anthelmintic resistance

Efficacy of anthelmintics in horses and donkeys in Ireland: An in vivo and in vitro study

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Date presented: 
Friday 3 June 2016
Abstract

Strongyles are the most important parasite group infecting equids. Management of these parasites has relied on intensive use of anthelmintics, however, resistance has developed against all drug classes and is becoming a major practical problem in many countries. Resistance to the benzimidazole (BZ) group is geographically widespread and resistance to pyrental has also been reported. Today the macrocyclic lactones (ML) class of drugs has become the most commonly used drug, but evidence of emerging resistance (i.e. shortened egg reappearance period (ERP)) has been identified in many countries. A variety of tests are available to monitor anthelmintic efficacy but most of them are expensive, laborious and time consuming. The aim of this project was to determine the efficacy of anthelmintic drugs used in eight equine groups in Ireland. The anthelmintic efficacy was determined by calculating the percentage reduction in the faecal egg count (FEC) between the group mean at Day 0 and Day 14 post-treatment (FECRT). In addition FECs were also calculated at two week intervals for up to 16 weeks after anthelmintic drug administration to determine the ERP for BZ, ivermectin and moxidectin. ERP was defined when the group arithmetic mean FEC exceeded 10% of the group arithmetic mean FEC at Day 0.The larval development assay (LDA) was used to detect resistance to BZ in two groups of horses and the larval migration inhibition assay (LMIA) was also performed to measure the sensitivity to ivermectin in two groups of donkeys and moxidectin in two horse farms and two groups of donkeys. The results of FECRT indicate BZ-resistance on both farms; FECR of 86% and 61%, an ERP of only two weeks and the EC50 for the LDA of 0.3 and 0.7 µg/ml, respectively. While MLs were still effective in all cases with a FECR >95% and the EC50 for the LMIA ranging from 0.06 to 0.38 µg/ml the ERP ranged from only 4 to 10 weeks. Overall the results from this study indicate that BZ was ineffective but both ivermectin and moxidectin are still effective in all groups. However, the reduced ERP results for the MLs would suggest that these products are less effective compared to label claims - a shortened ERP is believed to be an early indicator of resistance.

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Development of the larval migration inhibition test for comparative analysis of ivermectin sensitivity in cyathostomin populations

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Claire McArthur, Ian G Handel, Ailie Robinson, Jane Hodgkinson, Barend MdeC Bronsvoort, Faith A. Burden, Ray Kaplan, Jacqui. B. Matthews. June 2015. Development of the larval migration inhibition test for comparative analysis of ivermectin sensitivity in cyathostomin populations. Veterinary Parasitology.

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Publication date: 
20 June 2015
DOI number: 
10.1016/j.vetpar.2015.06.019
Abstract

Cyathostomins are the most prevalent parasitic pathogens of equids worldwide. These nematodes have been controlled using broad-spectrum anthelmintics; however, cyathostomin resistance to each anthelmintic class has been reported and populations insensitive to more than one class are relatively commonplace. The faecal egg count reduction test (FECRT) is considered the most suitable method for screening anthelmintic sensitivity in horses, but is subject to variation and is relatively time-consuming to perform. Here, we describe a larval migration inhibition test (LMIT) to assess ivermectin (IVM) sensitivity in cyathostomin populations. This test measures the paralysing effect of IVM on the ability of third stage larvae (L3) to migrate through a pore mesh. When L3 from a single faecal sample were examined on multiple occasions, variation in migration was observed: this was associated with the length of time that the L3 had been stored before testing but the association was not significant. Half maximal effective concentration (EC50) values were then obtained for cyathostomin L3 from six populations of horses or donkeys that showed varying sensitivity to IVM in previous FECRTs. Larvae from populations indicated as IVM resistant by FECRT displayed significantly higher EC50 values in the LMIT than L3 from populations classified as IVM sensitive or L3 from populations that had not been previously exposed to IVM or had limited prior exposure. The analysis also showed that EC50 values obtained using L3 from animals in which IVM faecal egg count reduction (FECR) levels had been recorded as <95% were significantly higher than EC50 values obtained using L3 from animals for which FECR was measured as >95%. For one of the populations, time that had elapsed since IVM administration had an effect on the EC50 value obtained, with a longer time since treatment associated with lower EC50 values. These results indicate that the LMIT has value in discriminating IVM sensitivity amongst cyathostomin populations, but several factors were identified that need to be taken into account when executing the test and interpreting the derived data.

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A Potential Novel Anthelmintic? The Cysteine Proteases Show Potent Anthelmintic Activity Against Cyathostomins In Vitro

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Laura Peachey, Jacqui. B. Matthews, Gina L. Pinchbeck, J. Benkhe, Faith A. Burden, Jane Hodgkinson. September 2014. A Potential Novel Anthelmintic? The Cysteine Proteases Show Potent Anthelmintic Activity Against Cyathostomins In Vitro. Equine Veterinary Journal. 46. 23.

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Publication date: 
8 September 2014
Volume: 
46
Page numbers: 
23
DOI number: 
10.1111/evj.12323_52
Abstract

Reasons for performing study
Anthelmintic resistance is a global problem and constitutes a major threat to the welfare of equids worldwide. The cyathostomins are the most numerous and pathogenic gastrointestinal nematode (GIN) of equids in the developed world. Cyathostomins show widespread resistance to 2 out of 3 of the major classes of anthelmintic and recently there are reports of reduced efficacy to the potent macrocyclic lactones (MLs). None of the 3 novel classes of anthelmintic that have emerged in the last decade are licensed for use in equids. The cysteine proteases (CPs) are plant proteins that have shown potent activity against GINs in vivo in sheep and pigs.

Objectives
This study aimed to evaluate the anthelmintic effect of the CP papain on cyathostomins in vitro using the egg hatch assay (EHA) and larval migration inhibition test (LMIT).

Methods
Samples of cyathostomin eggs and third stage larvae were collected and cultured from a population of equids that have recently shown reduced ML efficacy in vivo. The EHA and LMIT were performed on repeated samples with increasing concentrations of papain. Dose–response curves were plotted and PROBIT analysis performed on the data to give EC-50 values (concentration that gives 50% of the maximal response).

Results
Papain caused a dose dependent inhibition of both egg hatch and larval migration. The EC-50 values were 2 μmol/l and 100 μmol/l in the EHA and LMIT respectively, indicating a more potent effect on egg hatch.

Conclusions
The CP papain shows potent anthelmintic activity against cyathostomins in vitro. Good evidence of anthelmintic effect against GINs in other host species is supportive of its potential use in equids. Further work is indicated to evaluate safety and in vivo efficacy.

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An evidence-based approach to the evaluation of ethnoveterinary medicines against strongyle nematodes of equids

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Laura Peachey, Gina L. Pinchbeck, Jacqui. B. Matthews, Faith A. Burden, Mulugeta Getachew, Claire Scantlebury, Jane Hodgkinson. March 2015. An evidence-based approach to the evaluation of ethnoveterinary medicines against strongyle nematodes of equids. Veterinary Parasitology.

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Publication date: 
25 March 2015
Abstract

Cyathostomins are the most important gastrointestinal nematode infecting equids. Their effective control is currently under threat due to widespread resistance to the broad spectrum anthelmintics licenced for use in equids. In response to similar resistance issues in other helminths, there has been increasing interest in alternative control strategies, such as bioactive plant compounds derived from traditional ethnoveterinary treatments. This study used an evidence-based approach to evaluate the potential use of plant extracts from the UK and Ethiopia to treat cyathostomins. Plants were shortlisted based on findings from a literature review and additionally, in Ethiopia, the results of a participatory rural appraisal (PRA) in the Oromia region of the country. Systematic selection criteria were applied to both groups to identify five Ethiopian and four UK plants for in vitro screening. These included Acacia nilotica (L.) Delile, Cucumis prophetarum L., Rumex abyssinicus Jacq., Vernonia amygdalina Delile. and Withania somnifera (L.) Dunal from Ethiopia and Allium sativum L. (garlic), Artemisia absinthium L., Chenopodium album L. and Zingiber officinale Roscoe. (ginger) from the UK. Plant material was collected, dried and milled prior to hydro-alcoholic extraction. Crude extracts were dissolved in distilled water (dH2O) and dimethyl sulfoxide (DMSO), serially diluted and screened for anthelmintic activity in the larval migration inhibition test (LMIT) and the egg hatch test (EHT). Repeated measures ANOVA was used to identify extracts that had a significant effect on larval migration and/or egg hatch, compared to non-treated controls. The median effective concentration (EC-50) for each extract was calculated using PROBIT analysis. Of the Ethiopian extracts A. nilotica, R. abyssinicus and C. prophetarum showed significant anthelmintic activity. Their lowest EC-50 values were 0.18 (Confidence interval (CI): 0.1-0.3), 1.1 (CI: 0.2-2.2) and 1.1 (CI: 0.9-1.4) mg/ml, respectively. All four UK extracts, A. sativum, C. album, Z. officinale and A. absinthium, showed significant anthelmintic activity. Their lowest EC-50 values were 1.1 (CI: 0.9-1.3), 2.3 (CI: 1.9-2.7) and 0.3 (CI: 0.2-0.4) mg/ml, respectively. Extract of A. absinthium had a relatively low efficacy and the data did not accurately fit a PROBIT model for the dose response relationship, thus an EC-50 value was not calculated. Differences in efficacy for each extract were noted, dependent on the assay and solvent used, highlighting the need for a systematic approach to the evaluation of bioactive plant compounds. This study has identified bioactive plant extracts from the UK and Ethiopia which have potential as anthelmintic forages or feed supplements in equids.

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Pyrantel resistance in two herds of donkey in the UK

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Edwina Lawson, Faith A. Burden, Hany Elsheikha. January 2015. Pyrantel resistance in two herds of donkey in the UK. Veterinary Parasitology.

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Publication date: 
7 January 2015
DOI number: 
10.1016/j.vetpar.2014.12.026
Abstract

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.

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Clinical trial on the efficacy of moxidectin oral gel formulation on donkeys naturally infected by cyathostominae

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Date presented: 
Saturday 7 February 2015
Abstract

Donkeys and horses share several parasites including the small strongyles, Cyathostominae. Moxidectin (MOX), a compound of macrocyclic lactones, has a wide range of ecto and endoparasitic activity in many species. For horses, MOX is available as oral gel formulation that provides excellent and long-lasting efficacy against nematodes such as large and small strongyles. There is a paucity of data available on the efficacy of anthelmintics used in donkeys (Veneziano et al., 2011). Therapeutics, such as antiparasitic compounds, are often administered to donkeys on the basis of dosage and intervals recommended for horses, because very few drugs have donkey-specific label indications (Grosenbaugh et al., 2011). The objective of the present study was to evaluate the field efficacy and Egg Reappearance Period (ERP) of MOX oral gel up to 84 days at horse dose against natural infection of Cyathostominae in donkeys.

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The P-glycoprotein inhibitor ketoconazole causes a reversion to ivermectin sensitivity in cyathostomins in vitro

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Laura Peachey, Jacqui. B. Matthews, Gina L. Pinchbeck, Faith A. Burden, Nikki Stradling, Jane Hodgkinson. The P-glycoprotein inhibitor ketoconazole causes a reversion to ivermectin sensitivity in cyathostomins in vitro. Presented at British Society for Parasitology Spring Meeting.

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Date presented: 
Monday 7 April 2014
Abstract

Anthelmintic resistance is a major veterinary and public health issue globally, of most concern is the level of resistance to the macrocyclic lactones. Recent studies have identified a role in resistance for the ATP binding cassette (ABC) drug transporters, P-glycoproteins (P-gps). This study demonstrates the effect of the P-gp inhibitor ketoconazole on the efficacy of ivermectin (IVM) against equid cyathostomin larvae using the larval migration inhibition test (LMIT). Third stage cyathostomin larvae (L3) were cultured from two populations; 1) with recent history of IVM resistance in vivo and 2) naive to anthelmintic exposure. The sensitivity to IVM in each group (n=8) was characterised using the LMIT. The IVM LMIT was repeated for each sample with and without the addition of 10µM ketoconazole. Probit analysis was performed on grouped data from each population to give LC-50 values. The LC-50 value for IVM in Populations 1 and 2 was 4.9 and 2.4µg/ml respectively indicating that Population 1 has a resistant phenotype in comparison to Population 2. Addition of 10µM ketoconazole to IVM in Population 1 caused a drop in LC-50 value from 5.8 to 1.6µg/ml. In Population 2 the effect of the addition of ketoconazole was negligible (1.1 to 0.9µg/ml). This study demonstrates that the P-gp inhibitor ketoconazole causes reversion to a sensitive phenotype in IVM-resistant cyathostomins, inferring that P-gps play a role in their resistance to IVM. This work will be corroborated by investigation into P-gp genes and their expression in cyathostomins.

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The P-glycoprotein inhibitor ketoconazole causes a reversion to sensitivity in ivermectin resistant cyathostomins in vitro

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Laura Peachey, Jacqui. B. Matthews, Gina L. Pinchbeck, Faith A. Burden, Jane Hodgkinson. The P-glycoprotein inhibitor ketoconazole causes a reversion to sensitivity in ivermectin resistant cyathostomins in vitro. Presented at Anthelmintics: From Discovery to Resistance. (5 February - 7 February 2014). San Francisco, USA.

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Date presented: 
Thursday 6 February 2014
Abstract

Anthelmintic resistance is a growing problem in both the developed and developing world; of most concern is the level of resistance detected against the potent macrocylic lactone (ML) anthelmintics. To identify and target a common mechanism of resistance to anthelmintics would allow potential modification of existing drugs, and may even enable the prediction and prevention of the development of resistance to novel drugs. There is a growing body of evidence that P-glycoproteins (P-gps) are involved in resistance to the MLs in many parasitic nematodes of humans and veterinary species (Ardelli et al, 2011). P-gps belong to class two of the ATP binding cassette (ABC) transporter protein superfamily; they are responsible for the active removal of xenobiotic compounds from cells. The cyathostomins are gastrointestinal nematodes of equids that cause significant pathology. Recently resistance to MLs has been described in cyathostomins (Molento et al, 2008), its mechanisms have not yet been elucidated.

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Common helminth infections of donkeys and their control in temperate regions

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Jacqui. B. Matthews, Faith A. Burden. March 2013. Common helminth infections of donkeys and their control in temperate regions. Equine Veterinary Education.

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Publication date: 
18 March 2013
DOI number: 
doi: 10.1111/eve.12018
Abstract

Roundworms and flatworms that affect donkeys can cause disease. All common helminth parasites that affect horses also infect donkeys, so animals that co-graze can act as a source of infection for either species. Of the gastrointestinal nematodes, those belonging to the cyathostomin (small strongyle) group are the most problematic in UK donkeys. Most grazing animals are exposed to these parasites and some animals will be infected all of their lives. Control is threatened by anthelmintic resistance: resistance to all 3 available anthelmintic classes has now been recorded in UK donkeys. The lungworm, Dictyocaulus arnfieldi, is also problematical, particularly when donkeys co-graze with horses. Mature horses are not permissive hosts to the full life cycle of this parasite, but develop clinical signs on infection. In contrast, donkeys are permissive hosts without displaying overt clinical
signs and act as a source of infection to co-grazing horses. Donkeys are also susceptible to the fluke, Fasciola hepatica. This flatworm can be transmitted, via snails and the environment, from ruminants. As with cyathostomins, anthelmintic resistance is increasing in fluke populations in the UK. A number of the anthelmintic products available for horses do not have a licence for use in donkeys, and this complicates the design of parasite control programmes. As no new equine anthelmintic classes appear to be near market, it is important that the efficacy of currently effective drugs is maintained. It is important that strategies are used that attempt to preserve anthelmintic efficacy. These strategies should be based on the concept that the proportion of worms in a population not exposed to anthelmintic at each treatment act as a source of ‘refugia’. The latter is an important factor in the rate at which resistance develops. Thus, it is imperative that parasite control programmes take into account the need to balance therapy to control helminth-associated disease with the requirement to preserve anthelmintic effectiveness.

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