Real time PCR for the diagnosis of benzimidazole resistance in trichostrongylids of sheep

Abstract

This report describes a new molecular method for the diagnosis of benzimidazole susceptibility or resistance in three main species of trichostrongylids of sheep ( Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus vitrinus). This assay is based on the use of real time polymerase chain reaction (PCR) to detect mutations of residue 200 on isotype 1 of β -tubulin. The technique allows calculation of the proportion of each allelic variant as it combines kinetic (real time quantitative) PCR with allele-specific amplification and requires no post-PCR processing. The level of resistance in the population is determined by the proportion of the β -tubulin codon 200 TAC allele. Hence, it was observed that the proportion of the resistant allele in susceptible strains ranged between 24% and 32.3%; in resistant strains this value increased to between 71.3% and 86.3%. Furthermore, there was a good correlation between real time PCR, faecal egg count reduction test, egg hatch assay and conventional allele-specific PCR, in both resistant and susceptible strains. A sensitive, rapid, highly reproducible and inexpensive technique for detecting resistance to benzimidazoles in a worm population has been developed.