Behavioral fingerprints predict insecticide and anthelmintic mode of action.

Novel invertebrate-killing compounds are required in agriculture and medicine to overcome resistance to existing treatments. Because insecticides and anthelmintics are discovered in phenotypic screens, a crucial step in the discovery process is determining the mode of action of hits. Visible whole-organism symptoms are combined with molecular and physiological data to determine mode of action. However, manual symptomology is laborious and requires symptoms that are strong enough to see by eye. Here, we use high-throughput imaging and quantitative phenotyping to measure Caenorhabditis elegans behavioral responses to compounds and train a classifier that predicts mode of action with an accuracy of 88% for a set of ten common modes of action. We also classify compounds within each mode of action to discover substructure that is not captured in broad mode-of-action labels. High-throughput imaging and automated phenotyping could therefore accelerate mode-of-action discovery in invertebrate-targeting compound development and help to refine mode-of-action categories.

An in vitro larval migration assay for assessing anthelmintic activity of different drug classes against Ascaris suum.

In vitro methods have been developed for the detection of anthelmintic resistance in a range of nematode species. However, the life cycle of Ascaris suum renders the commonly used egg hatch assay and larval development assay unusable. In this study we developed a combined multi-well culture and agar gel larval migration assay to test the effect of benzimidazole and tetrahydropyrimidin/imidazothiazole anthelmintics against nine isolates of A. suum collected from locations in China and Denmark. Drugs tested were thiabendazole, fenbendazole, mebendazole, levamisole, and pyrantel. The percentages of larvae that migrated to the surface of each treated and control well were used to calculate the drug concentration which inhibits 50% of the larvae migration (EC50). The values of EC50 of thiabendazole, fenbendazole, mebendazole, levamisole, and pyrantel against A. suum isolates ranged 74-150, 4.9-13.9, 2.3-4.3, 358-1150 and 1100-4000nM, respectively. This combined multi-well culture and agar gel larval migration assay was a sensitive bioassay for anthelmintic activity and could serve as an in vitro method to detect for lowered drug efficacy against A. suum or possibly to screen for anthelmintic drug candidates.

Molecular mechanisms for anthelmintic resistance in strongyle nematode parasites of veterinary importance.

Veterinarians rely on a relatively limited spectrum of anthelmintic agents to control nematode parasites in domestic animals. Unfortunately, anthelmintic resistance has been an emerging problem in veterinary medicine. In particular, resistance has emerged among the strongyles, a group of gastrointestinal nematodes that infect a variety of hosts that range from large herbivores to small companion animals. Over the last several decades, a great deal of research effort has been directed toward developing an understanding of the mechanisms conferring resistance against the three major groups of anthelmintics: macrocyclic lactones, benzimidazoles, and nicotinic agonists. Our understanding of anthelmintic resistance has been largely formed by determining the mechanism of action for each drug class and then evaluating drug-resistant nematode isolates for mutations or differences in expression of target genes. More recently, drug efflux pumps have been recognized for their potential contribution to anthelmintic resistance. In this mini-review, we summarize the evidence for mechanisms of resistance in strongyle nematodes.

[ANTHELMINTIC SUBSTANCES: MAIN CLASSES, PROBLEMS, TRENDS IN DEVELOPMENT AND PROSPECTS].

The review chronologically considers the main classes of the currently available anthelminthic substances: early anthelmintic compounds, benzimidazoles, imidazolthiazoles, tetrahydropyrimidines, avermectins and milbemycins, and salicylanilides. Great attention is paid to novel substances (emodepside, monepantel, derquantel, tribendimidine) and promising developments. Some aspects of the molecular mechanisms of action of anthelmintics, their resistance, and alternative dehelmintization methods are discussed.

Cross-sectional survey of parasite control practices on Thoroughbred and Standardbred training yards in New Zealand.

REASONS FOR PERFORMING STUDY: There is growing concern worldwide regarding anthelmintic resistance in equine parasites. In order to improve parasite control practices and reduce the selection for resistant parasites, baseline data are required. OBJECTIVES: To describe the current parasite management and control practices used for racehorses. STUDY DESIGN: Cross-sectional survey. METHODS: Thoroughbred and Standardbred trainers were surveyed online regarding demographics, parasite control methods, grazing management and quarantine, and the use of faecal egg counts (FEC), with questions stratified by horse type, i.e. racehorses (horses in training) and spellers (racehorses on a break from training), and industry (Thoroughbred and Standardbred). Multivariable logistic regression was used to examine associations with FEC use. RESULTS: In total, 234 respondents completed the survey for an estimated response rate of 16%. In total, 50.5% of trainers treated horses on an interval treatment strategy and treated a median of 6 (interquartile range (IQR) 4-7) and 6 (IQR 4-8) times annually for Thoroughbred and Standardbred racehorses, respectively. A total of 62.5% (130/208) of respondents reported seeking veterinary advice for deworming products, and FEC had been done by 20.1% (39/194) of respondents. The odds of a trainer doing FEC were 4 times higher if the trainer had consulted a veterinarian, compared with those that had not. CONCLUSIONS: This study has highlighted an industry-wide overuse of anthelmintic products and few trainers were using surveillance-based control strategies. The relationship between veterinarians and trainers should be explored further to enhance information dissemination and implement effective control strategies, to maintain horse health and delay the advance of anthelmintic resistance.

Characterisation of the inflammatory cytokine response to anthelmintic treatment in ponies.

REASONS FOR PERFORMING STUDY: Anthelmintic treatments have been associated with local inflammatory reactions. Since each class of anthelmintic has unique mechanisms of action affecting different subpopulations of parasites, we hypothesised that they will also induce characteristic proinflammatory responses. OBJECTIVES: To determine the effect of anthelmintic class on the proinflammatory response post treatment. STUDY DESIGN: Ponies naturally infected with cyathostomins and other parasites after pasture grazing were left untreated or treated with representatives of 3 different classes of anthelmintics: fenbendazole (benzimidazole); pyrantel tartrate (pyrimidine); and moxidectin (macrocyclic lactone). All were monitored for the expression of proinflammatory genes in the peripheral blood using real-time PCR. METHODS: The ponies were divided into 4 treatment groups: Group 1 (n = 4) were untreated controls; Group 2 (n = 5) received 5 daily doses of fenbendazole (10 mg/kg bwt); Group 3 (n = 4) received daily treatment of pyrantel tartrate 2× (2.65 mg/kg bwt); and Group 4 (n = 5) received a single dose of moxidectin (400 µg/kg bwt). Blood samples were collected daily for 2 weeks to determine the effect of deworming on proinflammatory gene expression. Faecal egg counts were used to evaluate the efficacy of each drug. RESULTS: While treatment with the benzimidazole significantly reduced egg counts up to 14 days post treatment, it also stimulated proinflammatory gene expression. Treatment with pyrantel salt also reduced faecal egg counts with less of a proinflammatory response. Treatment with the macrocyclic lactone was the most successful in reducing faecal egg counts and produced no signs of increased proinflammatory cytokine expression. CONCLUSIONS: This study revealed pronounced differences in the cytokine responses to anthelmintic treatment. This inflammatory reaction may play a role in the development of parasitic disease post anthelmintic treatment.

The Conqueror Worm: recent advances with cholinergic anthelmintics and techniques excite research for better therapeutic drugs.

The following account is based on a review lecture given recently at the British Society of Parasitology. We point out that nematode parasites cause very widespread infections of humans, particularly in economically underdeveloped areas where sanitation and hygiene are not adequate. In the absence of adequate clean water and effective vaccines, control and prophylaxis relies on anthelmintic drugs. Widespread use of anthelmintics to control nematode parasites of animals has given rise to the development of resistance and so there is a concern that similar problems will occur in humans if mass drug administration is continued. Recent research on the cholinergic anthelmintic drugs has renewed enthusiasm for the further development of cholinergic anthelmintics. Here we illustrate the use of three parasite nematode models, Ascaris suum, Oesophagostomum dentatum and Brugia malayi, microfluidic techniques and the Xenopus oocyte expression system for testing and examining the effects of cholinergic anthelmintics. We also show how the combination of derquantel, the selective nematode cholinergic antagonist and abamectin produce increased inhibition of the nicotinic acetylcholine receptors on the nematode body muscle. We are optimistic that new compounds and combinations of compounds can limit the effects of drug resistance, allowing anthelmintics to be continued to be used for effective treatment of human and animal helminth parasites.

Treatment of echinococcosis: albendazole and mebendazole--what else?

The search for novel therapeutic options to cure alveolar echinococcosis (AE), due to the metacestode of Echinococcus multilocularis, is ongoing, and these developments could also have a profound impact on the treatment of cystic echinococcosis (CE), caused by the closely related Echinococcus granulosus s.l. Several options are being explored. A viable strategy for the identification of novel chemotherapeutically valuable compounds includes whole-organism drug screening, employing large-scale in vitro metacestode cultures and, upon identification of promising compounds, verification of drug efficacy in small laboratory animals. Clearly, the current focus is targeted towards broad-spectrum anti-parasitic or anti-cancer drugs and compound classes that are already marketed, or that are in development for other applications. The availability of comprehensive Echinococcus genome information and gene expression data, as well as significant progress on the molecular level, has now opened the door for a more targeted drug discovery approach, which allows exploitation of defined pathways and enzymes that are essential for the parasite. In addition, current in vitro and in vivo models that are used to assess drug efficacy should be optimized and complemented by methods that give more detailed information on the host-parasite interactions that occur during drug treatments. The key to success is to identify, target and exploit those parasite molecules that orchestrate activities essential to parasite survival.

Anthelmintic drugs and nematicides: studies in Caenorhabditis elegans.

Parasitic nematodes infect many species of animals throughout the phyla, including humans. Moreover, nematodes that parasitise plants are a global problem for agriculture. As such, these nematodes place a major burden on human health, on livestock production, on the welfare of companion animals and on crop production. In the 21st century there are two major challenges posed by the wide-spread prevalence of parasitic nematodes. First, many anthelmintic drugs are losing their effectiveness because nematode strains with resistance are emerging. Second, serious concerns regarding the environmental impact of the nematicides used for crop protection have prompted legislation to remove them from use, leaving agriculture at increased risk from nematode pests. There is clearly a need for a concerted effort to address these challenges. Over the last few decades the free-living nematode Caenorhabditis elegans has provided the opportunity to use molecular genetic techniques for mode of action studies for anthelmintics and nematicides. These approaches continue to be of considerable value. Less fruitful so far, but nonetheless potentially very useful, has been the direct use of C. elegans for anthelmintic and nematicide discovery programmes. Here we provide an introduction to the use of C. elegans as a 'model' parasitic nematode, briefly review the study of nematode control using C. elegans and highlight approaches that have been of particular value with a view to facilitating wider-use of C. elegans as a platform for anthelmintic and nematicide discovery and development.

Present-day anthelmintics and perspectives on future new targets.

In absence of vaccines for the majority of helminths, chemotherapy is still the mainstay for controlling human helminthiases. However, a limited number of drugs are available in the market to combat parasitic helminths in human. Besides, the development and spread of drug resistance have declined the use of most currently available anthelmintics. Clearly, availability of new anthelmintic agents will be essential in the next few years. More research into the mechanisms of drug actions and their targets are eminent for the discovery and development of novel anthelmintic agents. Recent drug discovery techniques mostly rely on mechanism-based screening of compounds on heterologously expressed targets in bacterial, mammalian or yeast cells. Although this is usually a successful approach, it is money- and time-consuming; meanwhile, pharmaceutical companies prefer the tested target that is chosen based on basic research. The nervous system is the site of action of several chemotherapeutics including pesticides and antinematode drugs; accordingly, the nervous system continues to be a promising target. Recent advances in exploring helminths' nervous system, neurotransmitters and receptors have paved the way for the development of potential agents targeting the nervous system and its components.

A survey of helminth control practices on sheep farms in Great Britain and Ireland.

A telephone survey of 600 farmers throughout Great Britain and Ireland was conducted in order to characterise helminth control practices, and identify factors correlated with perceived anthelmintic failure. Overall, 93% of surveyed farmers routinely treated their sheep against nematodes, 67% against liver fluke and 58% against tapeworms. Anthelmintic resistance in nematodes was perceived by farmers to be present on 10% of farms. Farmers who dosed ewes at mating were more likely to have observed anthelmintic failure, than those who were aware of national guidelines on parasite control. However, objective assessment of anthelmintic efficacy had only been undertaken on 19% of farms. Ewes were treated at mating and lambing on 63% and 62% of farms, respectively. On average, lambs were treated 3.6 times annually, depending on geographical region and on dates of lambing and finishing. Although 'quarantine' treatments were widely administered to bought-in stock, these were appropriately applied in only 3% of cases. This study provides baseline data against which the impact of future anthelmintic information campaigns can be assessed; it will facilitate the development of rational, farm-level mathematical models in support of sustainable parasite control, and will aid in the design of farm management practices that prolong the effective lifespan of novel classes of anthelmintic.

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.

Anthelmintic drugs.

C. elegans is sensitive to the majority of anthelmintic drugs that are used against parasitic worm infections of humans and livestock. This has provided the opportunity to use molecular genetic techniques in the worm for mode of action studies. These approaches continue to be of considerable value to the field of parasitology. In addition, there are numerous examples of anthelmintic drugs providing exceptionally useful pharmacological tools to delineate fundamental aspects of cell signalling in C. elegans. This has primarily been achieved through the use of anthelmintics in forward genetic screens followed by the mapping and characterization of genes that confer altered susceptibility to the drug. Less fruitful so far, but nonetheless useful, has been the direct use of C. elegans for anthelmintic discovery programmes. In this brief review we provide an introduction to the use of C. elegans as a 'model parasite', outline the actions of the main classes of anthelmintics, and highlight approaches that have been of particular value.

Study (1991 to 2001) of drug-resistant Population B small strongyles in critical tests in horses in Kentucky at the termination of a 40-year investigation.

Population B, drug-resistant small strongyles have been studied in naturally infected horses in Kentucky for more than 40 years. These parasites first were found to be resistant to phenothiazine (PTZ) and thiabendazole (TBZ), later to other parasiticides. Studies have been on evaluation of antiparasitic efficacy of several compounds, especially the benzimidazoles, against Population B small strongyles in clinical (field) tests (1959-1983) on the commercial farm of origin and in clinical and critical tests (1966-2001) at the University of Kentucky (UK) research farm. Research on these nematodes through 1990 has been published. The current paper presents data on efficacies of various anthelmintics (mostly TBZ) against these and other internal parasites in critical tests done between 1991 and 2001. These were the last critical tests in the UK horses; the entire herd was terminated in 2005. Population B small strongyles were established in horses on a pasture at the UK research farm on Old Lot 4 in 1966, and a satellite of this group was relocated to Field 24 in 1987. The last treatment of any of the horses in clinical tests on pasture was 22 years for Old Lot 4 (mostly benzimidazoles) and 5 years for Field 24 (TBZ) before the last critical test in 2001. Antiparasitic compounds (all paste formulations) administered orally in critical tests (n = 36) reported in this paper were TBZ (@ 44 mg/kg), pyrantel pamoate (PRT @ 6.6 mg base/kg), PTZ (@ 55 mg/kg), fenbendazole (FBZ @ 5 mg/kg), oxfendazole (OFZ @ 10 mg/kg), and oxibendazole (OBZ @ 10 mg/kg). The drug given and number of horses treated from Old Lot 4 were TBZ (18), PRT (3), PTZ (2), FBZ (2), OFZ (1), and OBZ (1) and from Field 24 were OFZ (1) and TBZ (8). Removal of small strongyles in Old Lot 4 was excellent for PRT, OFZ, and OBZ but much less for TBZ, PTZ, and FBZ. For the 16 species present in this lot, removal by TBZ was lowest for seven species (Coronocyclus (Cor.) coronatus, Cyathostomum (Cya.) catinatum, Cylicocyclus (Cyc.) nassatus, Cylicostephanus (Cys.) calicatus, Cylicostephanus goldi, Cylicostephanus longibursatus, and Cylicostephanus minutus). Of these seven species, lowest activity was found for five by PTZ and FBZ. One of the five resistant species was different for each of these two drugs. In Field 24, efficacy against small strongyles was excellent for the one foal treated with OFZ early (1992) in the study. TBZ initially had higher activity than in later years. Of the 12 small strongyle species present in this field, TBZ activity throughout the study was, in general, low for Cor. coronatus, Cys. goldi, and Cys. longibursatus, but it declined more or less progressively for Cya. catinatum, Cylicocyclus leptostomus, Cyc. nassatus, and Cys. calicatus over the study period. Cys. minutus were not present in high enough numbers to evaluate drug efficacy. Overall activity of TBZ on the group of small strongyles did not change; that is, susceptibility did not increase over time in Old Lot 4 where these parasites were not exposed to a benzimidazole for many years. However, in Field 24, where additional TBZ pressure was put on these parasites, efficacy not only did not increase but it decreased. From the data for small strongyles in the two groups of foals, eight species were considered benzimidazole resistant in varying degrees (most research on TBZ). Data on prevalence and drug activity on other internal parasite species besides small strongyles also are given.

[Therapy and anthelmintic resistance]. / Schemi terapeutici e antielmintico-resistenza.

Treatment against nematode parasites in sheep and goats is reviewed. The main risk factors for parasitic infection in these hosts are briefly outlined. The mechanism of action of the most important chemical groups (imidazothiazoles and pyrimidines, benzimidazoles/ pro-benzimidazoles and macrocyclic lactones) to which the modern anthelmintic drugs belong are illustrated and discussed with particular emphasis on possible selection of anthelmintic resistance. The need for strategic integrated control based also on the epidemiological patterns of parasitism, the composition and the production of the herd (milk, meat, wool) and the potency of the drugs are discussed. The importance of diet and potential alternative control measures (nematophagus fungi and natural compounds such as tannins) are illustrated.