A practical miracidial motility assay for assessing Fasciola hepatica sensitivity to compounds in vitro

Fasciola hepatica causes fasciolosis in livestock and humans worldwide, yet reliable tools to assess drug efficacy against the early developmental stages of this parasite are lacking. Here, we developed an automated miracidial motility assay (MMA) using the WMicroTracker ONE infrared detection system to quantify the sensitivity of F. hepatica miracidia to anthelmintic compounds including clorsulon (CLORS), closantel (CLOS), triclabendazole (TCBZ) and triclabendazole-sulphoxide (TCBZ-SO). Systematic optimisation of assay conditions, including inoculum size, observation window and solvent concentration yielded a reliable platform for evaluating the sensitivity of F. hepatica miracidia from diverse geographic isolates to these compounds. Our results demonstrated that three compounds (CLOS, TCBZ and TCBZ-SO) produced concentration-dependent motility inhibition, whereas CLORS had no effect. CLOS displayed the highest potency among isolates from New South Wales (NSW), Tasmania (TAS) and Victoria (VIC), whereas TCBZ and TCBZ-SO exhibited isolate-specific sensitivity patterns. Miracidial responses of the NSW and TAS isolates to TCBZ, TCBZ-SO and CLOS were also compared in vitro with those of newly excysted juveniles (NEJs) produced from the same isolates. Overall, the findings show that MMA provides a reproducible, host-independent and high-throughput phenotypic platform for assessing miracidial sensitivity to compounds.