Ivermectin – Old Drug, New Tricks?

Ligand-gated ion channels, particularly glutamate-gated chloride channels, are well characterised as the targets of IVM in nematodes and insects. Nematode genomes are helping to cast light on the diversity of ion-channel subunits in different parasite species of human and veterinary importance. Resistance to IVM is an increasing problem in the control of parasitic nematodes, and resolving the mechanisms is an important research priority. Recent studies in other biological systems suggest that IVM can affect a number of additional pathways. IVM may have novel applications in the treatment and control of important human diseases. Ivermectin is one of the most important drugs in veterinary and human medicine for the control of parasitic infection and was the joint focus of the 2015 Nobel Prize in Physiology or Medicine, some 35 years after its remarkable discovery. Although best described for its activity on glutamate-gated chloride channels in parasitic nematodes, understanding of its mode of action remains incomplete. In the field of veterinary medicine, resistance to ivermectin is now widespread, but the mechanisms underlying resistance are unresolved. Here we discuss the history of this versatile drug and its use in global health. Based on recent studies in a variety of systems, we question whether ivermectin could have additional modes of action on parasitic nematodes. Ivermectin is one of the most important drugs in veterinary and human medicine for the control of parasitic infection and was the joint focus of the 2015 Nobel Prize in Physiology or Medicine, some 35 years after its remarkable discovery. Although best described for its activity on glutamate-gated chloride channels in parasitic nematodes, understanding of its mode of action remains incomplete. In the field of veterinary medicine, resistance to ivermectin is now widespread, but the mechanisms underlying resistance are unresolved. Here we discuss the history of this versatile drug and its use in global health. Based on recent studies in a variety of systems, we question whether ivermectin could have additional modes of action on parasitic nematodes. Ivermectin (IVM) is one of the best known and most widely used antiparasitic drugs in human and veterinary medicine. From a fortuitous discovery on a Japanese golf course to a Nobel Prize, the impact of IVM on human health to date has been extraordinary. Notwithstanding the role of IVM in global food production, the Mectizan Donation Program has lifted the burden of onchocerciasis (river blindness) and, subsequently, lymphatic filariasis (elephantiasis), from millions of people in the poorest countries in the world, and set a precedent for the role of public–private partnerships in global health. However, despite extensive research since its discovery over 35 years ago, the mode of action of IVM in parasitic species remains unclear, as are the mechanisms of resistance that allow some pathogens to survive treatment and thus the implications for current and future control strategies. Intriguingly, IVM has a diverse range of effects in many different organisms, far beyond the endoparasites and ectoparasites it was developed to control. For example, IVM has been shown to regulate glucose and cholesterol levels in diabetic mice [1Jin L. et al.Selective targeting of nuclear receptor FXR by avermectin analogues with therapeutic effects on nonalcoholic fatty liver disease.Sci. Rep. 2015; 5: 17288Crossref PubMed Scopus (35) Google Scholar], to suppress malignant cell proliferation in various cancers [2Yin J. et al.DEAD-box RNA helicase DDX23 modulates glioma malignancy via elevating miR-21 biogenesis.Brain. 2015; 138: 2553-2570Crossref PubMed Scopus (53) Google Scholar], to inhibit viral replication in several flaviviruses [3Mastrangelo E. et al.Ivermectin is a potent inhibitor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drug.J. Antimicrob. Chemother. 2012; 67: 1884-1894Crossref PubMed Scopus (262) Google Scholar], and to reduce survival in major insect vectors of malaria and trypanosomiasis [4Pooda H.S. et al.Administration of ivermectin to peridomestic cattle: a promising approach to target the residual transmission of human malaria.Malaria J. 2014; 13 (1, 496)Google Scholar, 5Pooda S.H. et al.Decrease in survival and fecundity of Glossina palpalis gambiensis vanderplank 1949 (Diptera: Glossinidae) fed on cattle treated with single doses of ivermectin.Parasites Vectors. 2013; 6: 165Crossref PubMed Scopus (17) Google Scholar]. Clearly, much remains to be learned about this versatile drug, but the promise of more sustainable strategies for current helminth-control programmes and novel applications to improve and democratise human health, are compelling arguments to pursue this cause. In this article we review the current uses of IVM and discuss recent studies demonstrating a remarkably wide range of drug targets in different systems. We highlight some important but unresolved questions regarding drug mode of action and mechanism of resistance in parasitic nematodes, and suggest that recently available, high-quality genomic resources for parasitic helminths are the appropriate tools to answer to these longstanding questions. In 1970, microbiologist Satoshi Ōmura collected a soil sample from woods close to a golf course in Kawana, on the south east coast of Honshu, Japan [6Van Voorhis W.C. et al.Profile of William C. Campbell, Satoshi Omura, and Youyou Tu, 2015 Nobel Laureates in Physiology or Medicine.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 15773-15776Crossref PubMed Scopus (34) Google Scholar]. Ōmura isolated and cultured a Gram-positive bacterium, sample NRRL 8165–a then unknown species of Streptomyces, which was sent to William Campbell at Merck (along with 50 other strains of Streptomyces which were considered unusual in appearance or culture characteristics) to test for antiparasitic effects. NRRL 8165 cultures showed potent activity against Nematospiroides dubius (now known as Heligomosoides polygyrus) infection in mice, and the active components were purified, revealing a family of macrocyclic lactones. These naturally occurring compounds were named the avermectins (and the bacterium, Streptomyces avermitilis) to reflect the worm-free ‘averminous’ conditions they produced [7Burg R.W. et al.Avermectins, new family of potent anthelmintic agents: producing organism and fermentation.Antimicrob. Agents Chemother. 1979; 15: 361-367Crossref PubMed Scopus (746) Google Scholar, 8Campbell W.C. An introduction to the avermectins.N. Z. Vet. J. 1981; 29: 174-178Crossref PubMed Scopus (68) Google Scholar]. Naturally produced avermectins are a mixture of four compounds, avermectin A1, A2, B1, and B2, each of which exists as two variants, a and b [8Campbell W.C. An introduction to the avermectins.N. Z. Vet. J. 1981; 29: 174-178Crossref PubMed Scopus (68) Google Scholar, 9Campbell W.C. et al.Ivermectin: a potent new antiparasitic agent.Science. 1983; 221: 823-828Crossref PubMed Scopus (639) Google Scholar]. The ‘A’ and ‘B’ designations describe the presence of methoxy or hydroxy groups at position C5, while the superscripts 1 and 2 refer to the presence of a double bond between C22 and C23 or a hydrogen at C22 and hydroxy group at C23, respectively. The ‘a’ variants have secbutyl at C25, while the ‘b’ variants have isopropyl. These subtle differences in chemical structure were found to have significant functional consequences; while initial trials found that all four avermectins showed some efficacy against gastrointestinal nematodes of sheep, avermectins of the ‘B’ series showed highest activity [10Blair L.S. Campbell W.C. Efficacy of avermectin B1a against microfilariae of Dirofilaria immitis.Am. J. Vet. Res. 1979; 40: 1031-1032PubMed Google Scholar]. Further, when given orally, avermectin B1 was more active than B2, while with parenteral administration, avermectin B2 was more active than B1 [9Campbell W.C. et al.Ivermectin: a potent new antiparasitic agent.Science. 1983; 221: 823-828Crossref PubMed Scopus (639) Google Scholar]. On this basis, development of a commercial anthelmintic focused on the ‘B’ series and the chemical structure at the C22 and C23 positions. IVM is a chemically modified derivative of naturally produced avermectin B1, comprised of ∼80% 22,23-dihydro-avermectin B1a and ∼20% 22,23-dihydro-avermectin B1b [8Campbell W.C. An introduction to the avermectins.N. Z. Vet. J. 1981; 29: 174-178Crossref PubMed Scopus (68) Google Scholar, 9Campbell W.C. et al.Ivermectin: a potent new antiparasitic agent.Science. 1983; 221: 823-828Crossref PubMed Scopus (639) Google Scholar] (Figure 1), with potent activity against a broad spectrum of parasitic nematodes after both oral and parenteral administration. IVM is not active against flukes or tapeworms, but does have activity against various arthropods, including lice, mites, and some ticks. IVM has a wide safety margin in most mammals, although some dogs with a deletion mutation in MDR1, a P-glycoprotein that functions in the blood–brain barrier, are susceptible to neurological effects [11Mealey K.L. et al.Ivermectin sensitivity in collies is associated with a deletion mutation of the mdr1 gene.Pharmacogenetics. 2001; 11: 727-733Crossref PubMed Scopus (353) Google Scholar]. The potency of IVM against both endoparasites and ectoparasites led to the creation of the term ‘endectocide’ and this first drug of its kind was introduced to the animal health market by Merck & Co. in 1981 [12Omura S. Crump A. Ivermectin: panacea for resource-poor communities?.Trends Parasitol. 2014; 30: 445-455Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar]. New formulations of IVM for different livestock species and domestic pets were released almost every year, and, by the late 1980s, IVM was the largest selling animal health product in the world (http://merial.com/en/). A number of derivatives, such as eprinomectin (topical application for farm animals, with extended activity and no milk withdrawal [13Shoop W.L. et al.Eprinomectin: a novel avermectin for use as a topical endectocide for cattle.Int. J. Parasitol. 1996; 26: 1237-1242Crossref PubMed Scopus (161) Google Scholar]) and selamectin (topical application for small animals, with a wider safety margin than IVM in dogs with the MDR1 mutation [14Bishop B.F. et al.Selamectin: a novel broad-spectrum endectocide for dogs and cats.Vet. Parasitol. 2000; 91: 163-176Crossref PubMed Scopus (103) Google Scholar]), have been developed since, to great commercial success. Two additional macrocylic lactones of commercial importance, moxidectin and milbemycin oxime, belong to a closely related but distinct family of Streptomyces-derived anthelmintics called the milbemycins. The key similarities and differences between the avermectins and milbemycins have been described elsewhere [15Prichard R. et al.Moxidectin and the avermectins: consanguinity but not identity.Int. J. Parasitol. Drugs Drug Resist. 2012; 2: 134-153Crossref PubMed Scopus (207) Google Scholar]. The market for IVM has remained exceptionally strong in the livestock industry, particularly for the control of gastrointestinal roundworms, although it is also licensed to control bovine lungworm and various ectoparasites. IVM and other macrocyclic lactones are currently the most commonly used anthelmintics in the UK sheep industry [16Burgess C.G. et al.A survey of the trichostrongylid nematode species present on UK sheep farms and associated anthelmintic control practices.Vet. Parasitol. 2012; 189: 299-307Crossref PubMed Scopus (60) Google Scholar] and in the US cattle industry [17McArthur M.J. Reinemeyer C.R. Herding the U.S. cattle industry toward a paradigm shift in parasite control.Vet. Parasitol. 2014; 204: 34-43Crossref PubMed Scopus (30) Google Scholar]. They are also the most frequently used anthelmintics to control equine roundworms in the UK [18Relf V.E. et al.A questionnaire study on parasite control practices on UK breeding Thoroughbred studs.Equine Vet. J. 2012; 44: 466-471Crossref PubMed Scopus (44) Google Scholar, 19Stratford C.H. et al.A questionnaire study of equine gastrointestinal parasite control in Scotland.Equine Vet. J. 2014; 46: 25-31Crossref PubMed Scopus (35) Google Scholar]. Additionally, there is a large market for macrocyclic lactones in the control of parasitic nematodes and ectoparasites in domestic pets. IVM is licensed to control gastrointestinal roundworms (in combination with pyrantel in dogs) and the canine heartworm, Dirofilaria immitis. IVM is not active against the adult stages of D. immitis, but it is widely used to prevent disease by targeting the developing larvae following transmission from the mosquito. IVM is active during the first 6 weeks of infection, against the L3, L4, and juvenile adult, but does not risk the potentially catastrophic effects of dead and dying mature adult worms in the heart, which is key to its value in endemic areas [20Campbell W.C. Lessons from the history of ivermectin and other antiparasitic agents.Annu. Rev. Anim. Biosci. 2016; 4: 1-14Crossref PubMed Scopus (33) Google Scholar]. While the potential value of IVM in the livestock and companion animal health market was recognised from the start, there was very little financial incentive to produce IVM for the human health market. However, its efficacy against the filarial nematodes responsible for onchocerciasis and lymphatic filariasis, moved Dr Roy Vagelos, CEO of Merck & Co., to donate as much IVM (licensed as Mectizan) ‘as was needed, for as long as needed, to anyone who needed it’ [21Molyneux D.H. Ward S.A. Reflections on the Nobel Prize for Medicine 2015 – the public health legacy and impact of avermectin and artemisinin.Trends Parasitol. 2015; 31: 605-607Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar]. Since 1987, the Mectizan Donation Program has approved 1.4 billion treatments for the control and elimination of onchocerciasis, and 1.2 billion treatments (administered with albendazole, donated by GlaxoSmithKline) for the control and elimination of lymphatic filariasis (http://www.mectizan.org/resources/2014-annual-highlights). IVM does not kill adult Onchocerca volvulus, but a single oral dose (150 μg/kg) given annually suppresses microfilarial production and prevents disease progression [22Taylor H.R. Greene B.M. The status of ivermectin in the treatment of human onchocerciasis.Am. J. Trop. Med. Hyg. 1989; 41: 460-466Crossref PubMed Scopus (64) Google Scholar]. Similarly, in lymphatic filariasis, IVM monotherapy does not kill adult Wuchereria bancrofti but is microfilaricidal, although in this case the suppression of microfilaria production is too brief to interrupt disease transmission [23Diallo S. et al.Dose-ranging study of ivermectin in treatment of filariasis due to Wuchereria bancrofti.Lancet. 1987; 1: 1030Abstract PubMed Scopus (35) Google Scholar, 24Endeshaw T. et al.Presence of Wuchereria bancrofti microfilaremia despite 7 years of annual ivermectin monotherapy mass drug administration for onchocerciasis control: a study in north-west Ethiopia.Pathog. Glob. Health. 2015; 109: 344-351Crossref PubMed Scopus (5) Google Scholar, 25Richards Jr., F.O. et al.Mass ivermectin treatment for onchocerciasis: lack of evidence for collateral impact on transmission of Wuchereria bancrofti in areas of co-endemicity.Filaria J. 2005; 4: 6Crossref PubMed Scopus (10) Google Scholar]. However, when IVM is administered annually with albendazole, control is highly successful [26Ottesen E.A. et al.Strategies and tools for the control/elimination of lymphatic filariasis.Bull. World Health Organ. 1997; 75: 491-503PubMed Google Scholar]. Furthermore, recent studies found that a single dose of IVM administered with albendazole and diethylcarbamazine (DEC) resulted in complete clearance of microfilariae, which was maintained in all patients tested after 12 months (12 of 12 patients) and 24 months (6 of 6 patients) [27Thomsen E.K. et al.Efficacy, safety, and pharmacokinetics of coadministered diethylcarbamazine, albendazole, and ivermectin for treatment of Bancroftian filariasis.Clin. Infect. Dis. 2016; 62: 334-341Crossref PubMed Scopus (119) Google Scholar]. This compared to 1 of 12 patients who was microfilaria-free after a single dose of albendazole and DEC after 12 months. While the authors were unable to identify a pharmokinetic interaction between IVM and albendazole or DEC, the findings do suggest a novel synergistic effect, resulting in either permanent sterilisation or death of the adult stage of W. bancrofti. In addition to onchocerciasis and lymphatic filariasis, IVM also clears coinfection with a number of soil-transmitted helminths, including Ascaris lumbricoides and Strongyloides stercoralis, and some ectoparasites such as Sarcoptes scabies [28Freedman D.O. et al.The efficacy of ivermectin in the chemotherapy of gastrointestinal helminthiasis in humans.J. Infect. Dis. 1989; 159: 1151-1153Crossref PubMed Scopus (60) Google Scholar, 29Whitworth J.A. et al.A field study of the effect of ivermectin on intestinal helminths in man.Trans. R. Soc. Trop. Med. Hyg. 1991; 85: 232-234Abstract Full Text PDF PubMed Scopus (35) Google Scholar, 30Meinking T.L. et al.The treatment of scabies with ivermectin.N. Engl. J. Med. 1995; 333: 26-30Crossref PubMed Scopus (319) Google Scholar]. While the efficacy of IVM in treating a broad spectrum of parasitic infections is well established, its mode of action is less clear. At nanomolar concentrations, IVM affects nematode motility, feeding, and reproduction and acts via ligand-gated chloride channels, specifically those gated by glutamate [31Cully D.F. et al.Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans.Nature. 1994; 371: 707-711Crossref PubMed Scopus (578) Google Scholar, 32Yates D.M. et al.The avermectin receptors of Haemonchus contortus and Caenorhabditis elegans.Int. J. Parasitol . 2003; 33: 1183-1193Crossref PubMed Scopus (84) Google Scholar]. Glutamate-gated chloride channels (GluCls) are not present in vertebrates, and as such are thought to confer the broad safety margin of IVM. However, at micromolar concentrations, IVM can interact with a wider range of ligand-gated channels found in both invertebrates and vertebrates, including GABA, glycine, histamine, and nicotinic acetylcholine receptors (reviewed in [33Wolstenholme A.J. Rogers A.T. Glutamate-gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics.Parasitology. 2005; 131: S85-S95PubMed Google Scholar]). GluCls are expressed in nematode motor neuron commissures, lateral and sublateral nerve cords, and pharyngeal neurons [34Portillo V. et al.Distribution of glutamate-gated chloride channel subunits in the parasitic nematode Haemonchus contortus.J. Comparative Neurol. 2003; 462: 213N222Crossref Scopus (59) Google Scholar], and the effect of IVM on worm motility and feeding presumably relates to binding to GluCls at these sites [33Wolstenholme A.J. Rogers A.T. Glutamate-gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics.Parasitology. 2005; 131: S85-S95PubMed Google Scholar]. Functional GluCls are composed of five subunits, with native GluCls containing multiple subunit types [33Wolstenholme A.J. Rogers A.T. Glutamate-gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics.Parasitology. 2005; 131: S85-S95PubMed Google Scholar]. In the free-living nematode Caenorhabditis elegans there are six genes encoding GluCl subunits, of which glc-1 is the major target of IVM [35Hibbs R.E. Gouaux E. Principles of activation and permeation in an anion-selective Cys-loop receptor.Nature. 2011; 474: 54-60Crossref PubMed Scopus (790) Google Scholar, 36Ghosh R. et al.Natural variation in a chloride channel subunit confers avermectin resistance in Caenorhabditis elegans.Science. 2012; 335: 574-578Crossref PubMed Scopus (109) Google Scholar]. However, the GluCl family appears to be remarkably divergent in parasitic nematodes, even in closely related species. The gastrointestinal parasite of sheep, Haemonchus contortus, and the human hookworms, Necator americanus and Ancylostoma ceylanicum, reside in the same phylogenetic clade as C. elegans, yet all lack glc-1 orthologues [37Laing R. et al.The genome and transcriptome of Haemonchus contortus, a key model parasite for drug and vaccine discovery.Genome Biol. 2013; 14: R88Crossref PubMed Scopus (239) Google Scholar, 38Laing R. et al.Haemonchus contortus: genome structure, organization and comparative genomics.Adv. Parasitol. 2016; 93: 569-598Crossref PubMed Scopus (19) Google Scholar]. Functional GluCl channels can, however, be generated from different combinations of subunits, and differences in the distribution and composition of the GluCl channels may contribute to differences in IVM susceptibility of different nematode species (strikingly, A. ceylanicum exhibits a 40- to 300-fold greater susceptibility to IVM than does N. americanus – in vitro and in vivo studies respectively [39Richards J.C. et al.In vitro studies on the relative sensitivity to ivermectin of Necator americanus and Ancylostoma ceylanicum.Int. J. Parasitol. 1995; 25: 1185-1191Crossref PubMed Scopus (45) Google Scholar, 40Behnke J.M. et al.Sensitivity to ivermectin and pyrantel of Ancylostoma ceylanicum and Necator americanus.Int. J. Parasitol. 1993; 23: 945-952Crossref PubMed Scopus (26) Google Scholar]), as could differential sensitivity of the other ligand-channel types referred to above [33Wolstenholme A.J. Rogers A.T. Glutamate-gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics.Parasitology. 2005; 131: S85-S95PubMed Google Scholar]. IVM also interferes with nematode fertility, a finding that is best characterised from studies on filarial worms, where it has long been recognised that IVM inhibited production of microfilariae in utero [41Chavasse D.C. et al.The effect of repeated doses of ivermectin on adult female Onchocerca volvulus in Sierra Leone.Trop. Med. Parasitol. 1992; 43: 256-262PubMed Google Scholar]. Transcriptomic analysis has since identified changes in gene expression following exposure of female Brugia malayi to 100 nM–1 μM IVM in vitro [42Ballesteros C. et al.The effects of ivermectin on Brugia malayi females in vitro: a transcriptomic approach.PLoS Negl. Trop. Dis. 2016; 10: e0004929PubMed Google Scholar], with differentially expressed transcripts particularly enriched for those involved in female reproduction. Until recently, no GluCls had been reported in the nematode reproductive tract, so the effect of IVM on fecundity was thought to be indirect [33Wolstenholme A.J. Rogers A.T. Glutamate-gated chloride channels and the mode of action of the avermectin/milbemycin anthelmintics.Parasitology. 2005; 131: S85-S95PubMed Google Scholar]. However, analysis of the B. malayi genome showed that a GluCl subunit, avr-14, was present [43Moreno Y. et al.Ivermectin disrupts the function of the excretory-secretory apparatus in microfilariae of Brugia malayi.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 20120-20125Crossref PubMed Scopus (111) Google Scholar], and using specific RNA probes this transcript was localised to the reproductive tract of adult Brugia [44Li B.W. et al.High level expression of a glutamate-gated chloride channel gene in reproductive tissues of Brugia malayi may explain the sterilizing effect of ivermectin on filarial worms.Int. J. Parasitol. Drugs Drug Resist. 2014; 4: 71-76Crossref PubMed Scopus (27) Google Scholar]. avr-14 was most strongly expressed in embryonic stages of microfilariae, as well as the uterine wall of the female worm and, to a lesser extent, the male reproductive tract, an observation that may help in defining the mechanism underlying IVM induced sterility. As described in filarial nematodes, susceptibility to IVM can also vary between different life-stages of parasite, and there is growing evidence that interactions with the host immune response play a role in the activity of IVM. In B. malayi microfilariae, an antibody against a peptide derived from AVR-14-A was used to localise GluCl to the tissue surrounding the excretory–secretory (ES) apparatus only. IVM was proposed to cause a reduction in release of proteins from the ES vesicle, which may modulate host immune responses in vivo [43Moreno Y. et al.Ivermectin disrupts the function of the excretory-secretory apparatus in microfilariae of Brugia malayi.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 20120-20125Crossref PubMed Scopus (111) Google Scholar]. This hypothesis is consistent with findings in D. immitis microfilariae, where exposure to IVM in vitro resulted in increased binding of peripheral blood mononuclear cells and neutrophils [45Vatta A.F. et al.Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro.Vet. Parasitol. 2014; 206: 38-42Crossref PubMed Scopus (32) Google Scholar]. Also, for both D. immitis and O. volvulus microfilariae, the in vitro effects of IVM required much higher concentrations than in vivo [45Vatta A.F. et al.Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro.Vet. Parasitol. 2014; 206: 38-42Crossref PubMed Scopus (32) Google Scholar, 46Bennett J.L. et al.Pharmacology of ivermectin.Parasitol. Today. 1988; 4: 226-228Abstract Full Text PDF PubMed Scopus (42) Google Scholar], supporting a role for host immune function in the activity of IVM. IVM has been widely used in veterinary species for the prophylaxis and treatment of parasitic disease, often using a mass drug administration (MDA) strategy to protect all animals considered ‘at risk’. However, applying this blanket approach has resulted in rapid selection for parasitic nematodes that are capable of surviving drug treatment. Anthelmintic resistance is now a major global problem in the control of gastrointestinal roundworms of sheep, cattle, and horses [47Kaplan R.M. Vidyashankar A.N. An inconvenient truth: global worming and anthelmintic resistance.Veterinary Parasitol. 2012; 186: 70-78Crossref PubMed Scopus (524) Google Scholar], and there are now reports of IVM resistance in the canine heartworm, D. immitis [48Wolstenholme A.J. et al.The emergence of macrocyclic lactone resistance in the canine heartworm, Dirofilaria immitis.Parasitology. 2015; 142: 1249-1259Crossref PubMed Scopus (50) Google Scholar]. Concurrently, reports of reduced embryostatic effects of IVM on O. volvulus in Ghana and Cameroon have raised concerns that IVM resistance may evolve in human parasites [49Osei-Atweneboana M.Y. et al.Phenotypic evidence of emerging ivermectin resistance in Onchocerca volvulus.PLoS Negl. Trop. Dis. 2011; 5: e998Crossref PubMed Scopus (200) Google Scholar, 50Osei-Atweneboana M.Y. et al.Prevalence and intensity of Onchocerca volvulus infection and efficacy of ivermectin in endemic communities in Ghana: a two-phase epidemiological study.Lancet. 2007; 369: 2021-2029Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 51Nana-Djeunga H.C. et al.Reproductive status of Onchocerca volvulus after ivermectin treatment in an ivermectin-naive and a frequently treated population from Cameroon.PLoS Negl. Trop. Dis. 2014; 8: e2824Crossref PubMed Scopus (38) Google Scholar]. In light of the rapid rise and spread of IVM resistance in the veterinary field, MDA of IVM as the sole means of control for onchocerciasis might be deemed a risky strategy, and there are calls for more integrated approaches [52Makepeace B.L. et al.The case for vaccine development in the strategy to eradicate river blindness (onchocerciasis) from Africa.Expert Rev. Vaccines. 2015; 14: 1163-1165Crossref PubMed Scopus (18) Google Scholar]. While the potential impact of population structure and genetic diversity (with the potential bottleneck of vector transmission for the filarial nematodes) remain unclear, increased effort to develop sensitive markers of resistance is warranted. In C. elegans, IVM resistance involves a number of genes. In 2000, Dent et al., found simultaneous mutation of three GluCl genes, glc-1, avr-14, and avr-15, conferred high levels of IVM resistance, with little or no resistance provided by mutations in any two of the genes [53Dent J.A. et al.The genetics of ivermectin resistance in Caenorhabditis elegans.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2674-2679Crossref PubMed Scopus (312) Google Scholar]. The resistance phenotype was further modulated by mutations in the innexins, unc-7 and unc-9, which are essential components of gap junctions and are required for normal locomotion and egg laying, and in four dyf genes, osm-1, osm-5, dyf-11, and che-3, which have roles in sensory neuron function [54Urdaneta-Marquez L. et al.A dyf-7 haplotype causes sensory neuron defects and is associated with macrocyclic lactone resistance worldwide in the nematode parasite Haemonchus contortus.Int. J. Parasitol. 2014; 44: 1063-1071Crossref PubMed Scopus (36) Google Scholar]. A frameshift mutation in another dyf gene, dyf-7, has since been found to confer IVM resistance in two laboratory-selected isolates of C. elegans [54Urdaneta-Marquez L. et al.A dyf-7 haplotype causes sensory neuron defects and is associated with macrocyclic lactone resistance worldwide in the nematode parasite Haemonchus contortus.Int. J. Parasitol. 2014; 44: 1063-1071Crossref PubMed Scopus (36) Google Scholar]. In natural populations of C. elegans, a four-amino-acid deletion in glc-1 was found to confer abamectin and IVM resistance in multiple diverse populations, but other resistant populations lacked this mutation [36Ghosh R. et al.Natural variation in a chloride channel subunit confers avermectin resistance in Caenorhabditis elegans.Scien