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.     

A method for single pair mating in an obligate parasitic nematode

Parasitic nematode species have extremely high levels of genetic diversity, presenting a number of experimental challenges for genomic and genetic work. Consequently, there is a need to develop inbred laboratory strains with reduced levels of polymorphism. The most efficient approach to inbred line development is single pair mating, but this is challenging for obligate parasites where the adult sexual reproductive stages are inside the host, and thus difficult to experimentally manipulate. This paper describes a successful approach to single pair mating of a parasitic nematode, Haemonchus contortus. The method allows for polyandrous mating behaviour and involves the surgical transplantation of a single adult male worm with multiple immature adult females directly into the sheep abomasum. We used a panel of microsatellite markers to monitor and validate the single pair mating crosses and to ensure that the genotypes of progeny and subsequent filial generations were consistent with those expected from a mating between a single female parent of known genotype and a single male parent of unknown genotype. We have established two inbred lines that both show a significant overall reduction in genetic diversity based on microsatellite genotyping and genome-wide single nucleotide polymorphism. There was an approximately 50% reduction in heterozygous SNP sites across the genome in the MHco3.N1 line compared with the MoHco3(ISE) parental strain. The MHco3.N1 inbred line has subsequently been used to provide DNA template for whole genome sequencing of H. contortus. This work provides proof of concept and methodologies for forward genetic analysis of obligate parasitic nematodes.     

Genetics of mating and sex determination in the parasitic nematode Haemonchus contortus

Genetic analysis of parasitic nematodes has been a neglected area of research and the basic genetics of this important group of pathogens are poorly understood. Haemonchus contortus is one of the most economically significant livestock parasites worldwide and is a key experimental model for the strongylid nematode group that includes many important human and animal pathogens. We have undertaken a study of the genetics and the mode of mating of this parasite using microsatellite markers. Inheritance studies with autosomal markers demonstrated obligate dioecious sexual reproduction and polyandrous mating that are reported here for the first time in a parasitic helminth and provide the parasite with a mechanism of increasing genetic diversity. The karyotype of the H. contortus, MHco3(ISE) isolate was determined as 2n = 11 or 12. We have developed a panel of microsatellite markers that are tightly linked on the X chromosome and have used them to determine the sex chromosomal karyotype as XO male and XX female. Haplotype analysis using the X-chromosomal markers also demonstrated polyandry, independent of the autosomal marker analysis, and enabled a more direct estimate of the number of male parental genotypes contributing to each brood. This work provides a basis for future forward genetic analysis on H. contortus and related parasitic nematodes.     

Anthelmintic tolerance in free-living and facultative parasitic isolates of Halicephalobus (Panagrolaimidae)

SUMMARY Studies on anthelmintic resistance in equine parasites do not include facultative parasites. Halicephalobus gingivalis is a free-living bacterivorous nematode and a known facultative parasite of horses with a strong indication of some form of tolerance to common anthelmintic drugs. This research presents the results of an in vitro study on the anthelmintic tolerance of several isolates of Halicephalobus to thiabendazole and ivermectin using an adaptation of the Micro-Agar Larval Development Test hereby focusing on egg hatching and larval development. Panagrellus redivivus and Panagrolaimus superbus were included as a positive control. The results generally show that the anthelmintic tolerance of Halicephalobus to both thiabendazole and ivermectin was considerably higher than that of the closely related Panagrolaimidae and, compared to other studies, than that of obligatory equine parasites. Our results further reveal a remarkable trend of increasing tolerance from fully free-living isolates towards horse-associated isolates. In vitro anthelmintic testing with free-living and facultative parasitic nematodes offers the advantage of observing drug effect on the complete life cycle as opposed to obligatory parasites that can only be followed until the third larval stage. We therefore propose Halicephalobus gingivalis as an experimental tool to deepen our understanding of the biology of anthelmintic tolerance.     

Deep amplicon sequencing as a powerful new tool to screen for sequence polymorphisms associated with anthelmintic resistance in parasitic nematode populations

Parasitic gastrointestinal nematodes contribute to significant human morbidity and cause billions of dollars per year in lost agricultural production. Control is dependent on the use of anthelmintic drugs which, in the case of livestock parasites, is severely compromised by the widespread development of drug resistance. There are now concerns regarding the emergence of anthelmintic resistance in parasitic nematodes of humans in response to the selection pressure resulting from mass drug administration programs. Consequently, there is an urgent need for sensitive, scalable and accurate diagnostic tools to detect the emergence of anthelmintic resistance. Detecting and measuring the frequency of resistance-associated mutations in parasite populations has the potential to provide sensitive and quantitative assessment of resistance emergence from an early stage. Here, we describe the development and validation of deep amplicon sequencing as a powerful new approach to detect and quantify the frequency of single nucleotide polymorphisms associated with benzimidazole resistance. We have used parasite communities in sheep to undertake a proof-of-concept study of this approach. Sheep provide an excellent host system, as there are multiple co-infecting trichostrongylid nematode species, each likely with a varying prevalence of benzimidazole resistance. We demonstrate that the approach provides an accurate measure of resistance allele frequencies, and can reliably detect resistance alleles down to a frequency of 0.1%, making it particularly valuable for screening mutations in the early stages of resistance. We illustrate the power of the technique by screening UK sheep flocks for benzimidazole resistance-associated single nucleotide polymorphisms at three different codons of the β-tubulin gene in seven different parasite species from 164 populations (95 from ewes and 69 from lambs) in a single MiSeq sequencing run. This approach provides a powerful new tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations of both animals and humans.     

Understanding anthelmintic resistance: the need for genomics and genetics

Anthelmintic resistance is a major problem for the control of many parasitic nematode species and has become a major constraint to livestock production in many parts of the world. In spite of its increasing importance, there is still a poor understanding of the molecular and genetic basis of resistance. It is unclear which mutations contribute most to the resistance phenotype and how resistance alleles arise, are selected and spread in parasite populations. The main strategy used to identify mutations responsible for anthelmintic resistance has been to undertake experimental studies on candidate genes. These genes have been chosen predominantly on the basis of our knowledge of drug mode-of-action and the identification of mutations that can confer resistance in model organisms. The application of these approaches to the analysis of benzimidazole and ivermectin resistance is reviewed and the reasons for their relative success or failure are discussed. The inherent limitation of candidate gene studies is that they rely on very specific and narrow assumptions about the likely identity of resistance-associated genes. In contrast, forward genetic and functional genomic approaches do not make such assumptions, as illustrated by the successful application of these techniques in the study of insecticide resistance. Although there is an urgent need to apply these powerful approaches to anthelmintic resistance research, the basic methodologies and resources are still lacking. However, these are now being developed for the trichostrongylid nematode Haemonchus contortus and the current progress and research priorities in this area are discussed.     

The Emergence of Resistance to the Benzimidazole Anthlemintics in Parasitic Nematodes of Livestock Is Characterised by Multiple Independent Hard and Soft Selective Sweeps

Anthelmintic resistance is a major problem for the control of parasitic nematodes of livestock and of growing concern for human parasite control. However, there is little understanding of how resistance arises and spreads or of the “genetic signature” of selection for this group of important pathogens. We have investigated these questions in the system for which anthelmintic resistance is most advanced; benzimidazole resistance in the sheep parasites Haemonchus contortus and Teladorsagia circumcincta. Population genetic analysis with neutral microsatellite markers reveals that T. circumcincta has higher genetic diversity but lower genetic differentiation between farms than H. contortus in the UK. We propose that this is due to epidemiological differences between the two parasites resulting in greater seasonal bottlenecking of H. contortus. There is a remarkably high level of resistance haplotype diversity in both parasites compared with drug resistance studies in other eukaryotic systems. Our analysis suggests a minimum of four independent origins of resistance mutations on just seven farms for H. contortus, and even more for T. circumincta. Both hard and soft selective sweeps have occurred with striking differences between individual farms. The sweeps are generally softer for T. circumcincta than H. contortus, consistent with its higher level of genetic diversity and consequent greater availability of new mutations. We propose a model in which multiple independent resistance mutations recurrently arise and spread by migration to explain the widespread occurrence of resistance in these parasites. Finally, in spite of the complex haplotypic diversity, we show that selection can be detected at the target locus using simple measures of genetic diversity and departures from neutrality. This work has important implications for the application of genome-wide approaches to identify new anthelmintic resistance loci and the likelihood of anthelmintic resistance emerging as selection pressure is increased in human soil-transmitted nematodes by community wide treatment programs.     

Molecular markers and genetic diversity of Plasmodium vivax

Enhanced understanding of the transmission dynamics and population genetics for Plasmodium vivax is crucial in predicting the emergence and spread of novel parasite phenotypes with major public health implications, such as new relapsing patterns, drug resistance and increased virulence. Suitable molecular markers are required for these population genetic studies. Here, we focus on two groups of molecular markers that are commonly used to analyse natural populations of P. vivax. We use markers under selective pressure, for instance, antigen-coding polymorphic genes, and markers that are not under strong natural selection, such as most minisatellite and microsatellite loci. First, we review data obtained using genes encoding for P. vivax antigens: circumsporozoite protein, merozoite surface proteins 1 and 3α, apical membrane antigen 1 and Duffy binding antigen. We next address neutral or nearly neutral molecular markers, especially microsatellite loci, providing a complete list of markers that have already been used in P. vivax populations studies. We also analyse the microsatellite loci identified in the P. vivax genome project. Finally, we discuss some practical uses for P. vivax genotyping, for example, detecting multiple-clone infections and tracking the geographic origin of isolates.     

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.     

In silico analyses of neuropeptide-like protein (NLP) profiles in parasitic nematodes

Nematode parasite infections cause disease in humans and animals and threaten global food security by reducing productivity in livestock and crop farming. The escalation of anthelmintic resistance in economically important nematode parasites underscores the need for the identification of novel drug targets in these worms. Nematode neuropeptide signalling is an attractive system for chemotherapeutic exploitation, with neuropeptide G-protein coupled receptors (NP-GPCRs) representing the lead targets. In order to successfully validate NP-GPCRs for parasite control it is necessary to characterise their function and importance to nematode biology. This can be aided through identification of receptor activating ligand(s) via deorphanisation. Such efforts require the identification of all neuropeptide ligands within parasites. Here we mined the genomes of nine therapeutically relevant pathogenic nematodes to characterise the neuropeptide-like protein complements and demonstrate that: (i) parasitic nematodes possess a reduced complement of neuropeptide-like protein-encoding genes relative to Caenorhabditis elegans; (ii) parasite neuropeptide-like protein profiles are broadly conserved between nematode clades; (iii) five Ce-nlps are completely conserved across the nematode species examined; (iv) the extent and position of neuropeptide-like protein-motif conservation is variable; (v) novel RPamide-encoding genes are present in parasitic nematodes; (vi) novel Allatostatin-C-like peptide encoding genes are present in both C. elegans and parasitic nematodes; (vii) novel neuropeptide-like protein families are absent in C. elegans; and (viii) highly conserved nematode neuropeptide-like proteins are bioactive. These data highlight the complexity of nematode neuropeptide-like proteins and reveal the need for nomenclature revision in this diverse neuropeptide family. The identification of neuropeptide-like protein ligands, and characterisation of those with functional relevance, advance our understanding of neuropeptide signalling to support exploitation of the neuropeptidergic system as an anthelmintic target.     

Microsatellite analysis reveals marked genetic differentiation between Haemonchus contortus laboratory isolates and provides a rapid system of genetic fingerprinting

Many of the Haemonchus contortus isolates currently used for experimental work were originally derived from different regions of the world and are commonly exchanged between laboratories. In most cases, these are largely genetically uncharacterised other than the analyses conducted on specific genes of interest. We have used a panel of eight microsatellite markers to genetically characterise five different commonly used H. contortus isolates including MHco3 (ISE), the isolate chosen for full genome sequencing as part of the H. contortus genome project. There is an extremely high level of genetic differentiation between each of the isolates except the two which have a common origin, MHco1 (MOSI) and MHco3 (ISE). We have investigated the amplification of microsatellite markers from pooled DNA as a potential method for fingerprinting different isolates. Good estimates of the true allele frequencies can be made by amplification from either pooled adult DNA or bulk L3 DNA for seven out of the eight markers tested. Both single worm genotyping and bulk DNA fingerprinting revealed no genetic differentiation between adult worms in the host and larvae derived from faecal culture. Furthermore, none of the eight markers showed genetic changes when isolates were passaged through different individual hosts. Hence the microsatellite genotyping of bulk larval DNA samples provides a simple and rapid method to genetically define and monitor laboratory isolates, and to determine their relationship with particular field populations.     

Inference of population structure and patterns of gene flow in canine heartworm (Dirofilaria immitis)

Understanding the genetic variation within a parasitic species is crucial to implementing successful control programs and preventing the dispersal of drug resistance alleles. We examined the population genetics and structure of canine heartworm (Dirofilaria immitis) by developing a panel of 11 polymorphic microsatellite loci for this abundant parasite. In total, 192 individual nematodes were opportunistically sampled from 9 geographic regions in the United States and Mexico and genotyped. Population genetic analyses indicate the presence of 4 genetic clusters. The canine heartworm samples used in this study were characterized by low heterozygosity, with eastern and central North America experiencing high levels of reciprocal gene flow. Geographic barriers impede the movement of vectors and infected hosts west of the Rocky Mountains and south of the Central Mexican Plateau. This, combined with corridors of contiguous habitat, could influence the spread of drug resistance alleles.     

Microsatellite markers for the human nematode parasite Ascaris lumbricoides: development and assessment of utility

We describe 35 microsatellite markers from the human parasitic nematode Ascaris lumbricoides. We found 7 sex-linked markers and demonstrate that 26 autosomal loci can be scored reliably. These markers have high genetic variability and provide the tools to address multiple questions concerning the epidemiology, fine-scale genetic structure, host specificity, and mating systems of this parasite.     

Anthelmintic treatment alters the parasite community in a wild mouse host

Individuals are often co-infected with several parasite species, yet the consequences of drug treatment on the dynamics of parasite communities in wild populations have rarely been measured. Here, we experimentally reduced nematode infection in a wild mouse population and measured the effects on other non-target parasites. A single oral dose of the anthelmintic, ivermectin, significantly reduced nematode infection, but resulted in a reciprocal increase in other gastrointestinal parasites, specifically coccidial protozoans and cestodes. These results highlight the possibility that drug therapy may have unintended consequences for non-target parasites and that host–parasite dynamics cannot always be fully understood in the framework of single host–parasite interactions.     

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.     

Haemonchus contortus: characterization of a glutamate binding site in unselected and ivermectin-selected larvae and adults.

A specific ivermectin-sensitive, glutamate binding site has been identified in the parasitic nematode Haemonchus contortus. Glutamate binding in H. contortus was saturable and occurred in a single class of high-affinity binding sites which appeared to have pharmacological properties different from those of mammalian glutamate receptors. Adult and larval forms of H. contortus had dramatically different glutamate binding kinetics, the larvae showing nearly up to 200-fold higher Bmax values and up to 9-fold increases in Kd values compared to adults. Treatment of adult H. contortus with the anthelmintic, ivermectin, decreased the Bmax value for glutamate binding in the susceptible strain but not in the resistant parasites. Furthermore, selection for ivermectin resistance was associated with a significant increase in Bmax for glutamate binding in adults and a similarly significant increase in glutamate binding affinity in larvae. These results suggest that the H. contortus glutamate binding site identified in this study may be involved in the phenomenon of ivermectin resistance.     

Transport proteins of the ABC systems superfamily and their role in drug action and resistance in nematodes

The completion of a number of nematode genomes has provided significant information on ABC systems in these organisms. Nematodes have more ABC systems genes and greater diversity than do mammalian species. Class 1 and class 2 ABC systems, more commonly known as ABC transporters, are present. As in other organisms, nematode ABC systems are characterized by a highly conserved ATP-binding domain (ABC_2) and a less conserved transmembrane domain (ABC_TM1/TM1F). Studies of drug resistance in nematodes have suggested that ABC transporters are part of the resistance mechanism. Evidence in support of this has been obtained from genetic studies where an association between anthelmintic selection and ABC transporters was shown by comparisons between unselected and drug selected, or resistant, populations of parasitic nematodes. In drug resistant populations, genetic polymorphism and diversity, genotype patterns, and linkage disequilibrium were disrupted. Multidrug resistance (MDR) reversing agents that inhibit ABC function improve efficacy in sensitive nematode populations and restore sensitivity in resistant populations. Similar to the situation in clinical oncology, overexpression of ABC systems occurs in drug resistant and sensitive populations following drug exposure, particularly those in the P-glycoprotein (PGP) subfamily. Deletion or disruption of ABC genes, particularly PGP and the multidrug resistance associated protein (MRP), increases sensitivity to some drugs, particularly ivermectin. These studies provide evidence that ABC transporters play a role in drug action and resistance in nematodes.     

Speckle interferometry applied to pharmacodynamic studies: evaluation of parasite motility

The work reported here describes the application of the optical technique known as dynamic speckle interferometry to evaluate the motility of nematode parasites exposed to different anthelmintic drugs. This technique, a well proven tool for assessing the time evolution of different phenomena, is here successfully used to quantify parasite motility in pharmacodynamic assays. The characterization of the pharmacological properties of anthelmintic drugs is critical to optimize their use in parasite control. Besides, the evaluation of nematode motility is a relevant indicator of the pharmacodynamic effect of anthelmintic drugs. The application of this approach to study the motility of Haemonchus contortus (used as a model of nematode parasites) larvae exposed to different drugs is presented, showing its usefulness.Abbreviations ABZ albendazole - BZD benzimidazole - IVM ivermectin - LVM levamisole