechanism of IBD ................................................................. 16 2.6. Pathogenesis and pathological lesions ........................................................................ 16 2.7. Clinical findings of the disease .................................................................................... 21 2.8. Laboratory Diagnosis .................................................................................................. 22 2.8.1. Serological diagnosis .............................................................................................. 22 2.8.2. Virological diagnosis .............................................................................................. 23 2.8.3. Molecular characterization .................................................................................... 24 i 2.9. Treatment, prevention and control ............................................................................ 25 2.9.1. Treatment ................................................................................................................ 25 2.9.2. Prevention and Control .......................................................................................... 25 2.9.3. Vaccination failures and potential causes .............................................................. 28 2.10. Economic Importance of IBD ................................................................................... 34 2.11. The status of IBD in Ethiopia .................................................................................... 35 3. MATERIALS AND METHODS ...................................................................................... 37 3.1. Study Areas ................................................................................................................... 37 3.2. Study Population .......................................................................................................... 38 3.3. Study Design and Sampling Method .......................................................................... 38 3.4. Study Methodology ...................................................................................................... 39 3.4.1. Observation of clinical manifestation and post mortem examination .................... 39 3.4.2. Sample collection, transportation and preparation ................................................ 39 3.4.3. Molecular Analysis of the Samples ......................................................................... 41 3.4.4. On-farm Experimental Study and Determination of Antibody Titres ..................... 45 3.5. Data Management and Statistical Analysis ............................................................... 48 4. RESULTS ........................................................................................................................... 49 4.1. Clinical and Postmortem findings .............................................................................. 49 4.2. Results of Molecular Diagnosis ................................................................................... 50 4.3. Results of Experimental study .................................................................................... 51 5. DISCUSSION ..................................................................................................................... 53 6. CONCLUSION AND RECOMMENDATIONS ............................................................. 58 7. REFERENCES ................................................................................................................... 59 8. ANNEXS ............................................................................................................................757 ii ACKNOWLEDGEMENTS Above all, I praise the Merciful God Almighty through Jesus Christ, the only source of power and hope to my soul, who is guiding me in all aspects of my life. Hallelujah! Secondly, I sincerely acknowledge my academic advisor Dr. Teshale Sori for his time and patience to analyze the results, thorough editing and organizing this paper to be more informative and always ready to helped me through providing scientific suggestions for the completion of this work, and my co-advisor Dr. Fufa Abunna for his unreserved advice and his encouraging word 'Very good' for any effort I have done. My appreciation also goes to Dr. Berecha Bayissa (NVI) for giving me valuable comments and whenever we met, always interested to made our discussion epidemiological. Thirdly, I would like to thank Shok Singh for the provision of all poultry equipments and his assistance in feeding, watering, vaccinating and collecting blood samples in the whole period of the experiment and equally interested on the experiment. Without him this experiment work would have not come to its completion. I am also grateful to Addis Ababa University and Ethiopian Institute of Agricultural Research for partial funding the cost of the study and Debre Zeit Agricultural Research Center. My special thanks goes to Dr. Getnet Asefa and Dr. Solomon Chanyalew for facilitating the financial and technical activities. And to the National Veterinary Institute Research and Diagnostic Department for their permission to use their laboratory facilities during serology and molecular analysis of the samples. Finally, words fail to express my gratitude and love to my family, for their fervent prayers, tremendous support and encouragement throughout my life. iii LIST OF ABBREVIATIONS AA Amino acid Ab Antibody I-ELISA Indirect Enzyme-linked Immunosorbent assay AGID Agar gel immunodiffusion test Bp Base pair BF Bursa of Fabricius BALT Bronchial-associated lymphoid tissues CALT Conjunctiva-associated lymphoid tissues CAM Chorioallantoic membrane CEB Chicken embryo bursa CEF Chicken embryo fibroblasts CEKC Chicken embryo kidney cell CMI Cell mediated immunity CPE Cytopathic effect CSA Central statistical agency DFA Direct Fluorescent-Antibody Assay ELISA Enzyme linked Immunosorbent Assay FAO Food and agriculture organization GALT Gut-associated lymphoid tissues GC Germinal centre IBD Infectious Bursal disease IBDV Infectious Bursal disease virus Ig Immunoglobulin IFA Immunofluorescence Assay IFN Interferon IIF Indirect immunofluorescence IL Interleukin ILTV Infectious laryngo tracheitis virus MDAB Maternally derived antibodies iv MHC Major Histocompatibility complex class NAHDIC National Animal Health Diagnostic Investigation Center NCD Newcastle disease NVI National Veterinary Institute OD Optical density OIE International Animal Health Organization PCR Polymerase Chain Reaction PI Post infection qRT-PCR Quantitative real time RT-PCR RE Restriction enzymes RNA Ribonucleic acid IBD RFLP Restriction fragment length polymorphism. RPM Rotation per minute RT-PCR Reverse transcription polymerase chain reaction SAN Specific antibody negative S/P Sample positive SPF Specific pathogens free chickens SsRNA Single stranded RNA TCID50 Tissue culture infective dose 50 USA United States of America VIBD Virulent IBDV VNT Virus Neutralization Test VP Virus Protein VP2 Virus Protein2 VP1-VP4 Virus Protein1-4 VvIBDV Very Virulent Infectious bursal disease virus WRL World Referral Laboratory v