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A rapid and accurate assay for evaluating antibabesial drugs on a large scale is required for the discovery of novel chemotherapeutic agents against  Babesia  parasites. In the current study, we evaluated the usefulness of a fluorescence-based assay for determining the efficacies of antibabesial compounds against bovine and equine hemoparasites in  in vitro  cultures. Three different hematocrits (HCTs; 2.5%, 5%, and 10%) were used without daily replacement of the medium. The results of a high-throughput screening assay revealed that the best HCT was 2.5% for bovine  Babesia  parasites and 5% for equine  Babesia  and  Theileria  parasites. The IC 50  values of diminazene aceturate obtained by fluorescence and microscopy did not differ significantly. Likewise, the IC 50  values of luteolin, pyronaridine tetraphosphate, nimbolide, gedunin, and enoxacin did not differ between the two methods. In conclusion, our fluorescence-based assay uses low HCT and does not require daily replacement of culture medium, making it highly suitable for  in vitro  large-scale drug screening against  Babesia  and  Theileria  parasites that infect cattle and horses.

Optimization of a Fluorescence-Based Assay for Large-Scale Drug Screening against Babesia and Theileria Parasites