Examining equine anthelmintic efficacy using modified procedures

Abstract

With increasing evidence of anthelmintic resistance in the luminal stages of equine cyathostomins, the need to examine efficacy against encysted stages has risen. Encysted burdens are traditionally evaluated using mucosal digestion, wherein a 5% subsample of intestinal mucosa is processed and digested for enumeration. In the standard protocol, a single 2% aliquot of this subsample is counted for early third stage (EL3) and late third stage/mucosal fourth stage (LL3/L4) larvae. The first study investigated precision of larval counts from both the current gold standard procedure and a modified protocol where all larvae from the 5% tissue subsample were counted. In spring 2024, 12 untreated juvenile horses naturally infected with cyathostomins were enrolled, and triplicate 5% mucosal samples were collected from the cecum, ventral colon, and dorsal colon. For each sample, the standard 2% aliquot was counted as well as the remaining 5% tissue aliquot. Mixed models were constructed for counts, measuring proportion of variation for each fixed effect. Coefficients of variation (CV) were calculated to evaluate precision. The mean CV for the subsample (2%) and total (100%) counts by stage were as follows: EL3 (subsample) 100.7%, (total) 59.0%; L3/L4 (subsample) 112.7%, (total) 82.2%; total larvae (subsample) 85.7%, (total) 23.7%. Precision difference by aliquot amount varied significantly (P < 0.001). All fixed effects—foal, larval stage, aliquot size (P < 0 0.001), and organ (P = 0.003)—were statistically significant and contributed significant proportions of count variance; however, aliquot size (2% or 100%) provided the most (57.5%). These results suggest the standard mucosal digestion technique using the 2% count is nonrepresentative of overall larval burden and highly imprecise, warranting further investigation into how procedure can affect interpretation of data, particularly in the context of anthelmintic efficacy studies.