Comparison of ITS-2 rDNA nemabiome sequencing with morphological identification to quantify gastrointestinal nematode community species composition in small ruminant feces.

Mixed gastrointestinal nematode (GIN) infections are a common and significant cause of financial loss for small ruminant producers. Morphologic examination of third-stage larvae (L3) can be used to identify species composition in feces but has limitations due to the requirement for specialized expertise and the extensive time (8-15 d depending on method used) and labour involved. Moreover, differential development and survival of larvae during coproculture to the third stage often occurs. Deep amplicon sequencing of the ITS-2 rDNA locus of first-stage larvae (L1) allows for higher throughput with reduced specialist labour and reduces the risk of misidentification. Harvesting of L1 soon after hatching is also faster and further reduces labour as well as biases that can occur due to differential larval development and survival. This study compares the results of morphologic examination of L3 with those of ITS-2 rDNA deep amplicon sequencing of L1 from a set of pooled fecal samples. The proportions of eggs that were successfully recovered as larvae following culture to L3 and L1 were also compared. Larval recovery rate was significantly lower from L3 cultures than from L1 cultures (p < 0.001); eggs were 238.7 times less likely to develop to L3 than to L1 (95 % confidence interval for odds ratio 80.0-712.0). Significantly lower proportions of Teladorsagia circumcincta (odds ratio = 3.1, p = 0.008) and higher proportions of Trichostrongylus spp. (p = 0.009) were identified using morphologic examination of L3 compared with deep amplicon sequencing of L1 on the same samples. This is consistent with previous reports of differential survival of these species in L3 cultures. These results indicate that deep amplicon sequencing of L1 may reduce bias introduced by differential GIN survival to L3 in small ruminants.