Anthelmintic resistance in human helminths: Learning from the problems with worm control in livestock

During the past decade, the prevalence of anthelmintic resistance in some economically important helminths of sheep, goats and horses has increased dramatically. In some regions of Australia, South America and South Africa, anthelmintic resistance has become a serious threat to the survival of the sheep industry. Mass treatment programmes and exclusive reliance on anthelmintics for worm control in livestock are amongst the most important reasons for the development of anthelmintic resistance. In this article, Stanny Geerts, Gerald Coles and Bruno Gryseels draw the attention to a number of errors that have occurred in the control of helminths in livestock and that should be avoided in the control of worms in humans.     

Mechanism-based screening: discovery of the next generation of anthelmintics depends upon more basic research

The therapeutic arsenal for the control of helminth infections contains only a few chemical classes. The development and spread of resistance has eroded the utility of most currently available anthelmintics, at least for some indications, and is a constant threat to further reduce the options for treatment. Discovery and development of novel anthelmintic templates is strategically necessary to preserve the economic and health advantages now gained through chemotherapy. As the costs of development escalate, the question of how best to discover new drugs becomes paramount. Although random screening in infected animals led to the discovery of all currently available anthelmintics, cost constraints and a perception of diminishing returns require new approaches. Taking a cue from drug discovery programmes for human illnesses, we suggest that mechanism-based screening will provide the next generation of anthelmintic molecules. Critical to success in this venture will be the exploitation of the Caenorhabditis elegans genome through bioinformatics and genetic technologies. The greatest obstacle to success in this endeavour is the paucity of information available about the molecular physiology of helminths, making the choice of a discovery target a risky proposition.     

Drug resistance in veterinary helminths

At present, there is no effective alternative to chemical control of parasitic helminths where livestock are grazed intensively. Resistance to anthelmintics has become a major problem in veterinary medicine, and threatens both agricultural income and animal welfare. The molecular and biochemical basis of this resistance is not well understood. The lack of reliable biological and molecular tests means that we are not able to follow the emergence and spread of resistance alleles and clinical resistance as well as we need. This review summarizes some of the recent findings on resistance mechanisms, puts forward some recommendations for limiting its impact and suggests some priorities for research in this area.     

The Validation of Nematode-Specific Acetylcholine-Gated Chloride Channels as Potential Anthelmintic Drug Targets

New compounds are needed to treat parasitic nematode infections in humans, livestock and plants. Small molecule anthelmintics are the primary means of nematode parasite control in animals; however, widespread resistance to the currently available drug classes means control will be impossible without the introduction of new compounds. Adverse environmental effects associated with nematocides used to control plant parasitic species are also motivating the search for safer, more effective compounds. Discovery of new anthelmintic drugs in particular has been a serious challenge due to the difficulty of obtaining and culturing target parasites for high-throughput screens and the lack of functional genomic techniques to validate potential drug targets in these pathogens. We present here a novel strategy for target validation that employs the free-living nematode Caenorhabditis elegans to demonstrate the value of new ligand-gated ion channels as targets for anthelmintic discovery. Many successful anthelmintics, including ivermectin, levamisole and monepantel, are agonists of pentameric ligand-gated ion channels, suggesting that the unexploited pentameric ion channels encoded in parasite genomes may be suitable drug targets. We validated five members of the nematode-specific family of acetylcholine-gated chloride channels as targets of agonists with anthelmintic properties by ectopically expressing an ivermectin-gated chloride channel, AVR-15, in tissues that endogenously express the acetylcholine-gated chloride channels and using the effects of ivermectin to predict the effects of an acetylcholine-gated chloride channel agonist. In principle, our strategy can be applied to validate any ion channel as a putative anti-parasitic drug target.     

The diversity of ABC transporter genes across the Phylum Nematoda

It is estimated that one billion people globally are infected by parasitic nematodes, with children, pregnant women, and the elderly particularly susceptible to morbidity from infection. Control methods are limited to de-worming, which is hampered by rapid re-infection and the inevitable development of anthelmintic resistance. One family of proteins that has been implicated in nematode anthelmintic resistance are the ATP binding cassette (ABC) transporters. ABC transporters are characterized by a highly conserved ATP-binding domain and variable transmembrane regions. A growing number of studies have associated ABC transporters in anthelmintic resistance through a protective mechanism of drug efflux. Genetic deletion of P glycoprotein type ABC transporters in Caenorhabditis elegans demonstrated increased sensitivity to anthelmintics, while in the livestock parasite, Haemonchus contortus, anthelmintic use has been shown to increase the expression of ATP transporter genes. These studies as well as others, provide evidence for a potential role of ABC transporters in drug resistance in nematodes. In order to understand more about the family of ABC transporters, we used hidden Markov models to predict ABC transporter proteins from 108 species across the phylum Nematoda and use these data to analyze patterns of diversification and loss in diverse nematode species. We also examined temporal patterns of expression for the ABC transporter family within the filarial nematode Brugia malayi and identify cases of differential expression across diverse life-cycle stages. Taken together, our data provide a comprehensive overview of ABC transporters in diverse nematode species and identify examples of gene loss and diversification in nematodes based on lifestyle and taxonomy.     

The nervous systems of helminths as targets for drugs.

Processes that critically differentiate parasitic helminths and their hosts are obvious candidates for chemotherapeutic intervention. The recognition that neurobiology distinguishes helminths from their vertebrate hosts is due in part to the fact that several efficacious anthelmintics, derived generally from empirical screening, have been found to act selectively on the neuromuscular system of these parasites. In addition, basic physiological and pharmacological research has revealed considerable differences in the ways in which helminths and their hosts transmit information in the nervous system and respond to it in innervated tissues. Unfortunately, most of these differences have yet to be exploited in chemotherapy. The topics for this review include an analysis of mechanistic aspects of the pharmacology of anthelmintics that act on neuromuscular systems and a consideration of the prospects for discovery of novel drugs that act on this system.     

Contrasting views of animal healthcare providers on worm control practices for sheep and goats in an arid environment

A questionnaire survey was conducted to determine the worm control practices and anthelmintic usage of 150 key respondents involved in sheep and goat production in the arid Thal area of Pakistan. The information was collected by visiting farms, and interviewing the key respondents which included veterinary officers (n = 15), veterinary assistants (n = 51), traditional practitioners (n = 24), and small and large scale sheep/goat farm herders and owners (n = 60). Among all interviewed animal healthcare providers, the veterinary officers had the highest level of awareness of parasitic infection and advocated the use of modern available anthelmintics according to the predefined schedule. The farmers on the other hand, had the lowest level of knowledge about parasitic infections. They used modern anthelmintics at low frequencies (every six months) following an unusual practice of diluting the medicine. Veterinary assistants had a medium level of awareness about the parasitic infections using anthelmintic treatments when they deemed necessary rather than following a predefined treatment schedule. Traditional practitioners were also aware of parasitic infections and used traditional anthelmintics or a combination of the traditional and modern anthelmintics. The animal health providers had a different awareness and knowledge of parasitic infections which resulted in contrasting proposals for its' control. The farmers used worm control measures in accordance with their own views and those of animal healthcare advisors, combining modern and traditional treatments. This study provides the first insight into the differing views of those animal healthcare providers who form the basis for effective parasitic control within the sheep and goat industry of an arid region.     

Drug transfer into target helminth parasites

The pharmacokinetics of an anthelmintic drug includes the time course of drug absorption, distribution, metabolism and elimination from the host and determines the concentration of the active drug that reaches the location of the parasite. However, the action of the anthelmintic also depends on the ability of the active drug to reach its specific receptor within the target parasite. Thus, drug entry and accumulation in target helminths are important issues when considering how best to achieve optimal efficacy. Passive drug transfer through the external helminth surface is the predominant entry mechanism for most widely used anthelmintics and is discussed in this article. Despite the structural differences between the external surface of nematodes (the cuticle) and the external surface of cestodes and trematodes (the tegument), the mechanism of drug entrance into both types of helminth depends on the lipophilicity of the anthelmintic and this is the major physicochemical determinant for the drug to reach a therapeutic concentration in the target parasite. Understanding the processes that regulate drug transfer into helminth parasites is an important aspect in improving the control of parasites in human and veterinary medicine.     

Removal of adult cyathostomins alters faecal microbiota and promotes an inflammatory phenotype in horses

The interactions between parasitic helminths and gut microbiota are considered to be an important, although as yet incompletely understood, factor in the regulation of immunity, inflammation and a range of diseases. Infection with intestinal helminths is ubiquitous in grazing horses, with cyathostomins (about 50 species of which are recorded) predominating. Consequences of infection include both chronic effects, and an acute inflammatory syndrome, acute larval cyathostominosis, which sometimes follows removal of adult helminths by administration of anthelmintic drugs. The presence of cyathostomins as a resident helminth population of the equine gut (the “helminthome”) provides an opportunity to investigate the effect helminth infection, and its perturbation, has on both the immune system and bacterial microbiome of the gut, as well as to determine the specific mechanisms of pathophysiology involved in equine acute larval cyathostominosis. We studied changes in the faecal microbiota of two groups of horses following treatment with anthelmintics (fenbendazole or moxidectin). We found decreases in both alpha diversity and beta diversity of the faecal microbiota at Day 7 post-treatment, which were reversed by Day 14. These changes were accompanied by increases in inflammatory biomarkers. The general pattern of faecal microbiota detected was similar to that seen in the relatively few equine gut microbiome studies reported to date. We conclude that interplay between resident cyathostomin populations and the bacterial microbiota of the equine large intestine is important in maintaining homeostasis and that disturbance of this ecology can lead to gut dysbiosis and play a role in the aetiology of inflammatory conditions in the horse, including acute larval cyathostominosis.     

A survey in Louisiana of intestinal helminths of ponies with little exposure to anthelmintics

Ponies reared with minimal or no exposure to anthelmintics were surveyed for intestinal helminths in order to estimate prevalence and intensity of parasite populations unaltered by frequent exposure to anthelmintics. Thirty-seven mixed breed ponies of varying ages were examined. Thirty-four species of nematodes and 2 species of cestodes were found. Twenty-four of the nematode species (including 1 new species) were in the subfamily Cyathostominae (small strongyles). Eighty-seven percent of the total burden of adult small strongyles in the large intestine was composed of 10 species. By comparing the results of the present survey with those of recent surveys of horses from herds which had been subjected to treatments with anthelmintics, the effect of prolonged usage of anthelmintic treatment on the prevalence of individual species possibly can be estimated. The general ranking of the 10 most common cyathostome species was similar to those described in recent surveys of horses, suggesting that anthelmintic pressure does not affect the prevalence of most cyathostome species. The lack of anthelmintic treatment appeared not to affect prevalence rates for Anoplocephala perfoliata and Anoplocephala magna when compared to other studies. Conversely, prevalence rates for Strongylus spp., Triodontophorus spp., Craterostomum acuticaudatum, Oxyuris equi, and Parascaris equorum were higher than those reported for these species in recent studies of horses.     

The biochemical basis of anthelmintic action and resistance

The most commonly used modern anthelmintics include the benzimidazoles, the nicotinic agonists. praziquantel, triclabendazole and the macrocyclic lactones. These drugs interfere with target sites that are either unique to the parasite or differ in their structural features from those of the homologous counterpart present in the vertebrate host. The benzimidazoles exert their effect by binding selectively and with high affinity to the beta-subunit of helminth microtubule protein. The target site of the nicotinic agonists (e.g. levamisole, tetrahydropyrimidines) is a pharmacologically distinct nicotinic acetylcholine receptor channel in nematodes. The macrocyclic lactones (e.g. ivermectin, moxidectin) act as agonists of a family of invertebrate-specific inhibitory chloride channels that are activated by glutamic acid. The primary mode of action of other important anthelmintics (e.g. praziquantel, triclabendazole) is unknown. Anthelmintic resistance is wide-spread and a serious threat to effective control of helminth infections, especially in the veterinary area. The biochemical and genetic mechanisms underlying anthelmintic resistance are not well understood, but appear to be complex and vary among different helminth species and even isolates. The major mechanisms helminths use to acquire drug resistance appear to be through receptor loss or decrease of the target site affinity for the drug. Knowledge on the mechanisms of drug action and resistance may be exploitable for the development of new drugs and may provide information on ways to overcome parasite resistance, respectively.     

A novel member of the ligand-gated chloride channel gene family from Haemonchus contortus

Ligand-gated chloride channels (LGCCs) are key components of the nervous system of parasitic nematodes and important targets for anthelmintics. Here, we describe the isolation and characterization of a novel member of the LGCC gene family (HcLGCC1) from the parasitic nematode Haemonchus contortus. Sequence analysis revealed that the channel subunit encoded by HcLGCC1 is anion selective and a member of a group of channels characterized as having two Cys-loops in the N-terminal ligand-binding domain. Although the overall function of HcLGCC1 is presently unknown, the gene may play a key role in the early developmental stages of the parasite. Further investigations into the function of LGCCs, such as HcLGCC1, in parasitic nematodes should have implications for the discovery of new anthelmintic targets.     

The activity of drug-metabolizing enzymes and the biotransformation of selected anthelmintics in the model tapeworm Hymenolepis diminuta

The drug-metabolizing enzymes of some helminths can deactivate anthelmintics and therefore partially protect helminths against these drugs' toxic effect. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (albendazole, flubendazole, mebendazole) in the rat tapeworm Hymenolepis diminuta, a species often used as a model tapeworm. In vitro and ex vivo experiments were performed. Metabolites of the anthelmintics were detected and identified by HPLC with spectrofluorometric or mass-spectrometric detection. The enzymes of H. diminuta are able to reduce the carbonyl group of flubendazole, mebendazole and several other xenobiotics. Although the activity of a number of oxidation enzymes was determined, no oxidative metabolites of albendazole were detected. Regarding conjugation enzymes, a high activity of glutathione S-transferase was observed. A methyl derivative of reduced flubendazole was the only conjugation metabolite identified in ex vivo incubations of H. diminuta with anthelmintics. The results revealed that H. diminuta metabolized flubendazole and mebendazole, but not albendazole. The biotransformation pathways found in H. diminuta differ from those described in Moniezia expanza and suggest the interspecies differences in drug metabolism not only among classes of helminths, but even among tapeworms.     

The pharmacology of anthelmintics in livestock

The recent development of ivermectin, netobimin, closantel, triclabendazole and clorsulon as well as new ways of using established anthelmintics has significantly improved the potential for parasite control in livestock. It is of interest to note that potential control of F. hepatica has been particularly improved with four of these new anthelmintic drugs being fasciolicidal. In addition to an appreciation of the spectrum of action of the new anthelmintics, a considerable amount of information on the pharmaco-kinetic behaviour, metabolism and mode of action of these drugs has been published. New information on pharmacology of established anthelmintics has provided new insights into the biology of parasites, drug resistance and the options for improved chemotherapy by manipulation of pharmacokinetics and metabolism. Much recent pharmacological research has been concentrated on the BZs and this has been considered here in an attempt to provide a better understanding of this significant group of anthelmintics. The development of new delivery systems, such as intraruminal slow release devices, is potentially useful with most anthelmintics. So far, this new approach to parasite control has been applied most successfully with morantel tartrate.     

Using C. elegans Forward and Reverse Genetics to Identify New Compounds with Anthelmintic Activity

BackgroundThe lack of new anthelmintic agents is of growing concern because it affects human health and our food supply, as both livestock and plants are affected. Two principal factors contribute to this problem. First, nematode resistance to anthelmintic drugs is increasing worldwide and second, many effective nematicides pose environmental hazards. In this paper we address this problem by deploying a high throughput screening platform for anthelmintic drug discovery using the nematode Caenorhabditis elegans as a surrogate for infectious nematodes. This method offers the possibility of identifying new anthelmintics in a cost-effective and timely manner.Conclusions/SignificanceThe challenge of anthelmintic drug discovery is exacerbated by several factors; including, 1) the biochemical similarity between host and parasite genomes, 2) the geographic location of parasitic nematodes and 3) the rapid development of resistance. Accordingly, an approach that can screen large compound collections rapidly is required. C. elegans as a surrogate parasite offers the ability to screen compounds rapidly and, equally importantly, with specificity, thus reducing the potential toxicity of these compounds to the host and the environment. We believe this approach will help to replenish the pipeline of potential nematicides.     

Genetic variability following selection of Haemonchus contortus with anthelmintics

Genetic diversity in nematodes leads to variation in response to anthelmintics. Haemonchus contortus shows enormous genetic diversity, allowing anthelmintic resistance alleles to be rapidly selected. Anthelmintic resistance is now a widespread problem, especially in H. contortus. Here, I compare the genes involved in anthelmintic resistance in H. contortus with those that confer susceptibility or resistance on the free living nematode Caenorhabditis elegans. I also discuss the latest knowledge of genes associated with resistance to benzimidazoles, levamisole and the macrocyclic lactones and the need for DNA markers for anthelmintic resistance.     

Nematode cys-loop GABA receptors: biological function, pharmacology and sites of action for anthelmintics

Parasitic nematode infection of humans and livestock is a major problem globally. Attempts to control nematode populations have led to the development of several classes of anthelmintic, which target cys-loop ligand-gated ion channels. Unlike the vertebrate nervous system, the nematode nervous system possesses a large and diversified array of ligand-gated chloride channels that comprise key components of the inhibitory neurotransmission system. In particular, cys-loop GABA receptors have evolved to play many fundamental roles in nematode behaviour such as locomotion. Analysis of the genomes of several free-living and parasitic nematodes suggests that there are several groups of cys-loop GABA receptor subunits that, for the most part, are conserved among nematodes. Despite many similarities with vertebrate cys-loop GABA receptors, those in nematodes are quite distinct in sequence similarity, subunit composition and biological function. With rising anthelmintic resistance in many nematode populations worldwide, GABA receptors should become an area of increased scientific investigation in the development of the next generation of anthelmintics.     

Discovery of amino-acetonitrile derivatives, a new class of synthetic anthelmintic compounds

A new series of amino-acetonitrile derivatives (AAD) have been discovered that exhibit high anthelmintic activity against parasitic nematode species such as Haemonchus contortus and Trichostrongylus colubriformis. Significantly, these compounds also demonstrate activity against nematode strains resistant to the currently available broad-spectrum anthelmintics. The discovery, synthesis, structure–activity relationship and biological results are presented.     

Expression of ATP-binding cassette multidrug transporters in the giant liver fluke Fasciola gigantica and their possible involvement in the transport of bile salts and anthelmintics

ATP-binding cassette (ABC) transporters belong to one of the largest protein families that either import or export a wide spectrum of different substrates. Certain members of this superfamily have been implicated in multidrug resistance in various types of cancer as well as in pathogenic microorganisms. The role of ABC proteins in parasitic multidrug resistance becomes increasingly evident. However, studies on ABC transporters in helminths have been limited to MDR1 and MRP orthologues. In the present study, we reported, for the first time, the expression and localization of ABC proteins including orthologues of MDR1, MRP1, BCRP, and BSEP in the giant liver fluke Fasciola gigantica. Furthermore, the functional activities of these ABC transporters were characterized in isolated fluke cells using a fluorescent substrate, rhodamine. The results revealed the inhibition of rhodamine efflux by cyclosporin A, a potent inhibitor of ABC transporters. Interestingly, our data suggested that these proteins might play a role in the export of bile salts, in particular, taurocholate. Although, we did not observe any substantial changes in rhodamine transport in the presence of anthelmintics under experimental conditions, however, our findings altogether shed light on the possible involvement of several members of ABC proteins in the mechanism of drug resistance as well as detoxification process in helminths to survive inside their hosts.