Feasibility of genus-specific real-time PCR for the differentiation of larvae from gastrointestinal nematodes of naturally infected sheep.

Abstract

Results of real-time PCR analysis of coproculture third stage larvae (L3) using genus specific TaqMan minor groove binder probes were compared with the results of morphological differentiation of L3 after coprocultured and direct morphological worm differentiation from gastrointestinal samples of eight sheep with naturally acquired nematodes infections. Faecal egg counts prior to postmortem confirmed infections with trichostrongyles with a geometric mean count of 4828 eggs per gram for all sheep. Individual egg counts correlated positively with total worm counts (correlation coefficient 0.794). Five different nematode species and one genus were found in the abomasi and small intestines: Cooperia curticei, Haemonchus contortus, Nematodirus spp., Teladorsagia (Ostertagia) circumcincta, Trichostrongylus axei and Trichostrongylus colubriformis. Coproculture of faecal eggs yielded five of these, Cooperia spp., Haemonchus spp., Ostertagia/Teladorsagia spp. and Trichostrongylus spp. Comparison between morphological L3 and worm differentiation data showed high congruence (94%). The agreement between PCR analysis of L3 after coproculture and direct morphological worm differentiation was 84%. Thus, real-time PCR was found to be suitable as a speedy and reliable diagnostic tool for the assessment of gastrointestinal nematode infections of ruminants in the field.