Host genetic factors in susceptibility to mycobacterial disease in the African buffalo, Syncerus caffer

Thesis (PhD)--Stellenbosch University, 2014.ENGLISH ABSTRACT: Bovine tuberculosis (BTB) is a chronic, infectious disease found in domestic livestock and wildlife, and has serious biodiversity, economic and public health implications. African buffalo act as a wildlife reservoir of BTB, maintaining and transmitting the disease within the environment. The research presented in this thesis addresses the role of host genetic variation in resistance to BTB infection in African buffalo, and reviews the possible practical application of such information. Annual BTB prevalence within the African buffalo population in Hluhluwe iMfolozi Park, South Africa, was evaluated over a seven year period in order to define the extent of M. bovis infection. Prevalence changes over time suggest that the test and cull operation currently in place is performing successfully with respect to the original aims of the programme. A review of genetic studies of BTB in livestock and wildlife collated previous findings in this field and provided a collection of possible candidate genes and variants. It also highlighted a lack of research in wildlife, and the limitations of working with species with insufficient genetic data. To overcome the absence of whole-genome data, next-generation sequencing was performed on nine African buffalo, in order to identify novel genetic variants in this species. Upwards of 76 000 novel SNPs within gene regions were identified, and subsequent fluorescent genotyping of 173 SNPs showed a 57% validation rate. From the validated set, 69 SNPs located in genes related to the immune system were selected for association testing with BTB status in African buffalo, and were fluorescently genotyped in 868 individuals. Three SNPs, in the Solute Carrier family 7, member A13 (SLC7A13), Deleted in Malignant Brain Tumour-1 (DMBT1) and Interleukin 1 alpha (IL1α) genes, were identified as significantly associated with BTB status. Very little sequence information of the NRAMP1 (SLC11A1) gene was obtained from the next-generation sequencing performed, and this gene has been associated with brucellosis, salmonella and paratuberculosis in other animal species, making it an excellent candidate for BTB resistance. To characterise this gene in African buffalo, Sanger sequencing was performed to generate the complete coding region, and partially sequence the 5’UTR, intronic and 3’UTR regions. Fifteen novel polymorphisms and three microsatellites were identified within the gene. Finally, a review was prepared to assess the applicability of genetic information on BTB resistance to selective breeding programmes for African buffalo. Phenotypic, marker-assisted and genomic breeding strategies were discussed, with particular emphasis on their suitability to African buffalo. Identifying genes and variants involved in BTB resistance in African buffalo provides potential targets for drug or vaccine development, as well as information that could be incorporated into selective breeding programmes. This may support new management options for controlling the BTB epidemic in the game parks of South Africa, as an alternative to, or in conjunction with, lethal controlAFRIKAANSE OPSOMMING: Beestuberkulose (BTB) is ‘n chroniese, aansteeklike siekte wat in vee en wild voorkom en wat ernstige gevolge vir die ekonomie, biodiversiteit en openbare gesondheid inhou. Die Kaap-buffel is ‘n wild reservoir vir BTB wat die siekte onderhou en versprei in die omgewing. Die navorsing wat in hierdie tesis aangebied word fokus op die rol van gasheer genetiese variasie in die weerstand teen BTB infeksie in Kaap-buffels en gee ‘n oorsig van die moontlike praktiese toepassing van die resultate. Die jaarlikse BTB voorkomsyfer in die Kaap-buffel bevolking in die Hluhluwe iMfolozi Park in Suid-Afrika is oor ‘n tydperk van sewe jaar geëvalueer om die omvang van M. bovis infeksie te bepaal. Die verandering in voorkomsyfer oor tyd dui daarop dat die toets-en-slag operasie wat tans gebruik word die oorspronklike doelwitte van die program suksesvol bereik. ‘n Oorsig en vergelyking van vorige genetiese studies van BTB in vee en wild het ‘n versameling van moontlike kandidaatgene en –variante verskaf. Dit het ook die gebrek aan navorsing in wildediere uitgewys en die navorsingsbeperkinge wanneer ‘n spesie met onvoldoende genetiese data bestudeer word benadruk. Aangesien daar nie heel genoom data beskikbaar is nie, is volgende-generasie volgordebepaling van 9 Kaap-buffels gedoen om nuwe genetiese variasies in hierdie spesie te identifiseer. Meer as 76 000 nuwe enkel-nukleotied polimorfismes (ENPs) binne geen-areas is geïdentifiseer en die daaropvolgende genotipering van 173 ENPs het ‘n bevestigingskoers van 57% gehad. Vanuit die bevestigde stel ENPs is 69 gekies vir assosiasietoetse met BTB status in die Kaap-buffel en genotipering van 868 individue is gedoen. Drie ENPs, in die Solute Carrier family 7, member A13 (SLC7A13), Deleted in Malignant Brain Tumour-1 (DMBT1) en Interleukin 1 alpha (IL1α) gene, was beduidend geassosieer met BTB status. Baie min volgorde inligting van die NRAMP1 (SLC11A1) geen is verkry uit die volgende-generasie volgordebepaling. Aangesien hierdie geen voorheen met brucellose, salmonella en paratuberkulose in ander dierespesies geassosieer is, is dit ‘n uitstekende kandidaat vir BTB weerstand. Hierdie geen is in Kaap-buffels gekarakteriseer deur Sanger volgordebepaling van die volledige koderende, gedeeltelike 5’UTR, introniese en 3’UTR areas te doen. Vyftien nuwe polimorfismes en drie mikrosatelliete is geïdentifiseer. Ten slotte is ‘n oorsigstudie gedoen om die toepaslikheid van BTB genetiese weerstandsdata in selektiewe telingsprogramme van Kaap-buffels te evalueer. Fenotipiese, merkerbemiddelde en genomiese teling strategieë is bespreek, met spesifieke klem op die geskiktheid van die metodes vir Kaap-buffels. Identifisering van gene en variante wat betrokke is by BTB weerstand in die Kaap-buffel bied potensiële teikens vir medikasie of entstof ontwikkeling, sowel as inligting wat in selektiewe telingsprogramme gebruik kan word. Dit kan nuwe bestuursopsies vir die beheer van die BTB-epidemie in die parke van Suid-Afrika bied as 'n alternatief vir, of in samewerking met, dodelike beheermetodes