Phenotypic, biochemical and molecular analysis of Bacillus anthracis strains isolated during the outbreaks of anthrax in the Russian Federation, 2014-2016

In 2014 to 2016, despite effective measures to prevent an introduction and transmission of Anthrax in the Russian Federation, there were seven outbreaks of Anthrax in Volgograd, Rostov, Belgorod, Saratov regions, the Republic of Tatarstan, and also six outbreaks in reindeer population in two districts of Yamal-Nenets Autonomous Okrug where 2657 reindeers died. In this article we present some results of comprehensive characterization of genetic, biological features and phylogenetic relationship of Bacillus anthracis strains isolated during the outbreaks in Volgograd region, Yamal-Nenets Autonomous Okrug and from the soils of burial in Chuvash Republic during last 3 years. Here, we differentiated 11 strains as followed from growth morphology, mobility, Gram stain procedure, capsule in vivo and in vitro formation, sporulation, proteolytic, hemolytic, lecithinase, phosphatase, glycolytic activity, protocatechuic acid production, Congo red sorption from the medium, phage sensitivity, toxicity in vitro, plasmid profile, sensitivity to antibiotics recommended for use in veterinary medicine, virulence for mice. MLVA-typing of the anthrax strains was performed for 20 VNTR loci. It was shown that the main phenotypic and diagnostic features of anthrax strains differed insignificantly and, in general, corresponded to those of a typical B. anthracis strain. The most significant phenotypic differences were found in asporogenous and avirulent strain B. anthracis № 6017 isolated in 2016 from a Lappish reindeer dog. The B. anthracis strains isolated during one outbreak were grouped into separate clusters, and within the cluster some strains had insignificant differences in 1-2 loci. The strains isolated from the soils of burials in the Republic of Chuvashia and from the Lappish reindeer dog during the Yamal outbreak formed separate clusters. B. anthracis strains showed high epizootic risk due to pathogenicity factors expressed in vitro. The tests identified the presence of capsula and toxins, high hemolytic and proteolytic activity, protocatechuic acid synthesis, and high virulence for laboratory mice (at 6-1000 spores). These results confirm the necessity of continuous monitoring and evaluation of epizootic caution of anthrax burials and case sites (frost fields), and specific preventive anti-anthrax measures.