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.