Screening anthelmintic resistance to triclabendazole in Fasciola hepatica isolated from sheep by means of an egg hatch assay

Background: In the present study, the detection of anthelmintic resistance to triclabendazole (TCBZ) in sheep infected by Fasciola hepatica was studied using an egg hatch assay (EHA). F. hepatica eggs were recovered from bile and faeces of infected animals by isolates with different grade of anthelmintic resistance to TCBZ: i) a resistant isolate (RT); ii) a susceptible isolate (ST); iii) naturally infected sheep by a susceptible field strain (FST). In the EHA the percentage of hatched eggs were calculated according to the following concentrations of TCBZ diluted in dimethyl-sulfoxide (DMSO): 0.05, 0.2, 1, 5, and 25 mu g/ml. Results: In relation to the EHAs carried out with the eggs from bile of sheep infected by ST, differences were found in the percentage of hatched eggs between the control well, only with DMSO, and the two highest concentrations of TCBZ (5 and 25 mu g/m) (p < 0.05). However, when we tested the drug with the eggs from the bile of sheep infected by RT, the percentage of hatched eggs was similar among all concentrations. Since the range of hatching varied between isolates, we calculated the ratio of the results of each concentration to its control value confirming the higher hatching in RT than in ST. We developed an EHA with eggs recovered from faeces in order to avoid the slaughter of sheep. The results of the EHAs with the isolate ST showed differences in the percentage of hatching between the highest concentration (25 mu g/ml) and the control well (p < 0.05); however, these differences were not confirmed under field conditions with the strain FST. Conclusions: The ovicidal effect of TCBZ in F. hepatica eggs from bile was shown using a commercial formulation diluted in DMSO with a minimum concentration of 5 mu g/ml. However, in eggs recovered from faeces the results are not conclusive. The cleaning of eggs recovered from faeces is an important issue that should be reviewed and standardized before comparing results between susceptible and resistant isolates in this kind of EHA.