The response of Anisakis simplex (s. s.) to anthelmintics - Specific changes in xenobiotic metabolic processes.

Anisakiasis is a parasitic disease transmitted through the consumption of raw or undercooked fish and cephalopods that are infected with larvae of Anisakis simplex (sensu stricto) or Anisakis pegreffii. The purpose of this study was to investigate how A. simplex (s. s.) responds to the influence of anthelmintics such as ivermectin (IVM) and pyrantel (PYR). In vitro experiments were conducted using larvae at two developmental stages of A. simplex (s. s.) (L3 and L4) obtained from Baltic herring (Clupea harengus membras). Larvae were cultured with different concentrations of IVM or PYR (1.56, 3.125, and 6.25 µg/mL) for various durations (3, 6, 9, and 12 h) under anaerobic conditions (37 °C, 5% CO2). The gene expression of actin, ABC transporter, antioxidant enzymes, γ-aminobutyric acid receptors, and nicotinic acetylcholine receptors, as well as the oxidative status were analyzed. The results showed that A. simplex (s. s.) L3 stage had lower mobility when cultured with PYR compared to IVM. The analysis of relative gene expression revealed significant differences in the mRNA level of ABC transporters after treatment with IVM and PYR, compared to the control group. Similar patterns were observed in the gene expression of antioxidant enzymes in response to both drugs. Furthermore, the total antioxidant capacity (TAC) and glutathione S-transferase (GST) activity were higher in the treatment groups than in the control group. These findings suggest a relationship between the expression of the studied genes, including those related to oxidative metabolism, and the effectiveness of the tested drugs.

Pyrantel resistance in canine hookworms in Queensland, Australia.

Hookworms are the most common intestinal nematode parasites of dogs in Australia. The control of these parasites relies mostly on regular deworming with anthelmintics, with pyrantel-based dewormers being a relatively low cost and readily-available option for dog owners. Pyrantel resistance in canine hookworms in Australia was first reported in 2007, however pyrantel-based dewormers are still used against hookworm infection in dogs across Australia. The present study was conducted to evaluate the efficacy of pyrantel against hookworms infecting dogs housed in a shelter facility in Southeast Queensland which receives rescued or surrendered animals from greyhound rescue centres and dog shelters across this region. A total of 10 dogs were examined using the faecal egg count reduction test (FECRT). There was no reduction in FEC in any of the dogs following pyrantel treatment, with drug efficacies ranging from -0.9% to -283.3%. Given that these dogs originated from various sites across Southeast Queensland, the present study suggests that pyrantel resistance is widespread in this region, and hence this anthelmintic may not be a useful option for treatment of hookworm infections in dogs.

The first report of triple anthelmintic resistance on a French Thoroughbred stud farm.

This study assessed the anthelmintic resistance in strongylid nematodes against commonly used anthelmintic (AH) drugs in a French galloping racehorse stud farm from March to December 2023. Faecal egg count reduction tests (FECRTs) were conducted in three different groups of Thoroughbred yearlings (a group of 6 males, a group of 13 females and a group of 8 females and 3 males) following the new World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines. The efficacy of fenbendazole was tested in two groups once during the monitoring period (in March), the efficacy of ivermectin in 3 groups twice (in March-April and in November-December) and the efficacy of pyrantel in one group once (in May-June). For each FECRT, the 90% confidence interval of the percentage faecal egg count reduction was calculated using the hybrid Frequentist/Bayesian analysis method. The resistance in strongylids was observed to fenbendazole, pyrantel and ivermectin in all the groups in which these drugs were tested. The number of animals in each group was sufficient to reach ≥80% power for the resistance test. The results highlight the first case of triple AH resistance in strongylids in France. Further studies involving more farms and equids are required to assess the prevalence of AH resistance in France and refine recommendations for owners.

Preventive efficacy of six monthly oral doses of Simparica Trio®, Heartgard® Plus, and Interceptor® Plus against a macrocyclic lactone-resistant strain (ZoeLA) of heartworm (Dirofilaria immitis) in dogs.

BACKGROUND: Administration of four to six consecutive monthly doses of 24 µg/kg moxidectin alone shows high effectiveness in preventing the maturation of macrocyclic lactone (ML)-resistant heartworm strains, Dirofilaria immitis JYD-34 and ZoeLA. This laboratory study evaluated the efficacy of six consecutive monthly oral doses of Simparica Trio® (moxidectin/sarolaner/pyrantel) compared to six monthly doses of either Heartgard® Plus (ivermectin/pyrantel) or Interceptor® Plus (milbemycin oxime/praziquantel) against ML-resistant D. immitis ZoeLA strain. METHODS: Beagle dogs were inoculated with 50 third-stage (L3) D. immitis larvae (ZoeLA) 30 days prior to the first treatment. Dogs were randomized to treatment (six animals in each group) with six monthly oral doses of placebo, Simparica Trio, Heartgard Plus, or Interceptor Plus at their respective label doses. Microfilaria (MF) and antigen tests were conducted periodically, and efficacy was evaluated by necropsy for adult heartworms approximately 9 months after L3 inoculation. RESULTS: Adult heartworms were recovered from all six placebo dogs, with a geometric mean of 35.5 worms (range, 23-48). Five of the six dogs treated with Simparica Trio were infected with a geometric mean of 1.0 worms (range, 0-3), and all remained MF-negative. All Heartgard Plus-treated dogs (six) were infected with a geometric mean of 32.5 worms (range, 22-38); five of these dogs were MF-positive at day 236. All Interceptor Plus-treated dogs (six) were infected with a geometric mean of 22.8 worms (range, 10-34); five of these dogs were MF-positive at day 236. The efficacy of six consecutive doses with Simparica Trio, Heartgard Plus, and Interceptor Plus against ZoeLA was 97.2, 8.5, and 35.9%, respectively. Adult worm counts for the Simparica Trio-treated group were significantly lower (P < 0.0001) than placebo control, Heartgard Plus, and Interceptor Plus-treated groups. Adult worm counts for Heartgard Plus and Interceptor Plus were not significantly different from placebo (P > 0.05). CONCLUSIONS: Simparica Trio prevented microfilaremia in all dogs and was highly effective (97.2%) and significantly better than either Heartgard Plus (8.5%) or Interceptor Plus (35.9%) in preventing the development of the ZoeLA ML-resistant heartworm strain when administered for six consecutive months in this comparative laboratory efficacy study.

The narrow-spectrum anthelmintic oxantel is a potent agonist of a novel acetylcholine receptor subtype in whipworms.

In the absence of efficient alternative strategies, the control of parasitic nematodes, impacting human and animal health, mainly relies on the use of broad-spectrum anthelmintic compounds. Unfortunately, most of these drugs have a limited single-dose efficacy against infections caused by the whipworm, Trichuris. These infections are of both human and veterinary importance. However, in contrast to a wide range of parasitic nematode species, the narrow-spectrum anthelmintic oxantel has a high efficacy on Trichuris spp. Despite this knowledge, the molecular target(s) of oxantel within Trichuris is still unknown. In the distantly related pig roundworm, Ascaris suum, oxantel has a small, but significant effect on the recombinant homomeric Nicotine-sensitive ionotropic acetylcholine receptor (N-AChR) made up of five ACR-16 subunits. Therefore, we hypothesized that in whipworms, a putative homolog of an ACR-16 subunit, can form a functional oxantel-sensitive receptor. Using the pig whipworm T. suis as a model, we identified and cloned a novel ACR-16-like subunit and successfully expressed the corresponding homomeric channel in Xenopus laevis oocytes. Electrophysiological experiments revealed this receptor to have distinctive pharmacological properties with oxantel acting as a full agonist, hence we refer to the receptor as an O-AChR subtype. Pyrantel activated this novel O-AChR subtype moderately, whereas classic nicotinic agonists surprisingly resulted in only minor responses. We observed that the expression of the ACR-16-like subunit in the free-living nematode Caenorhabditis elegans conferred an increased sensitivity to oxantel of recombinant worms. We demonstrated that the novel Tsu-ACR-16-like receptor is indeed a target for oxantel, although other receptors may be involved. These finding brings new insight into the understanding of the high sensitivity of whipworms to oxantel, and highlights the importance of the discovery of additional distinct receptor subunit types within Trichuris that can be used as screening tools to evaluate the effect of new synthetic or natural anthelmintic compounds.

Competitive inhibition of the high-affinity choline transporter by tetrahydropyrimidine anthelmintics.

The high-affinity choline transporter CHT1 mediates choline uptake, the rate-limiting and regulatory step in acetylcholine synthesis at cholinergic presynaptic terminals. CHT1-medated choline uptake is specifically inhibited by hemicholinium-3, which is a type of choline analog that acts as a competitive inhibitor. Although the substrate choline and the inhibitor hemicholinium-3 are well-established ligands of CHT1, few potent ligands other than choline analogs have been reported. Here we show that tetrahydropyrimidine anthelmintics, known as nicotinic acetylcholine receptor agonists, act as competitive inhibitors of CHT1. A ligand-dependent trafficking assay in cell lines expressing human CHT1 was designed to search for CHT1 ligands from a collection of biologically active compounds. We found that morantel as well as other tetrahydropyrimidines, pyrantel and oxantel, potently inhibits the high-affinity choline uptake activity of CHT1 in a competitive manner similar to the inhibitor hemicholinium-3. They also inhibit the high-affinity choline transporter from the nematode Caenorhabditis elegans. Finally, tetrahydropyrimidines potently inhibit the high-affinity choline uptake in rat brain synaptosomes at a low micromolar level, resulting in the inhibition of acetylcholine synthesis. The rank order of potency in synaptosomes is as follows: morantel > pyarantel > oxantel (Ki = 1.3, 5.7, and 8.3 µM, respectively). Our results reveal that tetrahydropyrimidine anthelmintics are novel CHT1 ligands that inhibit the high-affinity choline uptake for acetylcholine synthesis in cholinergic neurons.

Anthelmintic resistance of horse strongyle nematodes to ivermectin and pyrantel in Lithuania.

BACKGROUND: With intensive use of anthelmintic drugs in recent decades, anthelmintic resistance (AR) in horse nematodes is becoming a growing issue in many countries. However, there is little available information about the parasites, treatment practices or AR in the horse population in Lithuania. The aim of this study was to assess the current situation of AR on horse farms in Lithuania. The study was conducted in 25 stables on horses with a strongyle faecal egg count (FEC) of ≥ 200 eggs per gram. A faecal egg count reduction test (FECRT) was performed on each farm after administration of ivermectin (IVM) or pyrantel (PYR). RESULTS: The efficacy of IVM was comparatively high, with 98.8% of 250 horses having a zero egg count 14 days after treatment. Two conditions were used to interpret the FECRT results for PYR: firstly, resistance was determined when FECR was < 90% and the lower 95% confidence interval (LCL) was < 80%, and secondly when in addition the upper confidence level (UCL) was < 95%. Under the first condition, resistance against PYR was found in five stables (25% of all tested herds), while when considering the UCL as well, resistance was only detected in two stables (8%). The FEC showed a significant (P < 0.01) difference between the treatment and control groups. Only cyathostomin larvae were detected in larval cultures derived from strongyle-positive faecal samples collected 14 days after treatment of a test group with PYR. CONCLUSIONS: This in vivo study showed that PYR resistance is prevalent on horse farms in Lithuania, while the efficacy of IVM still appears to be unaffected. However, further studies of ivermectin resistance are needed. These findings should guide the implementation of more sustainable management of strongyle infections in horses in Lithuania.

Field safety and efficacy of an orally administered combination of sarolaner, moxidectin and pyrantel (Simparica Trio®) for the prevention of angiostrongylosis in dogs presented as veterinary patients.

BACKGROUND: Infection with the cardiopulmonary nematode Angiostrongylus vasorum may cause severe disease in dogs, therefore prophylactic treatments are necessary to prevent infection in dogs at risk. A clinical field study was conducted to demonstrate the efficacy and safety of an oral combination of sarolaner, moxidectin and pyrantel (Simparica Trio®) for the prevention of A. vasorum infection in dogs (prevention study). A survey study was conducted concurrently to determine the infection pressure in the same areas. METHODS: Prevention and survey studies were both conducted at the same veterinary clinics in endemic hot spots for A. vasorum in Denmark and Italy. The prevention study was a randomized, placebo controlled, double masked study where 622 client-owned dogs were treated and tested at 30 days intervals for 10 months. In the survey study 1628 dogs that were at risk of infection and/or were suspected to be infected were tested by fecal and/or serological methods, and the percent of dogs positive for A. vasorum was calculated. RESULTS: In the prevention study, there were no adverse events related to treatment with Simparica Trio®. Two placebo-treated animals became infected with A. vasorum during the 10-month study period, while none of the dogs in the combination product-treated group became infected. In the survey study, 12.2% of the study dogs were found positive to A. vasorum, indicating high exposure to the parasite during the period of the prevention study. CONCLUSIONS: Monthly oral treatment with the combination of sarolaner, moxidectin and pyrantel (Simparica Trio®) was 100% effective in the prevention of natural infection with A. vasorum in dogs in highly endemic areas. In endemic areas, A. vasorum occurrence in dogs at risk is considerable.

Transcriptional responses in Parascaris univalens after in vitro exposure to ivermectin, pyrantel citrate and thiabendazole.

BACKGROUND: Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways. METHODS: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10-9 M, 10-11 M, 10-13 M), pyrantel citrate (10-6 M, 10-8 M, 10-10 M), thiabendazole (10-5 M, 10-7 M, 10-9 M) or without anthelmintics (control) at 37 °C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system. RESULTS: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj < 0.05 and log2 fold change ≥ 1 or ≤ - 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunit lgc-37, which was upregulated only in worms exposed to 10-9 M of ivermectin. CONCLUSIONS: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described in P. univalens and future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance.

Multiple drug resistance in the canine hookworm Ancylostoma caninum: an emerging threat?

BACKGROUND: The canine hookworm, Ancylostoma caninum is the most prevalent and important intestinal nematode parasite of dogs in the USA. Hookworms are typically well controlled by treatment with all commonly used anthelmintics that are approved for this use in dogs. However, in the past few years, cases of recurrent/persistent canine hookworm infections appear to have dramatically increased, suggesting that anthelmintic resistance (AR) may have evolved in this parasite. These cases are highly overrepresented by greyhounds, but multiple other breeds are also represented. The aim of this study was to characterize several of these suspected resistant isolates using in vitro, genetic and clinical testing to determine if these cases represent true anthelmintic resistance in A. caninum. METHODS: Fecal samples containing hookworm eggs from three cases of persistent hookworm infections; one from a greyhound, one from a miniature schnauzer and one from a hound-mix, were received by our laboratory. These were then used to establish infections in laboratory dogs and to perform egg hatch assays (EHA) and larval development assays (LDA) for detecting resistance to benzimidazoles and macrocyclic lactones, respectively. Additional EHA and LDA were performed on eggs recovered from the laboratory-induced infections. Fecal egg count reduction tests were performed to detect resistance to pyrantel. Deep amplicon sequencing assays were developed to measure the frequency of non-synonymous single nucleotide polymorphisms (SNP) at codons 167, 198 and 200 of the A. caninum isotype-1 ß-tubulin gene. RESULTS: Resistance ratios for the three A. caninum isolates tested ranged from 6.0 to > 100 and 5.5 to 69.8 for the EHA and LDA, respectively. Following treatment with pyrantel, reduction in faecal egg counts was negative or 0%. Deep amplicon sequencing of the isotype-1 ß-tubulin gene identified a high frequency of resistance-associated SNPs at codon 167 in all three resistant isolates and in two additional clinical cases. CONCLUSIONS: These data conclusively demonstrate multiple anthelmintic resistance in multiple independent isolates of A. caninum, strongly suggesting that this is an emerging problem in the USA. Furthermore, evidence suggest that these resistant hookworms originate from racing greyhound farms and kennels, though additional research is needed to confirm this.

Deciphering the molecular determinants of cholinergic anthelmintic sensitivity in nematodes: When novel functional validation approaches highlight major differences between the model Caenorhabditis elegans and parasitic species.

Cholinergic agonists such as levamisole and pyrantel are widely used as anthelmintics to treat parasitic nematode infestations. These drugs elicit spastic paralysis by activating acetylcholine receptors (AChRs) expressed in nematode body wall muscles. In the model nematode Caenorhabditis elegans, genetic screens led to the identification of five genes encoding levamisole-sensitive-AChR (L-AChR) subunits: unc-38, unc-63, unc-29, lev-1 and lev-8. These subunits form a functional L-AChR when heterologously expressed in Xenopus laevis oocytes. Here we show that the majority of parasitic species that are sensitive to levamisole lack a gene orthologous to C. elegans lev-8. This raises important questions concerning the properties of the native receptor that constitutes the target for cholinergic anthelmintics. We demonstrate that the closely related ACR-8 subunit from phylogenetically distant animal and plant parasitic nematode species functionally substitutes for LEV-8 in the C. elegans L-AChR when expressed in Xenopus oocytes. The importance of ACR-8 in parasitic nematode sensitivity to cholinergic anthelmintics is reinforced by a 'model hopping' approach in which we demonstrate the ability of ACR-8 from the hematophagous parasitic nematode Haemonchus contortus to fully restore levamisole sensitivity, and to confer high sensitivity to pyrantel, when expressed in the body wall muscle of C. elegans lev-8 null mutants. The critical role of acr-8 to in vivo drug sensitivity is substantiated by the successful demonstration of RNAi gene silencing for Hco-acr-8 which reduced the sensitivity of H. contortus larvae to levamisole. Intriguingly, the pyrantel sensitivity remained unchanged thus providing new evidence for distinct modes of action of these important anthelmintics in parasitic species versus C. elegans. More broadly, this highlights the limits of C. elegans as a predictive model to decipher cholinergic agonist targets from parasitic nematode species and provides key molecular insight to inform the discovery of next generation anthelmintic compounds.

In vitro evaluation of ivermectin, moxidectin, albendazole and pyrantel against cyathostomins of horses / Avaliação in vitro da ivermectina, moxidectina, albendazole e pirantel contra ciatostomíneos de equinos

Abstract Cyathostomins are the most prevalent nematodes of horses, and multidrug resistance has been reported worldwide. There is a need to implement alternative drug monitoring analytical tests. The objective of this study was to determine the consistency (5 repetitions) of the larval migration on agar test (LMAT) using ivermectin, moxidectin, pyrantel or albendazole against cyathostomin infective-stage larvae in eight different concentrations. LMAT showed a strong coefficient of determination (R2 > 0.91), between the test repetitions (n=5). The average 50% effective concentration (EC50) for ivermectin, moxidectin, pyrantel and albendazole were 0.0404, 0.0558, 0.0864 and 0.0988 nMol, respectively. The results of the EC50 for albendazole showed the greatest range of concentration. Ivermectin and moxidectin had the lowest in between-test variation. In the future, internationally certified susceptible isolates could be used for screening new drug candidates, or to follow up the pattern of drug efficacy from field populations.

Resumo Ciatostomíneos são os nematodas mais prevalentes em equinos e a resistência múltipla foi relatada em todo o mundo. Existe a necessidade de implementar o monitoramento dos produtos com testes analíticos alternativos. O objetivo deste estudo foi determinar a consistência (5 repetições) do teste de migração larval em ágar (TMLA) usando ivermectina, moxidectina, pirantel e albendazole contra larvas infectantes de ciatostomíneos em oito concentrações diferentes. O TMLA demonstrou um coeficiente de determinação (R2) acima de 0,91 entre as repetições do teste. A concentração efetiva para 50% (CE50) para ivermectina, moxidectina, pirantel e albendazole foi de 0,0404; 0,0558; 0,0864 e 0,0988 nMol, respectivamente. A CE50 do albendazole demonstrou a maior amplitude entre os testes. A ivermectina e a moxidectina tiveram as menores variações das doses entre as repetições. No futuro, isolados certificados susceptíveis poderão ser testados com o TMLA para indicação de novos produtos e mesmo para acompanhar o perfil de eficácia de populações do campo.

In vitro evaluation of ivermectin, moxidectin, albendazole and pyrantel against cyathostomins of horses.

Cyathostomins are the most prevalent nematodes of horses, and multidrug resistance has been reported worldwide. There is a need to implement alternative drug monitoring analytical tests. The objective of this study was to determine the consistency (5 repetitions) of the larval migration on agar test (LMAT) using ivermectin, moxidectin, pyrantel or albendazole against cyathostomin infective-stage larvae in eight different concentrations. LMAT showed a strong coefficient of determination (R2 > 0.91), between the test repetitions (n=5). The average 50% effective concentration (EC50) for ivermectin, moxidectin, pyrantel and albendazole were 0.0404, 0.0558, 0.0864 and 0.0988 nMol, respectively. The results of the EC50 for albendazole showed the greatest range of concentration. Ivermectin and moxidectin had the lowest in between-test variation. In the future, internationally certified susceptible isolates could be used for screening new drug candidates, or to follow up the pattern of drug efficacy from field populations.

Does evaluation of in vitro microfilarial motility reflect the resistance status of Dirofilaria immitis isolates to macrocyclic lactones?

BACKGROUND: Several reports have confirmed that macrocyclic lactone-resistant isolates of Dirofilaria immitis are circulating in the United States; however, the prevalence and potential impact of drug resistance is unknown. We wished to assess computer-aided measurements of motility as a method for rapidly assessing the resistance status of parasite isolates. METHODS: Blood containing microfilariae (MF) from two clinical cases with a high suspicion of resistance was fed to mosquitoes and the resultant L3 injected into dogs that were then treated with six doses of Heartgard® Plus (ivermectin + pyrantel; Merial Limited) at 30-day intervals. In both cases patent heartworm infections resulted despite the preventive treatment. Microfilariae isolated from these dogs and other isolates of known resistance status were exposed to varying concentrations of ivermectin in vitro and their motility assessed 24 h later using computer-processed high-definition video imaging. RESULTS: We produced two isolates, Yazoo-2013 and Metairie-2014, which established patent infections despite Heartgard® Plus treatments. Measurements of the motility of MF of these and other isolates (Missouri, MP3 and JYD-27) following exposure to varying concentrations of ivermectin did not distinguish between susceptible and resistant heartworm populations. There was some evidence that the method of MF isolation had an influence on the motility and drug susceptibility of the MF. CONCLUSIONS: We confirmed that drug-resistant heartworms are circulating in the southern United States, but that motility measurements in the presence of ivermectin are not a reliable method for their detection. This implies that the drug does not kill the microfilariae via paralysis.

Using a health-rating system to evaluate the usefulness of Caenorhabditis elegans as a model for anthelmintic study.

Soil-transmitted helminths (STHs) are intestinal parasitic nematodes that infect humans, and are transmitted through contaminated soil. These nematodes include the large roundworm (Ascaris lumbricoides), whipworm (Trichuris trichiura), and hookworm (Ancylostoma ceylanicum, Ancylostoma duodenale, and Necator americanus). Nearly 1.5 billion people (~24% of the population) worldwide are infected with at least one species of these parasites, burdening the poor, in particular, children and pregnant women. To combat these diseases, the WHO only recognizes four anthelmintic drugs, including the preferred drug, albendazole, for mass drug administration (MDA). These four drugs have a total of two different mechanisms of action, and, as expected, resistance has been observed. This problem calls for new drugs with different mechanisms of action. Although there is precedence for the use of Caenorhabditis elegans (C. elegans), a free-living nematode, as a model for drug screening and anthelmintic testing, their usefulness for such anthelmintic study is not clear as past research has shown that C. elegans did not show a strong response to albendazole, the MDA drug of choice, in comparison with various STHs under similar treatment. To further examine if C. elegans has the potential to be a good model organism for anthelmintic drug study, we employed a health rating scale in order to tease out potential effects of albendazole, and other anthelmintics, that may have been missed using a binary, dead/alive scale. Using the health-rating scale we found that although the worms may have not been dying, they were sick, showing dose responses to anthelmintic drugs, including albendazole, reinforcing C. elegans as a useful model for anthelmintic study.

Transient effects of levamisole on Brugia malayi microfilariae.

Levamisole is an anthelmintic drug that acts by activating nicotinic acetylcholine receptors at the nematode neuromuscular junction and causing paralysis. We measured the in vitro effects of levamisole on the motility of Brugia malayi microfilariae; after 2 h incubation the apparent IC50 was 2.68 mM. Lower drug concentrations, such as 1 mM, caused an immediate total paralysis that lasted for up to 1 h, but was completely reversed by 2 h of incubation. The 'recovered' parasites were still completely susceptible to application of a second nicotinic agonist, pyrantel.

In vitro levamisole selection pressure on larval stages of Haemonchus contortus over nine generations gives rise to drug resistance and target site gene expression changes specific to the early larval stages only.

There is some evidence that resistance to levamisole and pyrantel in trichostrongylid nematodes is due to changes in the composition of nicotinic acetylcholine receptors (nAChRs) which represent the drug target site. Altered expression patterns of genes coding for nAChR subunits, as well as the presence of truncated versions of several subunits, have been implicated in observed resistances. The studies have mostly compared target sites in worm isolates of very different genetic background, and hence the ability to associate the molecular changes with drug sensitivity alone have been clouded to some extent. The present study aimed to circumvent this issue by following target site gene expression pattern changes as resistance developed in Haemonchus contortus worms under laboratory selection pressure with levamisole. We applied drug selection pressure to early stage larvae in vitro over nine generations, and monitored changes in larval and adult drug sensitivities and target site gene expression patterns. High level resistance developed in larvae, with resistance factors of 94-fold and 1350-fold at the IC50 and IC95, respectively, in larval development assays after nine generations of selection. There was some cross-resistance to bephenium (70-fold increase in IC95). The expression of all the putative subunit components of levamisole-sensitive nAChRs, as well as a number of ancillary protein genes, particularly Hco-unc-29.1 and -ric-3, were significantly decreased (up to 5.5-fold) in the resistant larvae at generation nine compared to the starting population. However, adult worms did not show any resistance to levamisole, and showed an inverse pattern of gene expression changes, with many target site genes showing increased expression compared to the starting population. A comparison of the larval/adult drug sensitivity data with the known relationships for field-derived isolates indicated that the adults of our selected population should have been highly resistant to the drug if the larval/adult sensitivity relationships were in accordance with previous field isolates. Hence, our selected worms showed a life-stage drug sensitivity pattern quite different to that seen in the field. The present study has highlighted an association between drug target site changes and resistance to levamisole in H. contortus larvae. However, it has also highlighted the artificial nature of the larval selection method with levamisole, as the resistance phenotype and the associated molecular changes were only observed in the drug-pressured life stage. The study therefore reinforces the need for caution in extrapolating larval-based laboratory selection outcomes to field resistances.

Pyrantel resistance in two herds of donkey in the UK.

Resistance to currently available anthelmintics is a serious phenomenon which is prevalent globally. Cyathostomins are one of the major parasites, and are of primary concern in donkeys. There have been reports of emerging resistance to pyrantel, but the status of pyrantel resistance in donkey populations in the UK is largely unknown. This report investigates pyrantel resistance in two geographically isolated donkey herds in the South West of England. The first herd had suspected pyrantel resistance, with already established resistance to other anthelmintics. In the second herd the efficacy of pyrantel was not suspected at the time the study took place. Faecal Egg Count Reduction Test (FECRT) was carried out, revealing large scale resistance. Eighty one percent of the first herd and 73% of the second herd had a FEC of less than 95% after treatment, and anthelmintic resistance was confirmed using the World Association for the Advancement of Veterinary Parasitology guidelines. These findings indicate that anthelmintic resistance to pyrantel exists in both tested donkey populations and illustrate the continuing development of resistance through different classes of chemotherapeutics.

Anthelmintic resistance of intestinal nematodes to ivermectin and pyrantel in Estonian horses.

There is evidence of resistance in horses to anthelmintic treatment using ivermectin and pyrantel. However, little information is available about the parasites, treatment practices or anthelmintic resistance in the horse population in Estonia. In the present study, we examined 41 trotting and riding horses aged < 3 years from four stables in Estonia. Faecal samples were collected, and horses were selected for treatment if the nematode egg count per gram faeces exceeded 200. Horses (n= 32) that shed strongyle-type eggs were treated with pyrantel, whereas Parascaris equorum-positive animals received ivermectin. Up to 78% of horses required anthelmintic treatment and the efficiency of the anthelmintics was evaluated using a faecal egg count reduction test. Resistance of P. equorum was observed in 50% of horses treated with ivermectin and of strongyles in 27% of horses treated with pyrantel. Ivermectin treatment resulted in a mean reduction of 100% for strongyle eggs and an 89% reduction in P. equorum, and pyrantel-treated horses exhibited an 88% reduction in strongyle eggs. These results are considered to be the first indication of resistance to pyrantel, but further studies of ivermectin resistance are required. According to questionnaires completed by the owners of horses, resistance might be explained by a lack of evidence-based strategies, a strong preference for using ivermectin and possibly a subjective evaluation of the body weight of horses.

Nitazoxanide: nematicidal mode of action and drug combination studies.

Intestinal nematodes or roundworms (aka soil-transmitted helminths or STHs) cause great disease. They infect upwards of two billion people, leading to high morbidity and a range of health problems, especially in infected children and pregnant women. Development of resistance to the two main classes of drugs used to treat intestinal nematode infections of humans has been reported. To fight STH infections, we need new and more effective drugs and ways to improve the efficacy of the old drugs. One promising alternative drug is nitazoxanide (NTZ). NTZ, approved for treating human protozoan infections, was serendipitously shown to have therapeutic activity against STHs. However, its mechanism of action against nematodes is not known. Using the laboratory nematode Caenorhabditis elegans, we show that NTZ acts on the nematodes through avr-14, an alpha-type subunit of a glutamate-gated chloride ion channel known for its role in ivermectin susceptibility. In addition, a forward genetic screen to select C. elegans mutants resistant to NTZ resulted in isolation of two NTZ resistant mutants that are not in avr-14, suggesting that additional mechanisms are involved in resistance to NTZ. We found that NTZ combines synergistically with other classes of anthelmintic drugs, i.e. albendazole and pyrantel, making it a good candidate for further studies on its use in drug combination therapy of STH infections. Given NTZ acts against a wide range of nematode parasites, our findings also validate avr-14 as an excellent target for pan-STH therapy.