In vitro levamisole selection pressure on larval stages of Haemonchus contortus over nine generations gives rise to drug resistance and target site gene expression changes specific to the early larval stages only

Abstract

There is some evidence that resistance to levamisole and pyrantel in trichostrongylid nematodes is due to changes in the composition of nicotinic acetylcholine receptors (nAChRs) which represent the drug target site. Altered expression patterns of genes coding for nAChR subunits, as well as the presence of truncated versions of several subunits, have been implicated in observed resistances. The studies have mostly compared target sites in worm isolates of very different genetic background, and hence the ability to associate the molecular changes with drug sensitivity alone have been clouded to some extent. The present study aimed to circumvent this issue by following target site gene expression pattern changes as resistance developed in Haemonchus contortus worms under laboratory selection pressure with levamisole. We applied drug selection pressure to early stage larvae in vitro over nine generations, and monitored changes in larval and adult drug sensitivities and target site gene expression patterns. High level resistance developed in larvae, with resistance factors of 94-fold and 1350-fold at the IC50 and IC95, respectively, in larval development assays after nine generations of selection. There was some cross-resistance to bephenium (70-fold increase in IC95). The expression of all the putative subunit components of levamisole-sensitive nAChRs, as well as a number of ancillary protein genes, particularly Hco-unc-29.1 and –ric-3, were significantly decreased (up to 5.5-fold) in the resistant larvae at generation nine compared to the starting population. However, adult worms did not show any resistance to levamisole, and showed an inverse pattern of gene expression changes, with many target site genes showing increased expression compared to the starting population. A comparison of the larval/adult drug sensitivity data with the known relationships for field-derived isolates indicated that the adults of our selected population should have been highly resistant to the drug if the larval/adult sensitivity relationships were in accordance with previous field isolates. Hence, our selected worms showed a life-stage drug sensitivity pattern quite different to that seen in the field. The present study has highlighted an association between drug target site changes and resistance to levamisole in H. contortus larvae. However, it has also highlighted the artificial nature of the larval selection method with levamisole, as the resistance phenotype and the associated molecular changes were only observed in the drug-pressured life stage. The study therefore reinforces the need for caution in extrapolating larval-based laboratory selection outcomes to field resistances.