Probing in vivo parasite biology in vitro

肝吸虫(片形吸虫属)是一种寄生虫，感染各种哺乳动物，包括人和反刍动物，如牛、山羊和绵羊。在人类中，寄生虫导致肝片吸虫病，这是一种公认的被忽视的热带疾病，估计有1700万人被感染。这种蠕虫影响全球粮食安全，因为它破坏牲畜的健康和生产力，导致肝片吸虫病，估计每年在全球造成约30亿美元的损失。主要的担忧是：最近对英国流行率变化的估计预测，到2050年，肝片吸虫病的风险将达到前所未有的水平；农民依赖于药物的管理，这些药物由于耐药性而变得不那么有效。用于控制肝吸虫的主要药物是三氯苯咪唑(TCBZ)，它对成虫(生活在胆管中)和幼虫(幼虫在被植被包裹后，从肠道通过肝脏迁移到胆管，在此过程中造成很大损害)都有疗效。TCBZ抗药性威胁到世界许多地区畜牧业的可持续性，因此需要新的杀线虫剂和/或疫苗来帮助控制问题。对这种寄生虫的研究依赖于使用实验室宿主动物(通常是小鼠或大鼠)来进行寄生虫生物学的功能研究，因为不可能在实验室(体外)培养这种复杂的寄生虫。然而，我们已经开发出允许幼虫肝吸虫在体外维持较长时间并允许它们生长发育的方法。这为体外开展不同的肝吸虫生物学实验提供了机会，并有很大的潜力减少用于研究这种寄生虫的实验室宿主动物的数量。另外两项最新进展正在推动肝吸虫研究的扩大。第一个是公布其基因组序列，第二个是开发一种名为RNA干扰(RNAi)的方法，使我们能够关闭寄生虫中的单个基因并研究它们的功能(这一过程被称为功能基因组学)。将这一RNAi工具与我们不断发展的体外培养方法相结合，为在不需要使用宿主动物的情况下进行肝吸虫功能基因组学研究提供了巨大的机会。这样的平台可用于替代动物用于肝吸虫研究的许多方面，例如在行业内，该工具包将有助于筛选针对体外生长和发育的蠕虫的候选抗寄生虫药物，显著减少基于宿主动物的实验的需要。在研究界使用这种新的肝吸虫体外工具包之前，需要对其进行验证。在这里，我们建议确定体外吸虫与体内对应的吸虫(从宿主动物身上发现的吸虫)有多相似。为此，我们建议比较体外和体内吸虫的行为、形态、毒力蛋白和参与发育过程的各种基因。我们还将优化针对发育中的吸虫的RNAi方法，因为它们密切反映了高度破坏性的迁徙幼虫阶段。重要的是，我们提议开发和验证的工具包将很容易被其他实验室采用，并且将是翻译的，因为它可以直接用于行业内的药物发现项目，从而取代动物用于寄生虫学研究的各个方面。事实上，几家最大的动物保健公司已经表示有兴趣了解这项工作的进展，以便他们可以考虑采用该工具包进行研究。这样，我们建议开发的体外工具箱将促进发现新的吸虫控制方法，并将导致商业和非商业研究实验室内的实验室动物取代肝吸虫研究。

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.