LIVER FLUKE MOTOR FUNCTION AND PARASITE CONTROL: EXPLOITING A 'TARGET VALIDATION TOOLBOX' AS A DRUG SCREEN-INTERFACE FOR FLUKICIDE DISCOVERY

Fasciola species of parasitic worms, also known as liver fluke, cause a widespread disease of animals called fasciolosis. Fasciolosis has a profound, negative influence on animal health and welfare, undermining our food security and causing major losses to both food producers (farmers) and food processors estimated at ~$US3 billion/year worldwide and at ~£300 million/year in the UK. In the UK, the major impact of liver fluke is on agriculture, where losses at farm level are dramatic and were estimated to be £25-30 per infected animal in 2011; liver condemnation at abattoir due to liver fluke is 26% in cattle and 6% in sheep. A major concern relating to fasciolosis in the UK is the fact that climate change is causing an increasing risk of fasciolosis outbreaks. Indeed, estimates of changing liver fluke prevalence in the UK have forecast unprecedented levels of fasciolosis risk in parts of the UK by 2050.
Currently, there are no vaccines against liver fluke such that farmers rely on the administration of drugs. The main drug (flukicide) used to control liver fluke is triclabendazole (TCBZ) as it is the only drug with significant efficacy against both the adult worms (which lives in the bile ducts) and juveniles (which, after being swallowed encysted on vegetation, migrate from the intestine through the liver to the bile ducts, causing much damage in the process). The over-reliance on TCBZ for liver fluke control has led to drug-resistance, which has been reported in Australia and across Europe. Indeed, TCBZ resistance in the absence of new drug classes or vaccines threatens the sustainability of livestock farming in some UK regions. Clearly, there is a pressing need for new drugs to control liver fluke.
Most of the drugs which are used to control worm parasites do so by disrupting the ability of their nerves and muscle to work together and coordinate normal behaviour. In other words, the best drugs disrupt normal parasite motor function. The disruption of motor function would prevent juvenile fluke from completing their damaging migration and would prevent adult fluke from attaching to the host bile ducts, feeding and reproducing, quickly resulting in worm death. Therefore, a better understanding of liver fluke motor function will allow more informed approaches to new drug / flukicide discovery efforts.

Several recent developments associated with liver fluke have seeded this project. First, the liver fluke genome sequence is almost complete and data from the genome will include key information on their motor function. To exploit these data, we need to be able to identify which of the motor function genes / proteins identified from the genome sequence are critical to their survival. A key development here is our recent discovery that liver fluke are amenable to reverse genetics, a technology which allows us to select a gene in the worm and to switch it off (or silence it). We have now optimized this technology in liver fluke such that we can silence any gene from the worm. After gene silencing, a worm which behaves normally would indicate that the gene is not a good drug target whereas one which shows aberrant behaviour or dies would associate with a gene that has been validated as a good drug target. By using the genome sequence to identify genes involved in motor function in fluke and then by silencing these genes in turn, using reverse genetics, and monitoring the effect that this has on worm biology, we will create a pipeline of validated drug targets. Critical here is the ability to exploit these validated targets quickly for liver fluke control. To this end, this project involves the support of an industrial collaborator who will adopt validated drug targets into their drug screening programmes to facilitate the discovery and development of new flukicides. In this way, basic research on the liver fluke genome and biology is rapidly used to help discover new drugs for liver fluke control.

Fasciolosis has a profound, negative effect on animal health and welfare. It undermines food security and causes major losses to food producers/processors estimated at $US3 billion/year worldwide and at £300 million/year in UK. Losses at UK farm level were estimated to be £25-30 per infected animal in 2011 with associated liver condemnations at abattoir running at 26% in cattle and 6% in sheep. There are no liver fluke vaccines and the dependence on triclabendazole, the only drug with significant efficacy against both migrating juvenile and adult fluke, is unsustainable in the face of drug-resistance. Further, climate change is causing an increasing risk of fasciolosis outbreaks and modelling approaches have forecast unprecedented levels of fasciolosis in parts of the UK by 2050. The liver fluke genome is being sequenced and this project aims to capitalize on this resource to identify and validate new drug targets and to exploit these for flukicide discovery. Most leading anthelmintics target motor function effectors, components of nerve-muscle signalling systems that regulate normal behaviour. We propose to identify/annotate selected core motor function effectors in fluke, and to prioritize these using our target validation toolbox. Preliminary selections based on known 'druggability' and on the occurrence of aberrant phenotypes upon knockout/knockdown in related/model organisms will be screened for expression in both juvenile and adult fluke. Promising candidates will be scrutinized using our optimized RNA interference methods which are interfaced with an array of in vitro/ex vivo phenotypic screens, providing a pipeline of validated targets. Where RNAi dynamics are appropriate, in vivo RNAi screening will provide additional validation for up to five targets. Finally, within and beyond the lifetime of this LINK project, our industrial collaborator will move to translate validated and approved targets for flukicide discovery/development.

This research programme, through the discovery, validation and exploitation of novel drug targets in liver fluke, will help drive efforts towards new flukicides for liver fluke control. The project will showcase the direct translation of 'omics' technologies for parasite control and will inform industry, government, funding bodies and the general public about the development of rapid routes from basic biology to impact.
The non-academic beneficiaries relevant to this proposal include:
1. Pharmaceutical/Biotech Industries: Through this LINK proposal, the industrial partner has already committed to developing prioritized targets validated in this project and developing these to screens for flukicide discovery. In addition to those targets selected for exploitation by our industrial collaborator, genome annotation of motor function effectors in fluke will provide a catalogue of putative targets and 'validation toolbox' approval will provide a pipeline of validated targets open for exploitation by other Pharma. These data are relevant to those industries developing treatments for animal or human fasciolosis, and will include the potential for job creation and/or the employment of appropriately trained researchers. This LINK proposal will hasten the exploitation of basic science.
2. Local Farming/Agricultural Communities: Liver fluke costs UK farmers ~£25-30 per infected animal. Livestock producers will receive economic gain in the long-term through novel drugs that are free from resistance and therefore more effective. Alternative drugs will improve the sustainability of livestock production systems through reduced numbers of treatments, and an increase in animal health/welfare and productivity. Increased productivity will drive economic prosperity in all trades/businesses involved in the 'production to consumption' system.
3. International Farming/Agricultural Communities: The greatest humanitarian impacts of Fasciolosis are associated with agricultural areas in developing countries where the consequences can be devastating for poor rural communities. Fasciolosis is threatening the livelihoods of many farmers and their families who rely on livestock, not only for income, but for food. Further, liver fluke are posing growing problems as food-borne-pathogens in these areas. Novel flukicides will improve the health, well-being, and quality of life of those afflicted with fasciolosis.
4. Stakeholders and Policy Makers: UK-based government bodies [Department of Agriculture & Rural Development-Northern Ireland; Department for Environment, Food and Rural Affairs (DEFRA)] and Levy boards, and other representatives of the Agri-Food industry will benefit as it will provide an evidence-base for policy development and in addressing EU-directed changes in agricultural legislation.
5. Educational Sector: Local schools will benefit from this research by educating both primary and post-primary students about liver fluke biology, and through raised awareness of the importance of research in our society. In addition, research findings will form the basis of research-led teaching to undergraduate students at Queen's University and during invited speaker lectures delivered at both national and international undergraduate and postgraduate teaching institutes. The host-institute will benefit through an enhanced research profile.
6. General Public: Consumers are demanding safe, chemical residue-reduced food that is produced cost-effectively from animals maintained in a welfare-friendly environment. A novel anthelmintic that is not undermined by resistance will facilitate a reduction in drug use, therefore reducing food contamination. The environmental impact associated with intensive drug use will be reduced. The general public will also gain an understanding of one of the most significant diseases affecting the health of their local livestock and food security, and will have opportunity to engage with scientists at the coal-face.