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

Lead Research Organisation: Queen's University of Belfast
Department Name: Sch of Biological Sciences

Abstract

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 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.

Technical Summary

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.

Planned Impact

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 i 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.
 
Description Dramatically improved methods for the in vitro maintenance of liver fluke that facilitate the study of growth and development in this parasite without the need for laboratory animals; improved methods for the identification of new drug and vaccine targets for liver fluke control; discovery and of multiple new putative targets from the brain and muscle systems of liver fluke parasites; expansion of gene silencing technology to in vitro maintained fluke across distinct developmental stages; screening of several flukicidal compounds (supplied by our industrial partner, Merial) which proved to be more potent than the existing front-line flukicide, triclabendazole.
The in vitro platform developed during this project is now being exploited by Boehringer Ingelheim Animal Health (BIAH) to screen new putative flukicides against bot susceptible and resistant fluke. We are undertaking this work. BIAH wish to adopt the motility screening platform within a new laboratory in Athens GA - they are consulting with us on standard operating procedures.
Exploitation Route The methods are being widely adopted by the research community and will likely reduce the use of laboratory animals to maintain liver fluke and enhance drug target discovery and validation, improving progression for pipelines of new flukicides.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
Description The academia-industry collaboration grant (LINK) has facilitated the identification and testing of new putative flukicides which are being trialled. The methods developed in the grant have seeded subsequent research funding from major pharma (Boehringer Ingelheim Animal Health) to undertake in vitro testing of potential flukicide leads. Multiple active compounds have been identified against susceptible and resistant fluke isolates and are being actively used to guide business decisions around the progression (or otherwise) of candidates.
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description Boehringer Ingelheim Research Collaboration Funding
Amount £227,000 (GBP)
Funding ID Postgenomic Tools for Parasiticide Discovery 
Organisation Boehringer Ingelheim 
Department Boehringer Ingelheim
Sector Private
Country Canada
Start 06/2017 
End 05/2019
 
Description The Dynamics of Gene Silencing in Liver Fluke
Amount £57,000 (GBP)
Organisation Government of Northern Ireland 
Department Department for Employment and Learning Northern Ireland (DELNI)
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2017
 
Description The Neuropeptide System of the Liver Fluke
Amount £57,000 (GBP)
Organisation Government of Northern Ireland 
Department Department for Employment and Learning Northern Ireland (DELNI)
Sector Public
Country United Kingdom
Start 10/2014 
End 09/2017
 
Title Long-term in vitro maintenance of liver fluke juveniles 
Description Building on a handful of reports from the 1970's, we have developed simple methods enabling long-term maintenance and growth of juvenile liver fluke in vitro. By supplementing RPMI 1640 with chicken serum, we have been able to maintain fluke for more than 6 months, during which time they grow to around 3mm in length and develop visible internal reproductive structures and complex gut branching, and other morphological changes that reflect those seen during development of liver fluke parasites in vivo. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact These methods enable long-term maintenance of fluke in vitro, conducive to investigating impacts of gene silencing and drug screens on fluke biology. These methods may also have '3Rs' impacts, by reducing usage of animals essential for passage of liver fluke parasites; we have now obtained NC3Rs funding (NC/N001486/1) to explore this. 
 
Title Motility analysis using ImageJ wrMTrck plugin 
Description Fluke are videoed using a dark field microscope and post capture analysis carried out using ImageJ and wrMTrck plugin for changes in worm motility (body length changes per minute). This is a method adapted for use in fluke from methodology published for C. elegans and involved altering wrMTrck settings to allow accurate analysis of fluke motility. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? No  
Impact This assay has enabled us to accurately analyse changes in fluke motility and is regularly used for compound screening and as part of functional genomic experiments to determine impacts of gene knockdown. 
 
Description Boehringer Ingelheim-link Consortium 
Organisation Boehringer Ingelheim
Country Germany 
Sector Private 
PI Contribution Identify putative control targets for liver fluke and generate reverse genetic data to validate those with potential as new targets for flukicides. Screen chemical leads provided by Boehringer Ingelheim in liver fluke functional bioassays.
Collaborator Contribution Expertise on target prioritisation and chemical leads with some flukicide activity for testing in functional bioassays for liver fluke.
Impact McVeigh et al 2018 International Journal for Parasitology: Drugs and Drug Resistance
Start Year 2017
 
Description Merial-Link Consortium 
Organisation Sanofi
Department Merial Plc
Country Global 
Sector Private 
PI Contribution Queen's University Belfast team identify putative control targets for liver fluke and generate reverse genetic data to validate those with potential as new targets for flukicides. Merial provide chemical compound libraries and expertise on target prioritization.
Collaborator Contribution Merial have provided chemical leads with some flukicide activity for testing in our functional bioassays for liver fluke
Impact 1. McVeigh et al (2012) International Journal for Parasitology-Drugs and Drug Resistance. 2. McCammick et al (2016) Parasites & Vectors 9(1):46
Start Year 2013
 
Description A-level DNA Technology Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Exhibited at a DNA Technology careers event as part of Northern Ireland Science Festival. Spoke to school pupils about our BBSRC-funded molecular biology applications, and routes into a career in molecular biology research.
Year(s) Of Engagement Activity 2016
 
Description Anthelmintics - From Discovery to Resistance 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster Presentation entitled 'Liver Fluke-Specific Tegument Proteins: Evaluation as Novel Control Targets'

The liver fluke (Fasciola spp.) costs the global agri-food industry an estimated $3.2 billion every year and up to 17 million humans are infected. Resistance to triclabendazole (the flukicide of choice) is increasing, contributing to the need for novel drugs and / or vaccines. A proteomic study (Wilson et al. 2011; Int. J. Parasitology, 41, 1347 - 1359) identified five Fasciola-specific tegument proteins of unknown function (designated Fh-TEG-1 to Fh-TEG-5). These proteins could represent appealing targets due to their potential for surface expression and their lack of homology to known host proteins. Using real-time PCR expression of these genes was monitored in newly excysted juvenile (NEJ) F. hepatica over the 3-week period post-excystment. It was found that only Fh-teg-3 and Fh-teg-5 were expressed immediately following excystment, although during subsequent in vitro maintenance, Fh-teg-1 and Fh-teg-5 showed rapid upregulation, with expression peaking 72-96 hours post-excystment. Importantly, all five tegumental genes are expressed in adults. These expression data mirror independent RNAseq data for both immature-stage and adult F. hepatica. RNA interference (RNAi) was used to knockdown Fh-teg-1 and Fh-teg-5 in NEJs across a variety of time points to ascertain their importance to worm biology in vitro. No aberrant survival phenotypes have been detected during the 3-week in vitro maintence of RNAi worms. Ongoing studies on Fh-Teg RNAi-worms are examining tegument structure using electron microscopy and motility using agar-slurry based bioassays. Concurrent efforts to generate recombinant Fh-TEG-1 and Fh-TEG-5 to facilitate vaccine trials are underway.
Year(s) Of Engagement Activity 2014
 
Description Anthelmintics from discovery to resistance - San Francisco/San Diego, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Fasciola spp. trematodes continue to inhibit the productivity of global ruminant agriculture, causing lost revenue on a billion dollar scale. Liver fluke are also responsible for zoonotic infections of humans, with an estimated 17 million humans in the developing world affected by fascioliasis. We are reliant on a single flukicide (triclabendazole) active against both adult and invasive juvenile life stages, a drug for which resistance has been documented in both veterinary and human infections. Despite these issues, no new broad-spectrum fasciolicides are in development, and no preventative vaccines are available. In an effort to contribute to drug discovery efforts, we have developed RNA interference (RNAi)-based functional assay pipelines for juvenile liver fluke, with which we aim to identify / validate new molecular targets that are essential to liver fluke biology. Amongst our targets of interest are three calmodulin-like genes (CaMs 1-3). These CaMs are expressed in myocytons throughout juvenile worms and have profound RNAi phenotypes that implicate functions in both development and motility. Silencing of CaMs 1-3 yielded juveniles that developed significantly more slowly in vitro than untreated/control-treated worms, and also migrated more rapidly through an agar-based motility assay than untreated/control-treated worms. Significantly, both of these RNAi phenotypes were recapitulated following treatment with trifluoroperazine, a known inhibitor of calmodulin-Ca2+ binding, providing independent validation of the RNAi data. These findings represent the first studies of CaM function in whole fluke and suggest that CaMs represent valid targets for novel fasciolicides. Experiments aiming to establish the impacts of these phenotypes on liver fluke infectivity in vivo are ongoing.
This work was supported by a DARD studentship grant to EMM and BBSRC grants BB/H009477/1 and BB/K009583/1.
Year(s) Of Engagement Activity 2014,2016
 
Description Anthelmintics from discovery to resistance- Tampa, Florida 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral presentation describing a functional genomics toolbox to support flukicide discovery. Abstract: Fasciola spp. liver fluke are important veterinary pathogens with global impacts on human and animal health. The absence of a liver fluke vaccine and increasing anthelmintic resistance threaten the sustainability of liver fluke control and underscore the need for novel flukicides. This work showcases the development of a target validation platform for liver fluke utilising in vitro culture methods, whole mount in situ hybridisation (WISH) and RNA interference (RNAi). Recent genome and transcriptome data provide opportunities for novel target identification. In addition, our progress with in vitro culture/maintenance of F. hepatica offers a much-improved ability to maintain juvenile fluke for extended periods (>1 year) and to characterise mediators of fluke growth and development across key life stages using functional genomic tools. Functional studies are informed by target localization such that we have developed WISH methods that support analysis of transcript expression in F. hepatica newly excysted juveniles (NEJs) and growing juveniles (4 weeks) in diverse tissues, including muscle and nerve. The in silico confirmation of a functional RNAi pathway in all life stages of F. hepatica highlights its validity as a reverse genetics tool. Here we have analysed RNAi dynamics in growing and non-growing F. hepatica and show that the platform will facilitate the identification of new intervention targets in fluke. Our data also show that initiating RNAi in growing F. hepatica juveniles increases the need for knockdown optimization on a target-by-target basis, prior to phenotypic evaluations. Although this restricts throughput, target transcript knockdown is commonly robust in both early-stage juvenile and juvenile fluke; gene silencing is possible in adult fluke, but is technically more challenging. Advances in methods for the in vitro culture and functional genomics studies on F. hepatica juveniles bridges the gap between genomics and functional studies that are critical to target validation and drug discovery pipelines.
Year(s) Of Engagement Activity 2017,2018
 
Description Anthelmintics: Discovery to Resistance II (San Diego) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral presentation on 'Stimulating growth - a boost to functional genomics approaches in liver fluke'

Fasciola hepatica, the liver fluke, is recognised globally as a significant agri-food industry burden, and now increasingly as a human health issue with around 17 million people thought to be infected. In recent years efforts to identify novel targets for drugs / vaccines have focused on understanding gene and protein expression in infective life stages of the liver fluke. To assess the candidature of these targets we used RNA interference to silence potential target genes in infectious life stages of the liver fluke. Unfortunately, these efforts were hampered by a lack of available in vitro functional biology platforms, and so we initially sought to improve these techniques. We achieved over 60% survival in vitro of juvenile fluke over six months, whilst simultaneously triggering growth, such that juveniles grew to an average of 38.5x their original size, with a few fluke growing to >220x their original size. Juveniles exhibited extensive gut, tegument and reproductive tissue development, similar to that seen in vivo. This fluke maintenance platform offers the first opportunity to apply functional genomics tools to examine growth and development in juvenile liver fluke which we have found to be supported by proliferative cells known as neoblasts. Validation of the platform has shown that RNAi persists much longer in non-growing compared to growing juveniles. The transcript recovery seen in growing worms was reduced via repeated, transient dsRNA exposure which subsequently resulted in increased protein knockdown compared to that seen in non-growing juveniles. Additionally, we report the first gene knockdown in F. hepatica adults following microinjection-based delivery of dsRNA to trigger RNAi. These enhanced methodologies provide a growing toolbox for the interrogation of fluke biology and the validation of new control targets.
Year(s) Of Engagement Activity 2016
URL http://www.extension.iastate.edu/registration/events/conferences/membrane/index.html
 
Description BBSRC-LINK Project Meetings 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Type Of Presentation paper presentation
Geographic Reach Local
Primary Audience Industry/Business
Results and Impact QUB appraised Merial of ongoing research progress; Merial suggested future directions and notified QUB of progress on relevant drug development processes.

NA
Year(s) Of Engagement Activity 2013,2014,2015,2016
 
Description Balmoral Exhibit at Agricultural Show 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact We hosted an interactive exhibit at the Balmoral Agricultural Show. As well as displaying live parasites of farm animals to the public and farming industry, we also distributed flyers and information sheets on parasites and parasitic diseases. These included information sheets aimed at school pupils, as well as more targeted flyers for farmers that included disease and control information. NC3Rs and BBSRC logos were displayed.
Year(s) Of Engagement Activity 2018
 
Description Boehringer Ingelheim meeting in Belfast May 2018 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Met with industrial delegate for discussion on in vitro maintenance platform and how this can be used alongside compound screening and fluke bioassays.
Year(s) Of Engagement Activity 2018
 
Description British Society for Parasitology Spring Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presented research data to peers, funders and stakeholders.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014
 
Description British Society of Parasitology Conference (Cambridge) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral Presentation on 'Fasciola hepatica: in vitro maintenance and evaluation of unique tegumental proteins as novel control targets'

The liver fluke (Fasciola spp.) costs the global agri-food industry ~$3.2 billion every year and up to 17 million humans are infected. Resistance to triclabendazole (the flukicide of choice) is increasing, contributing to the need for novel drugs and / or vaccines. We have been investigating five Fasciola-specific tegument proteins (designated Fh-TEG-1 to Fh-TEG-5) that have targetappeal due to their potential for surface expression and their lack of homology to host proteins. Using qPCR these genes were monitored in newly excysted juveniles (NEJs) over the 3-week period post-excystment -only Fh-teg-3 and Fh-teg-5 were expressed immediately following excystment, although during subsequent in vitro maintenance, Fh-teg-1 and Fh-teg-5 showed rapid upregulation. Importantly, all five tegumental genes are expressed in adults; mirroring independent RNAseq data for both immature-stage and adult F. hepatica. RNA interference (RNAi) of Fh-teg-1 and Fh-teg-5 in NEJs across a variety of time points was used to ascertain their importance to worm biology in vitro. No aberrant survival phenotypes were detected during in vitro maintenance. Ongoing studies on Fh-Teg RNAi-worms are examining tegument structure using electron microscopy and generating recombinant Fh-TEG-1 and Fh-TEG-5 to facilitate vaccine trials. For many helminth parasites, the inability to maintain them in vitro for extended periods limits opportunities for experimental manipulation. Here we also report our efforts to maintain liver fluke in vitro to advance the exploitation of its susceptibility to RNAi-based interventions. So far, we are able to maintain in vitro juvenile worms in excess of 80 days and adult worms over 1 week.
Year(s) Of Engagement Activity 2014
 
Description British Society of Parasitology Conference (Liverpool) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster Presentation on 'Neoblast proliferation supports growth and longevity of in vitro maintained Fasciola hepatica juveniles'

The liver fluke Fasciola hepatica is the cause of considerable economic and health burdens around the world with an estimated global cost to the agriculture industry of $3.2 billion and 17 million humans infected. The infective juvenile stage of this neglected tropical disease causing parasite has long been the focus for research into novel drug and vaccine targets. However, with ever increasing amounts of genomic/proteomic information about this parasite now available there is need for novel functional biology methods. A significant drawback to functional studies in liver fluke is the lack of research into the stimulation of growth and development in vitro. Here, we report development of a culture system that stimulates growth and development of juvenile fluke, supporting 80% survival for more than 200 days (more than two fold longer than in any previous study). These juveniles underwent developmental changes consistent with those reported in vivo, including elements of tegumental, reproductive and gut development towards more adult-like morphologies. This growth and development was supported by proliferation of neoblast-like cells (putative totipotent stem cells), which originate throughout the parenchyma. These cells then migrate, differentiating into mature cells in distinct tissues. These data represent enhanced methods for maintaining juvenile fluke through simulation of in vivo developmental triggers, and will enable study of previously intractable developmental changes in juvenile fluke. This will facilitate in vitro study of developmental processes and evaluation of potential drug/vaccine targets.
Year(s) Of Engagement Activity 2015
 
Description Dissemination of fluke culture methods at Thailand Networking Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Workshop on: A One Health Approach to Helminth Control in South East Asia. Included invited delegates from Thailand, India, Cambodia, Malaysia, Myanmar and Laos. Presented data on in vitro platforms for liver fluke developed in Belfast through BBSRC and NC3Rs funding. Discussions around developing similar methods for Opisthorchis in region.
Year(s) Of Engagement Activity 2017
 
Description Exhibit at Balmoral Agricultural Show 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact We hosted an interactive exhibit at the Balmoral Agricultural Show. As well as displaying live parasites of farm animals to the public and farming industry, we also distributed flyers and information sheets on parasites and parasitic diseases. These included information sheets aimed at school pupils, as well as more targeted flyers for farmers that included disease and control information. All flyers included the BBSRC logo, and highlighted the essential role of BBSRC funding in our work.
Year(s) Of Engagement Activity 2017
 
Description Invited Speaker at British Society for Parasitology Conference- Aberystwyth, Wales 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited speaker 30 minute oral presentation describing tools and resources available to aid in drug target identification in Fasciola hepatica. Abstract: Liver fluke species are pervasive parasites, renowned for undermining the productivity of farmed ruminants and for causing a neglected tropical zoonosis. Despite their sustained economic toll on agricultural production systems, a restricted panel of effective flukicides and mounting evidence for widespread drug resistance, research resources/tools for Fasciola species parasites have languished in the 20th Century, hindering the biological scrutiny needed to underpin drug and vaccine target discovery and validation. Further, after infection peak virulence associates with the migrating juvenile, a stage that has defied biological interrogation based on the challenge of juvenile worm recovery from infected livers at appropriate scale. The last decade has seen a slow, but remarkable progression in the resources and tools available for liver fluke, promoting confidence that these can help advance the development of new control agents. Coincident with liver fluke definitive host promiscuity is the largest helminth genome reported to date that along with improving transcriptomic resources provide new opportunities for in silico discovery. Gene silencing is robust during in vitro maintenance for diverse target genes and a growing panel of bioassays boast refined phenotypic readouts. In vitro maintenance supports long-term laboratory culture, facilitating experimentation on the juvenile, the key life stage for control. Here we will consider how these new tools are supporting advances in our understanding of two elements core to juvenile liver fluke biology: (i) their stem cell-like neoblasts that support the rapid growth and development displayed by migrating worms. These cells drive fluke virulence, offer important new avenues for control and potential tools for the heterologous expression of target genes; (ii) their neurobiology that underpins the sensory and motor coordination needed to support migration from the intestine to the bile ducts. The importance of nerve/muscle targets to nematode parasite control supports the hypothesis that fluke nerve and muscle cells are likely to provide a rich source of flukicide targets.
Year(s) Of Engagement Activity 2018
 
Description Irish Society of Parasitology (Dublin) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Oral Presentation on 'Growing the functional genomics toolbox for liver fluke'

Infections caused by Fasciola hepatica, the liver fluke, have severe impacts on the agri-food industry, and are increasingly recognised in humans. In recent years efforts to identify novel targets for drugs / vaccines have focused on understanding gene and protein expression in infective life stages of the liver fluke. We have focused on evaluating these targets using RNA interference to silence potential target genes in infectious life stages of the liver fluke. An impediment to this work was the limited survival of juvenile fluke in vitro. To resolve this issue we recently developed an in vitro maintenance and growth platform for juvenile liver fluke that allows us to maintain and develop juvenile fluke for months. We sought to investigate if juvenile in vitro growth led to greater transcript and protein knockdown following RNAi treatment. Validation of the platform has shown that RNAi persists longer in non-growing newly excysted juveniles (NEJs) compared to growing NEJs. The transcript recovery seen in growing worms was reduced via repeated, transient dsRNA exposure which subsequently resulted in increased protein knockdown compared to that seen in non-growing juveniles. Additionally, we report the first gene knockdown in F. hepatica adults following microinjection-based delivery of dsRNA to trigger RNAi. These enhanced methodologies provide a growing toolbox for the interrogation of fluke biology and the validation of new control targets.
Year(s) Of Engagement Activity 2016
URL http://www.irishparasitology.com/news.html
 
Description Liver Fluke impact on farms 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Policymakers/politicians
Results and Impact Much discussion afterwards in a workshop format with stakeholders/farmers groups and policy makers within DARD.

DARD requested to see data from presentation; increased participation in on-farm survey
Year(s) Of Engagement Activity 2012
 
Description Merial / Boehringer_Ingelheim meeting in Belfast, September, 2017 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Meeting with 2 industrial delegates, presented in vitro maintenance platform, sparked interest in various in vitro assays we can provide.
Year(s) Of Engagement Activity 2017
 
Description Merial / Boehringer_Ingelheim meeting in Cape Cod, March, 2017 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Meeting with 2 industrial delegates, updated on the in vitro maintenance platform, discussed in vitro assays we can perform prior to testing compound in animal models.
Year(s) Of Engagement Activity 2017
 
Description Merial Annual Discovery Lecture (Lyon, France) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited to deliver Annual Merial Discovery Lecture in 2013 in Lyon. Merial and Sanofi employees from facilities in UK, Germany, France and USA in attendance.

increased engagement with industry
Year(s) Of Engagement Activity 2013
 
Description Merial Discovery Lecture - Atlanta, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Stimulated much discussion and increased engagement with industry

Increased interaction with various components of Merial and Sanofi researchers and decision makers across diverse facilities. Lecture on live feed to other international Merial sites.
Year(s) Of Engagement Activity 2012
 
Description NemaTours Meeting 
Form Of Engagement Activity Scientific meeting (conference/symposium etc.)
Part Of Official Scheme? No
Type Of Presentation keynote/invited speaker
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact BBSRC-funded research data sparked a 20 min Q&A session after invited presentation; much additional engagement by academic groups keen to adopt methods reported at meeting

Increased awareness/understanding amoungst academic peers
Year(s) Of Engagement Activity 2014
 
Description Oral presentation at British Society for Parasitology Conference- Aberystwyth, Wales 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral presentation entitled 'G protein-coupled receptors (GPCRs) in the liver fluke, Fasciola hepatica.' Abstract- GPCRs are established drug targets in human medicine. Despite their considerable appeal as anthelmintic targets, poor understanding of GPCR diversity and function in parasitic helminths has thwarted progress towards GPCR-targeted anti-parasite drugs. To facilitate GPCR research in the liver fluke, Fasciola hepatica, we have generated the first profile of GPCRs from the F. hepatica genome. Our dataset describes 147 high confidence GPCRs, representing the largest cohort of GPCRs, and the largest set of in silico ligand-receptor predictions, yet reported in any parasitic helminth. All GPCRs fall within the established GRAFS nomenclature, comprising three glutamate, 135 rhodopsin, two adhesion, five frizzled, one smoothened, and one secretin GPCR. Amongst rhodopsins were 18 highly diverged receptors that maintained core rhodopsin signatures, but lacked significant similarity with non-flatworm sequences, providing a new sub-group of potential flukicide targets. Seventy-six orthologous sequences in other flatworm genomes identified these as new members of existing groups (PROF1/Srfb, Rho-L, Rho-R, Srfa, Srfc) of flatworm-specific rhodopsins. These receptors imply flatworm specific GPCR functions, and/or co-evolution with unique flatworm ligands, and could facilitate the development of selective anthelmintics. Liver fluke homologues of deorphanised rhodopsins displayed sequence conservation of ligand binding domains. These data enabled high confidence ligand-receptor matching of 17 receptors activated by acetylcholine, neuropeptide F/Y, octopamine or serotonin. RNA-Seq analyses showed expression of 101 GPCRs across various developmental stages, with the majority expressed most highly in the pathogenic, intra-mammalian, juvenile parasites. These data identify a broad complement of GPCRs in F. hepatica, including rhodopsins likely to have key functions in neuromuscular control and sensory perception, as well as frizzled and adhesion/secretin families implicated, in other species, in growth, development and reproduction. This catalogue of liver fluke GPCRs provides new opportunities to study flatworm biology and to advance anthelmintic discovery. Ongoing work aims to localise sites of GPCR gene expression through in situ hybridisation and investigate GPCR functions through RNA interference (RNAi).
Year(s) Of Engagement Activity 2018
 
Description Oral presentation at British Society for Parasitology Conference- Aberystwyth, Wales 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral presentation entitled 'Neuropeptide biology in Fasciola hepatica.' Abstract- Increasing resistance to existing flukicides continues to compromise the sustainable control of liver fluke. New flukicides are needed to suppress the impact of fluke infections in animals and humans. G-protein coupled receptors (GPCRs) are established targets for drugs used in human medicine. Within the GPCR complement of the liver fluke, Fasciola hepatica, at least 47 are putative peptide receptors with many of the associated ligands likely to be neuropeptides (npps). Neuropeptidergic-signalling systems are evolutionarily ancient and have been shown to play key roles in a variety of fundamental processes including motility, reproduction and development. Here we report the in silico discovery of the putative npp gene complement of F. hepatica. Thus far, we have identified 37 putative npp genes by using a combination of degenerative search strings involving common npp motifs as well as using reciprocal BLAST searches using previously identified putative npp genes as queries against genomic and transcriptomic datasets. Using the 'new Tuxedo' package (HISAT2, Stringtie and Ballgown), we have assessed the expression of these genes across the intra-mammalian life stages. Further, we have optimised a planarian wholemount in situ protocol for F. hepatica and used it to examine the spatial expression patterns of putative neuropeptide-F/Y-like gene transcripts. We show that these have distinct expression patterns suggesting differential functions. Expression and localisation information will be used to underpin functional genomics approaches such as RNA interference (RNAi) to generate functional data and, where possible, ligand- receptor pairings. These will provide impetus to anthelmintic/flukicide discovery efforts.
Year(s) Of Engagement Activity 2018
 
Description Participation in NI Science Festival STEM story time event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Volunteered to lead STEM story time event which was organised by Northern Ireland Science Festival. 43 school aged children aged 4-8 and their parents were engaged in a reading of 'super worm' followed by a short discussion on my job as a parasitologist and the importance of parasites to NI agriculture. This was followed by a show and tell event were parasite specimens were shown and the children were able to look down microscopes at live C. elegans and plant parasitic nematodes.
Year(s) Of Engagement Activity 2017,2018
 
Description Poster presentation at British Society for Parasitology Conference- Aberystwyth, Wales 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Poster presentation entitled, 'Neoblast-like cells of Fasciola hepatica.' Abstract- The liver fluke, Fasciola hepatica, exerts a significant burden on global livestock production and poses an emerging threat to human health. Combining this with the increasing resistance to triclabendazole, the frontline drug, makes the search for new flukicides critical to efforts to combat the disease. The stem cells of the liver fluke, similar to the neoblasts that give planarians their regenerative ability, could be valuable sources of novel drug targets that disrupt the growth and development of the parasite in the mammalian host. Using transcriptomic datasets generated from schistosomes, we have identified 92 homologues of the 128 genes down-regulated in irradiated schistosomes (i.e. those in which proliferative cells were destroyed), and show that the majority of these show up-regulation in in vivo parasites versus parasites cultured in vitro, correlating with our growth-rate dynamics data. We also demonstrate for the first time the use of fluorescence activated cell sorting (FACS) to investigate cell populations of the fluke at a single-cell level and show that we can obtain a single cell suspension of F. hepatica cells that includes viable proliferating cells. With further optimization and in combination with stem-cell ablation techniques including irradiation and anti-proliferative drugs, this technique will facilitate the isolation of neoblast-like cells from the fluke and seed omics approaches to interrogation of their biology.
Year(s) Of Engagement Activity 2018
 
Description STEM Outreach Activity (W5 - Belfast) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact STEM Speed Networking Event

Speed networking session where students (in groups of 5) from Campbell College, Belfast would ask questions about my career and how I got to where I am today over 5 minutes.
Year(s) Of Engagement Activity 2016
 
Description STEM outreach activity in primary life sciences 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Class of primary 6 (10 year old) students were engaged for talk and discussion about worms (parasites and free living) and the impacts they have. Students were given samples to look at under the microscope and told about how we work on them in the lab (in vitro). We discuss genetics and how this contributes to phenotypes and played a decision based "game" where students gained or lost points based on genes they had selected. Students performed a practical experiment of extracting DNA.
Year(s) Of Engagement Activity 2018
 
Description School visit (Belfast High School, Newtownabbey, County Antrim) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Delivered a practical session and accompanying talk on DNA electrophoresis to 20 sixth form pupils. Also talked about the global burden of parasitic disease and our BBSRC-funded efforts to research some of those diseases. Pupils engaged with the material and were shocked that they generally were unaware of such important diseases of humans and animals. The school reported an increased interest in requests for research-based work experience.
Year(s) Of Engagement Activity 2015,2017,2018
 
Description Science Cafe Event - Launch of GWWC in Northern Ireland 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Prolonged discussions on impacts of parasites and research approaches to sustained control

Public engagement in science - the importance of parasitology research
Year(s) Of Engagement Activity 2014
 
Description Sir Hans Sloane Lecture (The Ulster Museum) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Subsequent feedback from various Schools wanting visits and further discussion on the topic

School teachers and pupils from across Norther Ireland informed about BBSRC-funded parasitology research and its importance
Year(s) Of Engagement Activity 2014
 
Description World Association for the Advancement of Veterinary Parasitology Conference (Liverpool) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Oral Presentation on 'An in vitro platform for the interrogation of Fasciola hepatica development'

Fasciola hepatica liver fluke causes considerable economic losses to the agriculture industry of at least US$3.2 billion annually and poses a significant human health burden with over 17 million people infected. The infective juvenile stage of this neglected tropical disease causing parasite has long been the focus for research into novel drug and vaccine targets as it causes the acute form of the disease and is a key drug/vaccine target for effective liver fluke control. With rapidly improving genomic/transcriptomic data for fluke there is a pressing need for effective functional genomics tools that facilitate research on liver fluke biology. A hurdle to gene function studies is the inability to maintain developing liver fluke in vitro for extended periods. Here we report the development of a culture system that facilitates juvenile fluke growth and development; supporting 80% survival for more than 200 days (more than two fold longer than in any previous study). These juveniles display developmental changes in the tegument, reproductive system and gut that are consistent with those reported from flukes recovered from hosts in vivo. We have found that juvenile fluke growth and development is supported by the proliferation of neoblast-like cells (putative totipotent stem cells), which originate throughout the parenchyma. Once formed, these cells migrate, differentiating into mature cells in distinct tissues. These advances in monitoring in vitro development are now facilitating the evaluation of genes thought to be involved in growth and development and have the potential to enable their control target candidature to be evaluated using RNAi-based gene silencing. Here we examine the utility of RNAi as a method to probe gene function in developing fluke and optimize methods for sustained gene knockdown and functional studies on aspects of worm development. We have found that a single exposure to RNAi triggers enables effective but transient knockdown in fluke that are developing rapidly. However, multiple RNAi trigger exposure protocols enable prolonged gene silencing studies that facilitate the study of fluke developmental biology. Here we report the use of this platform to probe the function of genes associated with development.
Year(s) Of Engagement Activity 2015
 
Description World Association for the Advancement of Veterinary Parasitology, Liverpool, UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact G protein coupled receptors (GPCRs) are established drug targets in human medicine. In helminths, GPCRs are receptors for both classical and peptidergic neurotransmitters, where they are involved in core neuromuscular functions that have led to their adoption as promising targets for new anthelmintics. Most flatworm GPCR data are derived from Schistosoma spp. blood fluke, while GPCRs have also been mined from cestode and planarian genomes. Several GPCRs have been deorphanised, again primarily in schistosomes. No GPCRs have yet been described in the liver fluke Fasciola hepatica. This study employs Hidden Markov Models (HMMs) based around the GRAFS GPCR classification scheme to annotate at least 95 GPCRs from a draft F. hepatica genome. These comprise frizzled-, glutamate-, rhodopsin-, and secretin-like sequences; notably, adhesion-like GPCRs appear absent. Homology analyses divide the rhodopsin family into aminergic GPCRs (including putative adrenergic, cholinergic, dopaminergic and serotonergic receptors), peptidergic GPCRs (including receptors for allatostatin-, angiotensin-, capa-, neuropeptide F/Y-, and tachykinin-like peptides), and orphan receptors including putative representatives of the flatworm-specific PROF1 family, previously described in schistosomes, cestodes and planaria. Notably, the Fasciola PROF1 complement appears contracted relative to schistosomes. Our dataset also includes 11 putative flatworm-specific 7 transmembrane receptors without homologues in other genera. This work represents the first description of GPCRs in liver fluke. These are druggable targets of value for discovery screens, and will also enable analyses of fluke neuromuscular functions. We are currently engaged in efforts to map the temporal and spatial expression patterns of these receptors, alongside functional/validation experiments using gene-silencing methods. Funded by BBSRC grant BB/K009583/1.
Year(s) Of Engagement Activity 2015