INVESTIGATION OF TICK-BORNE PATHOGENS RESISTANCE MARKERS USING NEXT GENERATION SEQUENCING RESEARCH DISSERTATION for MSc degree in Life Sciences Submitted to the DEPARTMENT OF LIFE AND CONSUMER SCIENCES UNIVERSITY OF SOUTH AFRICA by AUBREY D CHIGWADA Student No: 61366943 SUPERVISOR: DR TM MASEBE CO-SUPERVISOR: DR NO MAPHOLI JULY 2021 i DECLARATION Name: Aubrey Dickson Chigwada Student number: 61366943 Degree: Master of Science in Life Sciences Dissertation title: INVESTIGATION OF TICK-BORNE PATHOGENS RESISTANCE MARKERS USING NEXT GENERATION SEQUENCING I, Aubrey D Chigwada, declare that investigation of tick-borne pathogens resistance markers using next-generation sequencing is my work, and sources I have used have been acknowledged by complete references. I submit this thesis for a Master of Science in life science at the college of agriculture and environmental science at the University of South Africa and have not been submitted to any other university. JULY 2021 -------------------------------- --------------------------- Aubrey Dickson Chigwada Date ii ACKNOWLEDGEMENTS Firstly, I would like to thank God almighty for the strength to carry on and a gift of life. Words cannot express my deepest gratitude to my supervisor Dr. TM Masebe for her professional guidance, encouragement, support, corrections, instructions, patience, and technical skills thought- out this work. I am forever indebted to your mentorship; you are a role model and an inspiration to me. I would like to extend my sincere appreciation to my Co-supervisor Dr. NO Mapholi for her insightful comments and suggestions. To the woman in research funds used for this project, I am grateful. Much gratitude to Dr. Ramganesh Selvarajan for his mentorship and training from the basics to the actual Miseq sequencing, which made it possible to sequence for this work. Would like to thank Dr. Ogola for taking his time to run data and teaching me how to analyze, use algorithms, and interpret the results. Your input shaped my laboratory techniques and data analysis skills. Thank you for the time and sacrifice you made, for this, I will forever be grateful. Additionally, I would like to thank our research team members ‘TICK GROUP’ Dr. Mbizeni Sikhumbuzo, Dr. Tambani Tshifhiwa. and Ms. Moholola Nthabiseng for their helpful contributions. Thank you to Kgomotso Chokoe, Itumeleng Setshedi, and Sharon Lerooibaaki laboratory technicians for their warm accommodation and support in the laboratory. My gratitude also goes out to my family and friends for their encouragement and support all through my studies. iii TABLE OF CONTENTS DECLARATION ..................................................................................................................................................................... ii ACKNOWLEDGEMENTS ..................................................................................................................................................... iii TABLE OF CONTENTS ........................................................................................................................................................ iv TABLE OF FIGURES ........................................................................................................................................................... vii LIST OF TABLES ................................................................................................................................................................. xii LIST OF ACRONYMS ......................................................................................................................................................... xiii CHAPTER 1 ............................................................................................................................................................................ 1 1.1 GENERAL INTRODUCTION ...................................................................................................................................... 1 1.2 RATIONALE OF THE STUDY .................................................................................................................................... 4 1.3 HYPOTHESIS .............................................................................................................................................................. 6 1.5 AIMS AND OBJECTIVE.............................................................................................................................................. 6 1.5.1 THE SPECIFIC OBJECTIVES: .................................................................................................................................. 6 CHAPTER 2 LITERATURE REVIEW .................................................................................................................................... 7 2.1 Ticks of veterinary and medical importance ................................................................................................................... 8 2.1.1 Amblyomma species ............................................................................................................................................... 8 2.1.2 Argasidae species ................................................................................................................................................... 9 2.1.3 Hyalomma species .................................................................................................................................................. 9 2.1.4 Rhipicephalus species ............................................................................................................................................. 9 2.1.5 Ixodes species ....................................................................................................................................................... 10 2.2 The life cycle of ticks ................................................................................................................................................... 11 2.3 Tick-borne diseases of cattle ......................................................................................................................................... 13 2.3.1 Bacterial tick-borne pathogens ............................................................................................................................... 13 2.4 Control and prevention ................................................................................................................................................. 16 2.5 Treatment of tick-borne bacterial diseases ..................................................................................................................... 17 2.6 Resistance development of bacteria to antimicrobial agents ........................................................................................... 17 2.6.1 Mechanisms of action of antibiotics and resistance development. ............................................................................ 17 2.7 Diagnostic techniques for tick-borne diseases ................................................................................................................ 23 2.8 Molecular diversity and functional resistance biomarkers in bacterial communities ........................................................ 24 2.9 SUMMARY................................................................................................................................................................. 25 CHAPTER 3 BACTERIAL COMMUNITIES AND THEIR FUNCTIONAL ANTIBIOTIC RESISTANT MARKERS IN AMBLYOMMA AND HYALOMMA TICKS. ............................................................................................................................ 26 iv 3.1 ABSTRACT ................................................................................................................................................................ 27 3.2 INTRODUCTION ........................................................................................................................................................ 29 3.3 METHODOLOGY ....................................................................................................................................................... 32 3.3.1 Ethics statement .................................................................................................................................................... 32 3.3.2 Sampling site ........................................................................................................................................................ 33 3.3.3 Tick samples collection and processing .................................................................................................................. 34 3.3.4 Tick lysis and DNA extraction ............................................................................................................................... 34 3.3.5 Library sample preparation for 16S rRNA metagenomic sequencing ....................................................................... 35 3.3.6 Library sequencing ................................................................................................................................................ 37 3.3.7 Sequences analysis of 16S rRNA bacterial data ...................................................................................................... 37 3.3.8 Functional resistance biomarker analysis. ..............................................................................................................