Application of molecular biology methods for the detection and genotyping of Babesia canis

Canine babesiosis is a widespread tick-borne infectious disease caused by intraerythrocytic protozoa Babesia canis. Over the past two decades, the number of cases of canine babesiosis in Europe has been increasing significantly, and higher numbers of cases have been observed in Lithuania as well. As the disease spreads rapidly, greater attention is needed for accurate diagnosis and effective treatment. Diagnostic methods commonly used in veterinary clinics, such as blood smears, are not always sensitive enough to identify the infection or do not allow for accurate identification of the pathogen. We aimed to investigate the genetic diversity of B. canis strains isolated from infected dogs in Lithuania using sequence analysis of B. canis 18S rRNA and Bc28.1 gene fragments. Blood samples were collected from 107 symptomatic dogs in veterinary clinics from spring to autumn of 2024. PCR tests were performed for both 18S rRNA and Bc28.1 genes. B. canis DNA was detected in 62 samples (66.34%) out of 107. Sequence analysis of the 18S rRNA gene fragment showed two variable sites in 81–82 nucleotide positions, dividing B. canis into three different genotypes. Bc28.1 gene fragment analysis identified four gene sequence variants, which were classified into three genotypes based on two nucleotide polymorphisms. The results highlight discrepancies between diagnoses made in veterinary clinics using blood smears and those obtained through molecular methods. Furthermore, they demonstrate the potential of molecular techniques for detecting genetic variability among B. canis strains, which could improve diagnostic accuracy and epidemiological surveillance.