Use of multiple locus variable number tandem repeats analysis in systematics of the causative agent of brucellosis

Abstract

Methods that allow one to carry out molecular genetic differentiation of strains of the same species are acquiring increasing importance in the modern system of struggle with brucellosis. The multiple locus variable number tandem repeats analysis (MLVA) method was selected for molecular genetic differentiation up until the strain level and simultaneous establishment of genetic relationship of the studied strains. The aim of the study was typing (by the MLVA method) of strains of three pathogenic to humans Brucella species with the analysis of stability of selected loci, identification of differences between them and conformity of the data, obtained by this method and by phenotypic method of Brucella differentiation, for use of the MLVA method in systematics of the causative agent of brucellosis. Materials and methods. Twenty-six Brucella strains, which are reference (n = 15); vaccine (n = 2); field strains of three pathogenic Brucella species, including B. melitensis (n = 3), B. abortus (n = 2), and B. suis (n = 2); and isolates (n = 2) of unknown taxonomic position were typed by the MLVA method with nine pairs of primers for known variable loci for the Brucella genome. Analysis of the stability of selected loci, their differences according to the Hunter-Gaston discrimination index (HGDI), and conformity of the data obtained with phenotypic methods of identification was conducted. Results. The MLVA method confirms the results of phenotypic methods of identification and the stability of selected loci in most reference and vaccine strains with a high variability HGDI index (0.9969) for all loci. A dendrogram based on the data of the MLVA typing allowed us to allocate the Brucella strains into related clusters according to their taxonomic species and biovar position and development of 25 genotypes. The B. melitensis strains generated a cluster related to the B. melitensis 63/9 biovar 2 reference strain. Australian Brucella 83-4 and Brucella 83-6 isolates from mouse like rodents generated a cluster distant from other Brucella strains. Conclusions. MLVA is a promising method of Brucella strain differentiation with known and undetermined taxonomic status for their systematics and creation of the MLVA genotype catalogue, which will favor a qualitative improvement of the system of epidemiological surveillance for this infection in Russia.