A molecular tool for species identification and benzimidazole resistance diagnosis in larval communities of small ruminant parasites

This report describes a molecular method for determining in a first step the generic composition of a nematode community and in a second step, the resistance of each species to benzimidazole (BZ). We first established a polymerase chain reaction (PCR) linked to a restriction fragment length polymorphism strategy using the isotype 1 beta-tubulin gene. This method overcame the limitations of morphological identification of larval stages of trichostrongylid nematode species. Geographically distant isolates from the three main gastrointestinal species in temperate zones, Teladorsagia circumcincta, Haemonchus contortus, and Trichostrongylus colubriformis, were distinguished using this method. We then used an allele-specific PCR (AS-PCR) to detect mutations of residue 200 of the beta-tubulin, which is implicated in BZ resistance. The sequences of several samples confirmed the BZ-resistance genotype determined by AS-PCR. The ability to process large numbers of samples simultaneously makes this PCR-based strategy particularly suitable for epidemiological studies. It may also be useful for monitoring the emergence of resistant alleles in nematode communities.