Genotypic diversity of Bacillus anthracis from 2014 to 2015 in the Kruger National Park

Abstract

Anthrax is one of the most well-known, yet underestimated zoonotic diseases in the world, remaining endemic on the African continent. Here the disease not only impacts upon the health of animals and humans, but also on the livelihoods of rural communities. Even so, the disease remains neglected in terms of funding and attention, which results in the under-reported of anthrax in Africa. This study aims to discuss the reasons for under-reporting of anthrax, and the factors impeding anthrax control in Africa. We provide an updated distribution map of anthrax-endemic regions across the continent to investigate under-reporting. Furthermore, we highlight the benefits which could be gained by researchers, in Africa and abroad, through enhancing the diagnostic and research capabilities on the continent. Special attention is given to multi-locus variable number of tandem repeat assay (MLVA), a genotyping technique which has already delivered much insight into the dynamics of anthrax disease, and the diversity of Bacillus anthracis. This study employed MLVA to provide insight into the genotypic diversity of anthrax in Kruger National Park (KNP), and the potential mechanisms which drive such outbreaks. To achieve this goal, we utilised MLVA, employing 31 markers, to investigate anthrax outbreaks from 2014-2015 in KNP. Briefly, isolates were confirmed to be B. anthracis using classical microbiology and real-time PCR investigation. Extracted B. anthracis DNA was subjected to multiplex PCR, with the resultant fragments being separated by size using capillary electrophoresis. These data were then utilised to cluster B. anthracis into lineages, and genotypes. Bacillus anthracis isolates (n=81) were obtained from carcasses and the environment, in the Pafuri, Houtboschrand and Mahlangeni regions of the KNP, between 2014 and 2015. All isolates belonged to the A-clade, consistent with findings in the KNP since 1990. Phylogenetic analysis indicated that the isolates belonged to five distinct genotypes, with genotypes 1 and 2 (GT1 and GT2) dominating, present in both 2014 and 2015. GT1 and GT2 belonged to the A1 sub-clade, and the A.Br.005/006 single nucleotide polymorphism (SNP) sub-group. GT3, belonging to the A3 (SNP A.Br.Aust94) sub-clade, has been reported in the KNP in previous outbreaks, and was isolated from vulture faeces in 2015. GT4 (P15-53) and GT5 (P15-54), each representing a single isolate, presented as atypical B. anthracis. Three VNTR markers, including the virulence factor on pX01, could not be detected in P15-53, while two VNTR markers could not be detected in P15-54. An additional investigation, into the potential of using an MLVA7 protocol as a first line assay, was performed. The results indicated that while such a protocol was capable of distinguishing between the 5 genotypes present in this study, it could not differentiate between A and B-clade isolates. Investigation of B. anthracis isolates from the environment, in the vicinity of anthrax-infected carcasses, provided insight into the complex epidemiology of the disease. Firstly, we provided evidence supporting necrophagous fly transmission of anthrax, since isolates collected from carcasses, blowflies, and vegetation in the vicinity, were of the same genotype. Secondly, we investigated the distribution of GT1, with two isolates (H15-01 & M15-01) occurring in Mahlangeni, and Houtboschrand, between 150 km and 200 km away from of the main outbreak location in Pafuri. Dissemination of GT1, by for example water-related dispersal of B. anthracis, was eliminated due to the topography of Pafuri, Mahlangeni, and Houtboschrand, as well as the distance involved. Alternative explanations are either that GT1 was dispersed over this distance by vultures, or that GT1 had been present in all three areas and had not been detected previously. Finally, we report here the first evidence of B. anthracis being shed in the milk of two Aepyceros melampus (impala) females.