RESUMO
Chemical treatments are the main strategy to control gastrointestinal nematodes in sheep, and the emergence of anthelmintic resistance, as consequence, results in control failures and leads to economic losses. Thus, molecular tests may constitute an excellent tool for the early detection of anthelmintic resistance-related mutations. Thus, a polymerase chain reaction (PCR)-based genotyping assay followed by polyacrylamide gel electrophoresis (PAGE) was developed to detect polymorphisms in exon 11 of the acetylcholine receptor monepantel-1 gene (mptl-1) that were previously associated with monepantel resistance through a genome-wide study in Haemonchus contortus. DNA samples recovered from individual and pooled third-stage larvae from two susceptible field-derived isolates and five (three in vivo-derived and two field-derived) resistant populations were used. New polymorphisms, including a 6-bp deletion and a 3-bp insertion, were detected in resistant individuals. These indels, confirmed using sequencing of cloned PCR products, are predicted to result in amino acid changes in transmembrane domain 2 (TMD2) of the MPTL-1 protein. The two susceptible isolates showed only the presence of the wild-type allele (100%), whereas lower frequencies of the wild-type allele were detected in monepantel-resistant populations (11.1 to 66.7%). These findings report new polymorphisms in the mptl-1 gene, validate the results obtained through genomic mapping for monepantel resistance, and provide a PCR-based assay to genotype indels located in exon 11 of mptl-1 in H. contortus.
