Genotyping and phylogenetic placement of Bacillus anthracis isolates from Finland, a country with rare anthrax cases

Abstract

Background: Anthrax, the zoonotic disease caused by the gram-positive bacterium Bacillus anthracis, is nowadays rare in northern parts of Europe including Finland and Scandinavia. Only two minor outbreaks of anthrax in 1988 and in 2004 and one sporadic infection in 2008 have been detected in animals in Finland since the 1970's. Here, we report on two Finnish B. anthracis strains that were isolated from spleen and liver of a diseased calf related to the outbreak in 1988 (strain HKI4363/88) and from a local scrotum and testicle infection of a bull in 2008 (strain BA2968). These infections occurred in two rural Finnish regions, i.e., Ostrobothnia in western Finland and Päijänne Tavastia in southern Finland, respectively.

Results: The isolates were genetically characterized by PCR-based methods such as multilocus variable number of tandem repeat analysis (MLVA) and whole genome-sequence analysis (WGS). Phylogenetic comparison of the two strains HKI4363/88 and BA2968 by chromosomal single nucleotide polymorphism (SNP) analysis grouped these organisms within their relatives of the minor canonical A-branch canSNP-group A.Br.003/004 (A.Br.V770) or canonical B-branch B.Br.001/002, respectively. Strain HKI4363/88 clustered relatively closely with other members of the A.Br.003/004 lineage from Europe, South Africa, and South America. In contrast, strain BA2968 clearly constituted a new sublineage within B.Br.001/002 with its closest relative being HYO01 from South Korea.

Conclusions: Our results suggest that Finland harbors both unique (autochthonous) and more widely distributed, common clades of B. anthracis. We suspect that members of the common clades such as strains HKI4363/88 have been introduced only recently by anthropogenic activities involving importation of contaminated animal products. On the other hand, autochthonous strains such as isolate BA2968 probably have an older history of their introduction into Finland as evidenced by a high number of single nucleotide variant sites in their genomes.

Keywords: Bacillus anthracis; Comparative genomics; Finland; Multiple locus variable number of tandem repeat analysis (VNTR, MLVA); Single nucleotide polymorphism (SNP); Whole genome sequencing (WGS).

Fig. 1

Position of strains HKI4363/88 and BA2968 in UPGMA cluster analysis based on 31 B. anthracis MLVA markers. Positions of strains HKI4363/88 (a) and BA2968 (b) (indicated in red) among their closest MLVA-relatives within a data-set of 976 B. anthracis isolates are shown

Fig. 2

Unrooted phylogenetic tree of representative B. anthracis strains derived from chromosomal SNPs. The evolutionary history of B. anthracis strains was inferred from 1,548 non-homoplasious chromosomal SNPs by using the Maximum Likelihood method based on the Tamura-Nei model [22]. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Colors indicate canSNP groups A.Br.003/004 (A.Br.V770, green), B.Br.CNEVA (purple) or B.Br.001/002 (yellow), respectively. Bootstrap values (based on 1000 replications) are given as percentages at branch nodes. The right column indicates the country of origin and canSNP group information of representatives (in brackets)