Exploiting stem cell biology for liver fluke control

Liver fluke (Fasciola species) are flatworm parasites that infect diverse mammals including humans and ruminant livestock such as cattle, goats and sheep. In humans, the parasite causes the disease fascioliasis which is a neglected tropical disease with an estimated 17 million people believed to be infected. The worm impacts global food security as it undermines the health and productivity of livestock in which it causes fasciolosis, estimated to cause losses of ~$US3 billion/year worldwide. Major concerns are that: the Animal and Plant Health Agency list liver fluke as the most commonly diagnosed helminth parasite of sheep and cattle in UK; recent estimates of changing prevalence in the UK have forecast unprecedented levels of fasciolosis risk by 2050; farmers rely on the administration of drugs which are becoming less effective due to drug resistance.

Adult worms live in the bile ducts and juveniles occur encysted on vegetation; after being swallowed the juveniles excyst and migrate from the intestine through the liver to the bile ducts. The juvenile worm is the key pathogen, causing profound damage to the liver as it moves from the intestine to the bile duct. It is this stage that can kill lambs and sheep and the stage against which there is only one effective drug, triclabendazole (TCBZ). Critically, TCBZ-resistance threatens the sustainability of livestock farming in many regions of the world such that new flukicides and/or a vaccine are needed.

Key impediments to research studies on liver fluke have included the lack of good genomic resources, the absence of a model system for functional studies and the reliance on host animal-based in vivo experiments to inform fluke biology. The new bioinformatic resources as well as the ability to culture the juvenile fluke in the laboratory outside of a host, mean that experiments on fluke developmental biology are now possible, stimulating this project. Our ability to culture growing juveniles, for sustained timeframes, and to selectively disrupt gene targets provides an opportunity to transform approaches to liver fluke biology and the discovery of new drug targets.

We have found that liver fluke stem cells are critical to their growth and development and as such, are an appealing resource for new drug targets. Whilst the critical role played by stem cells in chemotherapy and drug resistance is established in the cancer field, their role in parasite-drug interactions and potential exploitation in parasite control have not been investigated. Our preliminary data expose a critical role for these stem cells in liver fluke growth and indicate that they play a role in parasite-drug interaction, encouraging their exploitation for the discovery of new drug targets. In this project, supported by an industrial partner, we propose to discover key genes involved in liver fluke stem cell biology and to evaluate their potential as novel drug targets for new flukicides.

To do this we propose to generate new bioinformatics resources on the genes expressed in juvenile liver fluke stem cells. We will also evaluate the importance of stem cells to parasite-drug interactions to help inform their role in how liver fluke parasites tolerate/recover from drug exposure. We will study the relationships between parasite drug sensitivities and stem cell biology and the consequences of disrupting liver fluke stem cells on parasite responses to drug treatments. Finally, we will evaluate a sub-set of liver fluke stem cell genes for their potential as new drug targets using the functional genomics tools we have established.

Prioritized and validated targets will be entered into new drug screens by our industrial collaborator to initiate the discovery of new drugs to control liver fluke. The approaches taken here have direct relevance to liver fluke and indirect relevance to other parasitic worms that cause a diverse range of important diseases of animals and humans.

Fasciola hepatica (liver fluke) is a threat to food security globally through infection of cattle, sheep and goats. In UK, on-farm losses are ~£25-30/infected animal, the Animal and Plant Health Agency list liver fluke as the most commonly diagnosed helminth parasite of sheep and cattle, and available indicators/models predict increasing distribution and prevalence. The absence of a vaccine and increasing drug resistance mean that current control is unsustainable.

The juvenile worm is the key pathogen, causing profound damage to the liver during its migration to the bile duct. The juvenile displays remarkable growth/development that is critical to disease progression and establishment. We propose that undermining associated stem-cell driven growth/development provides a compelling control option. This proposal is motivated by the fact we have: (i) new transcriptomic data for juvenile liver fluke; (ii) developed a robust in vitro RNA interference (RNAi) platform for juvenile fluke; (iii) developed methods to maintain and grow juvenile fluke in vitro long-term; and, (v) developed methods to disperse and dysregulate selected fluke cells, including the neoblast-like stem cells that drive parasite growth/development.

To this end we will: generate data on the neoblasts of juvenile liver fluke using subtractive transcriptomics approaches on control and neoblast-depleted juveniles as well as single cell transcriptomic datasets; evaluate the importance of fluke-neoblasts to parasite-drug interactions by investigating the relationship between drug sensitivity and neoblast dynamics, and establishing the consequences of dysregulating normal neoblast dynamics on flukicide responses; evaluate selected neoblast markers for their potential as new flukicide targets using RNAi - we propose to screen >30 candidate targets and select at least five prioritized and validated targets for further development by our industrial collaborator in this Industrial Partnership proposal.

Through the discovery, validation and exploitation of novel drug targets in liver fluke, this project will help drive efforts towards new flukicides for parasite 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.

Non-academic beneficiaries relevant to this proposal include:

1. Pharmaceutical/Biotech Industries: Through this IPA proposal, the industrial partner has committed to providing financial resource, chemical tools to help interrogate new drug targets in fluke and the commitment to develop prioritized targets validated in this project into screens for new flukicide discovery. In addition to those targets selected for exploitation by our industrial collaborator, relevant industry can benefit from the catalogue of putative targets identified. These data are relevant to industries developing treatments for animal/human fasciolosis, and will include the potential for job creation and/or employment of trained researchers. This IPA proposal will hasten the exploitation of basic science research.

2. Local Farming/Agricultural Communities: Liver fluke costs UK farmers ~£25-30/infected animal. Livestock producers will receive economic gain in the long-term through more-effective drugs that treat drug-resistant fluke. New drugs will improve the sustainability of livestock production systems through reduced treatments, and increased animal health/welfare and productivity. Increased productivity will drive economic prosperity in all trades/businesses in the 'production to consumption' ecosystem.

3. International Farming/Agricultural Communities: Major 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 farmers and their families who rely on livestock, not only for income, but for food. Further, liver fluke pose 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 [Dept. of Agriculture, Environment & Rural Affairs (DAERA); Dept. for Environment, Food and Rural Affairs (DEFRA)], Levy boards, and other representatives of the Agri-Food industry will benefit as the work will feed an evidence-base for policy developments associated with animal health and welfare.

5. Educational Sector: Local schools will benefit through the education of both primary and post-primary students on parasites, and through raised awareness of the importance of research in society. Also, research findings will form the basis of research-led teaching to undergraduate and postgraduate 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: Beef and dairy product supplies, their quality, environmental impact, price and welfare of the animals from which they are produced are affected by fasciolosis, reducing its prevalence will be of benefit to society. Consumers are demanding safe, chemical residue-reduced food that is produced cost-effectively from animals maintained in a welfare-friendly environment. Novel flukicides that are not undermined by resistance will facilitate a reduction in drug use, 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.