Assay methods for in vitro and in vivo anti-Babesia drug efficacy testing: Current progress, outlook, and challenges

•Babesiosis: A disease of veterinary importance with an empty drug pipeline.•There is a wide gap in the treatment of animal babesiosis.•Empty anti-Babesia drug pipeline was mainly due to the absence of HTS drug system.•SG I fluorescence assay has helped to fulfil the anti-Babesia drug discovery gap.•Establishing of anti- B. gibsoni, B. canis, and B. ovis HTS drug system is required. Absence of an effective high-throughput drug-screening system for Babesia parasites is considered one of the main causes for the presence of a wide gap in the treatment of animal babesiosis when compared with other hemoprotozoan diseases, such as malaria. Recently, a simple, accurate, and automatic fluorescence assay was established for large-scale anti-Babesia (B. bovis, B. bigemina, B. divergens, B. caballi and T. equi) drug screening. Such development will facilitate anti-Babesia drug discovery, especially in the post-genomic era, which will bring new chemotherapy targets with the completion of the Babesia genome sequencing project currently in progress. In this review, we present the current progress in the various assays for in vitro and in vivo anti-Babesia drug testing, as well as the challenges, highlighting new insights into the future of anti-Babesia drug screening.