Use of developmental temperature and gastrointestinal tract location to isolate pure Trichostrongylus vitrinus from mixed, naturally acquired trichostrongylid infections in sheep

Objective To isolate Trichostrongylus vitrinus from naturally‐acquired mixed trichostrongylid infections (predominantly Trichostrongylus spp. and Teladorsagia circumcincta) in sheep using differential larval development temperatures and the anatomical location of adults. Methods Faeces were collected from ewes with mixed, naturally acquired trichostrongylid infection, and incubated at low temperatures (4°C or 8°C) for 20–68 days depending on temperature. Harvested infective larvae were passaged through worm‐free lambs, from which the first 5 m of small intestine was collected and adult worms recovered. Purity of infection with T. vitrinus was assessed by examination of spicules on 100 adult male worms. Eggs of recovered adult female worms were cultured at 25°C and harvested larvae were re‐passaged through worm‐free lambs. Results Low temperature incubation (≤8°C) successfully exploited variation in the developmental temperature required by pre‐parasitic stages of small intestinal nematodes, allowing T. vitrinus to develop while others did not. Differential organ harvest from infected sheep allowed isolation of T. vitrinus from other gastrointestinal nematodes, with amplification of pure T. vitrinus achieved by passage through a further generation of lambs. Conclusion A successful method for the isolation of pure T. vitrinus from mixed infections is described. The resulting pure strain of T. vitrinus has been amplified and is held by two institutions for use in research (CSIRO reference SARDI 2011 strain).