Molecular detection and genotypes characterization of Canine babesiosis causative agent Babesia canis

Canine babesiosis is a widespread tick-borne disease caused by haematozoan parasites of the genus Babesia. The vast majority of clinical babesiosis cases in dogs in Europe is caused by Babesia canis. Canine babesiosis has become quite frequent in Lithuania during the past decade. Babesiosis caused by B. canis may range from mild to severe disease in dogs. Such difference in the virulence of B. canis strains is associated with genetic heterogeneity among B. canis strains. The aim of study was to investigate the genetic diversity of B. canis strains isolated from naturally infected dogs in Lithuania using PCR-RFLP assay and sequence analysis based on 18S rRNR and Bc28.1 genes. In total 138 blood samples from dogs suspected of babesiosis were collected in Lithuania during 2016-2017. DNA was isolated from EDTA-anticoagulated whole blood. Positive 18S rRNA and Bc28.1 gene PCR products were digested with HincII and MboI restriction enzymes, respectively. Results of RFLP analysis were evaluated by agarose gel electrophoresis. Selected positive samples were sequenced and then analyzed using the Mega 6.0 software. B. canis genotypes were distinguished on the basis on two nucleotide (GA→AG) substitutions observed in 18S rRNA gene sequences. Based on PCR-RFLP and sequence analyses of 18S rRNA gene four B. canis genotypes were identified: 15,18 % samples contained 18S rRNA-A genotype (AG), 0,89 % 18S rRNA-B (GA), 83,04 % 18S RNA-A/B (RR) and 0,89 % C (RA) genotype. Two genotypes of B. canis were detected by RFLP analysis of Bc28.1 gene. The vast majority of the B. canis strains were of the genotype Bc28.1-B (83.18%;), followed by genotype Bc28.1-A (17.9 %). A total of fifteen single nucleotide polymorphisms in partial Bc28.1gene of B. canis were identified by sequence analysis. The distribution of B. canis Bc28.1- genotypes in Lithuania was similar to South – West Europe, but different from North – East Europe findings. [...]Biologijos katedraGamtos mokslų fakultetasLietuvos sveikatos mokslų universitetas. Veterinarijos akademijaVytauto Didžiojo universiteta