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Parasitological Responses of Onchocerca Volvulus to Ivermectin Treatment and Genetic Analysis of Beta Tubulin Gene

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Parasitological Responses of Onchocerca Volvulus to Ivermectin Treatment and Genetic Analysis of Beta Tubulin Gene University of Ghana http://ugspace.ug.edu.gh PARASITOLOGICAL RESPONSES OF ONCHOCERCA VOLVULUS TO IVERMECTIN TREATMENT AND GENETIC ANALYSIS OF BETA TUBULIN GENE BY GYAN, ERNEST TAWIAH (10363209) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF PHILOSOPHY (M.PHIL) ZOOLOGY DEGREE UNIVERSITY OF GHANA JUNE, 2013 University of Ghana http://ugspace.ug.edu.gh DECLARATION I do hereby declare that all supervised experimental works described in this thesis were carried out by myself with the exception of references made to other people’s works published or not and have all been duly acknowledged. This thesis has never been submitted anywhere else for the award of similar or different degree neither in whole nor in part. ………………………………………….. GYAN, ERNEST TAWIAH (STUDENT) ………………………………………….. DR. MIKE YAW OSEI-ATWENEBOANA (SUPERVISOR) ………………………………………….. DR. FRED ABOAGYE-ANTWI (SUPERVISOR) i University of Ghana http://ugspace.ug.edu.gh DEDICATION TO My dearest mother, Georgina Sarkodie, for her love, care, support and for giving me a reason to go to bed and wake up each day; My late father for challenging me to aspire for a higher education; My wife, Linda Gyan, for her perpetual love, review and support; and My boss, Dr. Mike Yaw Osei-Atweneboana, for being a father in supporting me in all aspects of my life and for mentoring me in the field of medical parasitology and molecular epidemiology. Thanks to you all for giving me a foundation for a brighter future ii University of Ghana http://ugspace.ug.edu.gh ACKNOWLEDGEMENT My undying gratitude and sincere thanks go to my supervisor, Dr. Mike Yaw Osei- Atweneboana of Water Research Institute (WRI) of the Council for Scientific and Industrial Research (CSIR), for his guidance, supervision, support and for receiving logistics from his laboratory and research grant for the entire masters’ programme. I would like to say a big thank you to my supervisor, Dr. Fred Aboagye-Antwi of the Department of Animal Biology and Conservation Science (DABCS) of the University of Ghana, for his guidance and supervision for a successful completion of this research. I am grateful to my colleagues; Kwadwo Kyereme Frempong, Edward Jenner Tettevi, Francis Balungnaa Dhari Veriegh, Buhari Adamu Hamidu and Samuel Armoo for their immense assistance in data acquisition from the field, data entry, laboratory processing of samples, proofreading of my draft thesis and for contributing suggestions during my presentations. My unreserved appreciation goes to Dr. Edward Dwumfour of World Bank, Mr. Isaac Tetteh Dautey (Retired) of Japan Embassy, Dr. Yaw Opoku Ankomah (Retired) and Mr. Kweku Amoako-Atta deGraft-Johnson (Retired) of CSIR-Water Research Institute (WRI) for their advice, motivation and for facilitating my process of being part of the prestigious team of the Medical Parasitology and Molecular Epidemiology Group, headed by Dr. Mike Yaw Osei- Atwenebonana of CSIR-Water Research Institute, that played a great role in this research work. iii University of Ghana http://ugspace.ug.edu.gh I would like to thank the management and staff of the Department of Animal Biology and Conservation Science (DABCS) of the University of Ghana, CSIR-Water Research Institute and Noguchi Memorial Institute for Medical Research (NMIMR) for their administrative support for my research project and the entire masters’ programme. I am grateful to the chiefs, elders, field staff, community volunteers and people of my study communities in the Tain and Kpandai districts in the Brong Ahafo and Northern regions respectively for their cooperation in making this study a success. iv University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS Contents Page DECLARATION ........................................................................................................................ i DEDICATION ........................................................................................................................... ii ACKNOWLEDGEMENT ....................................................................................................... iii TABLE OF CONTENTS ........................................................................................................... v LIST OF FIGURES ............................................................................................................... viii LIST OF TABLES .................................................................................................................... xi LIST OF PLATES .................................................................................................................. xiv LIST OF ABBREVITION ....................................................................................................... xv ABSTRACT ........................................................................................................................... xvii CHAPTER ONE ........................................................................................................................ 1 1.0 INTRODUCTION ....................................................................................................... 1 1.1 GENERAL INTRODUCTION ............................................................................... 1 1.2 JUSTIFICATION .................................................................................................... 6 1.3 OBJECTIVES OF THE STUDY ............................................................................ 7 CHAPTER TWO ....................................................................................................................... 8 2.0 LITERATURE REVIEW ............................................................................................ 8 2.1 Major Control Programmes of Onchocerciasis and Progress Made ........................ 8 2.2 Etiology and Distribution of Onchocerciasis ......................................................... 12 2.3 Onchocerciasis Pathology and Clinical Signs ....................................................... 14 v University of Ghana http://ugspace.ug.edu.gh 2.4 Onchocerciasis Control in Ghana .......................................................................... 17 2.5 Ivermectin, Uses and Genes Involved In Its Selection/Resistance ........................ 20 2.6 O. volvulus Responses and Issues of Sub-optimal Responses/Resistance to Ivermectin ......................................................................................................................... 23 2.7 Beta-Tubulin and its Role in Ivermectin Selection/Resistance ............................. 27 CHAPTER THREE ................................................................................................................. 29 3.0 METHODOLOGY .................................................................................................... 29 3.1 Study Area and Population .................................................................................... 29 3.2 Community Selection and Inclusion Criteria ........................................................ 30 3.3 Sample Size and Sampling Methods ..................................................................... 31 3.4 Study Design.......................................................................................................... 32 3.5 Ethical Consideration ............................................................................................ 32 3.6 Nodulectomies, Nodule Digestion and Adult O. volvulus Isolation...................... 33 3.7 Classification of O. volvulus IVM Response Status .............................................. 33 3.8 DNA Extraction ..................................................................................................... 34 3.9 Amplification of Fragment of ß-Tubulin Gene by Polymerase Chain Reaction (PCR) ……………………………………………………………………………………35 3.10 Agarose Gel Electrophoresis ................................................................................. 35 3.11 Gel extraction Protocol (Quick-Start protocol) ..................................................... 35 3.12 Genetic Diversity of O. volvulus, Indices and Statistical Analysis ....................... 36 CHAPTER FOUR .................................................................................................................... 38 vi University of Ghana http://ugspace.ug.edu.gh 4.0 RESULTS.................................................................................................................. 38 CHAPTER FIVE ..................................................................................................................... 86 5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS ............................ 86 5.1 DISCUSSION ........................................................................................................ 86 5.2 CONCLUSION ..................................................................................................... 95 5.3 RECOMMENDATIONS....................................................................................... 96 REFERENCES ........................................................................................................................ 97 APPENDICES ....................................................................................................................... 114 Appendix 1 ......................................................................................................................... 114 Additional Tables of Results .........................................................................................
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