Isolation and purification of 'Trichostrongylus vitrinus' from mixed naturally-acquired Trichostrongylid infections in sheep

Abstract

Extensive enquiries in 2011 confirmed that a pure strain of the sheep small intestinal parasite 'Trichostrongylus vitrinus' ('T. vitrinus') was no longer held in Australian laboratories. To provide for planned research, a pure strain of 'T. vitrinus' was isolated in the laboratory from mixed infections of sheep at Branxholme (South-west Victoria) and Kalangadoo (South-east South Australia). Parasite species confirmed on the properties included 'Teladorsagia circumcincta' ('Tel. circumcincta'), 'T. vitrinus', 'T. colubriformis', 'T. axei' and 'T. rugatus' (one property). Faecal samples were collected per rectum at each site in spring 2011. Faeces from each site were mixed in culture pots, covered and incubated at 4°C for 54-68 days, to prevent development of 'T. colubriformis' and 'T. rugatus' (Beveridge et a/.,1989). Harvested third stage larvae (L₃) comprised 'Trichostrongylus' spp. (undifferentiated), small numbers of 'T. axei', and 'Tel. circumcincta'. L₃ from each site were then passaged through housed worm-free Merino lambs to separate 'Tel. circumcincta' and 'T. axei' (abomasum parasites) from 'T. vitrinus'. Following patency lambs were euthanased and the contents of the proximal 5 metres of small intestine were washed and collected on a sieve (413pm), and examined microscopically. At least 100 male worms were examined for spicule morphology to confirm purity of 'T. vitrinus' infection, and all adult females recovered. Females from each site were mashed with a glass pestle in a small tube containing water to release eggs. The suspension containing eggs was mixed through "worm free" faeces, incubated at 25°C for 10 days and L₃ recovered. Recovered larvae were combined and amplified with further passage through housed "worm free" Merino lambs. An additional lamb was used to verify strain purity; no worms were recovered from the abomasum and all male worms from the small intestine were 'T. vitrinus'. This strain has been cryopreserved and is available for future research.