Evaluation of a larval development assay (DrenchRite) for the detection of anthelmintic resistance in cyathostomin nematodes of horses.

Abstract: A larval development assay (LDA, DrenchRite) was evaluated to determine the effectiveness of this method in detecting anthelmintic resistance in cyathostomin nematodes of horses. A total of 15 horse farms from Georgia and South Carolina (USA) and Population S ponies from the University of Kentucky (USA) were included in this study. Nematode eggs were extracted from pooled fecal samples and placed into the wells of a DrenchRite plate for testing against thiabendazole (TBZ), levamisole (LEV) and 2 ivermectin (IVM) analogs (IVM-1, IVM-2). After a 7-day incubation larvae in each well were counted and data were analyzed by logistic regression. Resistance status of each farm for different drugs was determined in a separate study using a fecal egg count reduction test. LDA were performed on the 15 farms once, however, the Population S cyathostomins were assayed on 3 separate occasions to estimate the consistency of results between assays. Mean TBZ LC50 for oxibendazole resistant, suspected resistant and sensitive farms were 0.2015, 0.1625, and 0.1355 microM, respectively. For LEV, mean LC50 for PYR resistant, suspected resistant and sensitive farms were 1.590, 1.8018 and 1.4219 microM, respectively. All 15 farms had worms susceptible to IVM; mean LC50 for IVM-1 and for IVM-2 were 7.5727 and 87.9718 nM, respectively. A linear mixed model was fitted to the data to determine the relationship between LC50 and LC95 and resistance status for each farm. No meaningful relations were found. Consistency of assays varied between drugs, being best for TBZ and worst for LEV and IVM-1. All farms in this study had benzimidazole-resistant nematodes; therefore usefulness of DrenchRite for discriminating susceptibility versus resistance to this drug class could not be accurately assessed. Moreover, since all farms tested were sensitive to IVM and resistance to this drug class has not yet been reported in cyathostomins, it is not possible to assess accurately the usefulness of DrenchRite LDA for detecting IVM resistance at this time. Assay results for LEV suggest that LEV in a LDA does not yield data that is useful in estimating PYR efficacy in vivo. Based on results for PYR/LEV, the current high prevalence of benzimidazole resistance, no known cases of IVM resistance, and the sometimes extreme variation in results seen in many of the assays, DrenchRite LDA cannot be considered a useful tool for the diagnosis of resistance in cyathostomins of horses at present.