Molecular characterisation of southern African Bacillus anthracis strains

Abstract

With important diseases including zoonotic diseases, it is necessary to find accurate and reliable techniques in the diagnosis of the causal agent. Bacillus anthracis the causal agent of anthrax has received a great deal of attention due to its negative association with biological warfare. Microbiological tests have routinely been used to confirm diagnoses of B. anthracis in suspected anthrax cases and to distinguish it from B. cereus and B. thuringiensis that also belong to the B. cereus group, along with B. anthracis. Multiple locus variable number tandem repeat (VNTR) analysis (MLVA) is the current, rapid, molecular assay of choice in typing B. anthracis strains. It relies on commonly practiced PCR based methods to target regions which differ in tandem repeat unit. Various MLVA panels, of which the first consisted of 8 VNTR markers, followed by the MLVA15 and MLVA25 panels have been used to differentiate anthrax strains and to evaluate the diversity of B. anthracis from different geographical areas. In this study, we investigated the use of 31 VNTR markers (combination of MLVA15 and 25 panels) to type B. anthracis isolates from southern Africa using both the capillary and agarose electrophoresis methods to determine the comparative value of each. The samples included B. anthracis sensu lato isolates from southern Africa (n=112), a clinical B. cereus isolate and 34F2 Sterne vaccine strain. This study indicated that the resolution using agarose gel electrophoresis does not allow the accurate separation of 6 VNTR loci with tandem repeat consisting of 6 bp or less, but that the remaining 25 VNTR loci are sufficient to type B. anthracis strains for the purpose of epidemiological study. Agarose electrophoresis is also the most cost effective and appropriate technique for the average African / developing country laboratory. Despite the fact that the 31 MLVA panel using capillary electrophoresis is not cost effective, it is a rapid and accurate method for B. anthracis typing. A comparison of the discriminative power of the four MLVA systems, using 8, 15, 25, and 31-markers clearly showed the superiority of the 31-marker MLVA. However cluster analysis of 113 B. cereus/ B. antrhacis sensu lato isolates from southern Africa indicated that the MLVA25 and MLVA31 panels are very similar and the latter only differentiated an additional 7 genotypes. As MLVA is known to reveal the genetic relationships within B. anthracis, we used the MLVA31 panel to also investigate whether it will differentiate isolates amongst the B. cereus group. The study revealed that MLVA alone may not be sufficient in resolving isolates from the B. cereus group, but is an effective tool in determining genetic and geographical distance and identifies isolates with anomalies that can be definitively identified with further study. Lastly, as blood smears are the most common method to diagnose anthrax and often the only available sample, 14 stained blood smear slides were evaluated as a source of DNA for the fingerprinting of anthrax using MLVA. This preliminary study indicated that typing of 31VNTR loci was only successful from freshly made stained blood smear slides. With stored, stained blood smears slide the quantity and quality of DNA from the slide was insufficient and had to be amplified using GenomiPhi and only amplified VNTR loci with fragment size smaller than 300 bp. This study identified factors that influenced fingerprinting / typing to be primarily the small amount of target DNA (anthrax spores) from a blood smear slide and the conditions of collection and storage. Results from this thesis, highlighted the use of MLVA for typing of B. anthracis and also identified areas that need further investigation.