Cryptosporidium movement in water - impact of eutrophication and climate change on the zoonotic disease agent

隐孢子虫是一种直到20世纪70年代中期才被发现的人类病原体; 1993年，密尔沃基有40万人在水处理失败后感染，从那时起，整个发达国家的大规模爆发（最近的瑞典，2010年）使寄生虫一直处于公众的视线中。受感染的宿主每天释放多达109个卵囊，发现单个卵囊会迫使关闭和失去饮用水供应（例如，2015年英格兰西北部，30万家庭受到影响）。在威尔士农村地区，不同的污染源尚不清楚，但很可能来自农村社区的养羊业。这些多种污染源很难诊断和监测，而且由于复杂方法的成本，几乎不可能消除。与感染剂量相比，卵囊的大量过量生产（只有10个卵囊可以开始感染）表明，在自然环境中，大多数卵囊被生物去除，可能是通过放牧和悬浮喂养无脊椎动物和原生生物。这些相互作用可能受到富营养化（与土地使用和气候变化有关）的不利影响，增加了隐孢子虫在发展中国家和发达国家“蓝色大理石”情况下的重要性。该项目将测试这些假设，使用下一代测序的组合，以确定隐孢子虫卵囊内的无脊椎动物放牧生物群落中的水富营养化和实验室实验，直接建立隐孢子虫清除和食草动物群落结构之间的联系，通过基于代理的模拟建模预测富营养化对隐孢子虫清除淡水的影响。利用在卡迪夫隐孢子虫生物反应器中体外培养的卵囊进行的实验室实验将确定相关无脊椎动物摄取、消化或运输卵囊的潜力。将使用免疫荧光染色、qPCR和感染性试验评估无脊椎动物清除卵囊的潜力。功能性反应，以及氮富营养化和浊度对重要食草动物清除的影响将被确定。实地研究将利用威尔士的地点（通过威尔士水和卡迪夫水研究中心确定），沿着利用标准技术建立的氮富营养化梯度（由于不同的土地使用等原因）。NGS与环境metabarcoding，和传统的筛选生物样品的生物相关的类别，将被用来建立水生群落的富营养化的反应，而隐孢子虫卵囊在这些样品中的环境丰度将估计从NGS数据。将建立威尔士研究地点隐孢子虫卵囊的感染性，并通过实验评估卵囊在不同环境条件（如洪水、气候变化）下的信念。

Cryptosporidium is a human pathogen unknown until the mid-1970s; in 1993 0.4 million people were infected in Milwaukee following a water treatment failure, and since then, large outbreaks throughout the developed world (most recently Sweden, 2010) have kept the parasite in the public eye. Infected hosts release up to 109 oocysts per day, and the discovery of a single oocyst forces closure and loss of drinking water supplies (e.g. NW England 2015, 0.3 million homes affected). In rural Wales, the different sources of contamination are unknown, but are likely to arise from sheep farming in rural communities. These multiple sources of contamination are difficult to diagnose and monitor, and almost impossible to eliminate due to costs of complex methodologies. The huge excess production of oocysts compared to the infective dose (only 10 oocysts can start an infection) suggests that in the natural environment, most oocysts are removed biotically, probably by grazing and suspension feeding invertebrates and protists. These interactions may be adversely affected by eutrophication (connected with land use and climate change), increasing the importance of Cryptosporidium in both the developing world and in 'Blue Marble' situations within the developed world. This project will test these hypotheses using a combination of Next Generation Sequencing to identify Cryptosporidium oocysts within communities of invertebrate grazing organisms in response to water eutrophication and laboratory experiments to directly establish the link between Cryptosporidium clearance and grazer community structure, linked by agent-based simulation modelling to predict the impact of eutrophication on Cryptosporidium clearance from freshwater. Laboratory experiments with oocysts grown in vitro in the Cardiff Cryptosporidium Bioreactor will establish the potential of relevant invertebrates to ingest, digest or transport oocysts. Immunofluorescence staining, qPCR and infectivity assays will be used to assess the potential of invertebrates for oocyst clearance. Functional responses, and the impact of nitrogen eutrophication and turbidity on clearance by important grazers will be established. Field studies will utilise sites in Wales (identified through Welsh Water and the Water Research Centre at Cardiff) along gradients of nitrogen eutrophication (due to e.g. different land use) established using standard techniques. NGS with environmental metabarcoding, and conventional screening of the biota samples into biologically relevant categories, will be used to establish the response of the aquatic community to eutrophication, while the environmental abundance of Cryptosporidium oocysts in these samples will be estimated from NGS data. The infectivity of Cryptosporidium oocysts from Welsh study sites will be established, and the faith of the oocysts under different environmental conditions (e.g. flooding, climate change) will be experimentally assessed.