Emodepside targets SLO-1 channels of Onchocerca ochengi and induces broad anthelmintic effects in a bovine model of onchocerciasis.

Onchocerciasis (river blindness), caused by the filarial worm Onchocerca volvulus, is a neglected tropical disease mostly affecting sub-Saharan Africa and is responsible for >1.3 million years lived with disability. Current control relies almost entirely on ivermectin, which suppresses symptoms caused by the first-stage larvae (microfilariae) but does not kill the long-lived adults. Here, we evaluated emodepside, a semi-synthetic cyclooctadepsipeptide registered for deworming applications in companion animals, for activity against adult filariae (i.e., as a macrofilaricide). We demonstrate the equivalence of emodepside activity on SLO-1 potassium channels in Onchocerca volvulus and Onchocerca ochengi, its sister species from cattle. Evaluation of emodepside in cattle as single or 7-day treatments at two doses (0.15 and 0.75 mg/kg) revealed rapid activity against microfilariae, prolonged suppression of female worm fecundity, and macrofilaricidal effects by 18 months post treatment. The drug was well tolerated, causing only transiently increased blood glucose. Female adult worms were mostly paralyzed; however, some retained metabolic activity even in the multiple high-dose group. These data support ongoing clinical development of emodepside to treat river blindness.

Nicotinic acetylcholine receptors: Ex-vivo expression of functional, non-hybrid, heteropentameric receptors from a marine arthropod, Lepeophtheirus salmonis.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels mostly located in the post-synaptic membrane of cholinergic synapses. The natural neurotransmitter is acetylcholine, but they are also the direct targets for neonicotinoids, chemicals widely used against ectoparasites, arthropod vectors and agricultural pests. There are significant concerns regarding adverse effects of neonicotinoids on beneficial insects. In arthropods, functional nAChRs made of α subunits have been expressed from Drosophila genes, and hybrid receptors (sometimes also referred to as chimeric receptors) using species-specific α subunits and vertebrate ß subunits have been expressed ex-vivo. Arthropod-specific nAChRs made of both α and ß subunits from the target species have not been expressed ex-vivo. The aim of the current study was to express such receptors in Xenopus oocytes using only genes from Lepeophtheirus salmonis, to characterize them and study their modulation. Genes encoding α and ß subunits of the nAChRs and three ancillary proteins, RIC-3, UNC-50 and UNC-74 were identified in the L. salmonis genome, subjected to RACE-PCR, cloned into an expression vector and the cRNA produced was then injected into Xenopus laevis oocytes. Co-expression of the ancillary proteins was essential for the successful expression of the L. salmonis nAChRs with both α and ß subunits. Two functional nAChRs were identified: Lsa-nAChR1 consisting of α1, α2, ß1 and ß2 subunits, reconstituted to one distinct receptor, while Lsa-nAChR2, consisting of α3, ß1 and ß2 subunits reconstitutes receptors with two distinct characteristics. Out of seven neonicotinoids tested, six worked as partial agonist of Lsa-nAChR1 while only three did so for Lsa-nAChR2. Four non-neonicotinoid compounds tested had no effect on either of the nAChRs. The study demonstrated that fully functional, non-hybrid nAChRs containing both α and ß subunits from an arthropod can be reconstituted ex-vivo by co-expression of essential ancillary proteins. Such models would be valuable for in-depth studies of effects by neonicotinoids and other compounds on target pests, as well as for studies of adverse effects on non-target arthropods.

Remedial Treatment of Corroded Iron Objects by Environmental Aeromonas Isolates.

Using bacteria to transform reactive corrosion products into stable compounds represents an alternative to traditional methods employed in iron conservation. Two environmental Aeromonas strains (CA23 and CU5) were used to transform ferric iron corrosion products (goethite and lepidocrocite) into stable ferrous iron-bearing minerals (vivianite and siderite). A genomic and transcriptomic approach was used to analyze the metabolic traits of these strains and to evaluate their pathogenic potential. Although genes involved in solid-phase iron reduction were identified, key genes present in other environmental iron-reducing species are missing from the genome of CU5. Several pathogenicity factors were identified in the genomes of both strains, but none of these was expressed under iron reduction conditions. Additional in vivo tests showed hemolytic and cytotoxic activities for strain CA23 but not for strain CU5. Both strains were easily inactivated using ethanol and heat. Nonetheless, given a lesser potential for a pathogenic lifestyle, CU5 is the most promising candidate for the development of a bio-based iron conservation method stabilizing iron corrosion. Based on all the results, a prototype treatment was established using archaeological items. On those, the conversion of reactive corrosion products and the formation of a homogenous layer of biogenic iron minerals were achieved. This study shows how naturally occurring microorganisms and their metabolic capabilities can be used to develop bio-inspired solutions to the problem of metal corrosion.IMPORTANCE Microbiology can greatly help in the quest for a sustainable solution to the problem of iron corrosion, which causes important economic losses in a wide range of fields, including the protection of cultural heritage and building materials. Using bacteria to transform reactive and unstable corrosion products into more-stable compounds represents a promising approach. The overall aim of this study was to develop a method for the conservation and restoration of corroded iron items, starting from the isolation of iron-reducing bacteria from natural environments. This resulted in the identification of a suitable candidate (Aeromonas sp. strain CU5) that mediates the formation of desirable minerals at the surfaces of the objects. This led to the proof of concept of an application method on real objects.

Monepantel irreversibly binds to and opens Haemonchus contortus MPTL-1 and Caenorhabditis elegans ACR-20 receptors.

Monepantel is a recently developed anthelmintic with a novel mode of action. Parasitic nematodes with reduced sensitivity to monepantel have led to the identification of MPTL-1, a ligand-gated ion-channel subunit of the parasitic nematode Haemonchus contortus, as a potential drug target. Homomeric MPTL-1 channels reconstituted in Xenopus oocytes are gated by µM concentrations of betaine and mM concentrations of choline. Measurement of reversal potentials indicated that the channel has a similar conductance for Na(+) and K(+) ions and does not permeate Ca(2+). Concentrations of monepantel (amino-acetonitrile derivative [AAD]-2225) >0.1 µM, but not its inactive enantiomer AAD-2224, induced channel opening in an irreversible manner. Currents elicited by monepantel alone were larger than the maximal current amplitudes achieved with betaine or choline, making monepantel a superagonist. Currents elicited by betaine or choline were allosterically potentiated by nM concentrations of monepantel and to a much smaller degree by AAD-2224. We have also reconstituted the Caenorhabditis elegans homomeric ACR-20 receptor in Xenopus oocytes. The acr-20 sequence has higher similarity to mptl-1 than acr-23, the primary target for monepantel mode of action in C. elegans. The ACR-20 channel is gated similarly as MPTL-1. Monepantel, but not AAD-2224, was able to induce channel opening in an irreversible manner at similar concentrations as for MPTL-1. Interestingly, the allosteric potentiation measured in the presence of betaine was much smaller than in MPTL-1 receptors. Together, these results establish the mode of action of monepantel in H. contortus and contribute to our understanding of the mode of action of this anthelmintic.

acr-23 Encodes a monepantel-sensitive channel in Caenorhabditis elegans.

Monepantel is a member of the recently identified class of anthelmintics known as the amino-acetonitrile derivatives (AADs). Monepantel controls all major gastro-intestinal nematodes in sheep including those that are resistant to the classical anthelmintics. Previous studies have shown that the Caenorhabditis elegans acr-23 and the Haemonchus contortus Hco-mptl-1 genes may be prominent targets of monepantel. With this discovery it became possible to investigate the mode of action of monepantel in nematodes at the molecular level. In the present study, we show that a C. elegans mutant acr-23 strain is fully rescued by expressing the wild-type acr-23 gene. Moreover, we present a new mutant allele, and characterize acr-23 alleles genetically. We also show that acr-23 is expressed in body wall muscle cells, and provide therefore a possible explanation for the paralysis caused by monepantel. Furthermore, genetic evidence suggests that the chaperone RIC-3 is required for expression of full monepantel resistance. Finally, we present reconstitution of the C. elegans ACR-23 receptor in Xenopus laevis oocytes and provide direct evidence of its modulation by monepantel. Conversely, co-injection of the chaperone RIC-3 had no impact for channel reconstitution in X. laevis oocytes. These results reinforce the involvement of the ACR-23 family in the mode of action of monepantel and advance our understanding of this new class of anthelmintics.

A new class of anthelmintics effective against drug-resistant nematodes.

Anthelmintic resistance in human and animal pathogenic helminths has been spreading in prevalence and severity to a point where multidrug resistance against the three major classes of anthelmintics--the benzimidazoles, imidazothiazoles and macrocyclic lactones--has become a global phenomenon in gastrointestinal nematodes of farm animals. Hence, there is an urgent need for an anthelmintic with a new mode of action. Here we report the discovery of the amino-acetonitrile derivatives (AADs) as a new chemical class of synthetic anthelmintics and describe the development of drug candidates that are efficacious against various species of livestock-pathogenic nematodes. These drug candidates seem to have a novel mode of action involving a unique, nematode-specific clade of acetylcholine receptor subunits. The AADs are well tolerated and of low toxicity to mammals, and overcome existing resistances to the currently available anthelmintics.

Nemacol is a small molecule inhibitor of C. elegans vesicular acetylcholine transporter with anthelmintic potential.

Nematode parasites of humans and livestock pose a significant burden to human health, economic development, and food security. Anthelmintic drug resistance is widespread among parasites of livestock and many nematode parasites of humans lack effective treatments. Here, we present a nitrophenyl-piperazine scaffold that induces motor defects rapidly in the model nematode Caenorhabditis elegans. We call this scaffold Nemacol and show that it inhibits the vesicular acetylcholine transporter (VAChT), a target recognized by commercial animal and crop health groups as a viable anthelmintic target. We demonstrate that it is possible to create Nemacol analogs that maintain potent in vivo activity whilst lowering their affinity to the mammalian VAChT 10-fold. We also show that Nemacol enhances the ability of the anthelmintic Ivermectin to paralyze C. elegans and the ruminant nematode parasite Haemonchus contortus. Hence, Nemacol represents a promising new anthelmintic scaffold that acts through a validated anthelmintic target.

Phylogenomics of ligand-gated ion channels predicts monepantel effect.

The recently launched veterinary anthelmintic drench for sheep (Novartis Animal Health Inc., Switzerland) containing the nematocide monepantel represents a new class of anthelmintics: the amino-acetonitrile derivatives (AADs), much needed in view of widespread resistance to the classical drugs. Recently, it was shown that the ACR-23 protein in Caenorhabditis elegans and a homologous protein, MPTL-1 in Haemonchus contortus, are potential targets for AAD action. Both proteins belong to the DEG-3 subfamily of acetylcholine receptors, which are thought to be nematode-specific, and different from those targeted by the imidazothiazoles (e.g. levamisole). Here we provide further evidence that Cel-ACR-23 and Hco-MPTL-1-like subunits are involved in the monepantel-sensitive phenotype. We performed comparative genomics of ligand-gated ion channel genes from several nematodes and subsequently assessed their sensitivity to anthelmintics. The nematode species in the Caenorhabditis genus, equipped with ACR-23/MPTL-1-like receptor subunits, are sensitive to monepantel (EC(50)<1.25 µM), whereas the related nematodes Pristionchus pacificus and Strongyloides ratti, which lack an ACR-23/MPTL-1 homolog, are insensitive (EC(50)>43 µM). Genome sequence information has long been used to identify putative targets for therapeutic intervention. We show how comparative genomics can be applied to predict drug sensitivity when molecular targets of a compound are known or suspected.

Haemonchus contortus acetylcholine receptors of the DEG-3 subfamily and their role in sensitivity to monepantel.

Gastro-intestinal nematodes in ruminants, especially Haemonchus contortus, are a global threat to sheep and cattle farming. The emergence of drug resistance, and even multi-drug resistance to the currently available classes of broad spectrum anthelmintics, further stresses the need for new drugs active against gastro-intestinal nematodes. A novel chemical class of synthetic anthelmintics, the Amino-Acetonitrile Derivatives (AADs), was recently discovered and the drug candidate AAD-1566 (monepantel) was chosen for further development. Studies with Caenorhabditis elegans suggested that the AADs act via nicotinic acetylcholine receptors (nAChR) of the nematode-specific DEG-3 subfamily. Here we identify nAChR genes of the DEG-3 subfamily from H. contortus and investigate their role in AAD sensitivity. Using a novel in vitro selection procedure, mutant H. contortus populations of reduced sensitivity to AAD-1566 were obtained. Sequencing of full-length nAChR coding sequences from AAD-susceptible H. contortus and their AAD-1566-mutant progeny revealed 2 genes to be affected. In the gene monepantel-1 (Hco-mptl-1, formerly named Hc-acr-23H), a panel of mutations was observed exclusively in the AAD-mutant nematodes, including deletions at intron-exon boundaries that result in mis-spliced transcripts and premature stop codons. In the gene Hco-des-2H, the same 135 bp insertion in the 5' UTR created additional, out of frame start codons in 2 independent H. contortus AAD-mutants. Furthermore, the AAD mutants exhibited altered expression levels of the DEG-3 subfamily nAChR genes Hco-mptl-1, Hco-des-2H and Hco-deg-3H as quantified by real-time PCR. These results indicate that Hco-MPTL-1 and other nAChR subunits of the DEG-3 subfamily constitute a target for AAD action against H. contortus and that loss-of-function mutations in the corresponding genes may reduce the sensitivity to AADs.

Monepantel allosterically activates DEG-3/DES-2 channels of the gastrointestinal nematode Haemonchus contortus.

Monepantel is the first drug of a new family of anthelmintics, the amino acetonitrile derivatives (AAD), presently used to treat ruminants infected with gastrointestinal nematodes such as Haemonchus contortus. Monepantel shows an excellent tolerability in mammals and is active against multidrug-resistant parasites, indicating that its molecular target is absent or inaccessible in the host and is different from those of the classic anthelmintics. Genetic approaches with mutant nematodes have suggested acetylcholine receptors of the DEG-3 subfamily as the targets of AADs, an enigmatic clade of ligand-gated ion channels that is specific to nematodes and does not occur in mammals. Here we demonstrate direct interaction of monepantel, its major active metabolite monepantel sulfone, and other AADs with potential targets of the DEG-3 subfamily of acetylcholine receptors. H. contortus DEG-3/DES-2 receptors were functionally expressed in Xenopus laevis oocytes and were found to be preferentially activated by choline, to permeate monovalent cations, and to a smaller extent, calcium ions. Although monepantel and monepantel sulfone did not activate the channels by themselves, they substantially enhanced the late currents after activation of the channels with choline, indicating that these AADs are type II positive allosteric modulators of H. contortus DEG-3/DES-2 channels. It is noteworthy that the R-enantiomer of monepantel, which is inactive as an anthelmintic, inhibited the late currents after stimulation of H. contortus DEG-3/DES-2 receptors with choline. In summary, we present the first direct evidence for interaction of AADs with DEG-3-type acetylcholine receptors and discuss these findings in the context of anthelmintic action of AADs.

An electrophysiological characterization of naturally occurring tobacco alkaloids and their action on human α4ß2 and α7 nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptor (nAChR) subtype-selective pharmacological profiles of tobacco alkaloids are essential for understanding the physiological effects of tobacco products. In this study, automated electrophysiology was used to functionally characterize the effects of distinct groups of tobacco alkaloids on human α4ß2 and α7 nAChRs. We found that, in tobacco alkaloids, pyridine as a hydrogen bond acceptor and a basic nitrogen atom at a distance of 4-7 Šare pharmacophoric elements necessary for molecular recognition by α4ß2 and α7 nAChRs with various degrees of selectivity, potency, and efficacy. While four alkaloids-nicotine, nornicotine, anabasine and R-anatabine-potently activated α4ß2, they were also weak agonists of α7 nAChRs. Nicotine was the most potent agonist of α4ß2, while anabasine elicited the highest activation of α7. None of the tobacco alkaloids enhanced nAChR activity elicited by the endogenous ligand acetylcholine; therefore, none was considered to be a positive allosteric modulator (PAM) of either α4ß2 or α7 nAChRs. In contrast, we identified tobacco alkaloids, such as the tryptophan metabolite 6-hydroxykynurenic acid, that decreased the activity of both α4ß2 and α7 nAChRs. Our study identified a class of alkaloids with positive and negative effects against human α4ß2 and α7 nAChRs. It also revealed human α4ß2 to be the principal receptor for sensing the most abundant alkaloids in tobacco leaves.

High level efficacy of lufenuron against sea lice (Lepeophtheirus salmonis) linked to rapid impact on moulting processes.

Drug resistance in the salmon louse Lepeophtheirus salmonis is a global issue for Atlantic salmon aquaculture. Multiple resistance has been described across most available compound classes with the exception of the benzoylureas. To target this gap in effective management of L. salmonis and other species of sea lice (e.g. Caligus spp.), Elanco Animal Health is developing an in-feed treatment containing lufenuron (a benzoylurea) to be administered prior to seawater transfer of salmon smolts and to provide long-term protection of salmon against sea lice infestations. Benzoylureas disrupt chitin synthesis, formation, and deposition during all moulting events. However, the mechanism(s) of action are not yet fully understood and most research completed to date has focused on insects. We exposed the first parasitic stage of L. salmonis to 700 ppb lufenuron for three hours and observed over 90% reduction in survival to the chalimus II life stage on the host, as compared to vehicle controls. This agrees with a follow up in vivo administration study on the host, which showed >95% reduction by the chalimus I stage. Transcriptomic responses of salmon lice exposed to lufenuron included genes related to moulting, epithelial differentiation, solute transport, and general developmental processes. Global metabolite profiles also suggest that membrane stability and fluidity is impacted in treated lice. These molecular signals are likely the underpinnings of an abnormal moulting process and cuticle formation observed ultrastructurally using transmission electron microscopy. Treated nauplii-staged lice exhibited multiple abnormalities in the integument, suggesting that the coordinated assembly of the epi- and procuticle is impaired. In all cases, treatment with lufenuron had rapid impacts on L. salmonis development. We describe multiple experiments to characterize the efficacy of lufenuron on eggs, larvae, and parasitic stages of L. salmonis, and provide the most comprehensive assessment of the physiological responses of a marine arthropod to a benzoylurea chemical.

The novel isoxazoline ectoparasiticide lotilaner (Credelio™): a non-competitive antagonist specific to invertebrates γ-aminobutyric acid-gated chloride channels (GABACls).

BACKGROUND: The isoxazolines are a novel class of parasiticides that are potent inhibitors of γ-aminobutyric acid (GABA)-gated chloride channels (GABACls) and, to a lesser extent, of inhibitory glutamate-gated chloride channels (GluCls). Lotilaner (Credelio™), a novel representative of this chemical class, is currently evaluated for its excellent ectoparasiticide properties. METHODS: In this study, we investigated the molecular mode of action and pharmacology of lotilaner. We report the successful gene identification, cDNA cloning and functional expression in Xenopus oocytes of Drosohpila melanogaster (wild type and dieldrin/fipronil-resistant forms), Lepeophtheirus salmonis (an ectoparasite copepod crustacean of salmon), Rhipicephalus microplus and Canis lupus familiaris GABACls. Automated Xenopus oocyte two-electrode voltage clamp electrophysiology was used to assess GABACls functionality and to compare ion channel inhibition by lotilaner with that of established insecticides addressing GABACls as targets. RESULTS: In these assays, we demonstrated that lotilaner is a potent non-competitive antagonist of insects (fly) GABACls. No cross-resistance with dieldrin or fipronil resistance mutations was detected, suggesting that lotilaner might bind to a site at least partly different from the one bound by known GABACl blockers. Using co-application experiments, we observed that lotilaner antagonism differs significantly from the classical open channel blocker fipronil. We finally confirmed for the first time that isoxazoline compounds are not only powerful antagonists of GABACls of acari (ticks) but also of crustaceans (sea lice), while no activity on a dog GABAA receptor was observed up to a concentration of 10 µM. CONCLUSIONS: Together, these results demonstrate that lotilaner is a non-competitive antagonist specific to invertebrate's γ-aminobutyric acid-gated chloride channels (GABACls). They contribute to our understanding of the mode of action of this new ectoparasiticide compound.

Anticancer properties of novel aminoacetonitrile derivative monepantel (ADD 1566) in pre-clinical models of human ovarian cancer.

Monepantel (MPL) is a new anthelmintic agent approved for the treatment of nematode infections in farm animals. As a nematicide, it acts through a nematode-specific nicotinic receptor subtype which explains its exceptional safety in rodents and mammals. In the present study, we evaluated its potential as an anticancer agent. In vitro treatment of epithelial ovarian cancer cells with MPL resulted in reduced cell viability, inhibition of cell proliferation and suppression of colony formation. Proliferation of human ovarian surface epithelial cells and other non-malignant cells were however minimally affected. MPL-induced inhibition was found to be independent of the acetylcholine nicotinic receptor (nAChR) indicating that, its target in cancer cells is probably different from that in nematodes. Analysis of MPL treated cells by flow cytometry revealed G1 phase cell cycle arrest. Accordingly, MPL treated cells expressed reduced levels of cyclins D1 and A whereas cyclin E2 expression was enhanced. Consistent with a G1 phase arrest, cellular levels of cyclin dependent kinases (CDKs) 2 and 4 were lower, whereas expression of CDK inhibitor p27(kip) was increased. In cells expressing the wild-type p53, MPL treatment led to increased p53 expression. In line with these results, MPL suppressed cellular thymidine incorporation thus impairing DNA synthesis and inducing cleavage of poly (ADP-ribose) polymerase (PARP-1). Combined these pre-clinical findings reveal for the first time the anticancer potential of monepantel.

Monepantel induces autophagy in human ovarian cancer cells through disruption of the mTOR/p70S6K signalling pathway.

We have recently shown that the novel anthelmintic drug monepantel (MPL) inhibits growth, proliferation and colony formation, arrests the cell cycle and induces cleavage of PARP-1 in ovarian cancer cell lines. Here we report on the mechanism behind the anticancer properties of MPL. The cytotoxic effect of MPL on ovarian cancer cells (OVCAR-3 and A2780) was investigated employing a panel of tests used for the detection of apoptosis and autophagy. Apoptosis and autophagy were defined by caspase activity, DNA-laddering, Annexin-V and acridine orange (AO) staining. Autophagy markers such as LC3B, SQSTM1/p62 and mammalian target of rapamycin (mTOR) pathway related proteins were assessed by western blotting and ELISA techniques. MPL did not activate caspases 3 or 8, nor did it alter the percentage of Annexin V positive stained cells. Failure to cause DNA laddering and the inability of z-VAD-fmk to block the MPL antiproliferative effects led to the ruling out of apoptosis as the mechanism behind MPL-induced cell death. On the other hand, accumulation of acidic vacuoles with distinct chromatin morphology and an increase in punctuate localization of green fluorescent protein-LC3B, and MPL-induced changes in the expression of SQSTM1/p62 were all indicative of MPL-induced autophagy. Consistent with this, we found inhibition of mTOR phosphorylation leading to suppression of the mTOR/p70S6K signalling pathway. Our findings provide the first evidence to show that MPL triggers autophagy through the deactivation of mTOR/p70S6K signalling pathway.

Recent advances in candidate-gene and whole-genome approaches to the discovery of anthelmintic resistance markers and the description of drug/receptor interactions.

Anthelmintic resistance has a great impact on livestock production systems worldwide, is an emerging concern in companion animal medicine, and represents a threat to our ongoing ability to control human soil-transmitted helminths. The Consortium for Anthelmintic Resistance and Susceptibility (CARS) provides a forum for scientists to meet and discuss the latest developments in the search for molecular markers of anthelmintic resistance. Such markers are important for detecting drug resistant worm populations, and indicating the likely impact of the resistance on drug efficacy. The molecular basis of resistance is also important for understanding how anthelmintics work, and how drug resistant populations arise. Changes to target receptors, drug efflux and other biological processes can be involved. This paper reports on the CARS group meeting held in August 2013 in Perth, Australia. The latest knowledge on the development of molecular markers for resistance to each of the principal classes of anthelmintics is reviewed. The molecular basis of resistance is best understood for the benzimidazole group of compounds, and we examine recent work to translate this knowledge into useful diagnostics for field use. We examine recent candidate-gene and whole-genome approaches to understanding anthelmintic resistance and identify markers. We also look at drug transporters in terms of providing both useful markers for resistance, as well as opportunities to overcome resistance through the targeting of the transporters themselves with inhibitors. Finally, we describe the tools available for the application of the newest high-throughput sequencing technologies to the study of anthelmintic resistance.

Worms--a "license to kill".

Worm infections can cause severe harm and death to both humans and numerous domestic and wild animals. Despite the fact that there are many beneficial worm species, veterinarians, physicians and parasitologists have multiple reasons to combat parasitic worms. The pros and cons of various approaches for the discovery of new control methods are discussed, including novel anthelmintics, vaccines and genetic approaches to identify novel drug and vaccine targets. Currently, the mainstay of worm control remains chemotherapy and prophylaxis. The importance of knowledgeable and wise use of the available anthelmintics is highlighted.

In vitro selection of Haemonchus contortus for benzimidazole resistance reveals a mutation at amino acid 198 of beta-tubulin.

Benzimidazoles were the first broad-spectrum anthelmintics and are still in use today against gastro-intestinal nematodes of ruminants such as Haemonchus contortus. Benzimidazoles block the polymerization of nematode microtubules. However, their efficacy is jeopardized by the spread of drug-resistant parasites that carry point mutations in beta-tubulin. Here we use a novel in vitro selection-in vivo propagation protocol to breed drug-resistant H. contortus. After 8 generations of selection with thiabendazole an in vitro resistance factor of 1000 was reached that was also relevant in vivo in infected sheep. The same procedure carried out with ivermectin produced only a moderate resistance phenotype that was not apparent in sheep. Cloning and sequencing of the beta-tubulin genes from the thiabendazole-resistant H. contortus mutants revealed all of the isotype 1 alleles, and part of the isotype 2 alleles, to carry the mutation glutamate(198) to alanine (E198A). An allele-specific PCR was developed, which may be helpful in monitoring the prevalence of alanine(198) encoding alleles in the beta-tubulin isotype 1 gene pool of H. contortus in the field.