Using droplet digital PCR for the detection of hco-acr-8b levamisole resistance marker in H. contortus.

The nematode Haemonchus contortus is one of the most prevalent and pathogenic parasites in small ruminants. Although usually controlled using anthelmintics, the development of drug resistance by the parasite has become a major issue in livestock production. While the molecular detection of benzimidazole resistance in H. contortus is well developed, the molecular tools and protocols are far less advanced for the detection of levamisole resistance. The hco-acr-8 gene encodes a critical acetylcholine susceptible subunit that confers levamisole-sensitivity to the receptor. Here, we report the development of a droplet digital PCR assay as a molecular tool to detect a 63 bp deletion in the hco-acr-8 that has been previously associated with levamisole resistance. Sanger sequencing of single adult H. contortus yielded 56 high-quality consensus sequences surrounding the region containing the deletion. Based on the sequencing data, new primers and probes were designed and validated with a novel droplet digital PCR assay for the quantification of the deletion containing "resistant" allele in genomic DNA samples. Single adult worms from six phenotypically described isolates (n = 60) and from two Swedish sheep farms (n = 30) where levamisole was effective were tested. Even though a significant difference in genotype frequencies between the resistant and susceptible reference isolates was found (p = 0.01), the homozygous "resistant" genotype was observed to be abundantly present in both the susceptible isolates as well as in some Swedish H. contortus samples. Furthermore, field larval culture samples, collected pre- (n = 7) and post- (n = 6) levamisole treatment on seven Swedish sheep farms where levamisole was fully efficacious according to Fecal Egg Count Reduction Test results, were tested to evaluate the frequency of the "resistant" allele in each. Frequencies of the deletion ranged from 35 to 80% in the pre-treatment samples, whereas no amplifiable H. contortus genomic DNA was detected in the post-treatment samples. Together, these data reveal relatively high frequencies of the 63 bp deletion in the hco-acr-8 both on individual H. contortus and field larval culture scales, and cast doubt on the utility of the deletion in the hco-acr-8 as a molecular marker for levamisole resistance detection on sheep farms.