cyathostomin

P-glycoproteins play a role in ivermectin resistance in cyathostomins

Citation

Laura Peachey, Gina L. Pinchbeck, Jacqui. B. Matthews, Faith A. Burden, A. Lespine, G. von Samson-Himmelstjerna, J. Krucken, Jane Hodgkinson. October 2017. P-glycoproteins play a role in ivermectin resistance in cyathostomins. International Journal for Parasitology: Drugs and Drug Resistance. 7:3.

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Publication details
Publication date: 
25 October 2017
Volume: 
7
Issue: 
3
Abstract

Anthelmintic resistance is a global problem that threatens sustainable control of the equine gastrointestinal cyathostomins (Phylum Nematoda; Superfamily Strongyloidea). Of the three novel anthelmintic classes that have reached the veterinary market in the last decade, none are currently licenced in horses, hence current control regimens focus on prolonging the useful lifespan of licenced anthelmintics. This approach would be facilitated by knowledge of the resistance mechanisms to the most widely used anthelmintics, the macrocyclic lactones (ML). There are no data regarding resistance mechanisms to MLs in cyathostomins, although in other parasitic nematodes, the ABC transporters, P-glycoproteins (P-gps), have been implicated in playing an important role. Here, we tested the hypothesis that P-gps are, at least in part, responsible for reduced sensitivity to the ML ivermectin (IVM) in cyathostomins; first, by measuring transcript levels of pgp-9 in IVM resistant versus IVM sensitive third stage larvae (L3) pre-and post-IVM exposure in vitro. We then tested the effect of a range of P-gp inhibitors on the effect of IVM against the same populations of L3 using the in vitro larval development test (LDT) and larval migration inhibition test (LMIT). We demonstrated that, not only was pgp-9 transcription significantly increased in IVM resistant compared to IVM sensitive L3 after anthelmintic exposure (p < 0.001), but inhibition of P-gp activity significantly increased sensitivity of the larvae to IVM in vitro, an effect only observed in the IVM resistant larvae in the LMIT. These data strongly implicate a role for P-gps in IVM resistance in cyathostomins. Importantly, this raises the possibility that P-gp inhibitor-IVM combination treatments might be used in vivo to increase the effectiveness of IVM against cyathostomins in Equidae.

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Equine Cyathostominae can develop to infective third-stage larvae on straw bedding

Citation

Sandy Love, Faith A. Burden, Eoghan McGirr, Louise Gordon, Matthew Denwood. August 2016. Equine Cyathostominae can develop to infective third-stage larvae on straw bedding. Parasites and Vectors.

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Publication date: 
31 August 2016
DOI number: 
10.1186/s13071-016-1757-1
Abstract

Background
Domesticated grazing animals including horses and donkeys are frequently housed using deep litter bedding systems, where it is commonly presumed that there is no risk of infection from the nematodes that are associated with grazing at pasture. We use two different approaches to test whether equids could become infected with cyathostomines from the ingestion of deep litter straw bedding.

Methods
Two herbage plot studies were performed in horticultural incubators set up to simulate three straw bedding scenarios and one grass turf positive control. Faeces were placed on 16 plots, and larval recoveries performed on samples of straw/grass substrate over 2- to 3-week periods. Within each incubator, a thermostat was set to maintain an environmental temperature of approximately 10 °C to 20 °C. To provide further validation, 24 samples of straw bedding were collected over an 8-week period from six barns in which a large number of donkeys were housed in a deep litter straw bedding system. These samples were collected from the superficial bedding at 16 sites along a “W” route through each barn.

Results
No infective larvae were recovered from any of the plots containing dry straw. However, infective cyathostomine larvae were first detected on day 8 from plots containing moist straw. In the straw bedding study, cyathostomine larvae were detected in 18 of the 24 samples. Additionally, in the two barns which were sampled serially, the level of larval infectivity generally increased from week to week, except when the straw bedding was removed and replaced.

Conclusions
We have demonstrated that equine cyathostomines can develop to infective larvae on moist straw bedding. It is therefore possible for a horse or donkey bedded in deep litter straw to become infected by ingesting the contaminated straw. This has implications for parasite control in stabled equids and potentially in housed ruminants, and further investigation is required in order to establish the relative infective pressure from pasture versus straw bedding.

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Papaya latex supernatant has a potent effect on the free-living stages of equid cyathostomins in vitro

Citation

Laura Peachey, Gina L. Pinchbeck, Jacqui. B. Matthews, Faith A. Burden, J.M. Behnke, Jane Hodgkinson. August 2016. Papaya latex supernatant has a potent effect on the free-living stages of equid cyathostomins in vitro. Veterinary Parasitology.

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Publication date: 
1 August 2016
DOI number: 
10.1016/j.vetpar.2016.07.036
Abstract

The control of equid gastrointestinal nematodes in developed countries, in particular the cyathostomins, is threatened by high levels of anthelmintic resistance. In recent years, there has been increasing interest in the evaluation of traditional ‘ethnoveterinary’ medicines as alternatives to chemical anthelmintics. The cysteine proteinases (CPs), a group of enzymes derived from fruits such as papaya (Carica papaya), pineapple (Ananas comosus) and figs (Ficus spp.), have shown good efficacy against adult stages of a range of parasitic nematodes, in vitro and in vivo. The efficacy of CPs against cyathostomins remains to be explored. In this study, the efficacy of a crude preparation of CPs, papaya latex supernatant (PLS), against the free-living stages of cyathostomins was evaluated using two in vitro tests, the egg hatch test (EHT) and the larval migration inhibition test (LMIT). It was demonstrated that PLS had a potent effect in the EHT, with EC-50 values in the range of 0.12-0.22 μM. At concentrations above 6.25 μM the eggs did not develop, below this concentration the L1 developed but they lost integrity of the cuticle upon hatching. These effects were inhibited by pre-incubation of PLS with the CP inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino butane) (E64), indicating that CPs were responsible for the anti-parasitic activity. A dose-dependent inhibition of migration of third stage larvae (L3) in the LMIT was demonstrated at higher concentrations of PLS, with EC-50 values in the range of 67.35-106.31 μM. Incubation of PLS with E64 prior to use in the LMIT did not reverse the anti-migratory effect, suggesting that CPs were not responsible for the reduced migration of cyathostomin L3 and that PLS also contains an additional active compound. This is the first report of PLS and/or CPs showing activity against the free-living stages of a parasitic helminth. In addition, it suggests that cyathostomins are highly sensitive to the effects of CPs and further evaluation of their efficacy against parasitic stages and in vivo are strongly indicated.

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

Citation

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

Citation

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 details
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

Citation

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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Quantifying the effects of individual animal characteristics and climatological factors on faecal worm egg count shedding in donkeys

Citation

Christopher J Corbett, Sandy Love, Giles T. Innocent, Ian McKendrick, Jacqui. B. Matthews, Faith A. Burden, Matthew Denwood. Quantifying the effects of individual animal characteristics and climatological factors on faecal worm egg count shedding in donkeys. Presented at British Society for Parasitology Spring Meeting.

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Presentation details
Date presented: 
Monday 7 April 2014
Abstract

Cyathostomins, the predominant parasitic nematodes of equids, have developed varying degrees of resistance to all three classes of anthelmintic licensed for use in horses. It is essential that the effectiveness of alternative methods of control for these pathogens are quantified, including incorporating climatic data and the commonly advocated practice of removal of faeces from pasture. Here, we obtained monthly faecal worm egg counts (FWEC, n=4,460 individual counts) from 803 donkeys based at The Donkey Sanctuary (Devon, UK). The dataset also included age, sex, field, FWEC history and previous anthelmintic administrations in each individual, as well as the pasture hygiene management method applied in the field where the donkey was grazed. FWEC were analysed alongside local climatic data using a generalised linear mixed model to assess associations between these variables and each observed monthly FWEC. The preferred model was identified using a model selection algorithm based on penalised likelihoods, and associated a 2.1% decrease in FWEC per day with air frost two calendar months ago (p<0.001) and a 38% lower FWEC in groups with twice weekly manual faecal removal compared to those with no faecal removal (p=0.004). Other weather effects, both alone and as interaction terms with the average FWEC of the field were included in the model, alongside individual FWEC history with anthelmintic administration as interaction terms and date as a single term. Our study identifies factors that may be useful as part of on-going predictive modelling based methods of improving targeted selective therapy.

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

Citation

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.

Authors
Presentation details
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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