Modified mucosal digestion procedure improves precision of encysted larval cyathostomin counts in horses

With rising anthelmintic resistance in adult cyathostomins, the most pervasive equine parasites, the need to assess larvicidal efficacy has increased, yet available methods remain limited. Encysted larvae are quantified using mucosal digestion, wherein a 5% subsample of intestinal mucosa is processed, and a 2% aliquot is counted for early third stage (EL3) and late third stage/mucosal fourth stage (LL3/L4) larvae. This method is imprecise and post-treatment larval differences are often not observed. The following study aimed to assess standard and modified protocol precision and sensitivity. In phase one, 24 naturally infected horses with unknown treatment history were euthanized and a single 5% mucosal sample was collected from each organ. Following digestion, triplicate 2% aliquots were examined for larval presence. In phase two, 12 untreated, naturally infected horses were euthanized and triplicate 5% mucosal tissue samples were collected from each organ. Each sample was digested and a 2% aliquot and the remaining digesta were examined. Coefficient of variation and Wilcoxon Signed-Rank tests were used to evaluate precision. The standard protocol had lower precision (p = 0.003) and sensitivity (16.6%) compared to the modified procedure (66.6%). Estimated counts were higher using the 2% procedure (p < 0.0001). The protocol used in model burden estimation contributed the largest proportion of variation (33.8%), compared to individual animal variation (5.8%). Results suggest the standard 2% aliquot technique is non-representative of overall burden and imprecise, warranting investigation into procedural modifications that improve precision, which directly affects interpretation in anthelmintic efficacy studies for existing and developing drugs.