Identifying the mechanism underlying the effect of neurotoxic spider venoms on the parasite Fasciola hepatica.

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The parasite, Fasciola hepatica (liver fluke), is a significant cause of disease in ruminants and is a major constraint on livestock production globally. A recent report from the Agriculture and Horticulture Development Board and Responsible Use of Medicines in Agriculture identified fasciolosis in the top five disease in both sheep and cattle in UK with estimated annual losses of at least £38 million.Control of liver fluke relies almost exclusively on the use of specific veterinary medicines or flukicides. There are a limited number of flukicides on the market and resistance to one of the most effective, triclabendazole, is widespread globally. The liver fluke life cycle relies on a mollusc intermediate host, Galba truncatula. Both G. truncatula and the extra-mammalian stages of the parasite are dependent on mild temperatures and rainfall. Liver fluke is predicted to become more widespread and prevalent as the climate changes. Farmers are becoming more dependent on the use of flukicides to control of fasciolosis and concomitantly, anthelmintic resistance is becoming a major impediment to fluke control. There is no prospect of a commercial vaccine for control of fasciolosis appearing in the near future, so development of novel drugs is vital to help control disease. There is considerable interest in the use of spider venom toxins as potential biopesticides for a range of insect pests but their effect on parasites is not well studied. In proof of principle studies, the applicants have recently shown that, in vitro, immature F. hepatica are susceptible to two neurotoxic spider venom proteins that are known to act in other systems on distinct voltage-gated ion channels. The aim of this project is to investigate the mechanisms underlying how these toxins exert their effect in F. hepatica. For confidentiality /IP reasons, we have named them as SVT1 & SVT2. This project fits into the BBSRC Agriculture and Food Security theme, to improve productivity, health and welfare in the livestock sector.Our working hypothesis is that SVT1/2 act on liver fluke voltage-gated ion channels and have potential as next generation flukicides. The objectives of the project are:1. To develop standardised in vitro toxicity assays and produce a panel of recombinant proteins that will be tested in those assays. We have access to well defined panels of fluke isolates of known susceptibility to triclabendazole. Results to date suggest that both susceptible and resistant isolates are equally susceptible to killing by STV1.2. Using in vitro cultures, investigate how the toxins reach their target tissue, via the gut or outer tegument. We will evaluate the stability of the recombinant proteins in the presence of fluke gut enzymes; cysteine proteases are particularly dominant in the fluke gut.3. To mine the F. hepatica genome for information about sodium and calcium gated ion channels. Preliminary analysis of the F. hepatica genome suggests the presence of a range of voltage-gated ion channels including sodium, calcium and potassium channels. 4. To identify the targets of the toxins using confocal scanning laser microscopy and in situ hybridisation.5. Once the targets have been identified, to establish functionality, gene silencing (RNA interference) will be used to interrogate target-engagement and inform mechanisms of action. This is a technique that is well established in the laboratory of partner 3.