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

## TL;DR

This paper introduces a new high-throughput assay to test the sensitivity of Fasciola hepatica larvae to drugs, revealing geographic and drug-specific variations in response.

## Contribution

The study introduces a novel automated motility assay for assessing drug sensitivity in Fasciola hepatica miracidia.

## Key findings

- CLOS, TCBZ, and TCBZ-SO caused concentration-dependent motility inhibition in miracidia.
- Geographic isolates showed varying sensitivity to TCBZ and TCBZ-SO.
- MMA is a reproducible, high-throughput platform for drug testing.

## Abstract

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.

Image 1

•High-throughput, phenotypic motility assay established for Fasciola hepatica larvae (miracidia).•Significant variation in miracidium responses to different drugs.•Marked geographical variability in miracidial responses to the same drug.•This assay should be adaptable to other motile developmental stages and trematode species.

High-throughput, phenotypic motility assay established for Fasciola hepatica larvae (miracidia).

Significant variation in miracidium responses to different drugs.

Marked geographical variability in miracidial responses to the same drug.

This assay should be adaptable to other motile developmental stages and trematode species.