Precision and spatial variation of cyathostomin mucosal larval counts

Cyathostomins are pervasive parasites of equids across the world. Larval stages encyst in the mucosa of the cecum, ventral and dorsal colon and can induce an inflammatory response leading to larval cyathostominosis, a life-threatening generalized typhlocolitis. Mucosal digestion is the only gold standard procedure for identifying and quantifying all larval stages. There is a lack of standardization of this technique and several aspects are ambiguous, such as precision of the method and the possibility of spatial variation of mucosal larval counts. The aim of this study was to estimate precision for enumeration of early third stage larvae (EL3) and late third stage/fourth stage (LL3/L4) larvae and investigate spatial variation of encysted counts within large intestinal organs. Six naturally infected and untreated horses aged 2-5 years were euthanized as part of an anthelmintic efficacy study, and the cecum (Cec), ventral colon (VC) and dorsal colon (DC) were collected. Each organ was rinsed, weighed, and visually separated into 3 equally sized sections. Two 5% tissue samples were collected from each section, a total of six replicates per organ. The mucosae were digested, and 2% examined under the microscope for presence of EL3 and LL3/L4 stage larvae. Overall, 59 % of the harvested larvae were EL3s, and 41 % were LL3/L4s. The ventral colons represented 45 % of the total organ weight, and contributed 37 and 41 % of the EL3s and LL3/L4s harvested, respectively. The Cec, representing only 27 % of the weight contributed 23 % of EL3s and 47 % of LL3/L4s. The DC represented 28 % of the total organ weight, and 28 % and 12 % of the total EL3s and LL3/L4s, respectively. Coefficients of variation varied from 33 to 183 % for EL3 counts and 38-245% for LL3/L4 counts. There were no statistically significant associations between EL3 counts and either organ or location. For LL3/L4 counts there were no statistically significant differences between the three locations within organs (p = 0.1166), but the DC had significantly lower counts than the other two organs (p < 0.0001). Increasing the number of mucosal replicates from each organ improved estimation, but required a considerably increased workload. In conclusion, mucosal larval cyathostomin counts are highly variable, complicating their use for treatment efficacy estimation.