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. 2012;6(3):e1534.
doi: 10.1371/journal.pntd.0001534. Epub 2012 Mar 6.

Distribution and molecular evolution of bacillus anthracis genotypes in Namibia

Affiliations

Distribution and molecular evolution of bacillus anthracis genotypes in Namibia

Wolfgang Beyer et al. PLoS Negl Trop Dis. 2012.

Abstract

The recent development of genetic markers for Bacillus anthracis has made it possible to monitor the spread and distribution of this pathogen during and between anthrax outbreaks. In Namibia, anthrax outbreaks occur annually in the Etosha National Park (ENP) and on private game and livestock farms. We genotyped 384 B. anthracis isolates collected between 1983-2010 to identify the possible epidemiological correlations of anthrax outbreaks within and outside the ENP and to analyze genetic relationships between isolates from domestic and wild animals. The isolates came from 20 animal species and from the environment and were genotyped using a 31-marker multi-locus-VNTR-analysis (MLVA) and, in part, by twelve single nucleotide polymorphism (SNP) markers and four single nucleotide repeat (SNR) markers. A total of 37 genotypes (GT) were identified by MLVA, belonging to four SNP-groups. All GTs belonged to the A-branch in the cluster- and SNP-analyses. Thirteen GTs were found only outside the ENP, 18 only within the ENP and 6 both inside and outside. Genetic distances between isolates increased with increasing time between isolations. However, genetic distance between isolates at the beginning and end of the study period was relatively small, indicating that while the majority of GTs were only found sporadically, three genetically close GTs, accounting for more than four fifths of all the ENP isolates, appeared dominant throughout the study period. Genetic distances among isolates were significantly greater for isolates from different host species, but this effect was small, suggesting that while species-specific ecological factors may affect exposure processes, transmission cycles in different host species are still highly interrelated. The MLVA data were further used to establish a model of the probable evolution of GTs within the endemic region of the ENP. SNR-analysis was helpful in correlating an isolate with its source but did not elucidate epidemiological relationships.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Distribution of genotypes isolated by year.
The total number of isolates per year and per GT. GTs 2, 3, 6, 9, 18, and 22 (highlighted in grey) were found both within and outside the ENP.
Figure 2
Figure 2. Percentage of isolates of the most prevalent GTs in the ENP per year.
Numbers are given for years 2005–2010 when most of the samples were collected. Years 1983, 1987 and 1988–2002, including sampling gaps in 1990, 1993, 1996–99, and 2001, are pooled into the first data point. Total sample sizes are shown below x-axis.
Figure 3
Figure 3. Maps of the Etosha National Park and general location of farms.
(A): Main roads, pans, camps and main gates of the ENP are shown. Locations where GTs 4, 6, or 9, and 22 were found are indicated by colored marks. GTs 4, 6, and 9 were given the same label to indicate they are probably the same outbreak strain. Green areas in (B) represent National Park areas.
Figure 4
Figure 4. Median probabilities and 95% credible intervals for genotypes sampled from the Etosha National Park.
GTs occurring ≤5 times are pooled into the "Other" category.
Figure 5
Figure 5. Genetic distance as a function of temporal distance and host species similarity.
Isolates of Bacillus anthracis collected from the ENP during the period 1983–2010 are included.
Figure 6
Figure 6. Confirmed cases of anthrax in the four most commonly affected species in the ENP.
The period between 1988–2010 is shown. Isolates prior to 1988 have been excluded from this figure, having been collected during only short field studies in the Park. The insert indicates the mean monthly rain fall (in l/m2) in the Okaukuejo region, in 1983–2010.
Figure 7
Figure 7. Minimum spanning tree of 377 Namibian isolates typed by MLVA.
Clustering of MLVA profiles was done using a categorical coefficient. The MLVA-GTs are displayed as circles with the appropriate GT number. The size of each circle symbolizes the number of isolates of this particular GT. GTs differing in only one marker are combined in a complex seen as a gray halo if at least 3 GTs fulfill this criterion. The marker(s) on which the mutation is located is shown. Distances between circles do not reflect the correct phylogenetic distances. The vaccine isolates are not included. The relations within this tree remain the same if an outgroup like the B-cluster (as used in Figure S1) is included (not shown).

References

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