Egg hatch test

The EHT was used for detecting BZ resistance, as recommended by the World Association for the Advancement of Veterinary Parasitology [61, 62]. The test was performed following a standardised procedure [63]. Nematode eggs were isolated in deionised water and examined under the microscope to ensure that embryonation had not yet begun. Solutions of thiabendazole (TBZ, T8904, Sigma-Aldrich) at final concentrations of 0.05, 0.1, 0.2, 0.3, and 0.5 μg.ml^− 1 were applied to wells of 24-well tissue-culture plates containing an aqueous suspension of nematode eggs (~ 100 eggs per well). All dilutions contained 0.5% dimethyl sulphoxide (DMSO, Sigma-Aldrich). Three replicates were evaluated for each TBZ concentration, and three control wells without anthelmintic (0.5% DMSO) were included. The plates were sealed and incubated at 27 °C for 48 h. One drop (~ 10 μl) of Lugol’s solution was then added to each well both to prevent further hatching of eggs and to inactivate developed larvae. The numbers of eggs (those that failed to form larvae and those containing larvae) and L₁ larvae in each well were determined under an inverted microscope.