Studies on the Resistance to Diminazene Aceturate in Babesia gibsoni In Vitro

Canine babesiosis caused by Babesia gibsoni is treated with diminazene aceturate (DA). DA can temporarily improve the clinical signs of canine babesiosis but is unable to eliminate the parasites from infected dogs, and relapses often occur. Therefore, it is believed that B. gibsoni might achieve the resistance against DA. However, there is no report clearly demonstrating the DA resistance in B. gibsoni. Therefore, in this study, a DA-resistant B. gibsoni strain was developed and the differences between the DA-resistant B. gibsoni strain and wild-type B. gibsoni were compared. First, a DA-resistant B. gibsoni strain was developed in vitro by the gradual increase of the DA concentration from 1 to 200 ng/ml. The parasites survived and proliferated in the medium containing 200 ng/ml DA, which is much higher than the 50% inhibitory concentration (IC50) of DA for B. gibsoni. Subsequently, these parasites were removed from erythrocytes and exposed directly to 200 ng/ml DA. They survived and invaded fresh erythrocytes, though wild-type B. gibsoni did not survive. Based on these results, the parasites cultured with 200 ng/ml DA were determined as a DA-resistant B. gibsoni strain. Thereafter, to investigate the characteristics of the DA-resistant B. gibsoni strain, the effects of other antibabesial drugs, including clindamycin, doxycycline, metronidazole and pentamidine, on the DA-resistant B. gibsoni strain were examined. The DA-resistant B. gibsoni strain showed strong resistance against pentamidine, and weak resistance against clindamycin and doxycycline. Moreover, the IC50 values of clindamycin, doxycycline and pentamidine for the DA-resistant strain at day 7 were higher than those for the wild-type B. gibsoni, respectively. These results indicated that the DA-resistant B. gibsoni strain could have resistance not only to DA, but also to other antibabesial drugs. Especially the DA-resistant B. gibsoni strain exhibited resistance against pentamidine, which shares similar structure with DA. In other protozoan, the mechanisms of drug resistance through mutations and/or amplification in drug transporters or drug targets were demonstrated. Therefore, the analysis for those metabolic pathways in the DA-resistant B. gibsoni strain will lead to elucidate the mechanism of the action of DA against B. gibsoni.Consequently, to characterize the DA-resistant B. gibsoni strain, the transcription levels of B. gibsoni heat shock protein 70 (BgHsp70) gene, which plays important roles in cell proliferation and the control of cellular function, was measured by quantitative real-time reverse transcription-polymerase chain reaction. In Plasmodium falciparum, Hsp70 has been proposed to contribute to the development of drug resistance. Therefore, the change in the transcription levels of the BgHsp70 gene was analyzed in DA resistance. During the development of the DA-resistant B. gibsoni strain, DA-resistant B. gibsoni variants, which were maintained in culture with DA from 1 to 175 ng/ml for more than 8 weeks, were also obtained. The copy number of the BgHsp70 transcripts in the DA-resistant variant cultured with 1 ng/ml DA was significantly lower than in wild-type B. gibsoni while those in DA-resistant variants increased with escalating doses of DA from 1 to 75 ng/ml, though they were lower than in wild-type B. gibsoni. Moreover, those in DA-resistant variants cultured with > 125 ng/ml DA were almost the same as wild-type B. gibsoni. It is hypothesized that the transcription levels of the BgHsp70 gene would be reduced during the selection of the DA-resistant B. gibsoni strain under the long-term weak pressure of DA, and then would be returned to the normal level after achieving resistance against DA. However, since the reason why the transcription levels of the BgHsp70 gene was reduced is still unclear, further study will be necessary to confirm this hypothesis. In conclusion, it was clearly demonstrated the development of DA resistance of B. gibsoni in vitro. The DA-resistant B. gibsoni strain obtained resistance against other antibabesial drugs. Moreover, the transcription levels of the BgHsp70 gene was reduced by the weak DA pressure and then recovered when B. gibsoni had achieved resistance against DA. However, the role of BgHsp70 for the DA resistance in B. gibsoni remains unclear. Further studies of BgHsp70 might prove to determine the mechanism of the DA resistance of B. gibsoni. Finally, the results obtained from this study could contribute to a better understanding of the DA resistance in B. gibsoni in vitro.