Probing in vivo parasite biology in vitro
Lead Research Organisation:
Queen's University Belfast
Department Name: Sch of Biological Sciences
Abstract
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.
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.
Technical Summary
Helminth parasites impose a huge burden on human health and agricultural productivity. Amongst the most important parasites of livestock are Fasciola species liver fluke that cause fasciolosis - also a neglected tropical disease. The predicted impact of climate change on fluke epidemiology and growing drug resistance emphasise the need for new flukicides / vaccines. Unfortunately, major impediments to research that advances new control target discovery / validation have included the lack of good bioinformatic resources, the absence of a model system for functional studies and the reliance on host animal-based in vivo experiments to inform fluke biology; the reliance on in vivo efficacy studies in host animals impedes significantly the drug discovery process.
The new liver fluke genome sequence and the development of functional genomics tools expose a compelling need for in vitro tools for juvenile fluke to facilitate studies of their biology. In this respect we have made significant advances in their in vitro maintenance that facilitate fluke survival for >7 months (with >70% survival). Although our methods facilitate marked growth and development of the gut, tegument and reproductive systems, these are slower than those seen in vivo. This project will build on these new resources and preliminary data to enhance in vitro fluke culture. The work will be underpinned by comparative studies (behavioural, morphological, pharmacological and transcriptomic) of in vitro and in vivo fluke to validate the utility of functional / phenotypic readouts.
This project aims to deliver an in vitro toolkit for liver fluke that is robust and translational (i.e. predictive of in vivo outcomes) and enables the study fluke developmental biology, the validation of new drug targets and the screening new drug candidates. Progress made here will contribute significantly to the replacement of animals for studies of parasite biology.
The new liver fluke genome sequence and the development of functional genomics tools expose a compelling need for in vitro tools for juvenile fluke to facilitate studies of their biology. In this respect we have made significant advances in their in vitro maintenance that facilitate fluke survival for >7 months (with >70% survival). Although our methods facilitate marked growth and development of the gut, tegument and reproductive systems, these are slower than those seen in vivo. This project will build on these new resources and preliminary data to enhance in vitro fluke culture. The work will be underpinned by comparative studies (behavioural, morphological, pharmacological and transcriptomic) of in vitro and in vivo fluke to validate the utility of functional / phenotypic readouts.
This project aims to deliver an in vitro toolkit for liver fluke that is robust and translational (i.e. predictive of in vivo outcomes) and enables the study fluke developmental biology, the validation of new drug targets and the screening new drug candidates. Progress made here will contribute significantly to the replacement of animals for studies of parasite biology.
Planned Impact
Beneficiaries of the Research
NC3Rs scheme: The proposed study is directly relevant to NC3Rs under the theme of animal replacement. We propose to remove the dependence on animal experiments for studies on parasite biology through the development of a new in vitro toolkit for liver fluke that will facilitate functional genomics, drug screening and resistance testing.
Associated animal use metrics: over the last 4 years ~86 papers per years use host animals (mice, rats, rabbits) for fluke research. A random selection of 25 of these, each from a distinct research group, was used to establish that a mean of 48 animals were used per study, meaning that ~4128 host animals/year are used in published studies on liver fluke. The numbers used in industry far exceed this; if we conservatively estimate that industry uses a minimum of 3x this number of animals, then >16,500 animals are used per year for liver fluke research. Removing ~40% which are needed for vaccine trials and life cycle maintenance leaves ~9600 animals being used in studies that this toolkit could replace. This work has the potential to deliver an in vitro toolkit that reduces small laboratory animal use by >9000/year representing a reduction in the numbers of animals used of >50%.
The ability of our fluke data to inform research on other parasites has the potential to dramatically increase the impact on animal experimentation. According to the Home Office 131,000 animals were used for parasitology research in the UK in 2013 and we estimate that one third of these were for helminth research. Based on recent published data ~40% of the latter are used for propagation and / or vaccine trials, and that half again could be replaced using robust in vitro parasite biology platforms, then this research project has the potential to reduce animal parasitology experiments in the UK by up to 11,000/year.
Importantly, various developments will serve to increase liver fluke research activity, including: the recent completion of the liver fluke genome sequence; the growth of fasciolosis as an emerging NTD; and, the increase in flukicide resistance in farm animals. As more researchers work on the growing problem of fasciolosis, the availability of a robust in vitro biology platform would mitigate the associated increases in the use of host animals for liver fluke research.
Industry will benefit through the use of more cost-effective and time-efficient platforms to interrogate parasite biology. This will hasten the early-stage discovery of new targets (from reverse genetic studies) or drugs (from chemical screens).
Livestock farms and farming communities will benefit through enhanced economic returns associated with reduced parasite burdens and healthier livestock. As an indirect result of this project, livestock producers will benefit from more efficient pipelines of drug discovery/validation. Increased productivity will enhance economic success in all 'farm to fork' associated businesses, bolstering the competitiveness of the UK Agri-Food Industry.
Stakeholders and policy makers will benefit through research-evidence that will help inform policy development. UK-based government bodies, Levy boards, and other representatives of the Agri-Food industry will benefit as this project will provide an evidence-base for policy development and addresses EU-directed changes in agricultural legislation, e.g. rapid drug-intervention to facilitate 'Sustainable Intensification'.
Resource-poor people infected with fluke are potential beneficiaries from improved treatments for the NTD, fascioliasis.
The general public could benefit from improved flukicides that reduce the numbers of treatments and, therefore, food residues. Further, those students and local schools where the research will be showcased will benefit from improved knowledge and understanding of parasites, new technologies to improve their control and efforts to reduce animal use in science.
NC3Rs scheme: The proposed study is directly relevant to NC3Rs under the theme of animal replacement. We propose to remove the dependence on animal experiments for studies on parasite biology through the development of a new in vitro toolkit for liver fluke that will facilitate functional genomics, drug screening and resistance testing.
Associated animal use metrics: over the last 4 years ~86 papers per years use host animals (mice, rats, rabbits) for fluke research. A random selection of 25 of these, each from a distinct research group, was used to establish that a mean of 48 animals were used per study, meaning that ~4128 host animals/year are used in published studies on liver fluke. The numbers used in industry far exceed this; if we conservatively estimate that industry uses a minimum of 3x this number of animals, then >16,500 animals are used per year for liver fluke research. Removing ~40% which are needed for vaccine trials and life cycle maintenance leaves ~9600 animals being used in studies that this toolkit could replace. This work has the potential to deliver an in vitro toolkit that reduces small laboratory animal use by >9000/year representing a reduction in the numbers of animals used of >50%.
The ability of our fluke data to inform research on other parasites has the potential to dramatically increase the impact on animal experimentation. According to the Home Office 131,000 animals were used for parasitology research in the UK in 2013 and we estimate that one third of these were for helminth research. Based on recent published data ~40% of the latter are used for propagation and / or vaccine trials, and that half again could be replaced using robust in vitro parasite biology platforms, then this research project has the potential to reduce animal parasitology experiments in the UK by up to 11,000/year.
Importantly, various developments will serve to increase liver fluke research activity, including: the recent completion of the liver fluke genome sequence; the growth of fasciolosis as an emerging NTD; and, the increase in flukicide resistance in farm animals. As more researchers work on the growing problem of fasciolosis, the availability of a robust in vitro biology platform would mitigate the associated increases in the use of host animals for liver fluke research.
Industry will benefit through the use of more cost-effective and time-efficient platforms to interrogate parasite biology. This will hasten the early-stage discovery of new targets (from reverse genetic studies) or drugs (from chemical screens).
Livestock farms and farming communities will benefit through enhanced economic returns associated with reduced parasite burdens and healthier livestock. As an indirect result of this project, livestock producers will benefit from more efficient pipelines of drug discovery/validation. Increased productivity will enhance economic success in all 'farm to fork' associated businesses, bolstering the competitiveness of the UK Agri-Food Industry.
Stakeholders and policy makers will benefit through research-evidence that will help inform policy development. UK-based government bodies, Levy boards, and other representatives of the Agri-Food industry will benefit as this project will provide an evidence-base for policy development and addresses EU-directed changes in agricultural legislation, e.g. rapid drug-intervention to facilitate 'Sustainable Intensification'.
Resource-poor people infected with fluke are potential beneficiaries from improved treatments for the NTD, fascioliasis.
The general public could benefit from improved flukicides that reduce the numbers of treatments and, therefore, food residues. Further, those students and local schools where the research will be showcased will benefit from improved knowledge and understanding of parasites, new technologies to improve their control and efforts to reduce animal use in science.