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

隐孢子虫是一种人类病原体，直到20世纪70年代中期才为人所知；1993年，密尔沃基有40万人在水处理失败后感染，自那以后，发达国家(最近的一次是瑞典，2010年)的大规模疫情使这种寄生虫继续出现在公众的视线中。受感染的宿主每天释放多达109个卵囊，发现一个卵囊就会迫使饮用水供应关闭和失去供应(例如，2015年英格兰西北部，30万户家庭受到影响)。在威尔士农村，不同的污染源尚不清楚，但很可能是来自农村社区的绵羊养殖。这些多重污染源很难诊断和监测，而且由于复杂的方法成本，几乎不可能消除。与感染剂量(只有10个卵囊可能引发感染)相比，卵囊的产量巨大过剩，这表明在自然环境中，大多数卵囊是通过生物方式去除的，可能是通过放牧和悬浮喂养无脊椎动物和原生动物。这些相互作用可能受到富营养化(与土地利用和气候变化有关)的不利影响，从而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。该项目将结合使用下一代测序来验证这些假设，以识别无脊椎动物放牧生物群落中的隐孢子虫卵囊，以响应水体富营养化，并通过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来预测富营养化对从淡水中清除隐孢子虫的影响。在加的夫隐孢子虫生物反应器中用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。免疫荧光染色、定量聚合酶链式反应和传染性分析将被用来评估无脊椎动物清除卵囊的潜力。将确定功能反应，以及氮富营养化和浑浊度对重要草食动物清除的影响。实地研究将利用威尔士的站点(通过威尔士水和加的夫的水研究中心确定)，沿着使用标准技术建立的氮富营养化梯度(由于不同的土地使用)。将使用环境元编码的NGS，以及对生物群样本进行生物相关类别的常规筛选，以确定水生群落对富营养化的反应，同时将根据NGS数据估计这些样本中隐孢子虫卵囊的环境丰度。将确定来自威尔士研究地点的隐孢子虫卵囊的传染性，并将通过实验评估不同环境条件(如洪水、气候变化)下的卵囊的可信度。

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.