Comparison of two in vitro methods for the detection of ivermectin resistance in Haemonchus contortus in sheep

Abstract

SummaryGastrointestinal parasitic nematodes in sheep cause severe economic losses. Anthelmintics are the most commonly used drugs for prophylaxis and therapy against parasitic helminths. The problem of drug resistance has developed for all commercially available anthelmintics in several genera and classes of helminths. In vitro and in vivo tests are used to detect anthelmintic resistance. Two in vitro methods (larval migration inhibition test and micromotility test) for the detection of ivermectin (IVM) resistance were compared using IVM-resistant and IVM-susceptible isolates of Haemonchus contortus. The degree of resistance for each test was expressed as a resistance factor (RF). The micromotility test was more sensitive for quantitatively measuring the degree of resistance between susceptible and resistant isolates. The RFs for this test for IVM and eprinomectin ranged from 1.00 to 108.05 and from 3.87 to 32.32, respectively.

Motility in the L3 stage is a poor phenotype for detecting and measuring resistance to avermectin/milbemycin drugs in gastrointestinal nematodes of livestock

“… Folz et al (1987) reported less than 70% reduction in motility of L3 of H. contortus following incubation with greater than 100 μM ivermectin. Dolinská et al (2016) also reported 49.7–97.8% maximum percent reduction in L3 motility of six isolates of H. contortus following incubation with ivermectin. The dose related levels of inhibition in motility reported in Dolinská et al (2016) and Folz et al (1987) should be interpreted with caution as concentrations of ivermectin greater than 100 μM were tested, which are above the solubility limit of this compound.…”

“… Dolinská et al (2016) also reported 49.7–97.8% maximum percent reduction in L3 motility of six isolates of H. contortus following incubation with ivermectin. The dose related levels of inhibition in motility reported in Dolinská et al (2016) and Folz et al (1987) should be interpreted with caution as concentrations of ivermectin greater than 100 μM were tested, which are above the solubility limit of this compound. Thus, the actual amount of drug the worms were in contact with cannot be accurately inferred.…”

“…Thus, the actual amount of drug the worms were in contact with cannot be accurately inferred. Additionally, Dolinská et al, 2016 tested 10-fold dilutions of ivermectin and eprinomectin and did not report 95% confidence intervals for calculated EC 50 and EC 99. Even with these limitations, it is still clear that AM compounds do not completely inhibit motility of L3 in vitro .…”

“…A number of systems have been developed to quantitatively measure motility and test anthelmintic activity in nematodes ( Bennett and Pax, 1986 , Smout et al, 2010 , Marcellino et al, 2012 , Storey et al, 2014 , Nutting et al, 2015 ). However, few studies have evaluated quantitative motility to detect AM resistance in GIN of livestock ( Demeler et al, 2010a , Demeler et al, 2010b , Dolinská et al, 2016 ). In this study, we tested four isolates each of Cooperia spp.…”

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

“…In vitro assays are regarded as the most efficient for the early detection of anthelmintic resistance. In this context, monitoring worm motility via the larval migration assay in H. contortus has been suggested (Demeler et al, 2012; Dolinská et al, 2016). However, the manual counting of larvae, which is labor-intensive, could be a reason why this test isn’t commonly employed in the field for detecting drug resistance.…”