Developing a 'validation portfolio' to exploit key virulence proteins in Fasciola species for parasite control
Lead Research Organisation:
Queen's University Belfast
Department Name: Sch of Biological Sciences
Abstract
The tropical liver fluke parasite Fasciola gigantica is one of the most important worm infections of livestock in Asia and Africa. The disease inflicts very significant losses especially in livestock in India, with infection levels reaching 55% in some regions. Recently-estimated costs for liver fluke induced losses associated with livestock in India fall within the range of US$1.95-4.78 billion per year. This is a huge burden to the largely agricultural Indian economy and directly impacts the productivity of large and small farm holdings alike, negatively impacting individual farmers and their families. Liver fluke infected buffalo show reduction in general health, weight gain, feed conversion efficiency and reproduction. The infective worm stage encysts on vegetation such that host animals inadvertently consume the cysts during grazing. Once in the gut, the juvenile worms hatch, burrow through the gut wall and penetrate the liver, ending up as adult worms in bile ducts where they feed on host blood, significantly diminishing the health of the animal and greatly reducing agricultural productivity. In the developed world liver fluke is mainly controlled using the chemical triclabendazole (TCBZ). This drug is an Achilles heel of liver fluke control as it is the only drug that kills both adult and pathogenic juveniles. New control strategies are urgently needed as anthelmintic resistance in parasitic worm populations is spreading globally, and agricultural communities in many parts of the developing world simply cannot afford short-lived, anthelmintic-based treatment options. Therefore, there is an urgent need to develop a cost effective, single-treatment control strategy based on vaccination. Numerous vaccine trials have been completed in liver fluke, many showing some success, but none have provided protection levels adequate for immediate commercialization. The problem is that since most leading vaccine candidates exist in large, related groups of proteins within parasites, it has been impossible to determine which is the best candidate from the group such that up until now, these decisions have not been subject to rigorous validation. To this end, this international collaborative project between laboratories in India and the UK will, for the first time, incorporate new fast throughput nucleotide and protein technologies to validate the candidate proteins and thereby assess if these leading vaccine candidates can be used to treat liver fluke parasites in different parts of the world, a key to successful commercial production. Once a vaccine target is confirmed as widely present in liver fluke populations, we will use another new technology called reverse genetics that can switch-off (or silence) a target in a parasite, and then confirm if this target is essential for parasite survival. If confirmed as important to parasite survival, we will subsequently use yeast laboratory cultures to produce the candidates for vaccine trials in livestock in India. Since this is a multi-discipline and multi-laboratory project, we have formed an International Science Advisory Board (ISAB) in order to manage and co-ordinate progress and to trouble-shoot. Members of the ISAB include a representative from a large Animal Pharmaceutical Company, a parasitologist with many years of industrial experience, a leading world authority on liver fluke vaccination and a representative from the industrial Biotechnology sector in India. The programme addresses the mission of the BBSRC, including treatment of diseases of livestock, application of new technologies and international collaboration. Validated trial vaccine(s) from this programme would form the basis for commercial development for treating liver fluke disease, and our technology strategy is directly transferable to other helminth parasites of animal and humans.
Technical Summary
Liver fluke impose a major impact on global economies through the reduction of animal welfare and agricultural productivity. Whilst Fasciola hepatica is most common in temperate regions, Fasciola gigantica is one of the most important parasites of ruminants in the tropics. Although current control measures rely on anthelmintics, their use is financially unsustainable in developing countries and is further undermined by increasing drug resistance, leaving profound deficiencies in liver fluke control. Although a number of putative vaccine target candidates have been tested in small and large animals and shown some success, none have provided protection levels adequate for commercialization. Motivated by recent successes in RNAi and proteomics methods developed in our laboratories, this project proposes to optimise, validate and interface gene silencing and proteomic platforms for various liver fluke life stages, and to exploit these technologies as a drug/vaccine target validation portfolio. To do this, we propose the generation of a F. gigantica transcriptome to kick-start a reverse-chemotherapy/reverse-vaccinology programme whereby the superfamilies of leading virulence proteins (control targets) in fluke are probed for gene variation across pooled geographical isolates and via sampling of liver fluke adults and juveniles; variation qualified using transcriptomics would be quantified using subproteomic methods. Target proteins present in the majority of pooled geographical isolates and individuals would be validated using an integrated gene silencing/proteomics platform to select the most promising candidates. The leading validated candidates (i.e. population-abundant candidates that are functionally important and which are not compensated for by other pathways) would be tested for vaccine candidature in animals challenged with F. gigantica infections. These efforts will provide validated vaccine candidates/drug targets for future control strategies.
Planned Impact
Parasitic helminths are a major threat to global food security, and have been estimated to cost the livestock industry worldwide more than $50 billion/annum. The treat-all application approach of broad-spectrum anthelmintics has been the primary method to control parasitic worms in livestock in the developed world for over 50 years, and has made the production of cheap and plentiful food to a growing world population feasible. New control strategies are urgently needed as anthelmintic resistance in parasitic worm populations is spreading globally, and this coupled to the lack of new anthelmintics, means that in some parts of the developed world intensive livestock farming will no longer be sustainable. In addition, agricultural communities in many parts of the developing world simply cannot afford short-lived anthelmintic based treatment strategies and therefore, also urgently require a cost effective single treatment such as anti-worm vaccines. This programme, by discovering and validating vaccine candidates and/or new drug targets will help inform industry, governments and funding bodies of the potential for developing a more effective control strategy for parasitic worm diseases of livestock. The programme will be managed by an International Scientific Advisory Board (ISAB) that will help the consortium to realise the impact of the research. The ISAB will significantly benefit from the inclusion of Prof. Terry Spithill, a leading international authority on the development of liver fluke vaccines and several key representatives from industry. Furthermore, Profs Brophy and Maule have a long association with the Animal Health industry via collaborative research contracts and joint studentships. Publications from the research will be co-ordinated by respective Press and Public Relations Departments in India and UK. The non-academic beneficiaries of this proposal will range from worldwide farming communities to a range of pharmaceutical and biotechnology industries that will have the opportunity to further develop and/or commercialize new anti-worm vaccines and or new validated flukicide targets. With respect to the reduction of anthelmintic use, the general public also demand safe, chemical residue reduced food, produced cost effectively from animals maintained in a welfare friendly environment. The impact benefits of this programme will be revealed via website, media, public awareness events such as Science Weeks, Agricultural Shows and conferences. Within India, data will be disseminated by Quarterly newsletter (in English and vernacular language) distributed to Dairy Co-operatives and Sheep Breeders; TANUVAS will host a dedicated website for the project. Furthermore, the transcriptomic and reverse genetic optimization data will facilitate gene discovery and functional evaluations that are relevant to organisations underpinning the treatment of human parasite diseases, noteworthy as over a billion people, mostly from developing nations, are infected with parasitic worms. The UK applicants have a long history of engaging the farming community through publications in Farming Journals and consultancy of Devolved bodies and attendance at Agricultural shows. Public communication of our research in the UK will also be via stands, generic talks by research staff and academic staff at UCAS visits and Open Days to prospective students and parents in respective departments of the applicants. The integrated design of the programme and the questions being addressed should facilitate generation of at least one major headline publication with further impact capabilities. The discovery and release of new molecular datasets generated using next generation sequencing will stimulate veterinary parasitology research and multiple research applications for a number of years. Aberystwyth will be responsible for a programme website that will advertise and make available the transcriptomic datasets generated during the programme.
Organisations
- Queen's University Belfast (Lead Research Organisation)
- Scottish Government (Co-funder)
- Department for International Development (Co-funder)
- ABERYSTWYTH UNIVERSITY (Collaboration)
- Aligarh Muslim University (Collaboration)
- Sanofi (Collaboration)
- Boehringer Ingelheim (Collaboration)
- Tamil Nadu Veterinary and Animal Sciences University (Collaboration)
Publications
Dalzell JJ
(2012)
Considering RNAi experimental design in parasitic helminths.
in Parasitology
Herron CM
(2022)
Developmental Regulation and Functional Prediction of microRNAs in an Expanded Fasciola hepatica miRNome.
in Frontiers in cellular and infection microbiology
LaCourse EJ
(2012)
The Sigma class glutathione transferase from the liver fluke Fasciola hepatica.
in PLoS neglected tropical diseases
McCammick EM
(2016)
Calmodulin disruption impacts growth and motility in juvenile liver fluke.
in Parasites & vectors
McCusker P
(2016)
Stimulating Neoblast-Like Cell Proliferation in Juvenile Fasciola hepatica Supports Growth and Progression towards the Adult Phenotype In Vitro.
in PLoS neglected tropical diseases
McVeigh P
(2014)
RNAi dynamics in Juvenile Fasciola spp. Liver flukes reveals the persistence of gene silencing in vitro.
in PLoS neglected tropical diseases
McVeigh P
(2012)
Parasite neuropeptide biology: Seeding rational drug target selection?
in International journal for parasitology. Drugs and drug resistance
Description | Vaccine trial and knowledge transfer to farming communities in India: The ambitious goal of translating antigen discovery, characterization and validation to vaccine trials in buffalo in India within a 3-year project was achieved: Based on CIDLID data. the Indian partners have completed (May 2014) a preliminary liver fluke vaccine trail in Buffalo. Animals were challenged with 1200 metacercariae each (35% viability). Necropsy showed statistically significant reductions in worm burden of 21.8%, 29.1%, 34.5% (fluke secretion [FS], sigma-GST [sGST], FS & sGST) in the three treatment groups. The combination of ES & sGST proved the highest (34.5%). With a 34.5% reduction the animals remained clinically healthy throughout the 9 month study period. Further work is ongoing with varied doses of antigen for improvement in the reduction of worm burden and a small scale field study in subsistence and organized farm conditions will pursue other key candidate antigens identified by the work (both FABP1 and 3 in addition to ES & sGST). If there is significant and practically useful levels of protection in these follow-on trials, the Indian partners at TANUVAS will develop and commercialize this new treatment. They have experience in the development and commercialization of vaccines within India. Multi-language information leaflets were prepared and distributed to farming communities in both North and South India: A video and pamphlets in multiple languages providing information for the on-farm treatment and control of helminth parasites were developed and distributed by the Indian partners in both North and South India. These were distributed to farming communities and to local veterinary practices across Tamil Nadu and in Northern India in Aligarh and surrounding areas. Training Workshops and Seminars for Field Veterinarians: Workshops at three veterinary stations in both North and South India leaves a legacy of knowledge transfer activities that will continue to impact farmers, both men and women, in the future. New functional genomics tools developed during the project have been transferred to two Indian Universities (TANUVAS in Chennai and Aligarh Muslim University), increasing their capabilites in molecular parasitology. Vaccine trials in buffalo were completed but the protections provided (30%) was disappointing. New molecular parasitology laboratories developed at Aligarh Muslim University and at TANUVAS, Chennai. The latter is a molecular diagnostic laboratory for zoonotic diseases that is serving the public. Research methods / tools / resources provided by UK laboratories: In addition to the scientific papers/reports and conference presentations (data below), the UK laboratories provided the Indian collaborators and wider research community with the following: The Aberystwyth team generated recombinant proteins (with storage plasmid clones) and polyclonal antibodies to a number of project identified liver fluke vaccine candidates. Recombinant forms of Sigma, Omega and Mu Class Glutathione transferases (GSTs), fatty acid binding proteins (FABPs) IV, V, VI and VII, Thioredoxin glutathione transferase and Leucine aminopeptidase were produced and polyclonal antisera raised to these proteins. All recombinant proteins have been produced for both F. hepatica and F. gigantica. These reagents are available to the research community. Proteomics methodologies have been developed to study major protein superfamilies in liver fluke (and other organisms): GSTs (Morphew et al. 2012), Cathepsin L (Morphew et al. 2011) and FABPs (Morphew et al. in preparation). The transcriptome of newly excysted juveniles (NEJs) from Fasciola gigantica support the research community by providing gene discovery and protein identification databases. The data generated from transcriptomics approaches will be deposited into public databases (NCBI Genbank) and online for publically accessible BLAST facilities to be hosted by IBERS, Aberystwyth. We also generated an optimized and validated RNAi platform for the in vitro and in vivo analysis of control (vaccine or chemotherapy) target candidature (established protocols for efficient and robust knockdown of all putative vaccine targets identified in proposal - these were relayed to Indian collaborators during research visits to UK and in workshops held in India - see below). Also established the first evidence for RNAi in the tropical liver fluke, Fasciola gigantica. The new protocols will be available to the research community via publication in PLoS Negl Trop Dis (McVeigh et al. 2014; McCammick et al., in preparation). Data/Sequences lodged in databases: New sequences of immuno-suppressor and vaccine candidates were posted by Aberystwyth in public access databases (NCBI Genbank). a) F. gigantica Sigma GST GenBank: AFX98103.1 b) F. gigantica Omega GST GenBank: AFX98105.1 c) F. hepatica Omega GST GenBank: AFX98104.1 d) F. hepatica Cathepin L1D GenBank: ACJ12894.1 Additional vaccine candidate sequences will be deposited by Aberystwyth post publication on the Fatty Acid Binding Protein (FABP) superfamily (Morphew et al. in preparation). a) F. gigantica FABP IV; b) F. gigantica FABP V; c) F. gigantica FABP VI; d) F. gigantica FABP VII; e) F. hepatica FABP IV; f) F. hepatica FABP V; g) F. hepatica FABP VI; h) F. hepatica FABP VII |
Exploitation Route | The Indian partners now have the methods in place to continue the work to develop a new liver fluke vaccine and are doing so. The resources and training provided in India has improved their capacity for research and this is moving in many different directions, including improved molecular disease diagnosstics facilities for the puiblic |
Sectors | Agriculture Food and Drink Education Healthcare |
Description | 1. Information on liver fluke control has been disseminated through workshops in North and South India - including training workshops with veterinarians; further information on liver fluke control has been distributed via multi-language pamphlets in both North and South India. 2. Researchers in India have been trained in the application of new functional genomics tools for vaccine discovery and are using these in Chennai and Aligarh (South and North India) towards the development of new parasite vaccines. 3. Grant supported the development of a new molecular diagnostic laboratory in Chennai (TANUVAS) that is undertaking leptospirosis screening for the public. New Partnerships / Links with Industry established as a result of the CIDLID funding: • The new scientific collaborations between Aligarh (AMU-North India), Chennai (South India), Aberystwyth (Wales) and Belfast (Northern Ireland) were developed through resource and technology exchanges and training. The CIDLID programme has provided leverage for the procurement of additional funds from various national agencies within India for the establishment of a central instrumentation facility (Aligarh and Chennai) and animal holding complex (Chennai) [see above for more details]. • The team at Queen's University Belfast developed formal collaborative links with Merial to exploit the RNAi technology developed during the project. These links have been formalized through a co-funded BBSRC-LINK programme (details below) to validate and exploit new flukicide targets for liver fluke control. This project started in July 2013 and will run for 3-years. This academia-industry link developed as a direct result of interaction with Dr Lance Hammerland (Merial, Atlanta, USA) an industrial member of the International Scientific Advisory Board (ISAB) established for this CIDLID grant. • Another member of the ISAB for this CIDLID grant, Professor Terry Spithill (LaTrobe University, Australia), provided some additional and novel putative vaccine targets for validation through a new collaboration. As part of the development of this collaboration, a PhD student (Paul McCusker) who worked on the RNAi-tools development aspect of this CIDLID grant spent 3-months training in Prof Spithill's laboratory in Melbourne on vaccine antigen production. Further, Prof. Spithill will undertake a research visit to Belfast for one month from 14 July 2014. • Prof. Brophy (PI at Aberystwyth) was appointed to the Parasitology Steering Committee for Meat Promotion Wales (HCC) Levy Body in 2011 and this has lead to a HCC Contract on Liver Fluke control in Wales (2012) and a BBSRC-iCASE application with HCC on fluke in 2013. Professor Brophy and Dr Morphew have been awarded a 2013 BBSRC Sparking Award to host a workshop on commercial opportunities on liver fluke control at the new Aberystwyth Innovation Campus. • A PDRA from the CIDLID consortium (Dr Morphew) has recently been appointed to a lectureship position at Aberystwyth. Another PDRA from this CIDLID consortium (Dr McVeigh) secured Gates Foundation funding to develop new diagnostic tests for parasite diseases. • Dr. M. Raman and Dr. G. Ravikumar invited Dr. Ramamoorthy Jayaraj, Senior Lecturer-Clinical Sciences, Faculty of Engineering, Health, Science and the Environment, Charles Darwin University, Australia to TANUVAS Chennai for additional advise on the BBSRC Fasciola research project (Januray 2013). • Dr. M. Raman and Dr. G. Ravikumar visited Mahidol University, Bangkok, Thailand for research discussions with Dr. Suda Riengrojpitak on the development of a Fasciola vaccine (October 2013). |
First Year Of Impact | 2011 |
Sector | Agriculture, Food and Drink,Healthcare |
Impact Types | Economic Policy & public services |
Description | Molecular Diagnosis Facility at TANUVAS, Chennai |
Geographic Reach | Local/Municipal/Regional |
Policy Influence Type | Influenced training of practitioners or researchers |
Impact | Establishment of a leptospirosis diagnosis laboratory at TANUVAS, Chennai. This is now used to serve the public in the area. |
Description | Capacity Building at Aligarh Muslim University (leveraged by CIDLID award) |
Amount | ₹97,800,000 (INR) |
Organisation | Ministry of Science and Technology India |
Sector | Public |
Country | India |
Start | 01/2012 |
End | 12/2017 |
Description | Dynamics of Cathepsin B Protease Expression in Liver Fluke |
Amount | £80,000 (GBP) |
Organisation | Alagappa University |
Sector | Academic/University |
Country | India |
Start | 08/2010 |
End | 08/2013 |
Description | Exploiting RNAi biotechnologies for enhanced parasite control in livestock |
Amount | £57,000 (GBP) |
Organisation | Department of Agriculture and Rural Development (DARDNI) |
Sector | Public |
Country | United Kingdom |
Start | 09/2011 |
End | 09/2014 |
Description | Outreach programme research grant (India) |
Amount | £1,375 (GBP) |
Organisation | Indian Council of Agricultural Research |
Sector | Charity/Non Profit |
Country | India |
Start | 03/2014 |
Description | The development of RNA interference tools for the validation of new control targets in the parasite, Fasciola hepatica |
Amount | £51,000 (GBP) |
Organisation | Professor John Glover Memorial Fund |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2011 |
End | 09/2014 |
Description | Towards Sustainable Parasite Control in Sheep |
Amount | £58,000 (GBP) |
Organisation | Department of Agriculture and Rural Development (DARDNI) |
Sector | Public |
Country | United Kingdom |
Start | 09/2010 |
End | 09/2013 |
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. |
Title | RNA interference in juvenile liver fluke |
Description | We developed methods for gene silencing in Fasciola hepatica, and in the tropical fluke, Fasciola gigantica (see McVeigh et al., 2014). These methods are relatively simple to apply, and bring liver fluke reverse genetics studies within reach of researchers with access to basic molecular biology facilities. |
Type Of Material | Technology assay or reagent |
Year Produced | 2011 |
Provided To Others? | Yes |
Impact | Publication (McVeigh et al., 2014); Methods were brought to Indian collaborators at Chennai and Aligarh through a series of visits and formal training workshops. Workshop presentations were attended by >100 students/faculty members. this increased the capacity for functional genomics research in the parasitology laboratories at Chennai (TANUVAS) and Aligarh. |
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 | CIDLID consortium |
Organisation | Aberystwyth University |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | > Led development of RNAi methodology. > Reverse genetic identification of targets for vaccine trials. |
Collaborator Contribution | > Aberystwyth: Sub-proteomic identification of target protein family diversity; Generated recombinant proteins for vaccine trials. > TANUVAS: Collected samples; developed pamphlets for farmers; organised interactions with field-stations and with field veterinarians in Tamil Nadu; performed vaccine trial in water buffalo. > AMU: Generated Fasciola gigantica metacercariae for other collaborators; developed fluke RNAi platform for use in India; developed pamphlets for farmers; organised interactions with field-stations and with field veterinarians in and North India; |
Impact | Publications, methods, development needs in India. |
Start Year | 2010 |
Description | CIDLID consortium |
Organisation | Aligarh Muslim University |
Country | India |
Sector | Academic/University |
PI Contribution | > Led development of RNAi methodology. > Reverse genetic identification of targets for vaccine trials. |
Collaborator Contribution | > Aberystwyth: Sub-proteomic identification of target protein family diversity; Generated recombinant proteins for vaccine trials. > TANUVAS: Collected samples; developed pamphlets for farmers; organised interactions with field-stations and with field veterinarians in Tamil Nadu; performed vaccine trial in water buffalo. > AMU: Generated Fasciola gigantica metacercariae for other collaborators; developed fluke RNAi platform for use in India; developed pamphlets for farmers; organised interactions with field-stations and with field veterinarians in and North India; |
Impact | Publications, methods, development needs in India. |
Start Year | 2010 |
Description | CIDLID consortium |
Organisation | Tamil Nadu Veterinary and Animal Sciences University |
Country | India |
Sector | Academic/University |
PI Contribution | > Led development of RNAi methodology. > Reverse genetic identification of targets for vaccine trials. |
Collaborator Contribution | > Aberystwyth: Sub-proteomic identification of target protein family diversity; Generated recombinant proteins for vaccine trials. > TANUVAS: Collected samples; developed pamphlets for farmers; organised interactions with field-stations and with field veterinarians in Tamil Nadu; performed vaccine trial in water buffalo. > AMU: Generated Fasciola gigantica metacercariae for other collaborators; developed fluke RNAi platform for use in India; developed pamphlets for farmers; organised interactions with field-stations and with field veterinarians in and North India; |
Impact | Publications, methods, development needs in India. |
Start Year | 2010 |
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 | 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: 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 CIDLID project meetings (India, North and South) |
Form Of Engagement Activity | A formal working group, expert panel or dialogue |
Part Of Official Scheme? | No |
Type Of Presentation | keynote/invited speaker |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Updated CIDLID consortium of project progress; discussed future directions and alternative approaches to problems. Alongside conference dedicated to the BBSRC research we ran a training workshop on liver fluke control for veterinarians (60+) in Erode (Tamil Nadu) in 2011 and a workshop on reverse genetic methods for fluke at Aligarh Muslim University (2013). Veterinarians from Ooty where there was a serious outbreak of fasciolosis contacted us about liver fluke control on farms; after the Aligarh meeting/workshop numerous students from Aligarh Muslim University contacted us to find out more about the research. |
Year(s) Of Engagement Activity | 2011,2013 |
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 | 2010,2011,2012,2013,2014,2015,2016,2017,2018 |
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 | Conference attendance; 'Molecular Helminthology An Integrated Approach' in San Antonio, Texas April 7-10, 2019 |
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 given by Professor Aaron Maule entitled, 'Neoblasts, growth and flukicide interplay in Fasciola hepatica' NC3Rs and BBSRC grants acknowledged |
Year(s) Of Engagement Activity | 2019 |
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 | Exhibitor QUB Research Impact Showcase |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Highlighted BBSRC-funded parasitology research to a public audience. NA |
Year(s) Of Engagement Activity | 2011 |
Description | Exhibitor at NI Universities Research Showcase (Stormont, Belfast) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Policymakers/politicians |
Results and Impact | Highlighted the importance of our BBSRC-funded parasitology/animal health research to local government. NA |
Year(s) Of Engagement Activity | 2013 |
Description | GIESSEN Germany Joint meeting of the 20th Anniversary Drug Design & Development Seminar (DDDS) 2019 of the German Society for Parasitology (DGP) & the LOEWE Center DRUID "Novel Drugs against Zoonotic and Poverty-Related Diseases - Bridging the Innovation Gap - " |
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 given by Professor Aaron Maule in Geisson, Germany 26th-29th March 2019. Abstract entitled, 'Juvenile liver fluke stem cells, growth dynamics and therapeutics' NC3Rs and BBSRC grants acknowledged |
Year(s) Of Engagement Activity | 2019 |
Description | ICOPA XII - Melbourne, Australia |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Cathepsin-like cysteine proteases represent some of the most highly expressed proteins in the liver fluke Fasciola hepatica, and are known to be responsible for core parasite functions including tissue penetration, feeding and immune evasion. The monophyletic lineage of F. hepatica cathepsin Ls (FheCLs) encompass at least 18 distinct endopeptidase sequences comprising five clades, which are distinguished structurally, and by temporal expression pattern during the fluke's invasion process. Flukes demonstrate coordinated neuromuscular activity during the processes of invasion and migration, controlled by a highly peptidergic nervous system. Although knowledge of neuropeptide diversity and function is increasing in parasitic flatworms, we know little about neuropeptide signal termination mechanisms. Here we present several lines of evidence in support of a role for FheCL in the termination of neuropeptide signalling. As well as immunocytochemical localisation of FheCL to nerves and muscle, we demonstrate that recombinant FheCL can cleave flatworm neuropeptides in vitro and that FheCL inhibitors can increase peptidergic excitation of F. hepatica muscle bioassays. We have previously demonstrated the importance of cathepsin proteases by employing RNA interference (RNAi) to silence both FheCL and FheCB transcripts in newly-excysted juvenile (NEJ) worms, preventing their ability to penetrate rat gut tissue. Using clade-specific siRNAs in our F. hepatica RNAi platform, here we present evidence in support of a role for clade 2 FheCLs in neuropeptide signal termination, since our clade 2-specific siRNAs resulted in an increase in neuropeptide immunoreactivity, as measured by quantitative confocal microscopy. This phenomenon was not seen with negative control siRNAs, nor siRNAs directed against other FheCL clades. These data provide the first evidence in support of a specific role for FheCL clade 2 enzymes in neuropeptide degradation. |
Year(s) Of Engagement Activity | 2010 |
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 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 | National Sheep Association Events (at Parklands Veterinary Care) - Outreach |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | National Sheep Association Q&A session and presentations - much lively discussion. Many farmers and veterinarians engaged in discussions and provided access to farms for sampling of sheep to gauge fluke levels across NI. |
Year(s) Of Engagement Activity | 2011,2012 |
Description | Oral Presentation World Association for the Advancement of Veterinary Parasitology (WAAVP) conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Virtual attendance at WAAVP giving online presentation entitled; 'Transcriptomic analysis reveals a novel role for the nervous system in regulating growth and development of Fasciola hepatica juveniles'. ABSTRACT: Fasciola spp. liver fluke are important global pathogens with significant impacts on veterinary and human medicine. 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. A significant improvement in functional genomic approaches underpinned by enhanced culture methods for Fasciola hepatica facilitates an in vitro platform for drug and vaccine target validation focussed on the pathogenic life stage of this parasite. Current in vitro culture methods promote growth and development of juvenile fluke by stimulating morphological and behavioural changes towards an adult-like form. However, slower rates of growth and development observed in vitro highlights limitations of current culture methods, whilst also offering a unique opportunity to study the pathogenic stage during critical growth phases. Transcriptomic analysis of in vitro and in vivo maintained F. hepatica juveniles finds that despite considerable size differences, 86% of transcripts are expressed at similar levels across maintenance treatments suggesting core biological functioning across these juveniles is comparable. Significant upregulation of neoblast-like stem cell markers suggests a higher cell proliferation rate in in vivo maintained juveniles directly correlating with a marked increase in their size. Differing cathepsin protease profiles of juveniles provides evidence of developmental delay in vitro and suggests these juveniles are yet to undergo a developmental switch from cathepsin-B to cathepsin-L expression. Significant downregulation of classical and peptidergic nervous system components in in vivo maintained juveniles highlights a previously undescribed role of the nervous system in modulating growth and development mechanisms in liver fluke. Combined, these datasets significantly enhance our ability to improve in vitro functional genomic platforms and emphasizes a cohort of drug targets focussed on growth and development of pathogenic juveniles to drive novel anthelmintic development. |
Year(s) Of Engagement Activity | 2021 |
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 | Royal Welsh Agricultural Society shows |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | Facilitated much engegement with the farming community, stakeholders and veterinarians. Follow up queries from farmers / farming community representatives |
Year(s) Of Engagement Activity | 2010,2011,2012,2013 |
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 | SafeFood Meeting, Teagasc, Dublin |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | Fasciola spp. liver fluke continue to impede animal health and productivity in the UK and worldwide, and are additionally recognised amongst the World Health Organisation's list of Neglected Tropical Diseases of humans in the developing world. A small cadre of benzimidazole anthelmintics shoulder the majority of the burden for combating Fasciola infections, given the current absence of alternative control options. Post-genomic reverse genetics technologies promise to accelerate both the discovery of novel control targets and our understanding of fundamental helminth biology, but have not yet been widely applied to Fasciola spp. systems. Focusing on some of the most commonly cited vaccine candidate proteins (including cysteine proteases, fatty acid binding proteins, glutathione transferases, and leucine aminopeptidases), we have developed RNA interference (RNAi) methodology in Fasciola hepatica, permitting the specific 'knockdown' of mRNA and protein targets. While we can demonstrate profound transcript and protein knockdowns, we have seen little indication that doing so results in reduced survival of juvenile F. hepatica, during maintenance in vitro of up to 28 days. We are currently developing target-specific functional assays, with which to investigate the biological essentiality, and thus control target potential, of proposed drug and vaccine target candidates. Funded by BBSRC grant BB/H009477/1. |
Year(s) Of Engagement Activity | 2012,2014 |
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 |