University of Neuchâtel Faculty of Sciences Institute of Biology Laboratory of Eco-Epidemiology Ticks and tick-borne pathogens at the interplay of game and livestock animals in South Africa Thesis presented to the Faculty of Sciences of the University of Neuchâtel for the Degree of Doctor of Sciences by Mirko Berggoetz Members of the Jury: Prof. Lise Gern (Thesis Director); Prof. Patrick Guerin (University of Neuchâtel); Prof. Lorenza Beati (Southern University, Georgia); Prof. Kurt Pfister (University of Munich); Dr Heinz Sager (Novartis Saint-Aubin) Index 1 Abstract .............................................................................................................................. 9 2 Introduction ...................................................................................................................... 13 2.1 Tick biology ................................................................................................................ 13 2.1.1 Rhipicephalus species ......................................................................................... 16 2.1.2 Amblyomma species........................................................................................... 18 2.1.3 Hyalomma species ............................................................................................. 19 2.1.4 Haemaphysalis species ....................................................................................... 19 2.1.5 Ixodes species ..................................................................................................... 20 2.1.6 Margaropus species ........................................................................................... 21 2.1.7 Argasidae species ............................................................................................... 21 2.2 Protozoan pathogens ................................................................................................ 22 2.2.1 Life-cycle of Babesia species .............................................................................. 22 2.2.2 Life-cycle of Theileria species ............................................................................. 24 2.2.3 Clinical manifestations of Babesia and Theileria species ................................... 25 2.2.4 Piroplasm species ............................................................................................... 25 2.3 Bacterial pathogens ................................................................................................... 36 2.3.1 Life cycle of Anaplasma species ......................................................................... 37 2.3.2 Life cycle of Ehrlichia species ............................................................................. 38 2.3.3 Clinical manifestations of Anaplasma and Ehrlichia species ............................. 40 2.3.4 Rickettsiales species ........................................................................................... 40 2.4 Tick-borne pathogen circulation between livestock and game animals ................... 45 2.5 Objectives .................................................................................................................. 49 3 Materials and methods .................................................................................................... 51 3.1 Study areas ................................................................................................................ 51 3.1.1 Free State provincial nature reserves and surrounding farms .......................... 52 5 3.1.2 Farms in the Bethal area, Mpumalanga Province .............................................. 60 3.1.3 Farms in the Thabazimbi and Lephalale areas, Limpopo Province .................... 61 3.2 Tick and blood sampling on wild and domestic ruminants ....................................... 63 3.2.1 Game capture and culling in the Free State Provincial Nature Reserves .......... 63 3.2.2 Game capture and hunting in the Mpumalanga and Limpopo Provinces: ........ 64 3.2.3 Domestic ruminants ........................................................................................... 65 3.2.4 Tick and blood collection .................................................................................... 65 3.3 Tick dissection and DNA extraction of salivary glands and blood samples ............... 66 3.3.1 Tick dissection .................................................................................................... 66 3.3.2 DNA extraction of the salivary glands ................................................................ 66 3.3.3 DNA purification of blood samples .................................................................... 67 3.4 Amplification of tick-borne pathogen DNA ............................................................... 67 3.5 Reverse line blot (RLB) Hybridisation ........................................................................ 69 3.6 Gene sequencing ....................................................................................................... 73 3.7 Data analysis .............................................................................................................. 73 3.7.1 Pathogens in host blood ..................................................................................... 73 3.7.2 Pathogens in tick salivary glands ........................................................................ 74 4 Results .............................................................................................................................. 75 4.1 Paper 1………………………………………………………………………………………………………………….75 4.2 Paper 2 ..................................................................................................................... 115 5 Discussion ....................................................................................................................... 145 6 Conclusion ...................................................................................................................... 153 7 Appendix ......................................................................................................................... 155 7.1 Appendix A: Geographic distribution of tick species ............................................... 155 7.2 Appendix B: Phylogenetic relationships of Babesia and Theileria spp. ................... 159 7.3 Appendix C: Phylogenetic relationships of Anaplasma and Ehrlichia spp. ............. 160 6 7.4 Appendix D: Novel pathogen-host and pathogen-vector combinations. .................... 161 7.5 Appendix E: Exposure of host species to pathogen species. ....................................... 162 8 References ...................................................................................................................... 165 9 Acknowledgements ........................................................................................................ 187 Photographer: VW: Virginie Wyss; MS: Melody Schmid; DS: Daniel Ston; MB: Mirko Berggoetz. 7 1 Abstract To evaluate the exposure of wild and domestic ungulates to tick borne-pathogens, and hence to study the pathogen exchange at the wildlife-livestock interface, blood from 181 individual hosts assigned to 18 species and 7364 ticks belonging to 13 species were collected. Samples originated from nine localities in four South African Provinces (Free State, Mpumalanga, Gauteng and Limpopo). Polymerase chain reaction followed by reverse line blotting and sequencing was used to screen host blood and tick salivary glands for protozoan pathogens of the genera Babesia and Theileria as well as for bacterial pathogens of the genera Anaplasma and Ehrlichia. From each individual infested host, a maximum of ten males and ten females of each tick species were dissected to isolate the salivary glands, this led to 2117 analysed ticks. Three hundred twenty nine ticks (15.5%), belonging to eight species, were infected and harboured 397 infections among which 57.7% were identified to species level and were assigned to 23 pathogen species. While, 110 / 181 individual hosts were infected and harboured 210 infections and 163 were identified to species level and belonged to 16 pathogen species. Screening such a large variety of host and tick species allowed describing 30 new host-pathogen combinations, involving ten pathogen species, and 23 new vector-pathogen combination which involved 14 pathogen species. Principal component analysis (PCA) assigned the 163 infections, identified to species level in host blood, to four groups. Three groups were associated to sheep, cattle and horses and their respective wild counterparts. Each group was characterized by high homogeneity in pathogen assemblage and host phylogenetic status. These groups characterized the most privileged transmission routes between and among wild and domestic ungulates. Within six localities, we sampled an equal number of wild and domestic animals (n = 128). On this dataset, once having controlled for the significant variation among localities, the infection prevalence and intensity of infection did not differ significantly between wild and domestic hosts. Interestingly, salivary glands from ticks infesting wild ruminants displayed significantly higher infection prevalence and pathogen mean density than salivary glands from ticks infesting livestock animals. This suggests that wild ungulates are more refractory to tick- borne pathogen infections than domestic ones, given that the infection prevalence and intensity of infection displayed similar values in