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A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinergic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B at the whole-worm level has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells in the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that the synergism is due to the cytoplasmic Ca2+ overload that is induced by the combination of levamisole opening Ca2+ permeable L-subtype nAChRs and the Ca2+ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca2+ overloads that accelerates cell-death of the enterocytes., Competing Interests: The authors declare no competing interests., (Copyright: © 2024 Williams et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: The use of insufficiently treated wastewater or Faecal sludge in agriculture raises concerns because of the pathogen content. Helminth eggs (HE) are one of the most crucial pathogens for ensuring public health and safety. Widely used disinfection treatment methods do not guarantee the complete inactivation of helminth eggs. The current study evaluated the effectiveness of anaerobic digestion and electrochemical process on helminth ( Ascaris suum ) egg inactivation., Methods: Lab-scale biochemical methane potential (BMP) assay was conducted by spiking A. suum eggs in a serum bottle. Total solid (TS), volatile solid (VS), pH, biogas production and its composition, and volatile fatty acids (VFA) were analyzed along with A. suum inactivation every third day for the initial 15 days and fifth day for 45 days. In the second set of experiments, a hypochlorite (4700 ppm) solution was generated by electrolysis of aqueous NaCl solution in a membrane-less electrochemical cell. The hypochlorite was diluted (940, 470, 235, and 156ppm) in wastewater, spiked with A. suum eggs and then examined for inactivation at regular intervals., Results: The results of the anaerobic digestion treatment documented 98% inactivation of A. suum eggs (0.15 eggs/mL) in 35 days and remained at 0.14 eggs/mL until day 45. Correlation analysis revealed a positive relationship between non-viable eggs and pH and a negative relationship with all the other parameters. Electrochemical treatment achieved 10% inactivation at 940 ppm concentration in 24h., Conclusions: This study revealed that the inactivation of A. suum eggs by anaerobic digestion or electrochemical treatment is a combined effect of more than one parameter., Competing Interests: No competing interests were disclosed., (Copyright: © 2024 Patil PP and Mutnuri S.)

Soil-transmitted helminths (STH) are important and widespread intestinal pathogens of humans and animals. It is presently unknown which inactivating procedures may be universally effective for safe transport, preservation, and disinfection of STH-contaminated specimens, and this lack of knowledge may expose laboratory staff to higher risk of laboratory-acquired infections (LAI's). There are limited data on the efficacy of commonly used disinfectants and fecal fixatives for inactivating the eggs of STH. This work tested five disinfectants for surface cleanup, four storage temperature conditions, and six transport/storage fixatives, to inactivate eggs of three species of STH of animal origin ( Ascaris suum "roundworm," Trichuris vulpis "whipworm" and Ancylostoma caninum "hookworm") as surrogates for human STH. Among disinfectants, exposure to 10% povidone-iodine for ≥5 min inactivated 100% of the three species tested, while 5 min exposure to 95% ethanol inactivated T. vulpis and A. caninum eggs. All of the fixatives tested had inactivation effects on A. caninum hookworm eggs within 24 h of exposure, except potassium dichromate, which required 48 h. 95% ethanol for ≥48 h inactivated eggs from all three STH species. Freezing at ≤-20°C for ≥24 h inactivated eggs of T. vulpis and A. caninum, but only freezing at -80°C for ≥24 h inactivated >99% eggs, including A. suum . This work provides an evidence base for health and safety guidelines and mitigation strategies for the handling, storage, and disposal of stool samples containing STH eggs in laboratory, health care, childcare, or veterinary settings. IMPORTANCE This study systematically evaluates common laboratory disinfectants and storage conditions for their effectiveness in inactivating the infective stages of soil-transmitted helminths (STH). Animal-infecting proxy species were chosen to represent three major groups of STH that infect humans: roundworms, whipworms, and hookworms. Previously published work in this area typically focuses on a particular inactivation method, either for a single STH species, or on a subset of closely related species. Because prediagnostic fecal specimens must be regarded as potentially infectious with a mix of species, such information may be of limited utility in a working laboratory. We provide a straightforward summary of storage and disinfection methods that can achieve complete inactivation across a range of STH species, which represents a significant advance for clinical, veterinary and research laboratory biosafety.

Background: The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding β-tubulin proteins, the principal targets of BZ drugs., Methodology and Principal Findings: First, the β-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different β-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations., Significance: This study demonstrated the presence of at least seven β-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the β-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of β-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible., Competing Interests: The authors have declared that no competing interests exist.

Parasitic infections are a widespread health problem and research of novel anthelmintic compounds is of the utmost importance. In this study we performed a virtual screening campaign by coupling ligand-based pharmacophore, homology modeling and molecular docking. The virtual screening campaign was conducted using a joined pool of the Drugbank database and a library of purchasable compounds in order to identify drug like compounds with similar pharmacological activity. Our aim was to identify compounds with a potential antihelmintic modulatory effect on nicotinic acetylcholine receptors (nAChR). We derived a 3D pharmacophore model based on the chemical features of known Ascaris suum nAChR modulators. To evaluate the in silico predictions, we tested selected hit compounds in contraction assays using somatic muscle flaps of the Ascaris suum neuromuscular tissue. We tested the modulatory effects of GSK575594A, diazepam and flumazenil hit compounds on nematode contractions induced by acetyl choline (ACh). The compound GSK575594A (3 μM) increased the E max by 21 % with the EC 50 dose ratio of 0.96. Diazepam (100 μM) decreased the E max by 15 % (1.11 g to 0.95 g) with the EC 50 ratio of 1.42 (shifted to the left from 11.25 to 7.93). Flumazenil decreased the EC 50 value (from 11.22 μM to 4.88 μM) value showing dose ratio of 2.30, and increased the E max by 4 % (from 1.54 g to 1.59 g). The observed biological activity was rationalized by molecular docking calculations. Docking scores were calculated against several binding sites within the Ascaris suum homology model. We constructed the homology model using the ACR-16 subunit sequence. The compound GSK575594A showed strong affinity for the intersubunit allosteric binding site within the nAChR transmembrane domain. The binding modes of diazepam and flumazenil suggest that these compounds have a comparable affinity for orthosteric and allosteric nAChR binding sites. The selected hit compounds displayed potential for further optimization as lead compounds. Therefore, such compounds may be useful in neutralizing the growing resistance of parasites to drugs, either alone or in combination with existing conventional anthelmintics., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Infection by nematodes is a problem for human health, livestock, and agriculture, as it causes deficits in host health, increases production costs, and incurs a reduced food supply. The control of these parasites is usually done using anthelmintics, which, in most cases, have not been fully effective. Therefore, the search for new molecules with anthelmintic potential is necessary., Methods: In the present study, we isolated and characterized molecules from the nematophagous fungus Pochonia chlamydosporia and tested these compounds on three nematodes: Caenorhabditis elegans; Ancylostoma ceylanicum; and Ascaris suum., Results: The ethyl acetate extract showed nematicidal activity on the nematode model C. elegans. We identified the major substance present in two sub-fractions of this extract as ketamine. Then, we tested this compound on C. elegans and the parasites A. ceylanicum and A. suum using hamsters and mice as hosts, respectively. We did not find a difference between the animal groups when considering the number of worms recovered from the intestines of animals treated with ketamine (6 mg) and albendazole (P > 0.05). The parasite burden of larvae recovered from the lungs of mice treated with ketamine was similar to those treated with ivermectin., Conclusions: The results presented here demonstrate the nematicidal activity of ketamine in vitro and in vivo, thus confirming the nematicidal potential of the molecule present in the fungus P. chlamydosporia may consist of a new method of controlling parasites.

Cholinergic agonists, like levamisole, are a major class of anthelmintic drugs that are known to act selectively on nicotinic acetylcholine receptors (nAChRs) on the somatic muscle and nerves of nematode parasites to produce their contraction and spastic paralysis. Previous studies have suggested that in addition to the nAChRs found on muscle and nerves, there are nAChRs on non-excitable tissues of nematode parasites. We looked for evidence of nAChRs expression in the cells of the intestine of the large pig nematode, Ascaris suum, using RT-PCR and RNAscope in situ hybridization and detected mRNA of nAChR subunits in the cells. These subunits include components of the putative levamisole receptor in A. suum muscle: Asu-unc-38, Asu-unc-29, Asu-unc-63 and Asu-acr-8. Relative expression of these mRNAs in A. suum intestine was quantified by qPCR. We also looked for and found expression of G protein-linked acetylcholine receptors (Asu-gar-1). We used Fluo-3 AM to detect intracellular calcium changes in response to receptor activation by acetylcholine (as a non-selective agonist) and levamisole (as an L-type nAChR agonist) to look for evidence of functioning nAChRs in the intestine. We found that both acetylcholine and levamisole elicited increases in intracellular calcium but their signal profiles in isolated intestinal tissues were different, suggesting activation of different receptor sets. The levamisole responses were blocked by mecamylamine, a nicotinic receptor antagonist in A. suum, indicating the activation of intestinal nAChRs rather than G protein-linked acetylcholine receptors (GARs) by levamisole. The detection of nAChRs in cells of the intestine, in addition to those on muscles and nerves, reveals another site of action of the cholinergic anthelmintics and a site that may contribute to the synergistic interactions of cholinergic anthelmintics with other anthelmintics that affect the intestine (Cry5B)., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Nematode parasites infect approximately 1.5 billion people globally and are a significant public health concern. There is an accepted need for new, more effective anthelmintic drugs. Nicotinic acetylcholine receptors on parasite nerve and somatic muscle are targets of the cholinomimetic anthelmintics, while glutamate-gated chloride channels in the pharynx of the nematode are affected by the avermectins. Here we describe a novel nicotinic acetylcholine receptor on the nematode pharynx that is a potential new drug target. This homomeric receptor is comprised of five non-α EAT-2 subunits and is not sensitive to existing cholinomimetic anthelmintics. We found that EAT-18, a novel auxiliary subunit protein, is essential for functional expression of the receptor. EAT-18 directly interacts with the mature receptor, and different homologs alter the pharmacological properties. Thus we have described not only a novel potential drug target but also a new type of obligate auxiliary protein for nAChRs., Competing Interests: The authors declare no competing interests.

Chicory (Cichorium intybus) is a bioactive forage rich in sesquiterpene lactones (SLs) with reported in vitro and in vivo anthelmintic activity in livestock. However, the on-farm adoption of chicory as an anthelmintic crop is limited and may be facilitated by using standardised industrial chicory material. Chicory root pulp is a by-product obtained from industrial chicory roots after inulin extraction and can potentially retain SLs. However, SL content and associated anthelmintic activity of chicory root pulp have not been investigated. Here, we evaluated the anthelmintic activity of SL-enriched extracts from chicory root pulp and forage chicory, and used untargeted metabolomics and molecular networking to identify potential anthelmintic molecules. Six different sources of chicory material were used: fresh chicory root pulp (from industrial chicory roots C. intybus var. sativum; "Root Pulp"), fresh leaves from chicory cv. Spadona (sampled on four occasions) and fresh leaves from chicory cv. Choice. The resulting extracts were tested for anthelmintic activity against the free-living nematode Caenorhabditis elegans and the pig nematode Ascaris suum. The cytotoxicity of the chicory extracts was evaluated on mammalian (Vero) cells. In the C. elegans assays, the Root Pulp was the most potent extract and induced paralysis in >95% of worms exposed to >250  μg extract/mL (EC 50  = 64.2 μg/mL). In the A. suum assays, the Root Pulp was also the most potent chicory extract to inhibit worm motility (EC 50 = 87.6  μg/mL), followed closely by two of the Spadona leaf extracts (EC 50  = 89.8  μg/mL and 112.2  μg/mL) The Root Pulp extract had the lowest cytotoxicity of all tested extracts towards mammalian cells, with a selectivity index of 5.37. Untargeted metabolomics revealed that chicory Root Pulp had a markedly different chemical profile in comparison with forage chicory extracts. Molecular networking confirmed several SLs and SL-derivatives mainly present in chicory root pulp, that may be responsible of its potent anti-parasitic activity. Bioactivity-based molecular networking of chicory root pulp and the most potent forage chicory extracts revealed a high predicted anthelmintic score for the guaianolide SL 11,13-dihydro-lactucopicrin. In conclusion, chicory root pulp showed potent and selective in vitro anthelmintic activity against C. elegans and A. suum, with low cytotoxicity in mammalian cells. The promising anthelmintic activity of chicory root pulp should be confirmed in vivo to further explore the potential of this agro-industrial by-product as a nutraceutical anthelmintic for livestock and as novel source of anti-parasitic compounds., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Benzimidazoles are commonly used for the control of veterinary nematodes. Resistance to benzimidazoles has been associated with three single nucleotide polymorphisms in the β-tubulin gene of common nematodes. However, these mutations are infrequent in the genus Ascaris spp., Methods: In order to determine mutations associated with benzimidazole resistance in Ascaris suum, worms were collected from slaughtered pigs and a partial region of the β-tubulin gene was sequenced., Results: All parasites showed the wildtype genotype for codons 167, 198, and 200 of the β-tubulin gene., Conclusions: This is the first report of genetic sequences associated with benzimidazole resistance in A. suum.

The neuromuscular system of parasitic nematodes has proven to be an efficient pharmacological target for antihelmintics. Some of the most frequently used antiparasitic drugs are agonists or antagonists of nicotinic acetylcholine receptors (nAChRs). The antinematodal mechanism of action of carvacrol involves the inhibition of parasite muscle contraction. We have examined the interaction of carvacrol with antinematodal drugs that are agonists of different subtypes of nAChRs and monepantel, which is a non-competitive antagonist of this receptor in A. suum. Additionally, we investigated the effect of carvacrol on the muscle type of nAChRs in the mammalian host. As orthosteric agonists of nAChR, pyrantel, morantel and befinijum lead to dose-dependent contractions of the neuromuscular preparation of Ascaris suum. Carvacrol 100 μM decreased the E max of pyrantel, morantel and bephenium by 29%, 39% and 12 %, 39 % and 12 % respectively. The EC 50 ratio was 3.43, 2.95 and 2.47 for pyrantel, morantel and bephinium, respectively. Carvacrol 300 u μM reduces the E max of pyrantel, morantel and bephenium by 71%, 80% and 75 %, 80 % and 75 % respectively. The EC 50 ratio for pyrantel, morantel and bephenium was 3.88, 3.19 and 4.83 respectively. Furthermore, carvacrol enhances the inhibitory effect of monepantel on A. suum contractions, which may have an effective clinical application. On the other hand, tested concentrations of carvacrol did not significantly affect the EFS-induced contractions of the rat diaphragm, indicating a lack of interaction with the postsynaptic nAChR at the muscle end plate in mammals, but the highest concentration (300 μM) caused a clear tetanic fade. Carvacrol exhibited a time and dose-dependent effect on the Rota-rod performances of rats with a high value of the ED 50 (421.6 mg/kg). In our research, carvacrol dominantly exhibited characteristics of a non-competitive antagonist of nAChR in A. suum, and enhances the inhibitory effect of monepantel. The combination of monepantel and carvacrol may be clinically very effective, and the carvacrol molecule itself can be used as a promising platform for the development of new anthelmintic drugs., Competing Interests: Declaration of Competing Interest The authors have declared that no competing interests exist., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Anaerobic digestion (AD) is a commonly used method of processing waste. Regardless of the type of the used digestate (fertilizer, feedstock in case of solid-state fermentation, raw-material in case of thermal treatment) effective pathogen risk elimination, even in the case of high pathogen concentration is essential. An investigation of the survival time and inactivation rate of the Salmonella Senftenberg W 775 , Enterococcus spp., and Ascaris suum eggs during thermophilic anaerobic digestion performed on laboratory scale and confirmation of hygienization in full-scale operation were performed in this study. Except for sanitization efficiency, the AD process performance and stability were also verified based on determination of pH value, dry matter content, acidity, alkalinity, and content of fatty acids. The elimination of pathogen was met within 6.06 h, 5.5 h, and about 10 h for the Salmonella Senftenberg W 775 , Enterococcus spp., and Ascaris suum , respectively in the laboratory trials. The obtained results were confirmed in full-scale tests, using 1500 m 3 Kompogas ® reactors, operating in MBT Plant located in Poland. Sanitization of the digestate was achieved. Furthermore, the process was stable. The pH value, suspended solids, and ammonium content remained stable at 8.5, 35%, and 3.8 g/kg, respectively. The acetic acid content was noted between almost 0.8 and over 1.1 g/kg, while the concentration of propionic acid was noted at maximum level of about 100 mg/kg. The AD conditions could positively affect the pathogen elimination. Based on these results it can be found that anaerobic digestion under thermophilic conditions results in high sanitation efficiency.

The aim of this study was to investigate the effect of a strategic deworming program on Ascaris suum infection levels and technical performance parameters in fattening pigs. Eighteen fattening stables were selected and divided into two groups. Group 1 consisted of 9 stables in which the fattening pigs tested seropositive for Ascaris, indicative for the presence of Ascaris eggs in the stable, whereas group 2 consisted of 9 stables in which the fattening pigs tested seronegative for Ascaris, indicating of a low or absent environmental contamination with Ascaris eggs. The production in each stable was monitored for a period of 7 consecutive fattening rounds. The first of these 7 fattening rounds (i.e. round 0), during which no intervention took place in the deworming strategy applied in the stable, served as a historical control. A deworming program using 200 mg/ml fenbendazole oral suspension in drinking water for 2 days every 6 weeks was implemented for a period of 6 consecutive fattening rounds. For each fattening round and for each stable, technical performance parameters including average daily growth, feed conversion ratio, days in fattening and the percentage of affected livers were obtained from the producers. Blood was collected from 10 randomly selected animals per stable at the end of each fattening round and evaluated for the presence of anti-Ascaris antibodies using 2 different serological tests, namely the AsHb- and the L3-Lung ELISA. The serological results obtained indicated a lower exposure of the animals to Ascaris after the implementation of a strategic deworming program. A significant decline in anti-Ascaris antibody levels was detectable in the stables that originally tested positive for Ascaris and was already visible after one treatment round. The outcomes of hierarchical linear mixed models indicated that the level of L3-Lung antibody reactivity was a significant predictor of decreased ADG, increased FCR and prolonged DIF for the Ascaris-positive herds, indicating an effect of Ascaris infections on productivity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The ongoing and widespread emergence of resistance to the existing anti-nematodal pharmacopeia has made it imperative to develop new anthelminthic agents. Historically, plants have been important sources of therapeutic compounds and offer an alternative to synthetic drugs. Monoterpenoids are phytochemicals that have been shown to produce acute toxic effects in insects and nematodes. Previous studies have shown nicotinic acetylcholine receptors (nAChRs) to be possible targets for naturally occurring plant metabolites such as carvacrol and carveol. In this study we examined the effects of monoterpenoid compounds on a levamisole sensitive nAChR from Oesophagostomum dentatum and a nicotine sensitive nAChR from Ascaris suum. We expressed the receptors in Xenopus laevis oocytes and used two-electrode voltage-clamp to characterize the effect of various compounds on these cys-loop receptors. At 100 μM the majority of these compounds acted as antagonists. Interestingly, further experiments revealed that both 0.1 μM and 10 μM menthol potentiated acetylcholine and levamisole responses in the levamisole sensitive receptor but not the nicotine sensitive receptor. We also investigated the effects of 0.1 μM menthol on the contractility of A. suum somatic muscle strips. Menthol produced significant potentiation of peak contractions at each concentration of acetylcholine. The positive allosteric modulatory effects of menthol in both in vivo and in vitro experiments suggests menthol as a promising candidate for combination therapy with cholinergic anthelmintics., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tenvermectin (TVM) is a novel 16-membered macrocyclic lactone antibiotics, which contains component TVM A and TVM B. However there is not any report on safety and clinical efficacy of TVM for developing as a potential drug. In order to understand the part of safety and clinical efficacy of TVM, we conducted the acute toxicity test, the standard bacterial reverse mutation (Ames) test and the clinical deworming test. In the acute toxicity studies, TVM, TVM A and ivermectin (IVM) were administrated once by oral gavage to mice and rats. Results showed that the oral LD 50 values of TVM, TVM A and IVM in mice were 74.41, 106.95 and 53.06 mg/kg respectively. The oral LD 50 values of TVM and TVM A in rats were determined to be 164.22 and 749.34 mg/kg respectively. TVM and IVM are moderately toxic substances, meanwhile the TVM A belongs to low toxic compounds, implying that the acute toxicity is highly related to the length of side chain of TVM at position C25. In the Ames test, results showed that TVM did not induce mutagenicity in Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without metabolic activation system, speculating that the mutagenicity is probably not related to the side chain at position C25 of 16-membered macrocyclic lactone antibiotics. In the efficacy trail of TVM against swine nematodes, growing pigs natural infection of Ascaris suum and Trichuris suis were treated with a single subcutaneous injection 0.3 mg/kg b.w.. Results showed that TVM and IVM had excellent effect in expelling Ascaris suum, and TVM had potential efficacy against Trichuris suis, however IVM had no effect on Trichuris suis. This study suggests that the side chain of TVM at position C25 may have important biological functions, which is one of the key sites of the studies on structure-activity relationship of 16-membered macrocyclic lactone compounds. TVM is a new compound exhibited some advantages worthy of developing., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Zolvix ® is a recently introduced anthelmintic drench containing monepantel as the active ingredient. Monepantel is a positive allosteric modulator of DEG-3/DES-2 type nicotinic acetylcholine receptors (nAChRs) in several nematode species. The drug has been reported to produce hypercontraction of Caenorhabditis elegans and Haemonchus contortus somatic muscle. We investigated the effects of monepantel on nAChRs from Ascaris suum and Oesophagostomum dentatum heterologously expressed in Xenopus laevis oocytes. Using two-electrode voltage-clamp electrophysiology, we studied the effects of monepantel on a nicotine preferring homomeric nAChR subtype from A. suum comprising of ACR-16; a pyrantel/tribendimidine preferring heteromeric subtype from O. dentatum comprising UNC-29, UNC-38 and UNC-63 subunits; and a levamisole preferring subtype (O. dentatum) comprising UNC-29, UNC-38, UNC-63 and ACR-8 subunits. For each subtype tested, monepantel applied in isolation produced no measurable currents thereby ruling out an agonist action. When monepantel was continuously applied, it reduced the amplitude of acetylcholine induced currents in a concentration-dependent manner. In all three subtypes, monepantel acted as a non-competitive antagonist on the expressed receptors. ACR-16 from A. suum was particularly sensitive to monepantel inhibition (IC 50 values: 1.6 ± 3.1 nM and 0.2 ± 2.3 μM). We also investigated the effects of monepantel on muscle flaps isolated from adult A. suum. The drug did not significantly increase baseline tension when applied on its own. As with acetylcholine induced currents in the heterologously expressed receptors, contractions induced by acetylcholine were antagonized by monepantel. Further investigation revealed that the inhibition was a mixture of competitive and non-competitive antagonism. Our findings suggest that monepantel is active on multiple nAChR subtypes., (Copyright © 2017. Published by Elsevier Ltd.)

Ascaris suum is an important intestinal nematode causing economic losses in swine. Anthelminthic treatment is used to control A. suum infections and is part of normal production practices. Treatment with anthelminthic agents results in expulsion of adult worms from the intestinal tract and ends further contamination of the environment with eggs. The present study was conducted to determine the effects of drug treatment on the embryonation of A. suum eggs collected from worms obtained from pigs treated with 4 different commercially available anthelmintics. The effects of treatment with abamectin, doramectin, ivermectin, flubendazole, or no treatment on embryonation of A. suum eggs collected from female A. suum expelled in the feces was determined. The embryonation of eggs obtained from pigs treated with abamectin, doramectin, and ivermectin was not significantly (P > 0.05) different from eggs from non-treated control pigs. In contrast, the embryonation of A. suum eggs collected from worms from pigs treated with flubendazole demonstrated inhibited development, and most eggs remained in the 1-cell stage (85.5%) and only 6.3% of eggs developed larvae. In another experiment, we examined the direct effects of doramectin and flubendazole added to solutions of A. suum eggs collected from non-treated control pigs. Egg cultures were exposed to direct in vitro treatment with 0.04-parts per million (ppm) doramectin or 1.0-ppm flubendazole for 24 hr (highest concentrations [C max ] of drugs in serum) and then embryonation and infectivity for mice was determined. Treatment of eggs in vitro did not significantly effect (P > 0.05) larval development or oral infectivity for mice. Our study demonstrates that flubendazole fed to pigs results in inhibited embryonation of A. suum eggs. However, direct treatment of A. suum eggs in culture for 24 hr with flubendazole did not inhibit embryonation or oral infectivity of in vitro treated eggs. Anthelmintic treatment of pigs in vivo with abamectin, doramectin, and ivermectin had no significant (P > 0.05) effect on embryonation of A. suum eggs, and 24 hr treatment with doramectin in vitro had no direct effects (P > 0.05) on embryonation or oral infectivity of A. suum eggs.

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 (EC 50 ). The values of EC 50 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The husbandry of pigs for meat production is a constantly developing industry. Most studies on the effects of Ascaris suum infection in pigs and its prevention with anthelmintics are over a decade old. We examined the effect of 2.5mg fenbendazole per kg bodyweight administered in drinking water for two consecutive days on A. suum infection 1 and 6 weeks after pigs arrived to fattening units. We hypothesised that the treatment would reduce the presence of A. suum-infections, improve the average daily weight gain of pigs, reduce the percentage of liver rejections in pens by 50% and increase the lean meat percentage at slaughter by 1%. The study included a placebo group (427 pigs) and a treatment group (420 pigs) spanning four different farms previously reporting ≥15% liver rejection. The treatment was given for 2 consecutive days 1 and 6 weeks after the pigs arrived to the fattening unit. Faecal samples were collected during weeks 1, 6 and 12 from all pigs and examined for A. suum eggs. Blood was collected during weeks 1 and 12 from a subgroup of the pigs and examined for anti-A. suum antibodies and clinical blood parameters. Data on liver rejection and lean meat percentage were collected post-mortem. The proportion of Ascaris seropositive pigs changed from 8.6% to 22.2% and 20.3% to 16.3% in the placebo and treatment group respectively. Fenbendazole reduced the presence of A. suum eggs in faeces the percentage of liver rejections by 69.8%. The treatment did not affect daily weight gain or lean meat percentage. Pigs with A. suum eggs in faeces at week 6 had a lower average daily weight gain of 61.8g/day compared with pigs without parasite eggs. Fenbendazole treatment may be a useful option for farms struggling with persistent A. suum problems and demonstrate a beneficial effect on the weight gain of the animals shedding eggs in faeces and result in fewer condemned livers at slaughter., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The screening of candidate compounds and natural products for anthelmintic activity is important for discovering new drugs against human and animal parasites. We previously validated in Caenorhabditis elegans a microfluidic device ('chip') that records non-invasively the tiny electrophysiological signals generated by rhythmic contraction (pumping) of the worm's pharynx. These electropharyngeograms (EPGs) are recorded simultaneously from multiple worms per chip, providing a medium-throughput readout of muscular and neural activity that is especially useful for compounds targeting neurotransmitter receptors and ion channels. Microfluidic technologies have transformed C. elegans research and the goal of the current study was to validate hookworm and Ascaris suum host-stage larvae in the microfluidic EPG platform. Ancylostoma ceylanicum and A. caninum infective L3s (iL3s) that had been activated in vitro generally produced erratic EPG activity under the conditions tested. In contrast, A. ceylanicum L4s recovered from hamsters exhibited robust, sustained EPG activity, consisting of three waveforms: (1) conventional pumps as seen in other nematodes; (2) rapid voltage deflections, associated with irregular contractions of the esophagus and openings of the esophogeal-intestinal valve (termed a 'flutter'); and (3) hybrid waveforms, which we classified as pumps. For data analysis, pumps and flutters were combined and termed EPG 'events.' EPG waveform identification and analysis were performed semi-automatically using custom-designed software. The neuromodulator serotonin (5-hydroxytryptamine; 5HT) increased EPG event frequency in A. ceylanicum L4s at an optimal concentration of 0.5 mM. The anthelmintic drug ivermectin (IVM) inhibited EPG activity in a concentration-dependent manner. EPGs from A. suum L3s recovered from pig lungs exhibited robust pharyngeal pumping in 1 mM 5HT, which was inhibited by IVM. These experiments validate the use of A. ceylanicum L4s and A. suum L3s with the microfluidic EPG platform, providing a new tool for screening anthelmintic candidates or investigating parasitic nematode feeding behavior., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Proanthocyanidins (PAC) are a class of plant secondary metabolites commonly found in the diet that have shown potential to control gastrointestinal nematode infections. The anti-parasitic mechanism(s) of PAC remain obscure, however the protein-binding properties of PAC suggest that disturbance of key enzyme functions may be a potential mode of action. Glutathione-S-transferases (GSTs) are essential for parasite detoxification and have been investigated as drug and vaccine targets. Here, we show that purified PAC strongly inhibit the activity of both recombinant and native GSTs from the parasitic nematode Ascaris suum. As GSTs are involved in detoxifying xenobiotic substances within the parasite, we hypothesised that this inhibition may render parasites hyper-susceptible to anthelmintic drugs. Migration inhibition assays with A. suum larvae demonstrated that the potency of levamisole (LEV) and ivermectin (IVM) were significantly increased in the presence of PAC purified from pine bark (4.6-fold and 3.2-fold reduction in IC50 value for LEV and IVM, respectively). Synergy analysis revealed that the relationship between PAC and LEV appeared to be synergistic in nature, suggesting a specific enhancement of LEV activity, whilst the relationship between PAC and IVM was additive rather than synergistic, suggesting independent actions. Our results demonstrate that these common dietary compounds may increase the efficacy of synthetic anthelmintic drugs in vitro, and also suggest one possible mechanism for their well-known anti-parasitic activity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background and Purpose: Control of nematode parasite infections relies largely on anthelmintic drugs, several of which act on nicotinic ACh receptors (nAChRs), and there are concerns about the development of resistance. There is an urgent need for development of new compounds to overcome resistance and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of a homomeric nAChR, ACR-16, from a nematode parasite., Experimental Approach: Using RT-PCR, molecular cloning and two-electrode voltage clamp electrophysiology, we localized acr-16 mRNA in Ascaris suum (Asu) and then cloned and expressed acr-16 cRNA in Xenopus oocytes. Sensitivity of these receptors to cholinergic anthelmintics and a range of nicotinic agonists was tested., Key Results: Amino acid sequence comparison with vertebrate nAChR subunits revealed ACR-16 to be most closely related to α7 receptors, but with some striking distinctions. acr-16 mRNA was recovered from Asu somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, the existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, bephenium, tribendimidine and pyrantel) did not activate Asu-ACR-16 (except for a small response to oxantel). Other nAChR agonists: nicotine, ACh, cytisine, 3-bromocytisine and epibatidine, produced robust current responses which desensitized at a rate varying with the agonists. Unlike α7, Asu-ACR-16 was insensitive to α-bungarotoxin and did not respond to genistein or other α7 positive allosteric modulators. Asu-ACR-16 had lower calcium permeability than α7 receptors., Conclusions and Implications: We suggest that ACR-16 has diverse tissue-dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds., (© 2016 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Chicory is a perennial crop that has been investigated as a forage source for outdoor-reared ruminants and pigs, and has been reported to have anthelmintic properties. Here, we investigated in vitro anthelmintic effects of forage chicory-extracts against the highly prevalent swine parasites Ascaris suum and Oesophagostomum dentatum. Methanol extracts were prepared and purified from two different cultivars of chicory (Spadona and Puna II). Marked differences were observed between the anthelmintic activity of extracts from the two cultivars. Spadona extracts had potent activity against A. suum third (L3) and fourth (L4) - stage larvae, as well as O. dentatum L4 and adults, whereas Puna II extracts had less activity against A. suum and no activity towards O. dentatum L4. Transmission-electron microscopy of A. suum L4 exposed to Spadona extracts revealed only subtle changes, perhaps indicative of a specific anthelmintic effect rather than generalized toxicity. Ultra-high liquid chromatography-mass spectrometry analysis revealed that the purified extracts were rich in sesquiterpene lactones (SL), and that the SL profile differed significantly between cultivars. This is the first report of anthelmintic activity of forage chicory towards swine nematodes. Our results indicate a significant anthelmintic effect, which may possibly be related to SL composition.

Soil-transmitted helminth infections in humans and livestock cause significant debility, reduced productivity and economic losses globally. There are a limited number of effective anthelmintic drugs available for treating helminths infections, and their frequent use has led to the development of resistance in many parasite species. There is an urgent need for novel therapeutic drugs for treating these parasites. We have chosen the ACR-16 nicotinic acetylcholine receptor of Ascaris suum (Asu-ACR-16), as a drug target and have developed three-dimensional models of this transmembrane protein receptor to facilitate the search for new bioactive compounds. Using the human α7 nAChR chimeras and Torpedo marmorata nAChR for homology modeling, we defined orthosteric and allosteric binding sites on the Asu-ACR-16 receptor for virtual screening. We identified four ligands that bind to sites on Asu-ACR-16 and tested their activity using electrophysiological recording from Asu-ACR-16 receptors expressed in Xenopus oocytes. The four ligands were acetylcholine inhibitors (SB-277011-A, IC50, 3.12 ± 1.29 μM; (+)-butaclamol Cl, IC50, 9.85 ± 2.37 μM; fmoc-1, IC50, 10.00 ± 1.38 μM; fmoc-2, IC50, 16.67 ± 1.95 μM) that behaved like negative allosteric modulators. Our work illustrates a structure-based in silico screening method for seeking anthelmintic hits, which can then be tested electrophysiologically for further characterization.

The objective of this study was to evaluate the effects of several different commercial disinfectants on the embryogenic development of Ascaris suum eggs. A 1-ml aliquot of each disinfectant was mixed with approximately 40,000 decorticated or intact A. suum eggs in sterile tubes. After each treatment time (at 0.5, 1, 5, 10, 30, and 60 min), disinfectants were washed away, and egg suspensions were incubated at 25˚C in distilled water for development of larvae inside. At 3 weeks of incubation after exposure, ethanol, methanol, and chlorohexidin treatments did not affect the larval development of A. suum eggs, regardless of their concentration and treatment time. Among disinfectants tested in this study, 3% cresol, 0.2% sodium hypochlorite and 0.02% sodium hypochlorite delayed but not inactivated the embryonation of decorticated eggs at 3 weeks of incubation, because at 6 weeks of incubation, undeveloped eggs completed embryonation regardless of exposure time, except for 10% povidone iodine. When the albumin layer of A. suum eggs remained intact, however, even the 10% povidone iodine solution took at least 5 min to reasonably inactivate most eggs, but never completely kill them with even 60 min of exposure. This study demonstrated that the treatment of A. suum eggs with many commercially available disinfectants does not affect the embryonation. Although some disinfectants may delay or stop the embryonation of A. suum eggs, they can hardly kill them completely.

Proanthocyanidins (PA) from shea (Vitellaria paradoxa) meal were investigated by thiolytic degradation with benzyl mercaptan and the reaction products were analysed by high performance liquid chromatography-mass spectrometry. These PA were galloylated (≈40%), contained only B-type linkages and had a high proportion of prodelphinidins (>70%). The mean degree of polymerisation was 8 (i.e. average molecular size was 2384Da) and epigallocatechin gallate (EGCg) was the major flavan-3-ol subunit in PA. Shea meal also proved to be a potentially valuable source for extracting free flavan-3-ol-O-gallates, especially EGCg (575mg/kg meal), which is known for its health and anti-parasitic benefits. Proanthocyanidins were isolated and tested for bioactivity against Ascaris suum, which is an important parasite of pigs. Migration and motility tests revealed that these PA have potent activity against this parasitic nematode., (Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.)

Ascariasis affects more than 1 billion people worldwide, mainly in developing countries, causing substantial morbidity. Current treatments for Ascaris infection are based on mass drug administration (MDA) with synthetic anthelmintic drugs such as albendazole, however continual re-infection and the threat of drug resistance mean that complementary treatment options would be highly valuable. Here, we screened ethanolic extracts from 29 medicinal plants used in Africa (Ghana) and the Caribbean (US Virgin Islands) for in vitro anthelmintic properties against Ascaris suum, a swine parasite that is very closely related to the human A. lumbricoides. A wide variety of activities were seen in the extracts, from negligible to potent. Extracts from Clausena anisata, Zanthoxylum zanthoxyloides and Punica granatum were identified as the most potent with EC50 values of 74, 97 and 164 μg/mL, respectively. Our results encourage further investigation of their use as complementary treatment options for ascariasis, alongside MDA., (© A.R. Williams et al., published by EDP Sciences, 2016.)

The levamisole-sensitive nicotinic acetylcholine receptor present at nematode neuromuscular junctions is composed of multiple different subunits, with the exact composition varying between species. We tested the ability of two well-conserved nicotinic receptor subunits, UNC-38 and UNC-29, from Haemonchus contortus and Ascaris suum to rescue the levamisole-resistance and locomotion defects of Caenorhabditis elegans strains with null deletion mutations in the unc-38 and unc-29 genes. The parasite cDNAs were cloned downstream of the relevant C. elegans promoters and introduced into the mutant strains via biolistic transformation. The UNC-38 subunit of H. contortus was able to completely rescue both the locomotion defects and levamisole resistance of the null deletion mutant VC2937 (ok2896), but no C. elegans expressing the A. suum UNC-38 could be detected. The H. contortus UNC-29.1 subunit partially rescued the levamisole resistance of a C. elegans null mutation in unc-29 VC1944 (ok2450), but did cause increased motility in a thrashing assay. In contrast, only a single line of worms containing the A. suum UNC-29 subunit showed a partial rescue of levamisole resistance, with no effect on thrashing., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Cinnamon (Cinnamomum verum) has been shown to have anti-inflammatory and antimicrobial properties, but effects on parasitic worms of the intestine have not been investigated. Here, extracts of cinnamon bark were shown to have potent in vitro anthelmintic properties against the swine nematode Ascaris suum. Analysis of the extract revealed high concentrations of proanthocyanidins (PAC) and trans-cinnamaldehyde (CA). The PAC were subjected to thiolysis and HPLC-MS analysis which demonstrated that they were exclusively procyanidins, had a mean degree of polymerization of 5.2 and 21% of their inter-flavan-3-ol links were A-type linkages. Purification of the PAC revealed that whilst they had activity against A. suum, most of the potency of the extract derived from CA. Trichuris suis and Oesophagostomum dentatum larvae were similarly susceptible to CA. To test whether CA could reduce A. suum infection in pigs in vivo, CA was administered daily in the diet or as a targeted, encapsulated dose. However, infection was not significantly reduced. It is proposed that the rapid absorption or metabolism of CA in vivo may prevent it from being present in sufficient concentrations in situ to exert efficacy. Therefore, further work should focus on whether formulation of CA can enhance its activity against internal parasites.

Essential plant oils (or their active principles) are safe to use and a potentially attractive alternative to current antiparasitic drugs. In the present study, we tested the effects of carvacrol on the isolated tissues of Ascaris suum and investigated potential interactions with other antiparasitic drugs. We used somatic muscle flaps for contraction assays, as well as for electrophysiological investigations. Carvacrol 300 μM highly significantly inhibited contractions caused by 1, 3, 10, 30, and 100 μM of ACh (p = 0.0023, p = 0.0002, p = 0.0002, p < 0.0001, and p < 0.0001). The control EC50 for acetylcholine was 8.87 μM (log EC50 = 0.95 ± 0.26), while R max was 2.53 ± 0.24 g. The EC50 of acetylcholine in the presence of 300 μM of carvacrol was 27.71 μM (log EC50 = 1.44 ± 0.28) and the R max decreased to 1.63 ± 0.32 g. Furthermore, carvacrol highly significant potentiates inhibitory effect of GABA and piperazine on the contractions induced by ACh. However, carvacrol (100 and 300 μM), did not produce any changes in the membrane potential or conductance of the A. suum muscle cell. While, 300 μM of carvacrol showed a significant inhibitory effect on ACh-induced depolarization response. The mean control depolarization was 13.58 ± 0.66 mV and decreased in presence of carvacrol to 4.50 ± 1.02 mV (p < 0.0001). Mean control Δg was 0.168 ± 0.017 μS, while in the presence of 300 μM of carvacrol, Δg significantly decreased to 0.060 ± 0.018 ΔS (p = 0.0017). The inhibitory effect on contractions may be the explanation of the antinematodal potential of carvacrol. Moreover, inhibition of depolarizations caused by ACh and reduction of conductance changes directly points to an interaction with the nAChR in A. suum.

Recent studies on the respiratory chain of Ascaris suum showed that the mitochondrial NADH-fumarate reductase system composed of complex I, rhodoquinone and complex II plays an important role in the anaerobic energy metabolism of adult A. suum. The system is the major pathway of energy metabolism for adaptation to a hypoxic environment not only in parasitic organisms, but also in some types of human cancer cells. Thus, enzymes of the pathway are potential targets for chemotherapy. We found that flutolanil is an excellent inhibitor for A. suum complex II (IC50 = 0.058 μM) but less effectively inhibits homologous porcine complex II (IC50 = 45.9 μM). In order to account for the specificity of flutolanil to A. suum complex II from the standpoint of structural biology, we determined the crystal structures of A. suum and porcine complex IIs binding flutolanil and its derivative compounds. The structures clearly demonstrated key interactions responsible for its high specificity to A. suum complex II and enabled us to find analogue compounds, which surpass flutolanil in both potency and specificity to A. suum complex II. Structures of complex IIs binding these compounds will be helpful to accelerate structure-based drug design targeted for complex IIs.

Although lacking an NADPH→NAD(+) transhydrogenase system, the essentially energetically anaerobic mitochondria of the adult intestinal nematode Ascaris suum display an inner membrane-associated NADH→NAD(+) transhydrogenation reaction. This reaction is considered to be reflective of a mechanism(s) that acts in catalyzing a transmembrane translocation of reducing equivalents from NADH in the intermembrane space to matrix NAD(+), thereby forming matrix NADH that would serve in electron transport. Ascarid mitochondrial lipoamide dehydrogenase rather than an NADH→NAD(+) transhydrogenase system has been viewed as the predominant source of inner membrane-associated NADH→NAD(+) transhydrogenation activity. However, the present study made apparent yet another source of mitochondrial, inner membrane-associated NADH→NAD(+) activity in A. suum , viz., NADH dehydrogenase. This was made evident via comparisons of the A. suum mitochondrial NADH→NAD(+) transhydrogenation, NADH dehydrogenase, and lipoamide dehydrogenase activities in terms of pH effects, thermal labilities, the involvement of NADH dehydrogenase in the activities of mitochondrial, membrane-associated rotenone-insensitive and rotenone-sensitive NADH-dependent cytochrome c reductases, and mitochondrial membrane versus mitochondrial soluble localizations. Studies of the responses of the NADH→NAD(+) transhydrogenation, rotenone-insensitive and rotenone-sensitive cytochrome c reductases, and lipoamide dehydrogenase activities to inhibition by copper and cadmium lent additional support to the catalysis of an NADH→NAD(+) transhydrogenation activity by NADH dehydrogenase. Collectively, the data presented are consistent with an additional physiological catalysis of an NADH→NAD(+) transhydrogenation in A. suum mitochondria by an inner membrane NADH dehydrogenase component of the rotenone-sensitive cytochrome c reductase system, i.e., the NADH dehydrogenase component of the electron transport system. Comparisons of the A. suum data with those from other essentially anaerobic helminth parasites as well as free-living eukaryotic mitochondrial systems are noted.

The cholinergic class of anthelmintic drugs is used for the control of parasitic nematodes. One of this class of drugs, tribendimidine (a symmetrical diamidine derivative, of amidantel), was developed in China for use in humans in the mid-1980s. It has a broader-spectrum anthelmintic action against soil-transmitted helminthiasis than other cholinergic anthelmintics, and is effective against hookworm, pinworms, roundworms, and Strongyloides and flatworm of humans. Although molecular studies on C. elegans suggest that tribendimidine is a cholinergic agonist that is selective for the same nematode muscle nAChR as levamisole, no direct electrophysiological observations in nematode parasites have been made to test this hypothesis. Also the hypothesis that levamisole and tribendimine act on the same receptor, does not explain why tribendimidine is effective against some nematode parasites when levamisole is not. Here we examine the effects of tribendimidine on the electrophysiology and contraction of Ascaris suum body muscle and show that tribendimidine produces depolarization antagonized by the nicotinic antagonist mecamylamine, and that tribendimidine is an agonist of muscle nAChRs of parasitic nematodes. Further pharmacological characterization of the nAChRs activated by tribendimidine in our Ascaris muscle contraction assay shows that tribendimidine is not selective for the same receptor subtypes as levamisole, and that tribendimidine is more selective for the B-subtype than the L-subtype of nAChR. In addition, larval migration inhibition assays with levamisole-resistant Oesophagostomum dentatum isolates show that tribendimidine is as active on a levamisole-resistant isolate as on a levamisole-sensitive isolate, suggesting that the selectivity for levamisole and tribendimidine is not the same. It is concluded that tribendimidine can activate a different population of nematode parasite nAChRs than levamisole, and is more like bephenium. The different nAChR subtype selectivity of tribendimidine may explain why the spectrum of action of tribendimidine is different to that of other cholinergic anthelmintics like levamisole.

The effect of a two-step process on the concentration of pathogens and indicator microorganisms in black water (0.9-1% total solids) was studied. The treatment combined auto-thermal aerobic digestion (ATAD) and ammonia sanitisation. First, the temperature of the black water was increased through ATAD and when a targeted temperature was reached (33, 41 and 45.5 °C studied), urea was added to a 0.5% concentration (total ammonia nitrogen >2.9 g L⁻¹). Escherichia coli and Salmonella spp. were reduced to non-detectable levels within 3 days following urea addition at temperatures above 40 °C, whereas when urea was added at 33 °C E. coli was still present after 8 days. By adding urea at temperatures of 40 °C and above, a 5 log10 reduction in Enterococcus spp. and a 3 log10 reduction in Ascaris suum eggs was achieved 1 week after the addition. With combined ATAD and ammonia treatment using 0.5% ww urea added at an aerobic digestion temperature >40 °C, black water was sanitised regarding the pathogens studied in 2 weeks of total treatment time.

Diethylcarbamazine is a drug that is used for the treatment of filariasis in humans and animals; it also has effects on intestinal nematodes, but its mechanism of action remains unclear. Emodepside is a resistance-busting anthelmintic approved for treating intestinal parasitic nematodes in animals. The novel mode of action and resistance-breaking properties of emodepside has led to its use against intestinal nematodes of animals, and as a candidate drug for treating filarial parasites. We have previously demonstrated effects of emodepside on SLO-1 K+-like currents in Ascaris suum. Here, we demonstrate that diethylcarbamazine, which has been proposed to work through host mediated effects, has direct effects on a nematode parasite, Ascaris suum. It increases activation of SLO-1 K+ currents and potentiates effects of emodepside. Our results suggest consideration of the combination of emodepside and diethylcarbamazine for therapy, which is predicted to be synergistic. The mode of action of diethylcarbamazine may involve effects on parasite signaling pathways (including nitric oxide) as well as effects mediated by host inflammatory mediators.

Ascaris lumbricoides is the most prevalent soil-transmitted helminth (STH) infection of human beings worldwide. Chemotherapy with synthetic anthelmintics such as albendazole, mebendazole, and pyrantel pamoate is the current method of treatment; however, the emergence of anthelmintic resistance could substantially decrease the efficacy of such treatments and the sustainability of STH control programs. Additionally, benzimidazoles are not recommended for pregnant women or children under age one. A blinded, controlled study was conducted to evaluate the efficacy of two microencapsulated, plant-based essential oil blends, TTN1013 (α-pinene, linalyl acetate, p-cymene, and thymol octanoate) and TTN1014 (α-pinene, linalyl acetate, p-cymene, and thymol acetate) as functional foods against Ascaris suum infection in pigs, an important pathogen that closely resembles human infections with A. lumbricoides. Four groups of 16 female, 21-24 day old, Yorkshire-cross pigs were treated daily with 0.5 or 1.0mg/kg TTN1013, 1.0mg/kg TTN1014, or 1.0mg/kg equivalent of empty capsules, delivered inside a cream-filled sandwich cookie for 14 weeks. Three days after the initiation of daily treatments, pigs were inoculated daily with A. suum eggs for four weeks. Pigs were weighed weekly and fecal egg counts (FEC) were conducted weekly starting five weeks after initial inoculation with A. suum eggs. Fourteen weeks after first infection with eggs, pigs were necropsied and worms were recovered, counted and separated according to sex. TTN1013 administered daily at a dose of 1.0mg/kg yielded a statistically significant reduction in total worm counts (76.8%), female worm counts (75.5%), FEC (68.6%), and worm volume (62.9%) when compared to control group. Reduction of total and female worm numbers and FEC were not significant for TTN1014 or at the 0.5mg/kg dose of TTN1013. All treatments were well-tolerated by all pigs and did not cause any adverse reactions. All pigs remained clinically normal and showed no signs of reduced intestinal health for the duration of treatment. Based on these results, TTN1013 shows promise as a daily supplement to reduce infection burdens of soil transmitted helminths in both pigs and human beings., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Eggs of Ascaris suum from pigs are highly resistant and commonly used as a conservative indicator of pathogen inactivation during slurry storage. Eggs of Ascaridia galli, the poultry ascarid, are also known to be highly resistant but the suitability as an indicator of pathogen inactivation has never been tested. Pig slurry has to be stored for several months to inactivate pathogens but chemical treatment of slurry may reduce this time. The suitability of A. galli as an indicator of slurry sanitation was tested by comparing the survival of eggs of A. suum and A. galli in pig slurry. In addition, the effect of urea treatment on inactivation of ascarid eggs in relation to storage time was also tested. Nylon bags with 10,000 eggs of either species were placed in 200 ml plastic bottles containing either urea-treated (2%) or untreated pig slurry for up to 120 days at 20°C, 6 days at 30°C, 36h at 40°C or 2h at 50°C. At all the temperatures in both slurry types, A. galli eggs were inactivated at a significantly faster rate (P<0.05) compared to A. suum eggs. For each 10°C raise in temperature from 20°C, T50 (time needed to inactivate 50% of eggs) for both types of eggs was reduced markedly. At all temperatures, viability of eggs of both species was significantly higher (P<0.05) in untreated slurry compared to urea-treated slurry except A. galli eggs at 20°C where no significant difference was detected. In untreated slurry, the levels of pH (6.33-9.08) and ammonia (0.01-1.74 mM) were lower (P<0.0001) compared to that of urea-treated slurry (pH: 8.33-9.28 and ammonia 1-13 mM). The study demonstrated that A. galli eggs are more sensitive to unfavourable conditions compared to A. suum eggs. The use of A. galli eggs as hygiene indicator may thus be suitable to assess inactivation of pathogens that are more sensitive than A. galli eggs. Addition of urea may markedly reduce the storage time of slurry needed to inactivate A. suum and A. galli eggs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Ascaris suum is one of the most prevalent nematode parasites in pigs and causes significant economic losses, and also serves as a good model for A. lumbricoides, the large roundworm of humans that is ubiquitous in developing countries and causes malnutrition, stunted growth and compromises immunity to other pathogens. New treatment options for Ascaris infections are urgently needed, to reduce reliance on the limited number of synthetic anthelmintic drugs. In areas where Ascaris infections are common, ethno-pharmacological practices such as treatment with natural plant extracts are still widely employed. However, scientific validation of these practices and identification of the active compounds are lacking, although observed effects are often ascribed to plant secondary metabolites such as tannins. Here, we extracted, purified and characterised a wide range of condensed tannins from diverse plant sources and investigated anthelmintic effects against A. suum in vitro. We show that condensed tannins can have potent, direct anthelmintic effects against A. suum, as evidenced by reduced migratory ability of newly hatched third-stage larvae and reduced motility and survival of fourth-stage larvae recovered from pigs. Transmission electron microscopy showed that CT caused significant damage to the cuticle and digestive tissues of the larvae. Furthermore, we provide evidence that the strength of the anthelmintic effect is related to the polymer size of the tannin molecule. Moreover, the identity of the monomeric structural units of tannin polymers may also have an influence as gallocatechin and epigallocatechin monomers exerted significant anthelmintic activity whereas catechin and epicatechin monomers did not. Therefore, our results clearly document direct anthelmintic effects of condensed tannins against Ascaris and encourage further in vivo investigation to determine optimal strategies for the use of these plant compounds for the prevention and/or treatment of ascariosis.

To evaluate the effects of pesticides to parasite eggs, Ascaris suum eggs were incubated with 5 different pesticides (1:1,500-1:2,000 dilutions of 2% emamectin benzoate, 5% spinetoram, 5% indoxacarb, 1% deltamethrin, and 5% flufenoxuron; all v/v) at 20℃ for 6 weeks, and microscopically evaluated the egg survival and development on a weekly basis. The survival rate of A. suum eggs incubated in normal saline (control eggs) was 90±3% at 6 weeks. However, the survival rates of eggs treated with pesticides were 75-85% at this time, thus significantly lower than the control value. Larval development in control eggs commenced at 3 weeks, and 73±3% of eggs had internal larvae at 6 weeks. Larvae were evident in pesticide-treated eggs at 3-4 weeks, and the proportions of eggs carrying larvae at 6 weeks (36±3%-54±3%) were significantly lower than that of the control group. Thus, pesticides tested at levels similar to those used in agricultural practices exhibited low-level ovicidal activity and delayed embryogenesis of A. suum eggs, although some differences were evident among the tested pesticides.

The in vitro effect of ivermectin lethal dose on the activity of trehalose-6-phosphate synthase (TPS) and phosphatase (TPP) and the expression of their mRNA (tps1, tps2, and tpp genes) in the muscle of adult female Ascaris suum was investigated. The presence of ivermectin in the medium caused a decrease in TPS and TPP activities during the experiment compared with the start and control groups. The exception was the group of worms grown for 8 hours in a IVM solution, in which there was a little higher TPS activity than in the control. Real-time qPCR analysis showed reduced expression of tps1 and tps2, and unchanged tpp expression after 20 hours of incubation relative to the expression at time zero. Relative to the appropriate control groups, the expression of tps2 gene was slight increased but the other two genes were reduced after 8-hours of IVM-treatment. Then the expression of all three genes was lower at the end of cultivation. The level of gene expression was positively correlated with the activity of specific enzymes. In the case of tpp gene there was only a weak correlation. Prolonged exposure to ivermectin was effective in lowering TPS and TPP activity and their mRNA expression. However, the drug did not block the pathway.

Nicotinic acetylcholine receptors (nAChRs) play a key role in the normal physiology of nematodes and provide an established target site for anthelmintics. The free-living nematode, Caenorhabditis elegans, has a large number of nAChR subunit genes in its genome and so provides an experimental model for testing novel anthelmintics which act at these sites. However, many parasitic nematodes lack specific genes present in C. elegans, and so care is required in extrapolating from studies using C. elegans to the situation in other nematodes. In this review the properties of C. elegans nAChRs are reviewed and compared to those of parasitic nematodes. This forms the basis for a discussion of the possible subunit composition of nAChRs from different species of parasitic nematodes. Currently our knowledge on this is largely based on studies using heterologous expression and pharmacological analysis of receptor subunits in Xenopus laevis oocytes. It is concluded that more information is required regarding the subunit composition and pharmacology of endogenous nAChRs in parasitic nematodes., (Copyright © 2013. Published by Elsevier Ireland Ltd.)

Inactivation tests of Ascaris eggs (Ae) were performed using hydrogen peroxide and a Fenton type nanocatalyst supported on activated carbon (AC) (FeOx/C). Blank inactivation tests were also carried out using H2O2 and H2O2/AC as oxidation systems. The FeOx/C nanocatalyst was synthesized through a novel hybrid method developed in this work. The method is based on the incipient impregnation technique, using isopropyl alcohol as dissolvent and chelating agent of the iron salt and the ultrasonic method. The supported nanocatalyst contained 2.61% w/w of total iron and the support 0.2% w/w. Transmission electron microscopy (TEM)-energy dispersive spectrometer (EDS) images permitted verification of the presence of finely dispersed FeOx nanoparticles, with sizes ranging from 19 to 63 nm. SEM-EDS analysis and TEM images also showed good dispersion of iron oxide nanoparticles, most probably maghemite; γ-Fe2O3, able to produce hydroperoxyl radical as reported in the literature. The FeOx/C nanocatalyst-H2O2 system showed an average Ae inactivation efficiency of 4.46% Ae/mg H2O2. This value is significantly higher than the result obtained using the support-H2O2 system and H2O2 alone and it is also better than data reported for the classical Fenton process (homogeneous phase) with or without UV light.

Ascaris suum and Ascaris lumbricoides are two closely related geo-helminth parasites that ubiquitously infect pigs and humans, respectively. Ascaris suum infection in pigs is considered a good model for A. lumbricoides infection in humans because of a similar biology and tissue migration to the intestines. Ascaris lumbricoides infections in children are associated with malnutrition, growth and cognitive stunting, immune defects, and, in extreme cases, life-threatening blockage of the digestive tract and aberrant migration into the bile duct and peritoneum. Similar effects can be seen with A. suum infections in pigs related to poor feed efficiency and performance. New strategies to control Ascaris infections are needed largely due to reduced treatment efficacies of current anthelmintics in the field, the threat of resistance development, and the general lack of new drug development for intestinal soil-transmitted helminths for humans and animals. Here we demonstrate for the first time that A. suum expresses the receptors for Bacillus thuringiensis crystal protein and novel anthelmintic Cry5B, which has been previously shown to intoxicate hookworms and which belongs to a class of proteins considered non-toxic to vertebrates. Cry5B is able to intoxicate A. suum larvae and adults and triggers the activation of the p38 mitogen-activated protein kinase pathway similar to that observed with other nematodes. Most importantly, two moderate doses of 20 mg/kg body weight (143 nM/kg) of Cry5B resulted in a near complete cure of intestinal A. suum infections in pigs. Taken together, these results demonstrate the excellent potential of Cry5B to treat Ascaris infections in pigs and in humans and for Cry5B to work effectively in the human gastrointestinal tract.

Two commonly used disinfectants, a quaternary ammonium salt and povidone-iodine, were tested for effectiveness against unembryonated Ascaris suum eggs. The quaternary ammonium salt (alkyl dimethyl benzyl ammonium chloride) had no effect on the Ascaris eggs (10 minutes and 22°C) when compared with the controls in water with egg viabilities of 88.8% ± 3.3% and 86.9% ± 6.2%, respectively. An additional quaternary ammonium salt, 2.5% benzethonium chloride, also had no effect. Phenol (5%) and cresol (3%) completely inactivated the eggs. Povidone-iodine at 100%, 50%, 10%, and 1% had no effect on the eggs at exposures of 5, 15, 30, 60, or 120 minutes (22°C) compared with the water controls., (Published by Mosby, Inc.)

Startect(®) is a novel anthelmintic combination of derquantel and abamectin. It is hypothesized that derquantel and abamectin interact pharmacologically. We investigated the effects of derquantel, abamectin and their combination on somatic muscle nicotinic acetylcholine receptors and pharyngeal muscle glutamate gated chloride receptor channels of Ascaris suum. We used muscle-strips to test the effects of abamectin, derquantel, and abamectin+derquantel together on the contraction responses to different concentrations of acetylcholine. We found that abamectin reduced the response to acetylcholine, as did derquantel. In combination (abamectin+derquantel), inhibition of the higher acetylcholine concentration response was greater than the predicted additive effect. A two-micropipette current-clamp technique was used to study electrophysiological effects of the anthelmintics on: (1) acetylcholine responses in somatic muscle and; (2) on l-glutamate responses in pharyngeal preparations. On somatic muscle, derquantel (0.1-30μM) produced a potent (IC50 0.22, CI 0.18-0.28μM) reversible antagonism of acetylcholine depolarizations. Abamectin (0.3μM) produced a slow onset inhibition of acetylcholine depolarizations. We compared effects of abamectin and derquantel on muscle preparations pretreated for 30min with these drugs. The effect of the combination was significantly greater than the predicted additive effect of both drugs at higher acetylcholine concentrations. On the pharynx, application of derquantel produced no significant effect by itself or on responses to abamectin and l-glutamate. Abamectin increased the input conductance of the pharynx (EC50 0.42, CI 0.13-1.36μM). Our study demonstrates that abamectin and derquantel interact at nicotinic acetylcholine receptors on the somatic muscle and suggested synergism can occur., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The goal of this study was to further develop an incubation-quantitative polymerase chain reaction (qPCR) method for quantifying viable Ascaris eggs by characterizing the detection limit and number of template copies per egg, determining the specificity of the method, and testing the method with viable and inactivated larvated eggs. The number of template copies per cell was determined by amplifying DNA from known numbers of eggs at different development stages; the value was estimated to be 32 copies. The specificity of the method was tested against a panel of bacteria, fungi, protozoa and helminths, and no amplification was found with non-target DNA. Finally, fully larvated eggs were inactivated by four different treatments: 254 nm ultraviolet light, 2,000 ppm NH(3)-N at pH 9, moderate heat (48 °C) and high heat (70 °C). Concentrations of treated eggs were measured by direct microscopy and incubation-qPCR. The qPCR signal decreased following all four treatments, and was in general agreement with the decrease in viable eggs determined by microscopy. The incubation-qPCR method for enumerating viable Ascaris eggs is a promising approach that can produce results faster than direct microscopy, and may have benefits for applications such as assessing biosolids.

Increased glycolysis is the principal explanation for how cancer cells generate energy in the absence of oxygen. However, in actual human tumour microenvironments, hypoxia is often associated with hypoglycemia because of the poor blood supply. Therefore, glycolysis cannot be the sole mechanism for the maintenance of the energy status in cancers. To understand energy metabolism in cancer cells under hypoxia-hypoglycemic conditions mimicking the tumour microenvironments, we examined the NADH-fumarate reductase (NADH-FR) system, which functions in parasites under hypoxic condition, as a candidate mechanism. In human cancer cells (DLD-1, Panc-1 and HepG2) cultured under hypoxic-hypoglycemic conditions, NADH-FR activity, which is composed of the activities of complex I (NADH-ubiquinone reductase) and the reverse reaction of complex II (quinol-FR), increased, whereas NADH-oxidase activity decreased. Pyrvinium pamoate (PP), which is an anthelmintic and has an anti-cancer effect within tumour-mimicking microenvironments, inhibited NADH-FR activities in both parasites and mammalian mitochondria. Moreover, PP increased the activity of complex II (succinate-ubiquinone reductase) in mitochondria from human cancer cells cultured under normoxia-normoglycemic conditions but not under hypoxia-hypoglycemic conditions. These results indicate that the NADH-FR system may be important for maintaining mitochondrial energy production in tumour microenvironments and suggest its potential use as a novel therapeutic target.

To determine the effects of kimchi extracts at different temperatures on larval development, Ascaris suum eggs were mixed with soluble part of 7 different brands of commercially available kimchi and preserved at either 5℃ or 25℃ for up to 60 days. A. suum eggs incubated at 25℃ showed marked differences in larval development between kimchi extract and control group. While all eggs in the control group completed embryonation by day 21, only 30% of the eggs in the kimchi extract group became embryonated by day 36 and about 25% never became larvated even at day 60. At 5℃, however, none of the eggs showed larval development regardless of the incubation period or type of mixture group. To determine the survival rate of A. suum eggs that showed no embryonation after being preserved at 5℃, eggs preserved in kimchi extracts for 14, 28, and 60 at 5℃ were re-incubated at 25℃ for 3 weeks in distilled water. While all eggs in the control group became larvated, eggs in the kimchi extract group showed differences in their embryonation rates by the incubation period; 87.4 % and 41.7% of the eggs became embryonated after being refrigerated for 14 days and 28 days, respectively. When refrigerated for 60 days, however, no eggs mixed in kimchi extract showed larval development. Our results indicate that embryogenesis of A. suum eggs in kimchi extract was affected by duration of refrigeration, and that all eggs stopped larval development completely in kimchi kept at 5℃ for up to 60 days.

Parasitic diseases have a devastating, long-term impact on human health, welfare and food production worldwide. More than two billion people are infected with geohelminths, including the roundworms Ascaris (common roundworm), Necator and Ancylostoma (hookworms), and Trichuris (whipworm), mainly in developing or impoverished nations of Asia, Africa and Latin America. In humans, the diseases caused by these parasites result in about 135,000 deaths annually, with a global burden comparable with that of malaria or tuberculosis in disability-adjusted life years. Ascaris alone infects around 1.2 billion people and, in children, causes nutritional deficiency, impaired physical and cognitive development and, in severe cases, death. Ascaris also causes major production losses in pigs owing to reduced growth, failure to thrive and mortality. The Ascaris-swine model makes it possible to study the parasite, its relationship with the host, and ascariasis at the molecular level. To enable such molecular studies, we report the 273 megabase draft genome of Ascaris suum and compare it with other nematode genomes. This genome has low repeat content (4.4%) and encodes about 18,500 protein-coding genes. Notably, the A. suum secretome (about 750 molecules) is rich in peptidases linked to the penetration and degradation of host tissues, and an assemblage of molecules likely to modulate or evade host immune responses. This genome provides a comprehensive resource to the scientific community and underpins the development of new and urgently needed interventions (drugs, vaccines and diagnostic tests) against ascariasis and other nematodiases., (©2011 Macmillan Publishers Limited. All rights reserved)