Abstract
Gastro-intestinal nematodes in ruminants, especially Haemonchus contortus, are a global threat to sheep and cattle farming. The emergence of drug resistance, and even multi-drug resistance to the currently available classes of broad spectrum anthelmintics, further stresses the need for new drugs active against gastro-intestinal nematodes. A novel chemical class of synthetic anthelmintics, the Amino-Acetonitrile Derivatives (AADs), was recently discovered and the drug candidate AAD-1566 (monepantel) was chosen for further development. Studies with Caenorhabditis elegans suggested that the AADs act via nicotinic acetylcholine receptors (nAChR) of the nematode-specific DEG-3 subfamily. Here we identify nAChR genes of the DEG-3 subfamily from H. contortus and investigate their role in AAD sensitivity. Using a novel in vitro selection procedure, mutant H. contortus populations of reduced sensitivity to AAD-1566 were obtained. Sequencing of full-length nAChR coding sequences from AAD-susceptible H. contortus and their AAD-1566-mutant progeny revealed 2 genes to be affected. In the gene monepantel-1 (Hco-mptl-1, formerly named Hc-acr-23H), a panel of mutations was observed exclusively in the AAD-mutant nematodes, including deletions at intron-exon boundaries that result in mis-spliced transcripts and premature stop codons. In the gene Hco-des-2H, the same 135 bp insertion in the 5′ UTR created additional, out of frame start codons in 2 independent H. contortus AAD-mutants. Furthermore, the AAD mutants exhibited altered expression levels of the DEG-3 subfamily nAChR genes Hco-mptl-1, Hco-des-2H and Hco-deg-3H as quantified by real-time PCR. These results indicate that Hco-MPTL-1 and other nAChR subunits of the DEG-3 subfamily constitute a target for AAD action against H. contortus and that loss-of-function mutations in the corresponding genes may reduce the sensitivity to AADs.
Highlights
Throughout the world, successful livestock production of ruminants is hampered by gastro-intestinal nematodes
Haemonchus contortus, the largest nematode found in the abomasum of sheep and cattle, is a blood-feeding parasite that causes severe anemia that can lead to the sudden death of the infected animal; H. contortus is highly susceptible to Amino-Acetonitrile Derivatives (AADs)
In order to elucidate the mode of action of the AADs, we have developed 2 independent H. contortus mutants with reduced sensitivity to monepantel (AAD-1566)
Summary
Throughout the world, successful livestock production of ruminants is hampered by gastro-intestinal nematodes. Haemonchus contortus in particular is responsible for substantial losses to the global sheep industry [1]. Haemonchus contortus is a blood-feeding nematode that inhabits the abomasum of sheep, producing in acute infections, severe anemia that can lead to the death of infected animals. Broad spectrum chemotherapy against gastro-intestinal nematodes is restricted to 3 anthelmintic classes: the benzimidazoles, such as albendazole and oxfendazole, the imidazothiazoles, including levamisole and tetramisole and the macrocyclic lactones (e.g. ivermectin, moxidectin, abamectin and doramectin). In some countries in the southern hemisphere, sheep farming is severely endangered by such populations [4], further increasing the need for a new class of anthelmintic [11]
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