Developing a 'validation portfolio' to exploit key virulence proteins in Fasciola species for parasite control

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.

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.

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.