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Investigating Rickettsia Africae Infection in Amblyomma Hebraeum Ticks in Mnisi, Bushbuckridge Municipality, South Africa

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Investigating Rickettsia Africae Infection in Amblyomma Hebraeum Ticks in Mnisi, Bushbuckridge Municipality, South Africa Investigating Rickettsia africae infection in Amblyomma hebraeum ticks in Mnisi, Bushbuckridge Municipality, South Africa By Estere Mazhetese Submitted in partial fulfilment of the requirements of the degree Magister Scientiae (Veterinary Science) in the Department of Veterinary Tropical Diseases Faculty of Veterinary Science University of Pretoria November 2019 Supervisor: Dr. Darshana Morar-Leather Co-supervisor: Professor Luis Neves Declaration I, Estere Mazhetese, declare that this dissertation hereby presented to the University of Pretoria for the Master of Veterinary Science degree is my own work and I have not presented it for any degree or award in any other university. All secondary material used was acknowledged and referenced as required by the University of Pretoria. This research project was approved by the Animal Ethics Committee of the University of Pretoria on 02/08/2018 and by the Department of Agriculture, Forestry and Fisheries on the 17/07/2018. 12/11/2019 Estere Mazhetese Date i Acknowledgements I would like to express my sincere gratitude and appreciation to the following individuals, institutions and organisations who worked tirelessly to make this research a success; My mentor, Dr Darshana Morar-Leather, University of Pretoria, Department of Veterinary Tropical Diseases (DVTD), for her continued support throughout the research project. She has been a pillar of strength and motivation. She also worked hard to get the necessary applications approved for the project to start and on critical evaluation of the research proposal and the dissertation. Professor Luis Neves, for all his technical and moral support throughout the period of this research and writing up. His support was of great importance in this research. Dr Hein Stoltsz who helped in organising field logistics of the research project. Ms Zinathi Lukanji and Mr Marcus Makgabo for the technical knowledge and support in all the aspects of the research. Ms Anna-Mari Bosman for the training and technical knowledge in the laboratory at the DVTD. Dr Charles Byaruhanga who worked tirelessly assisting with sequence alignments, generating phylogenetic trees and phylogenetic analysis for this project. Ms Jeanette Wentzel and Dr Ilana Van Wyk who assisted me with sample collection and laboratory procedures and for their moral support during the sample collection period. The Hans Hoheisen Wildlife Research Station for technical help in the field during sample collection, storage and primary sample processing. State veterinarians and ii veterinary technicians who helped with the technical aspects of sample collection and sample processing at Hans Hoheisen Wildlife Research Station and in processing of permits, they made sample collection possible. The DVTD Laboratories where further processing of the samples was done. Belgian Directorate General for Development Co-operation Framework FA4 (ITM/DECD), Belgium, for the funding, without the funding this research would not have been possible. DVTD students and colleagues for their technical advice and moral support. My husband, Aleck Matingwina who supported me throughout my study period, this made me perform to the best of my abilities. My best friend, Anna Rungwe for supporting me throughout the research project. My family and friends for their social support and for accepting my long stay away from home. Almighty God for granting me the opportunity, health and ability to do this Masters. iii Table of Contents Declaration .................................................................................................................. i Acknowledgements ..................................................................................................... ii List of Tables ............................................................................................................. vii List of Figures ............................................................................................................. ix List of Abbreviations ................................................................................................... x DISSERTATION SUMMARY ..................................................................................... 1 CHAPTER 1 ............................................................................................................... 3 GENERAL INTRODUCTION ...................................................................................... 3 1.1 Research aim ................................................................................................ 6 1.1.1 Research objectives ............................................................................... 6 1.2 Benefits arising from the research project ..................................................... 6 CHAPTER 2 ............................................................................................................... 7 LITERATURE REVIEW .............................................................................................. 7 2.1 History and classification of R. africae .......................................................... 7 2.2 Scientific classification of R. africae .............................................................. 9 2.3 Characteristics of R. africae ........................................................................ 11 2.4 Vectors of R. africae .................................................................................... 11 2.4.1 Amblyomma vectors of R. africae ......................................................... 11 2.4.2 Other tick species found carrying R. africae DNA ..................................... 12 2.5 Life cycle of A. hebraeum ............................................................................ 13 2.5 Hosts of A. hebraeum ................................................................................. 15 2.6 Transmission and distribution of R. africae and its vectors ......................... 15 2.7 Clinical signs in animal hosts ...................................................................... 18 2.7 Clinical signs in human hosts ...................................................................... 19 iv 2.7.1 African Tick Bite Fever (ATBF) ............................................................. 19 2.7.2 Risk factors of ATBF ............................................................................. 19 2.7.3 Pathogenesis of ATBF .......................................................................... 20 2.7.4 Clinical signs of ATBF........................................................................... 21 2.7.5 Diagnosis of ATBF in humans .............................................................. 23 2.7.6 Treatment of ATBF ............................................................................... 24 2.7.7 Prevention and Control of ATBF ........................................................... 25 CHAPTER 3 ............................................................................................................. 26 MATERIALS AND METHODS ................................................................................. 26 3.1 Study area ................................................................................................... 26 3.2 Ethics approval and biosecurity ................................................................... 27 3.3 Consent from community ............................................................................ 28 3.4 Study design ............................................................................................... 28 3.5 Calculation of sample size .......................................................................... 28 3.6 Tick collection in the study area .................................................................. 29 3.7 Incubation of engorged ticks and collection of egg masses ........................ 30 3.8 DNA extraction from adult ticks and larvae ................................................. 30 3.9 DNA extraction from egg masses ................................................................ 31 3.10 DNA quantification ................................................................................... 31 3.11 gltA PCR for the detection of Rickettsia genus ........................................ 31 3.12 Conventional PCR (cPCR) ....................................................................... 35 3.13 Sequences and phylogenetic analysis ..................................................... 36 3.14 Data analysis ........................................................................................... 37 3.15 Farmer feedback report ........................................................................... 37 CHAPTER 4 ............................................................................................................. 39 RESULTS ................................................................................................................. 39 v 4.1 Detection of R. africae infection rates in A. hebraeum adult ticks and larvae …………………………………………………………………………………......39 4.1.1 Real-time Rickettsia PCR (qPCR) to screen adult tick and larvae samples for gltA gene ........................................................................................ 39 4.1.2 Conventional PCR (cPCR) to screen DNA from adult ticks and larvae for the ompA gene .................................................................................................. 39 4.1.3 Phylogenetic analysis of the ompA gene isolated from adult ticks and larvae samples..................................................................................................
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