Abstract
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.
Highlights
Haemonchus contortus is one of the most pathogenic and commonly encountered haematophagous, parasitic gastrointestinal nematode (GIN) species, responsible for contributing to productivity and profit ability setbacks in the small-ruminant farming sector across the world (Charlier et al, 2014, 2020)
Due to the worrying state of highly pathogenic GINs, such as H. contortus, found exhibiting elevated resistance to benzimidazoles, macrocyclic lactones and monepantel (Hoglund et al, 2009, 2015, 2020), it is vitally important to maintain the efficacy of levamisole – currently the “last line of defense” drug against GIN infections in small ruminants in Sweden
In order to monitor the development of levamisole resistance by H. contortus on Swedish sheep farms, a molecular screening tool for the rapid detection and estimation of resistance-encoding alleles in field samples would be of immense use
Summary
Haemonchus contortus is one of the most pathogenic and commonly encountered haematophagous, parasitic gastrointestinal nematode (GIN) species, responsible for contributing to productivity and profit ability setbacks in the small-ruminant farming sector across the world (Charlier et al, 2014, 2020). The most effective and reliable measure to control H. contortus (as well as other GIN) infections is through the use of broad-range anthelmintic drugs. Levamisole is a broad-spectrum anthelmintic drug, used for the treatment of GIN-infected sheep beginning in the 1960s. It exerts its effect by targeting the nicotinic acetylcholine receptors (nAChRs) pre sent in nematode body-wall muscles, where activation of the receptor by levamisole causes spastic muscle paralysis that, subsequently in capacitates the worm, resulting in its expulsion from the host (Martin et al, 2012). Generally considered as a last-line drug in terms of efficiency, reports describing levamisole-resistant field H. contortus range from multiple-decades old (van Wyk et al, 1989; Waruiru, 1997) to quite recent (Almeida et al, 2010; Chaparro et al, 2017)
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