Molecular characterization of Mycobacterium bovis from livestock and wildlife in South Africa : genetic marker optimization and identification using whole genome sequence data

Abstract

Bovine tuberculosis (BTB) is a disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, which are bacteria that infect both domestic and wildlife host species. The zoonotic nature of the disease is of great concern globally. Over 50 million animals are infected all over the world, resulting in economic losses of approximately three billion United States dollars per year. Molecular typing techniques have greatly improved knowledge regarding the sources and modes of BTB transmission in many countries including South Africa. The current study was aimed at filling the knowledge gap regarding the epidemiology of the disease in livestock and wildlife species in the country for the purpose of effective control and management (in wildlife) as well as ultimate eradication (in livestock). Identification of a suitable typing technique for potential application in South Africa was the core purpose of the study. In an attempt to determine the manner of transmission within and between animal populations and map the geographical spread of this zoonotic disease, samples were collected from a variety of animal species throughout the country. In Chapter 2, molecular techniques (i.e. Restriction Fragment Length Polymorphism, spoligotyping and variable number tandem repeat) were used to identify and characterize Mycobacterium bovis infecting buffaloes in the Hluhluwe-iMfolozi Park (HiP) and three epidemiologically related game reserves over a 15-year time frame. The findings demonstrated that the majority of the M. bovis infections in buffaloes were as a result of a single strain which has persisted throughout the years with spill-over to other wildlife species. Evidence of further M. bovis infections due to strains previously undetected in the park was also shown. With a view to providing a simple and reliable method suitable for studying the epidemiology of BTB in the country, we describe, in Chapter 3, how we evaluated the utility of known VNTR sequences as epidemiological markers to describe the molecular epidemiology of BTB in South Africa, with special emphasis on the Kruger National Park (KNP) where there is evidence of clonal expansion of the C8 parental cattle strain. A set of VNTR loci that provided sufficient discriminatory power for application in epidemiological studies was identified. Potential use of VNTR typing in exploring evolutionary changes was also highlighted. To further establish the genetic diversity of the isolates and study the population structure of M. bovis, the loci were used to study a larger panel of M. bovis isolates from different regions (Chapter 4). The findings showed that besides cattle, at least 16 animal species contracted the infection, and highlighted a strong evidence of intra and inter-species transmission of M. bovis. Furthermore, the study also showed that despite the fact that a national control programme for BTB is applied in commercial cattle, there is a high diversity of M. bovis persisting in the country, highlighting the importance and need for intensified diagnostic testing and consideration of alternative control measures such as vaccination. Together with results obtained in Chapter 5, the study further indicated the presence of M. bovis infection in previously uninfected (or unknown status) game parks or reserves. Overall, three novel M. bovis strains (i.e. SB1474, SB2199 and SB2200) were identified and their patterns are now available in the international M. bovis database. We also describe for the first time in South Africa, M. bovis infection in a blue wildebeest (Comochaetes taurinus), nyala (Tragelaphus angasii) and giraffe (Giraffa camelopardalis); and a rare case of M. bovis infection causing pulmonary tuberculosis in a horse (Equus ferus caballus) is described in Chapter 6. To improve VNTR typing method for clonally linked isolates from the KNP, we sequenced whole genomes of local M. bovis isolates in Chapter 7. The tandem repeat loci identified were exploited for polymorphisms in M. tuberculosis complex isolates including M. bovis, with two of the five polymorphic loci identified showing potential discriminatory capabilities for clonally linked isolates. The epidemiological information gained in this investigation is particularly important in the context of the establishment of the Greater Limpopo Transfrontier Park (GLTFP) where the potential spread of BTB from Kruger National Park to Limpopo National Park (Mozambique) and Gonarezhou National Park (Zimbabwe) (or vice versa) is of great concern. The availability of the local M. bovis genomes provides an opportunity for future comprehensive sequence comparisons which may assist in further deducing the phylogenetic and evolutionary relationships of M. bovis isolates and efforts towards vaccine developments.