Molecular Identification of Babesiosis in Stray Dog at Chittagong Metropolitan Area, Bangladesh

An epidemiological study of babesiosis in stray dogs was conducted at Chittagong Metropolitan area, Bangladesh, for six months. Blood samples from 130 stray dogs were collected from nine randomly selected areas along with the information of age and sex. Blood samples were initially examined by Giemsa’s stained blood smear method and DNA was extracted and Babesia spp. were confirmed by amplifying 18S rRNA gene. The prevalence was determined 6.92% in microscopic techniques and 4.61% by PCR. Significantly higher frequency of babesiosis was found in adult (10.11%) and male dog (11.94%) than younger and female, respectively (p<0.05). Furthermore, we sought to develop a seminested PCR to detect and differentiate Babesia gibsoni (Asia genotype) from other Babesia sp. An outer primer pair was designed to amplify a ~340 bp fragment of the 18S rRNA genes. Forward primers were premeditated that would particularly amplify a smaller fragment from each organism in the seminested PCR. Seminested PCR revealed that, all parasitic isolates were identified as Babesia gibsoni (Asian type). Sequencing of the PCR products and analysis of sequence homology with other Babesia using NCBI BLAST our isolate CVASU/ BD/Babesia (KR349267) showed 95% nucleotide homology with other Bangladeshi Babesia gibsoni isolate BD 37 (LC008285) and Kolkata isolate (KJ142323). The phylogenetic tree shows our isolate is most closely related to but distinct from Kolkata isolate. CVASU/BD /Babesia and Kolkata isolate forms clade which indicates geographical location may have some influence on the distribution of the parasite. These results recommended that Babesia gibsoni (Asian type) parasites are widespread in stray dog in Chittagong Metropolitan area, Bangladesh. Further investigations are required to clarify the origin, vector and pathogenesis of these parasites tracking in dogs in Bangladesh.