Probing in vivo parasite biology in vitro

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Liver fluke (Fasciola species) are parasitic worms that infect diverse mammals including humans and ruminant livestock such as cattle, goats and sheep. In humans, the parasite causes the disease fascioliasis which is a recognised neglected tropical disease with an estimated 17 million people believed to be infected. The worm impacts global food security as it undermines the health and productivity of livestock in which it causes fasciolosis, estimated to cause losses of ~$US3 billion/year worldwide. Major concerns are that: recent estimates of changing prevalence in the UK have forecast unprecedented levels of fasciolosis risk by 2050; farmers rely on the administration of drugs which are becoming less effective due to drug resistance. The main drug used to control liver fluke is triclabendazole (TCBZ) and it has efficacy against both the adult worms (which live 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). TCBZ-resistance threatens the sustainability of livestock farming in many regions of the world such that new flukicides and/or a vaccine are needed to help control the problem.Research on this parasite relies on the use of laboratory host animals (commonly mice or rats) to enable functional studies on parasite biology as it is not possible to culture this complex parasite in the laboratory (in vitro). However, we have developed methods that allow the maintenance of juvenile liver fluke in vitro for extended periods of time and which allow their growth and development. This provides an opportunity to undertake diverse experiments on liver fluke biology in vitro and has much potential to reduce the numbers of laboratory host animals used for research on this parasite.Two other recent developments are promoting the expansion of research on liver fluke. The first is the publication of its genome sequence and the second is the development of a method called RNA interference (RNAi) which allows us to switch-off individual genes in the parasite and investigate their function (a process called functional genomics). Merging this RNAi tool with our evolving in vitro culture methods provides immense opportunities to undertake functional genomics research on liver fluke without the need to use host animals. Such a platform could be used to replace the use of animals for many aspects of liver fluke research, e.g. within industry, this toolkit would facilitate the screening of candidate anti-parasite drugs on worms growing and developing in vitro, significantly reducing the need for host animal-based experiments.Before this new in vitro toolkit for liver fluke can be used by the research community, it needs to be validated. Here we propose to ascertain how similar the in vitro fluke are to their in vivo counterparts (those recovered from host animals). To do this, we propose to compare the behaviour, morphology, virulence proteins and diverse genes involved in developmental processes between the in vitro and the in vivo fluke. We will also optimize the RNAi methods for the developing fluke as they closely mirror the highly damaging, migrating juvenile stage.Importantly, the toolkit we are proposing to develop and validate will be easily adopted by other laboratories and will be translational, in that it can feed directly into drug discovery projects within industry so replacing animal use for diverse aspects of parasitology research. Indeed, several of the largest animal health companies have expressed an interest in knowing about the progress of this work so they can consider adopting the toolkit for their research. In this way, the in vitro toolkit we propose to develop will enhance the discovery of new control methods for fluke and will result in the replacement of laboratory animals for liver fluke research within both commercial and non-commercial research laboratories.