Adaptation of a 96-well plate larval migration inhibition test for measuring the sensitivity of cyathostomins to macrocyclic lactone anthelmintics

Abstract

The use of macrocyclic lactone drugs for control of equine cyathostomins is threatened by increasing levels of resistance. Detection of changes in drug sensitivity is important for effective and sustainable management of cyathostomins, however, at present such detection relies on the use of the faecal egg count reduction test, which is known to be an insensitive method. The present study therefore aimed to examine the use of a 96-well plate larval migration inhibition test for detection of resistance to macrocyclic lactone drugs in cyathostomins. We optimised conditions for migration of larvae, and examined the effects of larval storage time on drug dose responses. The modified test was able to define the sensitivity of cyathostomin isolates to ivermectin and eprinomectin in terms of dose response curves, and IC50 and IC95 values. The IC95 showed much greater consistency than the IC50 with larvae that had been stored for different periods prior to the test. Comparisons between two isolates, which had both been defined previously as susceptible using faecal egg count reduction tests, showed more variation at the IC50 compared to the IC95. Limitations of the test included the degree of variation in control-well migration despite optimisation of migration incubation conditions, and the need to incorporate a method to determine the species composition of the larval populations to account for possible species differences in drug sensitivity among cyathostomins. Validation of the technique on reference susceptible and resistant isolates of known species composition is still required.