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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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Anaerobic fungi are a key unexplored taxa for optimizing lignocellulosic fibre utilisation in equines

Citation

Joan E. Edwards, David A van Doorn, Wilbert F Pellikaan, Jan Dijkstra, Henk Everts, Faith A. Burden, Hauke Smidt. Anaerobic fungi are a key unexplored taxa for optimizing lignocellulosic fibre utilisation in equines. Presented at INRA-Rowett 2016 Symposium.

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Date presented: 
Tuesday 21 June 2016
Abstract

The hindgut microbiota of equines enables them to utilize forage/grazing based diets which contain a substantial proportion of lignocellulosic fibres. These fibres are a structural barrier that gut bacteria need to overcome when accessing plant nutrients, as well as being a challenging and structurally complex substrate that can be utilized. The limited dietary energy available from these ‘natural’ diets however means that many equines are supplemented with energy-dense concentrate feeds in order to fulfil their dietary energy requirements. Use of energy-dense concentrate feeds however can change the equine hindgut microbiome, and lead to the development of gut-mediated diseases (i.e. fermentative acidosis, laminitis, colic and stomach ulcers). There is therefore a clear need to optimize the utilization of lignocellulosic fibres in the equine hindgut in order to minimize the need for dietary supplementation. The most effective of the fibre-degrading gut microbes, anaerobic fungi (phylum Neocallimastigomycota), are known to be a normal member of the equine gut microbiota. Despite this however, they have been largely overlooked in equine gut microbiology studies to date. Research being conducted within the EU funded EQUIANFUN project will therefore establish baseline knowledge of the phylogeny, community structure, physiology and nutritional impact of anaerobic fungi in the equine hindgut. The insights gained will inform the development of novel strategies to promote indigenous anaerobic fungal communities in the equine hindgut, enabling optimization of the use of dietary forage as an energy source in equids. Reduction of the use of energy–dense diets and applying targeted nutritional strategies for optimizing microbial health may counteract processes in the gastrointestinal tract that have been associated with disease. Anaerobic fungi therefore offer the potential to enable significant advances to be made in the optimisation of the nutrition, health and welfare of all domesticated equids.

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Reference intervals for biochemical and haematological parameters in mature domestic donkeys (Equus asinus) in the UK

Citation

Faith A. Burden, Mulugeta Getachew, Elizabeth Hazell-Smith, Valerie Patrick, Ryan Trawford, Harriet Brooks Brownlie. November 2015. Reference intervals for biochemical and haematological parameters in mature domestic donkeys (Equus asinus) in the UK. Equine Veterinary Education.

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Publication date: 
18 November 2015
DOI number: 
10.1111/eve.12512
Abstract

Reference intervals (RIs) for haematology and serum biochemistry for donkeys in a temperate climate have been previously published using blood sample results from the resident population of a large donkey shelter in the UK. Periodic review of reference intervals is recommended to ensure their applicability to the patient population and changes in laboratory methods and technologies. The current study aimed to revise the previously published haematology and serum biochemistry values for the adult domestic donkey (Equus asinus) in the UK in the light of a change in analytical equipment at the Donkey Sanctuary laboratory, but also to refine the demography of the sample population with respect to age, physiology and clinical history. Clinical pathology results from 138 clinically healthy mature (4–24 years inclusive) female and castrated male donkeys selected from the resident population of the Donkey Sanctuary, were analysed retrospectively. The animals were blood sampled during the period February 2008 to June 2011 as part of a routine health screen prior to rehoming. Results for a total of 38 biochemical and haematological parameters were analysed including 3 previously unreferenced parameters in addition to those assessed in the previous study. The new reference intervals and median values show very poor transferability with recently derived reference intervals for non-Thoroughbred horses and only limited transferability with reference intervals previously published for donkeys in the UK. Of particular note is a marked reduction in the upper reference limit for triglycerides of 2.8 mmol/l (from 4.3 mmol/l) since this parameter is used to decide when donkeys are at risk of developing hyperlipaemia. This study demonstrates the value of intermittent review of reference intervals and refinement of study populations. Notwithstanding the caution with which reference interval data from different laboratories should be compared, the lack of transferability of results between donkeys and horses highlights the importance of use of species-appropriate reference intervals for clinical decision-making.

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Residual and ovicidal efficacy of essential oil-based formulations in vitro against the donkey chewing louse Bovicola ocellatus

Citation

Bryony Sands, Lauren Ellse, Richard Wall. November 2015. Residual and ovicidal efficacy of essential oil-based formulations in vitro against the donkey chewing louse Bovicola ocellatus. Medical and Veterinary Entomology.

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Publication date: 
2 November 2015
DOI number: 
10.1111/mve.12148
Abstract

Essential oils have shown good experimental potential as novel veterinary ectoparasiticides. However, if they are to be used as veterinary products, they must be available in formulations that are suitable for practical application against specific ectoparasites. Here, the efficacies of formulations containing 5% (v/v) lavender or tea tree oil, in combination with two emulsifiers [a surfactant, 5% (w/v) N-lauroylsarcosine sodium salt (SLS), and a soluble polymer, 5% (w/v) polyvinylpyrrolidone (PVP)], with or without 10% coconut oil, were tested in contact bioassays against the donkey chewing louse Bovicola ocellatus (Piaget) (Phthiraptera: Trichodectidae). Residual activity was quantified in open and closed containers; ovicidal efficacy was also examined. Exposure to either of 5% (v/v) lavender or tea tree oils with SLS or PVP resulted in louse mortality of 100%, but when coconut oil was included as an excipient, significantly lower efficacy was recorded. However, the formulations became significantly less effective after 2 h in open containers and 40 h in closed containers. The results confirm that the residual activity of essential oils is relatively transitory and the addition of 10% coconut oil does not prolong the period of insecticidal activity by slowing essential oil evaporation. Too short a period of residual activity is likely to be a significant impediment to the effective practical use of essential oils. However, unlike many synthetic pediculicides, the essential oils tested here were highly ovicidal, which suggests that prolonged residual activity may not be essential to kill newly hatched nymphs after treatment.

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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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Nutritional Management of Hyperlipaemia

Citation

Andy Durham, Alexandra K. Thiemann. May 2015. Nutritional Management of Hyperlipaemia. Equine Veterinary Education.

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Publication date: 
26 May 2015
DOI number: 
10.1111/eve.12366
Abstract

Hyperlipaemia is a disease resulting from excessive mobilisation of triglyceride stores such that plasma clearance processes become overwhelmed. Consequently increased plasma triglyceride concentrations (>5.6 mmol/l), visibly cloudy plasma and a sick, anorexic or hypophagic subject follow. Epidemiological studies have identified many predisposing and triggering factors and the attentive carer or veterinary surgeon should be alert to such risk factors so that the disease can be prevented, or at least identified and treated at an early stage. Hyperlipaemic subjects are invariably in a negative energy balance, and nutritional management therefore plays a central role in both the prevention and resolution of the disease.

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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 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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