Optimization of a larval migration inhibition assay (LMIA) for determining in vitro susceptibility of equine cyathostomins to macrocyclic lactone anthelmintics, and evaluation of variation in species-specific drug tolerance levels using a 454 large-scale
Zarate Rendon, Daniel Alexis
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Cyathostomins are the most important parasites of horses worldwide. The macrocyclic lactones (ML) constitute the most effective drug class available. However, increasing numbers of suspected ML-resistance cases currently exist. No validated bioassay for ML-resistance in cyathostomins is currently available and the presence of multiple species is a complicating factor. The objective of this study was to optimize and validate a larval migration inhibition assay (LMIA) protocol to detect and measure levels of ML resistance/susceptibility in cyathostomins and determine the impact of species composition. We developed and optimized a LMIA protocol and validated a 454 sequencing protocol for species identification based on ITS-2 sequences. The dose response curves generated using four different analogs in two different parasite populations showed good consistency. The 454 protocol exhibited high sensitivity but did not represent the species abundance accurately. This limitation did not allow a valid evaluation of the levels of innate tolerance among species.