Canine babesiosis caused by Babesia canis sensu stricto became an emerging disease of dogs across Europe calling for attention also in countries where it was an only rare imported disease. An easy accessibility of molecular methods and the growing amount of sequencing data led to the description of intraspecific variability in 18S rDNA sequences designated as "genotypes". Using material from a homogenous cohort of dogs with microscopically confirmed canine babesiosis caused by B. canis, we evaluated Babesia intraspecific variability and amplification sensitivity of three different genes (18S rDNA, COI, Cytb) to assess their potential as diagnostic or phylogenetic markers. In raw sequencing data obtained, we observed at least 3 ambiguous positions in up to 86% of chromatograms within the ∼560 bp fragment of 18S rDNA suggesting the existence of several, not identical copies of this gene. Our COI haplotype analysis resulted in a star-like pattern indicating a recent origin of most haplotypes, but not supporting the existence of two dominant haplotypes. Similarly, the Cytb sequences obtained from samples with all variants of 18S rDNA were identical. We corroborate previous observations from three other European countries and bring the evidence of the existence of 18S rDNA paralogs in B. canis genome replacing currently used "genotype" theory.

CEITEC VFU University of Veterinary and Pharmaceutical Sciences Brno Palackého tř 1946 1 612 42 Brno Czech Republic; Department of Pathological Morphology and Parasitology University of Veterinary and Pharmaceutical Sciences Brno Palackého tř 1946 1 612 42 Brno Czech Republic; Biology Centre Institute of Parasitology Czech Academy of Sciences Branišovská 31 370 05 České Budějovice Czech Republic

CEITEC VFU University of Veterinary and Pharmaceutical Sciences Brno Palackého tř 1946 1 612 42 Brno Czech Republic; Department of Virology Veterinary Research Institute Hudcova 296 70 621 00 Brno Czech Republic

Department of Parasitology Institute of Genetics and Microbiology University of Wrocław Przybyszewskiego 63 51 148 Wrocław Poland

Vetlab Veterinary Diagnostic Laboratory Parkowa 13 51 616 Wrocław Poland

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