Development of a toolkit for African horse sickness: identification of Culicoides vectors from Namibia and detection of African horse sickness virus C van Zyl 22823964 Dissertation submitted in fulfilment of the requirements for the degree Magister Scientiae in Environmental Sciences at the Potchefstroom Campus of the North-West University Supervisor: Dr C Mienie Co-supervisor: Dr D Liebenberg Co-supervisor: Dr K Labuschagne November 2016 Table of Contents TABLE OF CONTENTS ......................................................................................................... II ACKNOWLEDGEMENTS .................................................................................................... IV PREFACE................................................................................................................................ V SUMMARY ............................................................................................................................. VI LIST OF TABLES ..................................................................................................................VII LIST OF FIGURES............................................................................................................... VIII ACRONYMS AND ABBREVIATIONS................................................................................. X CHAPTER 1: INTRODUCTION ......................................................................................... 12 1.1 AFRICAN HORSE SICKNESS..................................................................................... 12 1.1.1. AHS history ......................................................................................................... 12 1.1.2. Geographical distribution of AHS .................................................................... 13 1.1.3. Aetiology of the AHSV....................................................................................... 14 1.1.4. Pathogenesis of the disease ............................................................................ 16 1.2. VECTOR GENUS: CULICOIDES ............................................................................... 20 1.2.1. Life cycle, feeding and habitat of Culicoides species................................... 21 1.2.2. Morphology of Culicoides species................................................................... 22 1.2.3 Transmission of AHS .......................................................................................... 23 1.3 PERSPECTIVE AND OUTLINE OF THE STUDY ....................................................... 26 1.3.1 Problem statement .............................................................................................. 26 1.3.2 Aim and objectives .............................................................................................. 28 1.3.3 Outline of dissertation......................................................................................... 28 CHAPTER 2: CLASSIFICATION AND IDENTIFICATION OF CULICOIDES SPECIES ............................................................................................................................... 29 2.1 INTRODUCTION .......................................................................................................... 29 2.1.1 Culicoides in Namibia ......................................................................................... 29 2.2 CLASSIFICATION AND IDENTIFICATION OF CULICOIDES .................................... 34 2.2.1 Classification of Culicoides ................................................................................ 34 2.2.2 Morphological identification ............................................................................... 35 2.2.3 Molecular and phenotypic identification .......................................................... 37 2.3. MATERIALS AND METHODS .................................................................................... 39 2.3.1 Sample collection ................................................................................................ 39 2.3.2 Culicoides identification...................................................................................... 42 2.3.3 DNA extraction .................................................................................................... 42 2.3.4 MT-COI DNA amplification ................................................................................ 43 2.3.5 Cycle sequencing of amplicons ........................................................................ 43 2.3.6 Bioinformatic tools for data analyses ............................................................... 44 2.4 RESULTS AND DISCUSSION ................................................................................. 47 2.4.1. Morphologically identified Culicoides of Namibia.......................................... 47 2.4.2. MT-COI DNA amplification of Culicoides species ........................................ 49 2.4.3. DNA barcoding of Namibian Culicoides through sequencing of MT-COI gene ................................................................................................................................ 51 2.4.4. Alignment of sequences for the phylogenetic tree ....................................... 57 II CHAPTER 3: DEVELOPMENT OF A SIMPLIFIED NUCLEIC ACID DIAGNOSTIC TOOL FOR THE DETECTION OF AHSV ........................................................................ 66 3.1 METHODOLOGY FOR DIAGNOSTIC TOOL ........................................................ 66 3.2 LAMP PRINCIPLE...................................................................................................... 66 3.3 MECHANISMS OF LAMP ......................................................................................... 67 3.3.1 Non-cyclic step .................................................................................................... 67 3.3.2 Cyclic amplification ............................................................................................. 68 3.3.3 Applications of LAMP ......................................................................................... 69 3.3.4 AHSV and LAMP................................................................................................. 74 3.4 MATERIALS AND METHODS: DIAGNOSTIC TOOL ........................................... 76 3.4.1 Primer design....................................................................................................... 76 3.4.2 RT-LAMP and optimisation................................................................................ 77 3.4.3 Sensitivity of novel RT-LAMP............................................................................ 78 3.4.4 Evaluation of RT-LAMP sensitivity for in-field testing.................................... 78 3.5 RESULTS AND DISCUSSION ................................................................................. 81 3.5.1 Primer design....................................................................................................... 81 3.5.2 Optimisation of RT-LAMP .................................................................................. 82 3.5.3 Evaluation of assay sensitivity by comparing indirect and direct amplification of AHSV-infected Culicoides midges.................................................. 87 CHAPTER 4: CONCLUSION ............................................................................................. 90 4.1. The establishment of Culicoides barcodes of the MT-COI gene (Chapter 2) ......................................................................................................................................... 90 4.2 Developing a simplified diagnostic tool for the detection of AHSV in Culicoides (Chapter 3).................................................................................................. 91 REFERENCE LIST .............................................................................................................. 93 APPENDIX A ................................................................................................................... 120 APPENDIX B ................................................................................................................... 145 APPENDIX C ................................................................................................................... 148 III Acknowledgements I hereby wish to express my gratitude to the following persons and institutions for their contributions for this study to be successfully completed. I would like to thank my supervisors: Dr Charlotte MS Mienie for her continued guidance, valuable input, support and constructive advice during the study. Dr Danica Liebenberg-Weyers for always having time to help, for her support, understanding ear and motivation throughout the study. Dr Karien Labuschagne who helped me with the identification of the Culicoides specimens, sharing her expertise in the field and for being a co-supervisor. I would also like to thank following people at the Agriculture Research Council- Onderstepoort Veterinary Institute (ARC-OVI): Drs Gert Venter and Antoinette van Schalkwyk for sharing their valuable knowledge. Prof. Oriel Thekisoe for allowing me to work in his laboratory and his guidance during the learning of a new molecular method. Tania de Waal for also being willing to help day or night, it is highly appreciated. I want to thank my family: Words cannot express my appreciation for all your prayers, patience and love. My fiancé Marnus, who believed in me