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Recently, we reported that clofazimine (CF) has an anti-piroplasm activity, but it could not completely eliminate parasites in the host. The currently available anti-piroplasm drug, diminazene aceturate (DA), has sometimes been reported to have toxic side effects. In the present study, we evaluated the combination treatment with CF and DA against piroplasms both  in vitro  and  in vivo . Additionally, mRNA level and DNA amounts were analyzed in CF‒ and DA‒treated  Babesia bovis  by a qPCR. The CF–DA combination had additive effects on  Babesia bovis ,  B .  bigemina , and  B .  caballi  and synergistic effects on  Theileria equi . The CF–DA combination chemotherapies against  B. microti  in mice were more potent than their monotherapies. In the CF‒ and DA‒treated  B. bovis , CF dose-dependently down-regulated mRNA level and DNA amounts of extranuclear genes (AT-rich featured), whereas DA down-regulated only DNA amounts of extranuclear genes, but those of nuclear genes were slightly down- or up-regulated by CF and DA. In conclusion, the CF–DA combination has a higher efficiency against piroplasms than CF or DA monotherapies. CF and DA might have an AT-rich DNA-binding activity. All results suggest that the CF–DA combination chemotherapy will be a better choice to treat piroplasmosis instead of DA monotherapy.

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Chemotherapeutic efficacies of a clofazimine and diminazene aceturate combination against piroplasm parasites and their AT-rich DNA-binding activity on Babesia bovis