Development of the larval migration inhibition test for comparative analysis of ivermectin sensitivity in cyathostomin populations
Introduction
Cyathostomins are highly prevalent and potentially pathogenic parasitic nematodes found in the large intestine of horses and other equids worldwide. The cyathostomin group comprises around 50 species (Lichtenfels et al., 2008); however, little is known of the ecology of the individual species or how they interact with one another in the host or in external environment. Anthelmintic resistance (AR) is a major issue in this group of nematodes: resistance to benzimidazoles (BZ) is widespread and, in some areas, resistance to pyrantel, a member of the tetrahydropyrimidine (THP) class, is highly prevalent (Kaplan, 2002). Reduced sensitivity to the macrocyclic lactone (ML) anthelmintics, ivermectin (IVM) and moxidectin (MOX), has also been recorded in cyathostomin populations (Trawford et al., 2005, Trawford and Burden, 2009, Molento et al., 2008, Traversa et al., 2012, Relf et al., 2014). Multi-class resistance in single populations to BZ and THP class anthelmintics is also commonly reported (Kaplan et al., 2004, Canever et al., 2013, Lester et al., 2013). As no new anthelmintic classes are being developed for use in horses in the short to medium term, and reversion to anthelmintic sensitivity does not seem to readily occur in resistant nematode populations (Jackson and Coop, 2000), it is essential to preserve efficacy of the currently effective products. For these reasons, tests that facilitate decisions regarding anthelmintic treatment in horses will play an increasingly important role in control (Matthews, 2014). In this context, it is important to identify AR as soon as practically possible so that measures can be taken to prevent its spread (Tandon and Kaplan, 2004). The faecal egg count reduction test (FECRT) is currently the ‘gold standard’ non-invasive test for assessing anthelmintic efficacy in horses (Vidyashankar et al., 2012). This test is relatively labour intensive to implement: faecal samples for analysis need to be obtained on at least two occasions and it is often a challenge to obtain adequate numbers of horses with a faecal egg count (FEC) of sufficient magnitude to perform the test with high accuracy. The non-uniform distribution of eggs within and between faecal samples further complicates data analysis (Denwood et al., 2010). For these reasons, AR detection methods that are more efficient to perform and subject to less variability need to be investigated. For cyathostomins, such tests should focus on ML anthelmintics. This is because these products hold the major market-share worldwide and, as the prevalence of ML resistance is currently less advanced than with BZ and THP anthelmintics (Molento et al., 2012), tests that inform on sensitivity to ML are likely to have most impact on mitigating the spread of resistance. Molecular mechanisms leading to ML resistance in cyathostomins remain to be defined, so there are no molecular tests available, leaving bench-based (in vitro) tests as the remaining option.
The larval development test has been investigated for measuring anthelmintic sensitivity in cyathostomins; however, this test has not proven particularly informative in defining ML sensitivity levels (Tandon and Kaplan, 2004, Lind et al., 2005, Matthews et al., 2012). An alternative test that has been investigated for assessing anthelmintic sensitivity in ruminant nematode (Demeler et al., 2010, Demeler et al., 2012, Demeler et al., 2013) and in cyathostomin (van Doorn et al., 2010) populations is the larval migration inhibition test (LMIT). Here, we assessed the potential of the LMIT for measuring IVM sensitivity in cyathostomin larvae derived from different equine populations. This test was deemed appropriate for purpose because the major targets of IVM are ligand-gated chloride channels, which, when bound, result in nematode paralysis (Shoop et al., 1995) and hence will affect the ability of larvae to migrate through small pores of a filter. The cyathostomin populations examined in the current study were derived from groups of donkeys demonstrated previously, by FECRT, to exhibit differing levels of sensitivity to IVM in vivo (Trawford and Burden, 2009), or from equids administered with minimal or no ML treatments (Wood et al., 2012). The proportion of L3 that migrated through a pore filter at increasing concentrations of IVM were measured and the data compared amongst individual equids and between populations to inform on the value of this test.
Section snippets
Populations
Parasites from six equine populations were used: four consisted of donkeys and two of ponies. Three donkey herds (Populations A–C) were based at the UK Donkey Sanctuary (Devon, England, UK). The three populations were grazed separately on geographically distinct farms. Donkeys in Populations A and B had been demonstrated previously, by FECRT, to harbour cyathostomins that exhibited reduced sensitivity to IVM (Trawford and Burden, 2009) and MOX (Trawford et al., 2005). In Population C, the
Value of the LMIT results in defining IVM sensitivity amongst cyathostomin populations
When the test was run on seven occasions on different days using L3 derived from a single donkey, it was found that L3 stored for shorter periods in culture generally exhibited higher migration in the presence of IVM, but the differences observed between storage time points were not significant. The L3 migration values were then compared amongst the six cyathostomin populations. The percentage of times that <70% migration in the control wells was observed was 10%. When <70% migration was
Discussion
Macrocyclic lactone anthelmintics, such as IVM, that paralyse nematode somatic muscles, among other modes of action, have been assessed in vitro via their effect on larval motility or migration. Such tests are potential options for detecting anthelmintic resistance because they are cheap, relatively quick to perform, preclude host influences and, as they can be run over a concentration range, may provide reproducible parameters with which to measure phenotype (Demeler et al., 2013). Several
Authors’ contributions
J.M., I.H. and C.M. designed the study. C.M. and A.R. performed the LMIT. F.B. and J.H. provided some of the faecal samples. C.M. and I.H. performed the data analysis. J.M. and C.M. drafted the manuscript, I.H. generated the figures and the other authors provided comments to the manuscript and approved the final version.
Conflict of interest
None of the authors have an actual or potential conflict of interest, including financial, personal or other relationship that could inappropriately influence, or be perceived to influence, the work presented in this manuscript.
Acknowledgements
The authors would like to thank the UK Donkey Sanctuary and the Horse Trust for funding this project. They would also like to thank Mel Slote of the Norfolk Wildlife Trust for supplying the faecal samples from Populations E and F and Professor Janina Demeler (Free University of Berlin) for providing technical advice on development of the LMIT.
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These authors contributed equally to the work described in this manuscript.