Establishment of a robotic, high-throughput platform for the specific diagnosis of gastrointestinal nematode infections in sheep

[Display omitted] ► A robotic, high throughput molecular assay for diagnosis of infections with ovine gastrointestinal nematodes is assessed. ► This assay is used to investigate the epidemiology of gastrointestinal parasites of sheep in Australia. ► The molecular results are directly compared with those of routine larval culture. ► The results achieved by larval culture are influenced by the developmental success of different species. ► This molecular diagnostic platform represents a rapid, user-friendly diagnostic tool for routine diagnosis and epidemiology. The accurate diagnosis of strongylid nematode infections is central to investigating their epidemiology and for parasite control. To overcome major limitations in sensitivity or specificity of traditional methods, including faecal egg count (FEC) and/or larval culture (LC), we evaluated and established a semi-automated, high throughput multiplexed-tandem PCR (MT-PCR) platform for the diagnosis of gastrointestinal strongylid nematode infections in sheep, and established its diagnostic sensitivity (100%) and specificity (87.5%) based on the testing of 100 faecal DNA samples from helminth-free sheep and 30 samples from sheep with infections confirmed by necropsy. Subsequently, the platform was employed to test 219 faecal samples from sheep with naturally acquired infections from various geographical localities within Australia and the results compared with those from conventional LC using 139 of the 219 samples. The results obtained using both MT-PCR and LC correlated significantly for most nematodes examined, but revealed that Oesophagostomum venulosum and Chabertia ovina (parasites of the large intestine) were significantly under-represented in the LC results. The results showed that Trichostrongylus spp. (87%), Teladorsagia circumcincta (80%) and Haemonchus contortus (67%) had the highest prevalences, followed by O. venulosum (51%) and C. ovina (12%). The molecular-diagnostic platform established can be used for species- or genus-specific diagnosis of patent nematode infections within 24h (compared with 7–10days for LC), and is a sensitive and cost effective tool for routine application in research and service laboratories.