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Phenotypic and Molecular Characterization of Hard Ticks

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Phenotypic and Molecular Characterization of Hard Ticks i Phenotypic and Molecular Characterization of Hard Ticks (Acari: Ixodidae) Sampled from Wild Herbivores from Lake Nakuru and Tsavo National Parks in Kenya Muruthi Wanjira Carolyn Reg. 156/25663/2011 A Thesis submitted in partial fulfilment of the requirement for the award of the degree of Master of Science (Medical Biochemistry) in the School of Pure and Applied Sciences of Kenyatta University September 2015 ii DECLARATION I declare that this thesis is my original work and has not been presented for a degree in any other university for any other award. Signature..................................... Date......................................................... We confirm that the work reported here was carried out by the candidate under our supervision. Dr. Joseph N. Makumi Department of Biochemistry and Biotechnology Kenyatta University Signature..................................... Date.................................................... Dr. Steven M. Runo Department of Biochemistry and Biotechnology Kenyatta University Signature..................................... Date........................................................ iii DEDICATION This thesis is dedicated with love and respect to my loving dad, the late Benson Muruthi, Mum Susan Muruthi, Sister Zipporah Muruthi, Brother Christopher Muruthi, and Nephew Mark Muruthi and the late Dr. J.N Makumi for their continued encouragement, patience, love and huge support during the time I was carrying out the project. iv ACKNOWLEDGEMENT I take this opportunity to thank my supervisors the late Dr J. N. Makumi and Dr. Steven Runo for their advice, encouragement and contribution to this project. They have tirelessly supported and followed me from the beginning to the end of this work. I also thank Mr. Moses Otiende and Mr. Vincent Obanda (Kenya Wildlife Services) who co-ordinated my laboratory work. I express my sincere gratitude to Dr. Olivia Wesula Lwande (ICIPE) who introduced me to tick identification techniques and Mutura Kamawe (Institute of Primate Research) who introduced me to molecular techniques. My special thanks to personnel at the Veterinary Services Department Kenya Wildlife Service who introduced me to molecular techniques and assisted me in various activities in the laboratory. This includes, Ms Asenath Owour, Ms. Wambui Maina, Anthony Kipruto Busienei and Samuel Muthemba who provided support while working in the laboratory. My deepest appreciation goes to my late dad, Benson Muruthi who was always proud of me and was looking forward to the completion of the project. Mum, Susan Muruthi who has been my inspiration and has supported me in pursuing my dreams. My special thanks to my brother, Christopher Muruthi, sister, Zipporah Muruthi and nephew, Mark Muruthi for their special support and love throughout the project. v TABLES OF CONTENTS DECLARATION ..................................................................................................... i DEDICATION ....................................................................................................... iii ACKNOWLEDGEMENT ..................................................................................... iv TABLES OF CONTENTS ..................................................................................... v LIST OF TABLES ................................................................................................ vii LIST OF FIGURES ............................................................................................. viii LIST OF PLATES ................................................................................................. ix ABBREVIATIONS AND ACRONYMNS ............................................................ x ABSTRACT ........................................................................................................... xi CHAPTER ONE ..................................................................................................... 1 INTRODUCTION .................................................................................................. 1 1.1 Background ................................................................................................... 1 1.2 Problem Statement and Justification ............................................................. 3 1.3 Hypothesis ..................................................................................................... 4 1.4 Objectives ...................................................................................................... 4 1.4.1 General Objective ................................................................................... 4 1.4.2 Specific Objectives ................................................................................. 4 CHAPTER TWO .................................................................................................... 5 LITERATURE REVIEW ....................................................................................... 5 2.1 Systematics of ticks ....................................................................................... 5 2.1.1 Morphology of Hard ticks ...................................................................... 6 2.2 Biology of Ticks ............................................................................................ 7 2.3 Economic importance of ticks ....................................................................... 9 2.3.1 Impact on the livestock Industry ............................................................ 9 2.3.2 Impact on human beings ....................................................................... 11 2.4 Wildlife ticks ............................................................................................... 13 2.5 Distribution of ticks in Kenya ..................................................................... 15 2.6 Tick identification ....................................................................................... 16 vi CHAPTER THREE .............................................................................................. 20 MATERIAL AND METHODS ............................................................................ 20 3.1 Study design and sample size ...................................................................... 20 3.2 Study sites ................................................................................................... 21 3.3 Morphological identification of Ticks ......................................................... 21 3.4 Molecular analyses of tick ........................................................................... 22 3.4.1 DNA Isolation....................................................................................... 22 3.4.2 Polymerase Chain Reaction .................................................................. 23 3.4.3 Electrophoresis and purification of PCR products ............................... 23 3.4.4 Sequencing and analysis ....................................................................... 24 3.5 Congruency test ........................................................................................... 24 CHAPTER FOUR ................................................................................................. 25 RESULTS ............................................................................................................. 25 4.1 Morphological identification ....................................................................... 25 4.1.1 Unidentified ticks ................................................................................. 35 4.2 Identification of Ticks using PCR and gel electrophoresis ......................... 36 4.1.2 Phylogenetic analysis ........................................................................... 37 4.3 Morphological and molecular characterization of tick samples .................. 46 CHAPTER FIVE .................................................................................................. 48 DISCUSSION, CONCLUSIONS AND RECOMMENDATIONS ...................... 48 5.1 Discussion ................................................................................................... 48 5.2 Conclusions ................................................................................................. 53 5.3 Recommendations ....................................................................................... 54 5.4 Further Studies ............................................................................................ 54 REFERENCES ..................................................................................................... 55 APPENDICES ...................................................................................................... 62 APPENDIX 1 .................................................................................................... 62 APPENDIX 2 .................................................................................................... 63 vii LIST OF TABLES Table 4.1: Ticks identified by morphological keys in genus Amblyomma........26 Table 4.2: Ticks identified by morphological keys in genus Rhipicephalus......26 Table 4.3: Ticks identified by morphological keys in genus Hyalomma...........26 Table 4.4: Ticks identified by morphological keys in genus Dermacentor........26 Table 4.5: Different sizes of ITS2 as depicted in the gel electrophoresis ……..38 Table 4.6: Morphological and Phylogenetic Identification...................................46 viii LIST OF FIGURES Figure 4.1: GenBank blast search for R. pulchellus haplotype………………40
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