Genetic diversity among Bacillus anthracis soil isolates at fine geographic scales

Abstract

Environmental samples were collected from carcass sites during and after anthrax outbreaks in 2000 and 2001 in the bison (Bison bison) population within Wood Buffalo National Park and the Hook Lake Region north of Wood Buffalo National Park. Bacillus anthracis spores were isolated from these samples and confirmed using phenotypic characterization and real-time PCR. Confirmed B. anthracis isolates were typed using multiple-locus variable-number tandem repeat analysis (MLVA15) and single-nucleotide-repeat analysis (SNRA). B. anthracis isolates split into two clades based on MLVA15, while SNRA allowed some isolates between carcass sites to be distinguished from each other. SNRA polymorphisms were also present within a single carcass site. Some isolates from different carcass sites having the same SNRA type had divergent MLVA types; this finding leads to questions about hierarchical typing methods and the robustness of the fine-scale typing of Bacillus anthracis.

Fig 1

Graphical representation of MLVA genotypes (outer circles) and SNRA SGTs (inner circles) of spores collected from environmental samples within and near Wood Buffalo National Park in 2001 and 2002. MLVA types differed only at the pXO1 locus but were identical at 14 other loci. SGT types are described in Table 3.

Fig 2

Graphical representation of MLVA genotypes (outer circles) and SNRA SGTs (inner circles) of spores collected from environmental samples at a specific carcass site within Wood Buffalo National Park in 2001. Each inner circle represents an individual isolate from that site. Numbers in white represent spore counts per gram that were isolated. Areas that are bare soil are shaded brown; those with vegetation are shaded green. Location of bison which died during the 2001 outbreak is shown. Each grid square represents 1 m². (Adapted from reference .)

Fig 3

Graphical representation of MLVA genotypes (outer circles) and SNRA SGTs (inner circles) of spores collected from a specific carcass site from a 2001 outbreak. Numbers in white represent spore counts per gram. Red dots represent samples containing spores. Blue dots represent samples that did not contain spores. Total area of grid site is ∼80 m². (Adapted from an image by D. C. Dragon with permission.)

Fig 4

SNRA genetic relationships among genotypes based on MLVA genotypes. The mutational changes from dominant SGTs are shown. Lines linking SNRAs show mutation steps and do not represent lineages. These models are based on MLVA genotypes being more stable and used to anchor the dominant genotypes for analysis regardless of isolate location. Therefore, the two groups with specific MLVA types were split for SGT description. Detailed genotypic description is presented in Table 3.

Fig 5

SNRA genetic relationships among genotypes based on geographic isolation. The mutational changes from dominant SGT are shown. Lines linking SNRAs show mutation steps and do not represent lineages. These models are based on geographic isolation of the samples as opposed to using MLVA for anchoring. Therefore, the two geographical areas were split into two groups for SGT description. Detailed genotypic description is presented in Table 3.