Development of a Luciferase-Based In Vitro Assay to Evaluate the Efficacy of Anti-Cryptosporidial Drugs Against Cryptosporidium parvum

Background/Objectives: Cryptosporidium parvum is a major causative agent of cryptosporidiosis; however, progress in anti-cryptosporidial drug discovery has been hindered by the lack of robust and reproducible in vitro evaluation systems. In this study, we developed and optimized a luciferase-based in vitro assay to quantitatively monitor C. parvum growth in HCT-8 cells. Methods: Key experimental parameters affecting Infection efficiency were systematically examined, including sodium taurocholate treatment, timing of medium replacement, and serum concentration. Results: Sodium taurocholate significantly enhanced Parasite infectivity, and removal of non-invaded parasites at 3 h post-infection (hpi) resulted in approximately 2-fold and 3.7-fold increase in luciferase activity at 24 and 48 hpi, respectively, compared with untreated controls. In contrast, removal at 24 hpi led to only an approximately 2.5-fold increase at 48 hpi, consistent with stage-dependent differences in Parasite development. Morphological analyses confirmed Parasite differentiation from trophozoites to meronts, followed by progression toward sexual stages. Using the optimized assay system, we evaluated several anticoccidial compounds and demonstrated potent in vitro activity of monensin and its structural analog kijimicin, whereas diclazuril and toltrazuril exhibited limited efficacy. Conclusions: Collectively, this luciferase-based platform provides a reliable and quantitative tool for anti-cryptosporidial drug screening and will facilitate future therapeutic development against C. parvum.