Classification of Babesia canis strains in Europe based on polymorphism of the Bc28.1-gene from the Babesia canis Bc28 multigene family

•In this study, we first developed a PCR-RFLP molecular test (designated MT2) based on polymorphism of Bc28.1 gene from Babesia canis.•The use of Bc28.1-derived MT2 test to analyze the genetic diversity of strains in Europe revealed 3 Bc28.1-derived genotypes (A, B or 34) with an overall predominance of genotype B.•Our data showed that strains from genotype 34 were restricted to France and a great variation in the distribution of strains from genotypes A and B according longitudinal and latitudinal location in Europe.•These results are discussed in terms of diversity within Dermacentor reticulatus ticks subpopulations.•Their significance to the development of efficient vaccines against canine babesiosis in Europe is also discussed.

The vast majority of clinical babesiosis cases in dogs in Europe is caused by Babesia canis. Although dogs can be vaccinated, the level of protection is highly variable, which might be due to genetic diversity of B. canis strains. One of the major merozoite surface antigens of B. canis is a protein with a Mr of 28 kDa that belongs to the Bc28 multigene family, that comprises at least two genes, Bc28.1 and a homologous Bc28.2 gene. The two genes are relatively conserved but they are very distinct in their 3′ ends, enabling the design of specific primers. Sequencing of the Bc28.1 genes from 4 genetically distinct B. canis laboratory strains (A8, B, 34.01 and G) revealed 20 mutations at conserved positions of which three allowed the classification of B. canis strains into three main groups (A, B and 34.01/G) by RFLP. This assay was subsequently used to analyze blood samples of 394 dogs suspected of clinical babesiosis from nine countries in Europe. All blood samples were first analyzed with a previously described assay that allowed detection of the different Babesia species that infect dogs. Sixty one percent of the samples contained detectable levels of Babesia DNA. Of these, 98.3% were positive for B. canis, the remaining cases were positive for B. vogeli. Analysis of the Bc28.1 gene, performed on 178 of the B. canis samples, revealed an overall dominance of genotype B (62.4%), followed by genotypes A (37.1%) and 34 (11.8%). Interestingly, a great variation in the geographical distribution and prevalence of the three B. canis genotypes was observed; in the North-East genotype A predominated (72.1% A against 27.9% B), in contrast to the South-West where genotype B predominated (10.3% A against 89.7% B). In the central part of Europe intermediate levels were found (26.0-42.9% A against 74.0-57.1% B, from West to East). Genotype 34 was only identified in France (26.9% among 78 samples) and mostly as co-infection with genotypes A or B (61.9%). A comparative analysis of the classification of 35 B. canis strains in genotypes A and B using a previously described 18SrDNA-derived PCR-RFLP test revealed a partial but no direct correlation with the classification based on polymorphism of the Bc28.1-gene described here.