Anthelmintic resistance in gastrointestinal nematodes of alpacas (Vicugna pacos) in Australia.
ABSTRACT: Gastrointestinal nematodes (GINs) can cause significant economic losses in alpacas due to lowered production of fibre and meat. Although no anthelmintics are registered for use in alpacas, various classes of anthelmintics are frequently used to control parasitic gastroenteritis in alpacas in Australia and other countries. Very little is known about the current worm control practices as well as the efficacy of anthelmintics used against common GINs of alpacas. This study aimed to assess the existing worm control practices used by Australian alpaca farmers and to quantify the efficacy of commonly used anthelmintics against GINs of alpacas.An online questionnaire survey was conducted to assess current worm control practices on 97 Australian alpaca farms, with an emphasis on the use of anthelmintics. Of this group of 97 alpaca farms, 20 were selected to assess the efficacy of eight anthelmintics and/or their combinations (closantel, fenbendazole ivermectin, monepantel, moxidectin and a combination of levamisole, closantel, albendazole, abamectin) using the faecal egg count reduction test (FECRT). A multiplexed-tandem PCR (MT-PCR) was used to identify the prevalent nematode genera/species.The response rate for the questionnaire was 94% (91/97). Almost half of the respondents kept alpacas with sheep and cattle, and 26% of respondents allowed alpacas to co-graze with these ruminants. Although only 63% respondents perceived worms to be an important health concern for alpacas, the majority of respondents (89%) used anthelmintics to control GINs of alpacas. The commonly used anthelmintics were macrocyclic lactones, monepantel, benzimidazoles, levamisole, closantel and their combinations, and they were typically administered at the dose rate recommended for sheep. The FECRT results showed that a combination of levamisole, closantel, albendazole and abamectin was the most effective dewormer followed by single drugs, including monepantel, moxidectin, closantel, fenbendazole and ivermectin. Haemonchus spp. were the most commonly resistant nematodes followed by Trichostrongylus spp., Camelostrongylus mentulatus, Ostertagia ostertagi and Cooperia spp.This is the first study aimed at assessing worm control practices and efficacy of commonly used anthelmintics in alpacas in Australia. Our findings document the extent of anthelmintics resistance on Australian alpaca farms and identify those anthelmintics that are still effective against GINs of alpacas.
Project description:BACKGROUND: Parasitic nematodes can cause substantial clinical and subclinical problems in alpacas and anthelmintics are regularly used to control parasitic nematodes in alpacas. Although anthelmintic resistance has been reported in ruminants worldwide, very little is known about anthelmintic resistance in alpacas. The present study was carried out to confirm a suspected case of anthelmintic resistance in Haemonchus contortus in alpacas in Australia. METHODS: Post mortem examination of an alpaca was conducted to determine the cause of its death. To confirm a suspected case of macrocyclic lactone (ML) resistance in H. contortus in alpacas, a faecal egg count reduction test (FECRT) was performed using closantel (7.5 mg/kg) and ivermectin (0.2 mg/kg). Nematode species were identified by morphological and molecular methods. RESULTS: Post mortem examination of a 1-year-old female alpaca that had died following a brief period of lethargy, anorexia and recumbency revealed severe anaemia, hypoproteinaemia and gastric parasitism by adult Haemonchus contortus, despite recent abamectin (0.2 mg/kg) treatment. Based on these findings and the exclusive use of MLs in the herd over the preceding six years, ML resistance in parasitic nematodes of alpacas on this farm was suspected. FECRT revealed that the efficacy of closantel was 99% (95% CI 93-100), whereas that of ivermectin was 35% (95% CI 0-78), indicating that the treatment failure was likely due to the presence of ML-resistant nematodes. Larval culture of faecal samples collected following ivermectin treatment consisted of 99% H. contortus and 1% Cooperia oncophora, a result confirmed using a PCR assay. CONCLUSIONS: This study provides the first evidence of ML resistance in H. contortus in alpacas in Australia. Based on the extent of anthelmintic resistance in sheep gastrointestinal nematodes in Australia, veterinarians and alpaca owners should be encouraged to implement integrated parasite management strategies to improve nematode control in alpacas.
Project description:BACKGROUND: Few effective drugs are available for soil-transmitted helminthiases and drug resistance is of concern. In the present work, we tested the efficacy of the veterinary drug monepantel, a potential drug development candidate compared to standard drugs in vitro and in parasite-rodent models of relevance to human soil-transmitted helminthiases. METHODOLOGY: A motility assay was used to assess the efficacy of monepantel, albendazole, levamisole, and pyrantel pamoate in vitro on third-stage larvae (L3) and adult worms of Ancylostoma ceylanicum, Necator americanus and Trichuris muris. Ancylostoma ceylanicum- or N. americanus-infected hamsters, T. muris- or Ascaris suum-infected mice, and Strongyloides ratti-infected rats were treated with single oral doses of monepantel or with one of the reference drugs. PRINCIPAL FINDINGS: Monepantel showed excellent activity on A. ceylanicum adults (IC(50) = 1.7 µg/ml), a moderate effect on T. muris L3 (IC(50) = 78.7 µg/ml), whereas no effect was observed on A. ceylanicum L3, T. muris adults, and both stages of N. americanus. Of the standard drugs, levamisole showed the highest potency in vitro (IC(50) = 1.6 and 33.1 µg/ml on A. ceylanicum and T. muris L3, respectively). Complete elimination of worms was observed with monepantel (10 mg/kg) and albendazole (2.5 mg/kg) in A. ceylanicum-infected hamsters. In the N. americanus hamster model single 10 mg/kg oral doses of monepantel and albendazole resulted in worm burden reductions of 58.3% and 100%, respectively. Trichuris muris, S. ratti and A. suum were not affected by treatment with monepantel in vivo (following doses of 600 mg/kg, 32 mg/kg and 600 mg/kg, respectively). In contrast, worm burden reductions of 95.9% and 76.6% were observed following treatment of T. muris- and A. suum infected mice with levamisole (200 mg/kg) and albendazole (600 mg/kg), respectively. CONCLUSIONS/SIGNIFICANCE: Monepantel reveals low or no activities against N. americanus, T. muris, S. ratti and A. suum in vivo, hence does not qualify as drug development candidate for human soil-transmitted helminthiases.
Project description:The recently launched veterinary anthelmintic drench for sheep (Novartis Animal Health Inc., Switzerland) containing the nematocide monepantel represents a new class of anthelmintics: the amino-acetonitrile derivatives (AADs), much needed in view of widespread resistance to the classical drugs. Recently, it was shown that the ACR-23 protein in Caenorhabditis elegans and a homologous protein, MPTL-1 in Haemonchus contortus, are potential targets for AAD action. Both proteins belong to the DEG-3 subfamily of acetylcholine receptors, which are thought to be nematode-specific, and different from those targeted by the imidazothiazoles (e.g. levamisole). Here we provide further evidence that Cel-ACR-23 and Hco-MPTL-1-like subunits are involved in the monepantel-sensitive phenotype. We performed comparative genomics of ligand-gated ion channel genes from several nematodes and subsequently assessed their sensitivity to anthelmintics. The nematode species in the Caenorhabditis genus, equipped with ACR-23/MPTL-1-like receptor subunits, are sensitive to monepantel (EC(50)<1.25 µM), whereas the related nematodes Pristionchus pacificus and Strongyloides ratti, which lack an ACR-23/MPTL-1 homolog, are insensitive (EC(50)>43 µM). Genome sequence information has long been used to identify putative targets for therapeutic intervention. We show how comparative genomics can be applied to predict drug sensitivity when molecular targets of a compound are known or suspected.
Project description:In 2008, Novartis Animal Health developed a new class of anthelmintics, the amino-acetonitrile derivatives (AAD) of which monepantel is the most prominent compound. Monepantel was designed for the treatment of sheep against the parasitic nematode Haemonchus contortus. Because monepantel acts through a different mechanism, it is effective against nematodes that have acquired resistance to long-standing anthelmintics. In order to benefit from a maximum lifespan and efficacy of this new compound, the mode of action of monepantel needs to be understood. Studies on the model nematode Caenorhabditis elegans led to the identification of at least one target of monepantel: the monovalent cation channel ACR-23. Here we comment on the effects of monepantel on C. elegans and on the development of resistant parasitic nematode strains.
Project description:Gut -associated microbes ('gut microbiota') impact the nutrition of their hosts, especially in ruminants and pseudoruminants that consume high-cellulose diets. Examples include the pseudoruminant alpaca. To better understand how body site and diet influence the alpaca microbiota, we performed three 16S rRNA gene surveys. First, we surveyed the compartment 1 (C1), duodenum, jejunum, ileum, cecum, and large intestine (LI) of alpacas fed a grass hay (GH; tall fescue) or alfalfa hay (AH) diet for 30 days. Second, we performed a C1 survey of alpacas fed a series of 2-week mixed grass hay (MGH) diets supplemented with ?25% dry weight barley, quinoa, amaranth, or soybean meal. Third, we examined the microbial differences of alpacas with normal versus poor body condition. Samples from GH- and AH-fed alpacas grouped by diet and body site but none of the four supplements significantly altered C1 microbiota composition, relative to each other, and none of the OTUs were differentially abundant between alpacas with normal versus poor body conditions. Taken together, the findings of a diet- and body-site specific alpaca microbiota are consistent with previous findings in ruminants and other mammals, but we provide no evidence to link changes in alpaca body condition with variation in microbiota relative abundance or identity.
Project description:BACKGROUND:Eukaryotic pathogens, including Cryptosporidium, Giardia and Enterocytozoon, have been implicated in neonatal diarrhoea, leading to marked morbidity and mortality in the alpaca (Vicugna pacos) and llama (Lama glama) around the world. Australia has the largest population of alpacas outside of South America, but very little is known about these pathogens in alpaca populations in this country. Here, we undertook the first molecular epidemiological survey of Cryptosporidium, Giardia and Enterocytozoon in V. pacos in Australia. METHODS:A cross-sectional survey of 81 herds, comprising alpacas of 6 weeks to 26 years of age, were sampled from the six Australian states (Queensland, New South Wales, Victoria, South Australia, Tasmania and Western Australia) across the four seasons. PCR-based sequencing was employed, utilising genetic markers in the small subunit of the nuclear ribosomal RNA (SSU) and 60-kilodalton glycoprotein (gp60) genes for Cryptosporidium, triose-phosphate isomerase (tpi) gene for Giardia duodenalis and the internal transcribed spacer region (ITS) for Enterocytozoon bieneusi. RESULTS:PCR-based analyses of 81 faecal DNA samples representing 1421 alpaca individuals detected Cryptosporidium, Giardia and/or Enterocytozoon on 15 farms in New South Wales, Victoria and South Australia, equating to 18.5% of all samples/herds tested. Cryptosporidium was detected on three (3.7%) farms, G. duodenalis on six (7.4%) and E. bieneusi on eight (9.9%) in two or all of these three states, but not in Queensland, Tasmania or Western Australia. Molecular analyses of selected faecal DNA samples from individual alpacas for Cryptosporidium, Giardia and/or Enterocytozoon consistently showed that alpacas of ≤ 6 months of age harboured these pathogens. CONCLUSIONS:This first molecular investigation of Cryptosporidium, Giardia and Enterocytozoon in alpaca subpopulations in Australia has identified species and genotypes that are of likely importance as primary pathogens of alpacas, particularly young crias, and some genotypes with zoonotic potential. Although the prevalence established here in the alpaca subpopulations studied is low, the present findings suggest that crias are likely reservoirs of infections to susceptible alpacas and/or humans. Future studies should focus on investigating pre-weaned and post-weaned crias, and on exploring transmission patterns to establish what role particular genotypes play in neonatal or perinatal diarrhoea in alpacas and in zoonotic diseases in different states of Australia.
Project description:A lack of quantitative information on the species composition of parasite communities present in fecal samples is a major limiting factor for the sensitivity, accuracy and interpretation of the diagnostic tests commonly used to assess anthelmintic efficacy and resistance. In this paper, we investigate the ability of ITS-2 rDNA nemabiome metabarcoding to enhance fecal egg count reduction testing by providing information on the effect of drug treatments on individual parasite species. Application of ITS-2 rDNA nemabiome metabarcoding to fecal samples from ewes from over 90 flocks across western Canada revealed high gastrointestinal nematode infection intensities in many flocks with Haemonchus contortus being the most abundant species followed by Teladorsagia circumcincta and then Trichostrongylus colubriformis. Integration of ITS-2 rDNA nemabiome metabarcoding with pre- and post-treatment fecal egg counting revealed consistently poor efficacy of producer-applied ivermectin and benzimidazole treatments against H. contortus, but much better efficacy against T. circumcincta and T. colubriformis, except for in a small number of flocks. Integration of nemabiome ITS-2 rDNA metabarcoding with Fecal Egg Count Reduction Tests (FECRT), undertaken on farm visits, confirmed that ivermectin and fenbendazole resistance is widespread in H. contortus but is currently less common in T. circumcincta and T. colubriformis in western Canada. FECRT/nemabiome testing did not detect moxidectin resistance in any GIN species but suggested the early emergence of levamisole resistance specifically in T. circumcincta. It also revealed that although poor efficacy to closantel was relatively common, based on total fecal egg counts, this was due to its narrow spectrum of activity rather than the emergence of anthelmintic resistance. This study illustrates the value of ITS-2 rDNA nemabiome metabarcoding to improve fecal egg count resistance testing, perform large-scale anthelmintic resistance surveillance and direct more targeted rational anthelmintic use.
Project description:New compounds are needed to treat parasitic nematode infections in humans, livestock and plants. Small molecule anthelmintics are the primary means of nematode parasite control in animals; however, widespread resistance to the currently available drug classes means control will be impossible without the introduction of new compounds. Adverse environmental effects associated with nematocides used to control plant parasitic species are also motivating the search for safer, more effective compounds. Discovery of new anthelmintic drugs in particular has been a serious challenge due to the difficulty of obtaining and culturing target parasites for high-throughput screens and the lack of functional genomic techniques to validate potential drug targets in these pathogens. We present here a novel strategy for target validation that employs the free-living nematode Caenorhabditis elegans to demonstrate the value of new ligand-gated ion channels as targets for anthelmintic discovery. Many successful anthelmintics, including ivermectin, levamisole and monepantel, are agonists of pentameric ligand-gated ion channels, suggesting that the unexploited pentameric ion channels encoded in parasite genomes may be suitable drug targets. We validated five members of the nematode-specific family of acetylcholine-gated chloride channels as targets of agonists with anthelmintic properties by ectopically expressing an ivermectin-gated chloride channel, AVR-15, in tissues that endogenously express the acetylcholine-gated chloride channels and using the effects of ivermectin to predict the effects of an acetylcholine-gated chloride channel agonist. In principle, our strategy can be applied to validate any ion channel as a putative anti-parasitic drug target.
Project description: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 (IC50 values: 1.6?±?3.1?nM and 0.2?±?2.3??M). 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.
Project description:Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important "classical" anthelmintics like levamisole and pyrantel, as well as "novel" anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.