Grazing ruminants suffer from various helminth infections, particularly those caused by gastrointestinal nematode (GIN) parasites. Today, the most reliable treatment for these diseases relies on the use of anthelmintic pharmaceuticals. Ivermectin (IVM), belonging to the macrocyclic lactone (ML) class, is the most important anthelmintic drug used worldwide in veterinary and human medicine. Moxidectin (MOX), another ML, is more effective than IVM against diverse resistant isolates. Eprinomectin (EPR) is the only anthelmintic drug that does not necessitate a milk withdrawal period during lactation. Inevitably, intensive use of an anthelmintic class has selected drug-resistant parasite populations globally in many animal species. The standard method for detecting resistance, the Faecal Egg Count Reduction Test (FECRT), is susceptible to misinterpretation, leading to flawed management decisions that undermine parasite control efforts. Thus, there is a pressing need for robust resistance detection methods in field parasites. We investigated the potential of the WMicrotrackerTM (WMi) motility assay, previously unexplored in ML resistance assessment. The aim of this research was to evaluate its potential to discriminate susceptible from resistant nematodes. Then, we evaluated the suitability of such methods for assessing the drug tolerance status to IVM, MOX, and EPR of several C. elegans strains of known resistance status and tested its applicability on a field parasite of interest, H. contortus.
Methods
Larval Motility Assay (LMA)
The susceptibility to MLs of the C. elegans strains and H. contortus isolates was determined in a larval motility assay (LMA). Motility of nematodes was assessed from score activity recording using the WMicroTracker™ One. 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). The drugs were solubilized in DMSO, and the final concentration of DMSO in the assay was below 0.5 %.
LMA on C. elegans strains: The LMA on C. elegans measures the potency of anthelmintics in inhibiting motility in young adults. Synchronized young adults (40-50 per well) were seeded into a final volume of 200 μl M9 in a 96-flat well plate. Plates were incubated 25 min at 21°C to allow the worms to settle. Then, basal activity was measured for 30 minutes to normalize the movement activity in each well at the beginning of the assay. Immediately after drug treatment, each score activity was recorded for a 120-minute period. Motility percentages were calculated for each treated well as –fold induction relative to DMSO treated worms which was set to 100.
LMA on the H. contortus isolates: The LMA on H. contortus measures the potency of anthelmintics in inhibiting motility in infective L3 larvae (iL3). Prior to experiment, H. contortus larvae were treated to discard cuticle. Briefly, worms were incubated 20 minutes at 37°C, in tap water supplemented by NaCl 0.15 % and vigorously shacked by vortex every 5 minutes. To prevent larval aggregation, larvae were filtered through a 40 μm mesh in LB medium. Each well of a 96-flat well plate received 80 iL3 suspended in a 200 μl final volume of LB medium. Then, the plates were treated with compounds and subsequently incubated at 37°C for 24 hours within a humidified incubator. Following the incubation period, motility of the larvae (iL3) was restored by exposing them to light at room temperature for 5 minutes. Thereafter, the movement of worms within each well was recorded over a 15-minute duration using WMi technology. The motility of worms in each well was then standardized against the average motility of control wells to derive the motility inhibition values (%). Each value was the mean of triplicate and the experiments were performed at least 3 times.
Results
IVM efficacy in adult C. elegans
Dose-response curves Fig. 1B showed that IVM was able to alter worm motility of all strains. However, IVM displayed different potencies in affecting C. elegans motility. Indeed, similar potency of IVM to inhibit motility was observed for N2B and nhr-8 deficient worms as shown by the superposition of the two dose-response curves and by the comparable EC50 values (33.52 ± 8.89 nM and 29.26 ± 6.33 nM, respectively). In contrast, the dose-response curve was significantly shifted to the right for the IVM-selected strain, reflecting a decrease in susceptibility to IVM. Indeed, IC50 of IVM was 2.12-fold higher for IVR10 than wild-type (71.20 ± 26.49 nM and 33.52 ± 8.89 nM respectively, P<0.0001).
Impact of MOX and EPR on adult C. elegans motility
Table 1 shows that IVR10 strain was more tolerant to the three drugs tested compared with the wild-type strain with IVM being the most potent drug. The trend towards greater IVM efficacy on worm motility phenotype seems to be continuing also in nhr-8 deficient worm, however differences are not significant compared with EPR treatment. The less potent drug toward motility in this strain was MOX.
Motility assay to discriminate susceptible from resistant H. contortus in the field
The results of the dose-response experiments with IVM, MOX and EPR against both susceptible and resistant H. contortus isolates are shown in Fig 2. As expected, increasing concentrations of all drugs were able to alter worm motility of both strains. However, there was a significant difference on their potency. Firstly, all three drugs were more effective on the susceptible strain than on the resistant strain, as demonstrated by the lower IC50 values. Secondly, on both isolates, MOX was the most potent drug when compared to IVM and EPR as shown by the smaller IC50 within isolates (, P<0.0001 versus IVM treatment.). However, the degree of resistance to MOX for the resistant isolate was identical to that of IVM as demonstrated by the same resistance factor.
Dose-response curves indicated differences in Ivermectin (IVM) susceptibility among strains of C. elegans. Additionally, the investigation using Haemonchus contortus revealed significant differences in drug potency between susceptible and resistant isolates. The motility assay effectively discriminated susceptible from resistant isolates in both C. elegans and H. contortus. Our findings demonstrate, for the first time, the relevance of the motility assay developed by WMi as a functional indicator of resistance in nematodes, offering a promising avenue for detecting resistance to Macrocyclic Lactones (MLs).
bioRxiv version posted May 5, 2024 https://www.biorxiv.org/content/10.1101/2024.05.02.592150v1
Mélanie Alberich, Marie Garcia, Julie Petermann, Clara Blancfuney, Sophie Jouffroy, Philippe Jacquiet, Anne Lespine.