PS10-23. Genomic Identification of the Most Common Sheep Gastrointestinal Nematodes in Missouri.

Abstract Gastrointestinal nematode infections significantly impact sheep and goat production, making precise species identification crucial for effective parasite monitoring and management. Traditional microscopic identification of nematode larvae is labor-intensive, time-consuming, and requires specialized taxonomic expertise. Therefore, this study aimed to evaluate a molecular diagnostic approach based on amplification of the second internal transcribed spacer region (ITS2) of nuclear ribosomal DNA for identifying common gastrointestinal nematode larvae in sheep fecal samples. Fecal samples were collected from 25 sheep farms across Missouri. Samples from each farm were pooled separately and cultured for two weeks to allow nematode eggs to hatch and develop into third-stage larvae. Larvae were recovered using the Baermann technique and initially classified by morphology. The most frequently identified nematodes were Haemonchus contortus (44%), Teladorsagia circumcincta/Trichostrongylus spp. (33%), Oesophagostomum spp. (12%), Cooperia curticei (5%), Nematodirus spp. (2%), and Strongyloides spp. (2%), while 2% remained unidentified. Larval samples were pooled into a composite suspension containing approximately 1500 larvae/mL, and genomic DNA was extracted using the Qiagen DNeasy Plant Kit. Species-specific primers were designed to amplify the ITS2 region from the five most common nematode taxa: H. contortus, T. circumcincta, Trichostrongylus colubriformis, C. curticei, and Oesophagostomum spp. PCR amplification was performed using the Qiagen Type-it HRM PCR kit according to the manufacturer’s protocol, and amplified products were visualized to assess primer specificity and detection performance. The ITS2-based molecular assay demonstrated high analytical sensitivity, enabling the detection of low levels of parasite DNA, and showed strong specificity in differentiating among gastrointestinal nematode species. Compared with conventional microscopic examination, this molecular approach provides a faster, more reliable, and efficient method for species-level identification of gastrointestinal nematodes in sheep. These findings support the use of ITS2-targeted PCR as a sustainable diagnostic tool for parasite surveillance and management in small-ruminant production systems.