MLVA-16 typing of Brucella suis biovar 2 strains circulating in Europe

Swine brucellosis due to Brucella suis biovar 2 is an emerging disease in Europe, associated with increase of extensive swine farms and high density of infected wild boars. Since knowledge of predominant circulating strains is a prerequisite for any epidemiological study, accurate molecular typing procedures were applied to a collection of 176 B. suis isolates. By using suis-ladder multiplex PCR and PCR-RFLP analysis of omp2a, omp2b and omp31 genes, five haplotypes were identified among 160 biovar 2 isolates, with haplotypes 2d and 2e restricted to Portugal and Spain and haplotypes 2a, 2b and 2c widespread in Europe (except Portugal). MLVA based on 16 genetic markers (MLVA-16) revealed 126 genotypes, with 101 singletons, and grouped biovar 2 isolates in two clusters according to their geographic origins and haplotypes, defining the Iberian (Portugal and Spain) and the Central-European clonal lineages. In order to get insights on the evolutionary associations between B. suis lineages and their host species, an extended analysis was performed using a subset of 11 markers and publicly available data for 350 additional strains. This MLVA-11 analysis revealed a high genetic divergence amongst the 526 B. suis strains based on their hosts and highlighted the close relationship between strains from swine, wild boars and hares. Beyond corroborating the existence of Iberian and Central European biovar 2 clonal lineages and pointing to the evolution of biovar 2 Iberian clonal lineage from Central-European one by an allopatric speciation event, an ongoing colonization of Iberian Peninsula with specific MLVA-11 genotypes is also highlighted.