Sensitivity of Haemonchus contortus to anthelmintics using different in vitro screening assays: a comparative study

Abstract

Background Helminthiasis and resistance to commercial anthelmintic compounds are major causes of economic losses for livestock producers, resulting in an urgent need for new drugs and reliable in vitro screening tests capable of detecting potentially active products. Considering this, a series of novel benzimidazole derivatives (5-methylbenzimidazole 1,2-disubstituted, 5-carboxybenzimidazole, 5-methylbenzimidazole 2-one) was screened on exsheathed L3 (xL3) and on the adult stage of Haemonchus contortus (Kirby anthelmintic-susceptible McMaster isolate). Methods This work presents the set-up of an automated motility assay on the xL3 stage of H. contortus using an infrared tracking device (WMicrotracker One) together with a larval development test (xL3 to L4) and a motility assay on the adult stage of H. contortus. A comparative study of the sensitivity of these in vitro assays using commercial anthelmintics with different mechanisms of action was carried out, also evaluating anthelmintic activity of a series of novel benzimidazole derivatives. Results The automated xL3 assay had the great advantage of being able to analyze many compounds simultaneously, but it showed the limitation of having lower sensitivity, requiring higher concentrations of the commercial anthelmintics tested compared to those needed for the adult motility or development assays. Although none of the novel 1,2,5-tri-substituted benzimidazole derivatives could significantly decrease the motility of xL3s, one of them (1e) significantly affected the development of xL3s to L4, and five new compounds (1b, 1d, 1e, 2a and 2c) reduced the motility of H. contortus adult stage. Conclusions The analysis of the results strongly suggests that the in vitro xL3 to L4 development test, particularly for the L4 stage, could be closer to the pharmacological sensitivity of the adult stage of H. contortus (target of interest) for commercial anthelmintic selected, with different mechanisms of action, and for the series of benzimidazole derivatives assayed. Therefore, an automated motility assay on L4 using the infrared tracking device is being set up. Further studies will be conducted to evaluate the in vivo anthelmintic activity of the most active novel benzimidazole derivatives. Graphical Abstract

Larval motility assay using WMicrotracker™: a high throughput test to discriminate between resistance and susceptibility to anthelmintic drugs in nematodes

“…Therefore, we studied WMi application on H. contortus L3 larvae and compared ML efficacy in two isolates, one susceptible and one, collected in farm, suspected of being ML resistant. Indeed, the IC50 values obtained for IVM and MOX (Table 2) were consistent with the concentrations previously reported for motility assays in xL3s after 24 hours of incubation (Munguía et al, 2022;Taki et al, 2021). Then, lower efficacy was observed for the three drugs in the resistant isolates.…”

“…We recommend avoiding ML contact with multiple containers to improve ML availability during assay. (Munguía et al, 2022).…”

“…Motility of nematodes was assessed from score activity recording using the WMicroTracker™ One (WMi) from PhylumTech (Santa Fe, Argentina), which detects infrared microbeam interruptions due to worm movement in liquid media. The method used has been adapted from protocols previously described for C. elegans (Bianchi et al, 2015; Risi et al, 2019; Simonetta et al, 2009; Simonetta and Golombek, 2007) and H. contortus (Munguía et al, 2022). The capacity of IVM, MOX and EPR to inhibit the worm motility was measured in a dose dependent assay by adding the drugs at increasing concentration (0.0125-1 µM for C. elegans and 0.01-100 µM for H. contortus ).…”

Current Landscape of Methods to Evaluate Antimicrobial Activity of Natural Extracts

“…Active extracts or pharmaceuticals used against helminths are named anthelmintic; these drugs are used to regulate the uncontrollable reproduction of worms and are expected to eliminate the parasites either by killing them or stimulating their escape from the infected organism [ 132 ]. Unlike antiprotozoal activity, microscopy’s observation is the gold standard for identifying anthelmintic activity.…”

Drug Discovery and Development for Soil-Transmitted Helminthiasis: Current Anthelmentics and Compounds in the Pipeline

“…Because of substitution in these compounds would not meet the structure-activity relationships conditions necessary to bind to tubulins at the anthelmintic benzimidazole binding site. These new benzimidazole derivatives could bind tubulins at a different site from 2-methyl-benzimidazole carbamate, or present a different drug target, so further studies should be conducted to identify the pharmacological target of these potential anthelmintic compounds [96]. Another modification on the benzimidazole scaffold, a coumarin-benzimidazole hybrid (51), was found to paralyze earthworms equipotent to albendazole.…”