Drug-resistant Worms Research Articles

Thermal proteome profiling reveals Haemonchus orphan protein HCO_011565 as a target of the nematocidal small molecule UMW-868.

Parasitic roundworms (nematodes) cause destructive diseases, and immense suffering in humans and other animals around the world. The control of these parasites relies heavily on anthelmintic therapy, but treatment failures and resistance to these drugs are widespread. As efforts to develop vaccines against parasitic nematodes have been largely unsuccessful, there is an increased focus on discovering new anthelmintic entities to combat drug resistant worms. Here, we employed thermal proteome profiling (TPP) to explore hit pharmacology and to support optimisation of a hit compound (UMW-868), identified in a high-throughput whole-worm, phenotypic screen. Using advanced structural prediction and docking tools, we inferred an entirely novel, parasite-specific target (HCO_011565) of this anthelmintic small molecule in the highly pathogenic, blood-feeding barber’s pole worm, and in other socioeconomically important parasitic nematodes. The “hit-to-target” workflow constructed here provides a unique prospect of accelerating the simultaneous discovery of novel anthelmintics and associated parasite-specific targets.

Anthelmintic drug actions in resistant and susceptible C. elegans revealed by electrophysiological recordings in a multichannel microfluidic device

Many anthelmintic drugs used to treat parasitic nematode infections target proteins that regulate electrical activity of neurons and muscles: ion channels (ICs) and neurotransmitter receptors (NTRs). Perturbation of IC/NTR function disrupts worm behavior and can lead to paralysis, starvation, immune attack and expulsion. Limitations of current anthelmintics include a limited spectrum of activity across species and the threat of drug resistance, highlighting the need for new drugs for human and veterinary medicine. Although ICs/NTRs are valuable anthelmintic targets, electrophysiological recordings are not commonly included in drug development pipelines. We designed a medium-throughput platform for recording electropharyngeograms (EPGs)—the electrical signals emitted by muscles and neurons of the pharynx during pharyngeal pumping (feeding)—in Caenorhabditis elegans and parasitic nematodes. The current study in C. elegans expands previous work in several ways. Detecting anthelmintic bioactivity in drugs, compounds or natural products requires robust, sustained pharyngeal pumping under baseline conditions. We generated concentration-response curves for stimulating pumping by perfusing 8-channel microfluidic devices (chips) with the neuromodulator serotonin, or with E. coli bacteria (C. elegans’ food in the laboratory). Worm orientation in the chip (head-first vs. tail-first) affected the response to E. coli but not to serotonin. Using a panel of anthelmintics—ivermectin, levamisole and piperazine—targeting different ICs/NTRs, we determined the effects of concentration and treatment duration on EPG activity, and successfully distinguished control (N2) and drug-resistant worms (avr-14; avr-15; glc-1, unc-38 and unc-49). EPG recordings detected anthelmintic activity of drugs that target ICs/NTRs located in the pharynx as well as at extra-pharyngeal sites. A bus-8 mutant with enhanced permeability was more sensitive than controls to drug treatment. These results provide a useful framework for investigators who would like to more easily incorporate electrophysiology as a routine component of their anthelmintic research workflow.

Transcriptomic profiling of nematode parasites surviving vaccine exposure

Some nematode species are economically important parasites of livestock, while others are important human pathogens causing some of the most important neglected tropical diseases. In both humans and animals, anthelmintic drug administration is the main control strategy, but the emergence of drug-resistant worms has stimulated the development of alternative control approaches. Among these, vaccination is considered to be a sustainable and cost effective strategy. Currently, Barbervax® for the ruminant strongylid Haemonchus contortus is the only registered subunit vaccine for a nematode parasite, although a vaccine for the human hookworm Necator americanus is undergoing clinical trials (HOOKVAC consortium). As both these vaccines comprise a limited number of proteins, there is potential for selection of nematodes with altered sequences or expression of the vaccine antigens. Here we compared the transcriptome of H. contortus populations from sheep vaccinated with Barbervax® with worms from control animals. Barbervax® antigens are native integral membrane proteins isolated from the brush border of the intestinal cells of the adult parasite and many of those are proteases. Our findings provide no evidence for changes in expression of genes encoding Barbervax® antigens in the surviving parasite populations. However, surviving parasites from vaccinated animals showed increased expression of other proteases and regulators of lysosome trafficking, and displayed up-regulated lipid storage and defecation abilities that may have circumvented the effect of the vaccine. Implications for other potential vaccines for human and veterinary nematodes are discussed.

Yeast-Based High-Throughput Screens to Identify Novel Compounds Active against Brugia malayi

BackgroundLymphatic filariasis is caused by the parasitic worms Wuchereria bancrofti, Brugia malayi or B. timori, which are transmitted via the bites from infected mosquitoes. Once in the human body, the parasites develop into adult worms in the lymphatic vessels, causing severe damage and swelling of the affected tissues. According to the World Health Organization, over 1.2 billion people in 58 countries are at risk of contracting lymphatic filariasis. Very few drugs are available to treat patients infected with these parasites, and these have low efficacy against the adult stages of the worms, which can live for 7–15 years in the human body. The requirement for annual treatment increases the risk of drug-resistant worms emerging, making it imperative to develop new drugs against these devastating diseases.Methodology/Principal FindingsWe have developed a yeast-based, high-throughput screening system whereby essential yeast genes are replaced with their filarial or human counterparts. These strains are labeled with different fluorescent proteins to allow the simultaneous monitoring of strains with parasite or human genes in competition, and hence the identification of compounds that inhibit the parasite target without affecting its human ortholog. We constructed yeast strains expressing eight different Brugia malayi drug targets (as well as seven of their human counterparts), and performed medium-throughput drug screens for compounds that specifically inhibit the parasite enzymes. Using the Malaria Box collection (400 compounds), we identified nine filarial specific inhibitors and confirmed the antifilarial activity of five of these using in vitro assays against Brugia pahangi.Conclusions/SignificanceWe were able to functionally complement yeast deletions with eight different Brugia malayi enzymes that represent potential drug targets. We demonstrated that our yeast-based screening platform is efficient in identifying compounds that can discriminate between human and filarial enzymes. Hence, we are confident that we can extend our efforts to the construction of strains with further filarial targets (in particular for those species that cannot be cultivated in the laboratory), and perform high-throughput drug screens to identify specific inhibitors of the parasite enzymes. By establishing synergistic collaborations with researchers working directly on different parasitic worms, we aim to aid antihelmintic drug development for both human and veterinary infections.

Gene expression profiles of hair and wool sheep reveal importance of Th2 immune mechanisms for increased resistance to.

Management of gastrointestinal parasites is a critical issue for sheep producers worldwide. Increases in the prevalence of drug-resistant worms have complicated parasite control and increased economic losses. Therefore, other methods of parasite control need to be assessed, including the use of genetically resistant animals in breeding programs. Hair sheep breeds such as the St. Croix have greater parasite resistance than conventional wool breeds. However, the immune mechanisms that control parasite resistance in hair or wool breeds have not yet been fully determined, and information on cytokine expression profiles for both wool sheep selected for increased resistance and hair sheep is limited. Our objective was to investigate gene expression differences in 24 parasite-resistant hair and 24 susceptible wool sheep to identify immune effectors associated with resistance to . One-half of the lambs were infected and sacrificed at 3 or 27 d after infection. Remaining lambs were not infected. Breed differences in expression of genes associated with Th1 and Th2 immune responses in lymph nodes and abomasal tissue were determined. Th2-associated genes included IL-4, IL-13, IL-5, IgE, the α chain of the IL-4 receptor, and the α chain of the high-affinity IgE receptor (FcεRI). Th1-associated genes included interferon gamma (IFN-γ), the p35 subunit of IL-12 (IL-12 p35), and the β1 and β2 chains of the IL-12 receptor (IL-12 Rβ1 and IL-12 Rβ2, respectively). In both hair and wool sheep, infection with resulted in greater expression of IgE, IL-13, IL-5, and IL-12 p35 and somewhat reduced expression of IFNγ in lymph nodes. In abomasal tissue, parasite infection resulted in greater IgE, IL-13, FcεRI, and IL-12 p35 expression in infected lambs compared with control lambs. Between breeds, hair sheep had a stronger Th2 response after infection than wool sheep, with increased expression of IgE and IL-13 and decreased expression of IFNγ in lymph nodes and increased expression of IL-13 and decreased expression of IL-12 p35 in abomasal tissue. Expression of IL-4 in lymph nodes did not differ between hair and wool lambs, and IL-4, IL-5, IL-12 Rβ1, and IL-12 Rβ2 expression was too low to measure at the times sampled in abomasal tissue.

A randomized survey of medicinal plants used as natural remedies by the local people of Manikganj district of Bangladesh to treat intestinal worms

Soil-transmitted helminthes (STHs) affect nearly two billion people worldwide. The infections STHs cause are categorized as Neglected tropical diseases (NTDs), diseases that persist under poverty and almost exclusively in the developing world. Efforts to control the spread of these parasites include educational campaigns and school-based anthelminthic programs. With no vaccines to protect against these diseases, recurrent use of anthelminthic drugs is the predominant treatment. However, the necessity for repeated doses, the development of drug-resistant worms, and the risk of re-infection after treatment, make current interventions unsustainable, necessitating the development of novel therapeutics. This paper documents knowledge regarding traditional plants used to treat STHs, to preserve this knowledge existing within a rapidly urbanizing nation, as natural products remain an important part of the drug discovery process. We have documented remedies reported from villages within the Manikganj District of Bangladesh, compiling data on the medicinal remedies for STHs and the methods used to prepare them. A randomized survey asked participants to answer questions pertaining to natural remedies, their preparations, and use. Images and names of the identified natural remedies were collected, and current literature was used to identify the scientific names and chemical properties. Twenty-two of the plants named are from 18 different families and include some herbs that have been previously reported as having anthelminthic properties. To conserve traditional medicines, knowledge of both the plant populations and their preparatory methods should be preserved. Key words: Parasites, traditional medicine, soil-transmitted helminthes, Bangladesh, Manikganj, anthelminthic, neglected tropical diseases.

The in vitro diagnosis of anthelmintic resistance in cyathostomins

Cyathostomins are the primary parasitic pathogens of equids. For over 40 years, these nematodes have been controlled using broad spectrum anthelmintics. Three classes of anthelmintic are currently available for this use but, unfortunately, resistance to each of these has now been recorded in cyathostomin populations. As part of an optimal strategy to control cyathostomin infections in the field, it will be important to identify drug-resistant worms at as early a stage as possible. This objective needs to be supported by methodologies that will allow the accurate comparison of anthelmintic resistance in different nematode populations. At present, the faecal egg count reduction test is considered the most suitable method for initial screening for anthelmintic resistance in equine nematode populations. However, in its current state, this test lacks sensitivity. It is also costly and time-consuming to perform. Laboratory-based techniques, such as the egg hatch assay, larval development assay, larval migration inhibition assay and the larval feeding inhibition assay offer alternative options for assessing anthelmintic resistance in nematode populations. All of these tests have been investigated for their utility in measuring drug resistance in sheep nematode populations and some have proven useful. The egg hatch assay, larval development assay and larval migration inhibition assay have been investigated for use in measuring levels of drug resistance in equine nematode populations. However, at best, the results obtained thus far indicate that these tests require further refinement.

Challenges of nematode control in ruminants: Focus on Latin America

Gastrointestinal nematodes (GIN) of ruminants (cattle, sheep and goats) are ubiquitous and can cause severe injuries to infected animals and significant losses in farming revenues. GIN are able to survive severe environmental and host conditions, but mankind has developed a number of ingenious methods for parasite control. The commerce and use of modern anthelmintic drugs with a broad spectrum of activity has been a solid tool for nearly 40 years, however the continuous use of these drugs, has led to the selection of populations of drug-resistant worms worldwide. At present, the ever-growing agricultural systems in Latin America are facing many challenges and cannot rely on the far-reaching objective of parasitic elimination from the host or the environment. The lack of extensive programs for monitoring drug resistance exacerbates the negative consequences of reduced efficacy, which is evident in some areas with the increase in mortality rate even after treatment. Experts agree that new schemes of parasitic control are needed and should be based on the strategy of targeted selective treatment where affected hosts are identified and treated accordingly. In this article, we will focus our discussion on the challenges for the control of GIN in Latin America by 2020 imposed by reduced drug efficacy. We will evaluate phenotypic and molecular markers, methods for single-animal evaluation, and the implementation of schemes for anthelmintic treatment that address parasites in refugia.

Microarray analysis reveals difference in gene expression profiles of hair and wool sheep infected with Haemonchus contortus

Sheep infected with the abomasal parasite, Haemonchus contortus, have reduced growth rates, decreased wool production, and anemia, and heavy infections may result in death. Anthelmintic treatment can remove worms, but the cost of treatment and prevalence of drug-resistant worms has led to greater focus on genetic resistance of the host to parasitism. Variation in parasite resistance exists within and among sheep breeds, and Caribbean hair sheep have greater resistance than most conventional wool breeds. Our objective was to investigate differences in gene expression between 24 parasite-resistant hair and 24 susceptible wool lambs to determine genetic mechanisms involved in resistance to H. contortus. Half of the animals of each breed were infected and sacrificed at 3 or 27 days post-infection; the remaining animals were uninfected controls. Breed differences in abomasum and abomasal lymph node tissue gene expression were assessed using bovine cDNA microarrays. Over 60 transcripts differed between breeds for each tissue and infection status. Genes differentially expressed between hair and wool sheep 3 days PI were assessed for gene function and mechanisms for greater immune cell infiltration, abomasal tissue repair, Th17 response, and anticoagulation were present in parasite-resistant hair sheep. By 27 days PI, hair sheep had greater expression of genes involved in gut motility, inflammatory cytokines, and cell proliferation and differentiation compared to wool sheep. Changes in these processes indicate Caribbean hair sheep have a stronger inflammatory response when infected with H. contortus which may facilitate the increased parasite resistance observed in these sheep.

めん羊牧場における捻転胃虫症の被害と駆虫対策の検討

There was a high incidence of sudden death due to haemonchosis in a sheep herd, where a regular anthelmicticprogram using diethylcarbamazine had been conducted. When a new anthelmintic program using ivermectin (IVM) or levamisole (LEV) was conducted over three years, the mortality apparently decreased fromapproximately 20% to 2.8% in the third year. The anthelmintic effects were poor with minimal reduction in eggper gram (EPG) in the pour-on route of IVM, but were significantly higher with a major reduction in the average EPG from 5418.5 to 3.2 in the subcutaneous route of IVM and from 98.2 to 1.9 in the oral route of LEV afteradministration for one month. However, the anthelmintic effects after two months of administration in the subcutaneousroute of IVM were lower than those of the one-month interval, in which the reduction of EPG wasfrom 120.5 to 41.1, indicating no significance. Moreover, despite the significantly higher anthelmintic effectsat first administration of both IVM and LEV for each year, the EPGs were gradually decreased after the secondadministration. This may indicate the occurrence of drug-resistant worms.

Adult worm tegumental damage and egg-granulomas in praziquantel-resistant and -susceptible Schistosoma mansoni treated in vivo.

The effect of treatment with praziquantel (PZQ) on the tegument of adult Schistosoma mansoni worms and on liver egg-granulomas has been examined in mice infected with PZQ-resistant and -susceptible parasite isolates. Two PZQ-resistant S. mansoni isolates, one selected by passage in the laboratory under drug pressure and one from Senegal established from eggs excreted by an uncured patient, were compared with PZQ-susceptible control isolates. Scanning electron microscopic observations on the tegument of Schistosoma adult worms treated in vivo with PZQ showed that more severe damage was inflicted by PZQ on susceptible worms than on drug-resistant worms. Observations on the pathology of Schistosoma egg-granulomas in the liver of infected mice after treatment with PZQ indicated that eggs from susceptible control isolates were more sensitive to PZQ than those from drug-resistant isolates.

Protection against multiply drug-resistant and geographically distant strains of Haemonchus contortus by vaccination with H11, a gut membrane-derived protective antigen

H11 is a “hidden antigen” derived from adult Haemonchus contortus which is capable of conferring a high degree of protection against subsequent challenge. To be commercially useful, any vaccine based on this antigen must protect against field strains and drug-resistant worms. The results show that vaccination with H11 affords high levels of protection against the multiply drug-resistant (MDR) Lawes strain of Haemonchus contortus. Moreover, there was no difference in protection whether the H11 was extracted from drug-resistant or susceptible worms, indicating that multiple drug-resistance does not significantly alter the immunogenicity of H11. Ideally, a commercial vaccine based on this antigen should also be capable of protecting against strains from geographically separate areas, so that it is not necessary to manufacture “region-specific” vaccines. Vaccination with H11 purified from either Australian or U.K. Haemonchus contortus was effective in protecting against subsequent challenge with Australian larvae, indicating that one type of H11 should have world-wide application.

Schistosoma mansoni: Hycanthone/oxamniquine resistance is controlled by a single autosomal recessive gene

Individual schistosomes of an hycanthone/oxamniquinesensitive strain were crossed with individual schistosomes of the opposite sex and belonging either to the same sensitive population or to a different strain which exhibited high resistance to the two drugs. Schistosome crosses were performed by transfer of single worm pairs into the mesenteric veins of mice and the drug sensitivity/resistance of individual progeny worms was assessed using an in vitro test. Drug resistance behaved as an autosomal recessive trait, as shown by the results of the F1 and F2 generation and of the backcrosses. Drug-resistant worms appeared to be slightly less viable than their sensitive counterpart at all stages of the life cycle. The results are relevant for an interpretation of drug resistance and drug mechanisms and the approach used in this study may be applicable to different genetic markers in schistosomes.

Effect of benzimidazole drugs on tubulin in benzimidazole resistant and susceptible strains of Caenorhabditis elegans

An in vitro assay was used to determine efficacy and if side resistance was present to benzimidazole anthelmintics tested against Caenorhabditis elegans after selection with albendazole. Side resistance was present to all the benzimidazoles tested, except for oxibendazole and parbendazole. At a concentration of 1 mM, all of the drugs, except thiabendazole, were effective in killing 100% of the albendazole susceptible worms. Tubulin from albendazole resistant and susceptible C. elegans was isolated and run on polyacrylamide gels. Western blots with anti-tubulin antibody showed that the albendazole resistant strain had an altered tubulin. Electron microscopy of albendazole-treated drug resistant worms showed microtubules throughout the intestinal cells. Microtubules were not observed in albendazole-treated drug susceptible worms.

Enzymatic differences between hycanthone-resistant and sensitive strains of Schistosoma mansoni

1. 1. Hycanthone-sensitive and resistant adult worms of Schistosoma mansoni were found to have generally similar specific activities in ten enzymes of carbohydrate metabolism. 2. 2. Kinetic analyses revealed that pyruvate kinase, glucose-6-phosphate (G6P) dehydrogenase and malate dehydrogenase from both strains possessed similar Michaelis-Menten constants and were not inhibited by hycanthone. 3. 3. Hexokinase and lactate dehydrogenase from the drug-resistant strain were not inhibited by hycanthone and showed three to five times greater K m values than those from the drug-sensitive worms which were also inhibited by hycanthone. 4. 4. Hycanthone more drastically affected the V max of phosphofructokinase from the hycanthone-sensitive parasite. 5. 5. These data showed that the hycanthone inhibitable enzymes were generally from the drug-sensitive strain whereas the enzymes from drug-resistant worms are mostly hycanthone insensitive.