While the F200Y SNP in the beta-tubulin gene is most commonly associated with benzimidazole resistance in trichostrongylid nematodes, other SNPs as well as drug efflux pathways have been implicated in the resistance. The relative contributions of all these mechanisms are not understood sufficiently to allow expected drug efficacy to be inferred from molecular data. As a component of developing better means to interpret molecular resistance tests, the present study utilised a drug resistant Haemonchus contortus isolate which possesses two of the principal benzimidazole resistance SNPs (E198A and F200Y) in order to assess the relative degree of resistance conferred by the two SNPs. We exposed larvae to a range of thiabendazole concentrations in in vitro development assays, and collected the surviving L3 larvae at each drug concentration to establish sub-populations showing increasing levels of resistance. We then sequenced the isotype 1 beta-tubulin gene in pooled larval samples, and measured allele frequencies at the two SNP positions. The frequency of the resistance allele at the 198 position increased as the thiabendazole concentration increased, while the frequency of the resistance allele at the 200 position decreased. Genotyping of individual larvae showed that the highest drug concentration was associated with the removal of all genotypes except for homozygous resistance at the 198 position alongside homozygous susceptible at the 200 position. This indicates that, at least for larval life stages, the E198A SNP is able to confer higher levels of resistance to benzimidazole drugs than the F200Y SNP, and that the homozygosity at 198 in the highly resistant individuals is mutually exclusive with heterozygosity or resistant homozygosity at the 200 position. This study illustrates the need to understand the relative contributions of different resistance mechanisms in order to maximise the degree to which molecular tests are able to inform on drug resistance phenotype.
We used an enzyme induction approach to study the role of detoxification enzymes in the interaction of the anthelmintic compound naphthalophos with Haemonchus contortus larvae. Larvae were treated with the barbiturate phenobarbital, which is known to induce the activity of a number of detoxification enzymes in mammals and insects, including cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UDPGTs), and glutathione (GSH) S-transferases (GSTs). Cotreatment of larvae with phenobarbital and naphthalophos resulted in a significant increase in the naphthalophos 50% inhibitory concentration (IC 50 ) compared to treatment of larvae with the anthelmintic alone (up to a 28-fold increase). The phenobarbital-induced drug tolerance was reversed by cotreatment with the UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, probenecid, and sulfinpyrazone. Isobologram analysis of the interaction of 5-nitrouracil with naphthalophos in phenobarbital-treated larvae clearly showed the presence of strong synergism. The UDPGT inhibitors 5-nitrouracil, 4,6-dihydroxy-5-nitropyrimidine, and probenecid also showed synergistic effects with non-phenobarbital-treated worms (synergism ratio up to 3.2-fold). This study indicates that H. contortus larvae possess one or more UDPGT enzymes able to detoxify naphthalophos. In highlighting the protective role of this enzyme group, this study reveals the potential for UDPGT enzymes to act as a resistance mechanism that may develop under drug selection pressure in field isolates of this species. In addition, the data indicate the potential for a chemotherapeutic approach utilizing inhibitors of UDPGT enzymes as synergists to increase the activity of naphthalophos against parasitic worms and to combat detoxification-mediated drug resistance if it arises in the field.
Resistance to the anthelmintic drug monepantel (Zolvix®) has emerged in parasitic worms infecting sheep and goats. The mechanism of resistance in these cases is unknown. The drug targets nicotinic acetylcholine receptors belonging to the nematode-specific DEG-3 subfamily. We examined the receptor gene, Hco-mptl-1, in a highly Zolvix®-resistant and a -susceptible isolate of the parasitic nematode Haemonchus contortus. cDNA coding for the full length receptor protein (Hco-MPTL-1) was present in all clones prepared from a pool of susceptible larvae (21/21 clones) and approximately 50% of those from the resistant isolate (17/33). On the other hand, the remaining clones from the resistant isolate showed various mutations that resulted in truncated predicted proteins, missing at least one transmembrane domain. The most common mutation (11/33 clones) resulted in the retention of intron 15, a premature stop codon, and a truncated protein. Sequencing of intron 15 genomic DNA showed very few SNPs in susceptible larvae and in 12/18 clones from resistant larvae, alongside the presence of at least 17 SNPs in the remaining resistant clones. The present study shows that the highly resistant isolate has a number of mutations in the drug target gene that would most-likely result in a non-functional receptor, thus rendering the larvae insensitive to the drug. The presence of many wild-type sequences in this highly-resistant population suggests that there was a significant presence of heterozygotes in the survivors of the field drench treatment from which the isolate was derived, and hence that at least some of the mutations may be dominant. Alternatively, their presence may be due to the additional influence of mutations at another locus contributing to the resistance phenotype. The presence of multiple separate mutations in the Hco-mptl-1 gene in this viable field-derived worm isolate may at least partly explain why resistance to Zolvix® has arisen rapidly in the field.
Macrocyclic lactone (ML) drugs inhibit pharyngeal pumping, motility and egg laying in parasitic nematodes. Previous work has indicated that in vitro effects on worm feeding occurred at lower ivermectin concentrations than effects on worm motility, suggesting that the pharynx musculature was a more important target site for the ML drugs than somatic musculature. We have reassessed this issue of relative sensitivity by examining the response of drug-susceptible and -resistant adult Haemonchus contortus worms to abamectin in vitro using both feeding and motility assays. The motility assay involved observation of changes in the form and degree of movement of individual worms in response to the drug. A comparison of the data from the two assays indicated that worm motility was affected at drug concentrations below those required to inhibit feeding. Analysis of the motility data using different levels of sensitivity (varying in the degree to which they accounted for subtle vs. more profound changes in worm motility) provided an explanation as to why earlier reports had observed feeding to be the more sensitive target. Motility IC50 values shifted from being less than feeding IC50s to being greater than the feeding IC50s as the motility assay analysis method became less sensitive. The present study indicates that when sensitive worm motility assessment methods are utilised, worm motility is affected at lower abamectin concentrations than worm feeding, and hence highlights somatic musculature as a more important target site for this ML drug, and most likely for ML drugs in general.
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