Results of Practical Evaluation of the Optimized Method for Multilocus Sequence Analysis of Pathogenic Borrelia burgdorferi sensu lato Group

Abstract

The aim of the study was to test the possibility of identifying isolates of pathogenic Borrelia burgdorferi sensu lato by the specificity of linked sequences of their recA and ospA gene loci, i.e. using the optimized multilocus sequence analysis (MLSA) method.Materials and methods. 25 Borrelia isolates from adult hungry Ixodes ricinus ticks collected in the forest-steppe part of the Voronezh Region were studied. Isolates were obtained through seeding the mid-gut of ticks on BSK medium. Their primary identification was performed by analyzing the linked sequences of the recA and ospA gene loci with a total length of 360 bp. Selective control of species affiliation of Borrelia isolates was carried out according to the protocol of full MLSA via assessment of the nucleotide sequences of 6 genes (recA, ospA, rrs, hbb, groEL, fla) and the intergenic spacer rrf-rrl (total length of all 7 loci being 1187 bp) using the BLAST platform, Sequence scanner 2 and MEGA11 programs.Results and discussion. The heterogeneity of the nucleotide sequences of recA and ospA genes in 25 Borrelia isolates has been investigated. Construction of dendrograms has revealed at least 5 different sequence variants among the isolates. The similarity of isolates within each of these five groups, as well as their distinction from comparable linked sequences of other pathogenic species of the B. burgdorferi sensu lato complex is demonstrated. To confirm the results obtained, a set of isolates from each group was sampled using the full MLSA protocol. It has been established that five Borrelia species circulate in the studied ecosystems of the Voronezh Region: B. afzelii, B. garinii, B. bavariensis, B. burgdorferi sensu stricto, and B. valasiniana.

Keywords

• ixodidae tick-borne borrelioses
• mlsa
• identification of the pathogen
• laboratory diagnostics