cysteine protease

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 details
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.

Online references

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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