It was recently reported by Birkenheuer et al. that combination therapy with atovaquone and azithromycin showed favorable therapeutic effects on canine babesiosis caused by Babesia gibsoni (B. gibsoni). However, Matsuu et al. showed that treatment with atovaquone could possibly induce the emergence or in vivo selection of drug-resistant variants of B. gibsoni with nucleotide substitutions in cytochrome b (cytb) gene.
Under such conditions, we report the treatment with atovaquone and azithromycin of 4 naturally B. gibsoni infected dogs at Kagoshima University Veterinary Teaching Hospital. Initially, all dogs responded well to treatment and the expected improvements of clinical conditions and hematological parameters were obtained. However, 2 of the 4 cases showed a relapse of the disease at 42 and 56 days, respectively, after the initiation of therapy. The emergence of drug-resistant or in vivo selected variants was suspected in both cases. Accordingly, DNA sequencing analysis of the cytb gene was conducted using blood samples collected pre- and post-treatment. Compared to the standard sequence of cytb gene (DDBJ/GenBank/EMBL accession number, AB215096), cytb genes from the relapsed cases had different nucleotide sequences; one of them had an amino acid substitution (M121I) that has been reported to possibly be responsible for drug-resistance. Furthermore, B. gibsoni with M121I was a dominant type in one of the cases, even at the time of the primary onset of the disease.
From findings of clinical cases, variants of B. gibsoni with drug-resistance related sequences in the cytb gene are suspected to already exist in nature. We thus performed an epidemiological survey based on the cytb gene using blood samples collected from 58 cases in areas west of the Kanto area of Japan. Many types of single nucleotide substitution were detected; however, most induced synonymous amino acid substitutions. Nine non-synonymous amino acid substitutions were detected. Sites and frequency in the 58 cases were as follows; M43I (1.7%), M121I (3.4%), I136V (1.7%), V220I (5.2%), I226V (13.8%), A276V (1.7%), A290V (1.7%), I303V (5.2%) and P310S (1.7%). I226V, V220I and I303V were observed at relatively high incidences compared to those in other sites. As well, the three sites reported by Matsuu et al., M121I, V220I and I303V, were also detected in this study; M121I, which corresponds to the possible atovaquone binding site, was detected in 2 cases.
This study indicated that drug-resistant variants might be induced even in naturally infected dogs treated with atovaquone. B. gibsoni with the variant cytb gene already exists in nature, though its frequency is not high. Therefore, attention needs to be given to the appearance of drug-resistant variants in cases where atovaquone is used.