Molecular Characterization Of Bacillus Anthracis And Brucella Species From Southern Africa

Abstract

The main focus of this project was to characterise Brucella and Bacillus anthracis strains from certain areas of southern Africa using molecular techniques. Multiplex PCR assays (AMOS-PCR and Bruce-ladder PCR) were used to identify Brucella strains isolated from different hosts in Zimbabwe and selected strains were further characterised by whole genome sequencing using next generation sequencing (NGS). Bacillus anthracis strains from wildlife in the KNP region in South Africa were typed using 31 multiple locus variable number of tandem repeats (VNTR) analysis (31-MLVA) to monitor the spread, distribution and diversity of the strains. In this study we successfully identified Brucella abortus, B. canis and B. suis from the 16 Brucella isolates from Zimbabwe using AMOS and Bruce-ladder PCR assays. This is the first formal report of B. canis and B. suis in Zimbabwe, since it is documented that only B. abortus, B. melitensis and B. ovis have been reported in Zimbabwe. On the basis of the origin (sample source) and the results when characterising Brucella isolates with AMOS and Bruce-ladder PCR, three (ZW043, ZW046 and ZW053) strains from bovines, identified as B. suis (ZW043 and 046) and B. abortus (ZW053), were selected and sequenced to verify their accurate identity. Despite the variants observed in our genome sequences, whole-genome sequencing of these three isolates using the Illumina Miseq® (Illumina®) and their comparison to Brucella reference whole genome sequences have proven that the isolates were B. suis (ZW043 and ZW046) and B. abortus (ZW053) respectively, thus corresponding with the data obtained with the Bruce-ladder, AMOS PCR assays. As most laboratories in Africa lack adequate resources, expertise and infrastructure to do biotyping of pathogens under proper biosafety and biosecurity conditions, multiplex-PCR assays, and especially Bruce-ladder could be used in the identification of Brucella species. Bacillus anthracis isolates (n=72) collected from various anthrax outbreaks and regions in KNP during 2012 and 2013 anthrax outbreaks were analysed using MLVA. The 72 B. anthracis isolates in KNP belonged to 23 B. anthracis genotypes clonal genotypes were observed in each outbreak, while five animals appeared to be infected by multiple genotypes. Clonal genotypes were observed in each outbreak and from the spread of the genotypes over large distances vultures seem to be the primary vector transmitting the anthrax strains from one region to another. Eighteen out of the 23 genotypes obtained from all the isolates were circulating during the 2012 outbreak spanning 7 regions and 5 catchment areas (a basin shaped area where rivers drained their water) compared to 6 genotypes circulating from 2013 cases and environmental samples, from sites where animals were suspected to die of anthrax during 2012 to early 2013, that were confined mostly to 1 region and 1 catchment area with 2 outlying cases. Cluster analysis of 30-MLVA data grouped 72 isolates in the major genetic A-clade. No genotype grouped in the clade B in this study although B-clade genotypes played an important role in KNP during 1970-1981 but played a minor role from the major outbreak in the 1990 onwards. MLVA is an important epidemiological tool and was proven to be an efficient method to characterize the spread as well as identify possible vectors of anthrax in KNP this study.