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Exploration of Tick-Borne Pathogens and Microbiota of Dog Ticks Collected at Potchefstroom Animal Welfare Society

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Exploration of Tick-Borne Pathogens and Microbiota of Dog Ticks Collected at Potchefstroom Animal Welfare Society Exploration of tick-borne pathogens and microbiota of dog ticks collected at Potchefstroom Animal Welfare Society C Van Wyk orcid.org 0000-0002-5971-4396 Dissertation submitted in fulfilment of the requirements for the degree Master of Science in Environmental Sciences at the North-West University Supervisor: Prof MMO Thekisoe Co-supervisor: Ms K Mtshali Graduation May 2019 24263524 DEDICATION This thesis is dedicated to the late Nettie Coetzee. For her inspiration and lessons to overcome any obstacle that life may present. God called home another angel we all love and miss you. “We are the scientists, trying to make sense of the stars inside us.” -Christopher Poindexter i ACKNOWLEDGEMENTS My sincerest appreciation goes out to my supervisor, Prof. Oriel M.M. Thekisoe, for his support, motivation, guidance, and insightfulness during the duration of this project and been there every step of the way. I would also like to thank my co-supervisor, Ms. Khethiwe Mtshali, for her patience and insightfulness towards the corrections of this thesis. I would like to thank Dr. Stalone Terera and the staff members at PAWS for their aid towards the collection of tick specimens. For the sequencing on the Illumina MiSeq platform and metagenomic data analysis I would like to thank Dr. Moeti O. Taioe, Dr. Charlotte M.S. Mienie, Dr. Danie C. La Grange, and Dr. Marlin J. Mert. I would like to thank the National Research Foundation (NRF) for their financial support by awarding me the S&F- Innovation Masters Scholarship and the North-West University (NWU) for the use of their laboratories. To my friends and fellow students Terese, Jani, Bridget, Sanchez, Anna, Malitaba, Thankgod, Setjhaba, Diseko, Spha, and Innocentia thank you all for your support during this study. A special thanks to Thomas, Tessa, Rohan, Vivienne, and Bren for your support, motivation, guidance, and especially patience from the beginning when I met you guys up to now. ii My appreciation and thanks go out to my parents, Anita and Willie, my brother, Henry, and grandma, Elisabeth, for their support during the duration of my studies, believing in me every step of the way, and guiding me in the right direction when things seem impossible. My greatest appreciation goes out to God for granting me every opportunity in life and proving that anything is possible when you do it through Christ. As declared in Jeremiah 29:11, “For I know the plans I have for you, declares the LORD, plans to prosper you and not harm you, plans to give you hope and a future.” iii TABLE OF CONTENTS DEDICATION ............................................................................................................................. I ACKNOWLEDGEMENTS ......................................................................................................... II TABLE OF CONTENTS ........................................................................................................... IV LIST OF FIGURES .................................................................................................................... X LIST OF TABLES .................................................................................................................. XIII LIST OF PLATES ................................................................................................................... XV ABBREVIATIONS .................................................................................................................. XVI RESEARCH OUTPUTS ....................................................................................................... XVIII ABSTRACT ........................................................................................................................... XIX CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW ................................................ 21 1.1 Background .................................................................................................... 21 1.2 Ixodid ticks ...................................................................................................... 21 1.2.1 Systematics ...................................................................................................... 21 1.2.2 Morphology ....................................................................................................... 25 1.2.3 Biology and ecology ......................................................................................... 26 1.2.3.1 Host finding strategies ...................................................................................... 26 1.2.3.2 Attachment and blood feeding .......................................................................... 27 1.2.3.3 Developmental stages ...................................................................................... 28 1.2.4 Geographical distribution .................................................................................. 29 1.2.5 Tick control measures ....................................................................................... 29 1.2.6 Molecular identification of tick species .............................................................. 30 1.2.7 Influence of ticks on humans, livestock and companion animals ....................... 32 iv 1.2.8 Ticks infesting dogs in South Africa .................................................................. 32 1.2.8.1 Haemaphysalis elliptica Koch, 1844 ................................................................. 32 1.2.8.2 Rhipicephalus sanguineus Latreille, 1806 ......................................................... 33 1.3 Role of ticks as vectors of tick-borne diseases ........................................... 34 1.3.1 Transmission of bacteria from ticks to hosts ..................................................... 34 1.3.2 Transmission of pathogens from ticks to humans ............................................. 35 1.3.3 Epidemiology of tick-borne pathogens .............................................................. 36 1.3.4 Pathogens transmitted by ticks infesting dogs in South Africa .......................... 37 1.3.4.1 Anaplasma phagocytophilum ............................................................................ 40 1.3.4.2 Ehrlichia canis .................................................................................................. 42 1.3.4.3 Babesiosis species ........................................................................................... 45 1.3.4.4 Rickettsioses species ....................................................................................... 49 1.3.4.5 Coxiella burnetii ................................................................................................ 51 1.4 Microbiota of ticks .......................................................................................... 54 1.4.1 Symbiotic bacteria of ticks ................................................................................ 56 1.4.2 Functional role of tick microbiome ..................................................................... 56 1.4.3 Metagenomics .................................................................................................. 57 1.4.4 Next-generation sequencing technology ........................................................... 58 1.4.5 Illumina MiSeq sequencing platform and microbiome analysis ......................... 59 CHAPTER 2: JUSTIFICATION OF THE STUDY, AIM AND OBJECTIVES ............................ 61 2.1 Justification of the study ............................................................................... 61 2.2 Aim .................................................................................................................. 62 2.3 Objectives ....................................................................................................... 63 v 2.4 Hypothesis ...................................................................................................... 63 2.5 Dissertation outline ........................................................................................ 63 CHAPTER 3: MATERIALS AND METHODS .......................................................................... 65 3.1 Study area ....................................................................................................... 65 3.1.1 North-West Province......................................................................................... 65 3.1.2 Origin of dogs within the study area .................................................................. 68 3.2 Sample collection ........................................................................................... 70 3.3 Morphological identification of ticks ............................................................. 71 3.4 Molecular analysis of ticks ............................................................................ 73 3.4.1 DNA extraction ................................................................................................. 73 3.4.2 Polymerase chain reaction................................................................................ 73 3.4.3 Purification and sequencing of amplicons ......................................................... 76 3.5 Morphological identification of tick-borne pathogens ................................. 76 3.6 Molecular detection of tick-borne pathogens ............................................... 77 3.6.1 DNA extraction ................................................................................................. 77 3.6.2 Polymerase chain reaction...............................................................................
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