Tracking Marine sources of a Cholera outbreak using high throughput molecular methods on archival samples

霍乱弧菌是一种海洋细菌，生活在上层海洋中被称为浮游动物的微小动物表面并以其为食。浮游动物被洋流分散，因此霍乱弧菌可以通过这条潜在的传播感染的途径传播到不同的地区。当海洋表面温度升高，被称为浮游动物的微小动物数量增加时，弧菌的数量就会增加。在人类中，如果食用受污染的海鲜或在受污染的沃茨中洗澡，霍乱弧菌会导致霍乱，这是一种沿着皮肤感染、脑膜炎和败血症的肠道疾病。霍乱弧菌也可以生活在淡水或微咸水中，因此也可以感染饮用受污染淡水的人。霍乱弧菌有许多不同的变异形式。在温暖的气候中，流行性O 1和0139变体存在并流行。这些会引起严重的胃肠道疾病，导致死亡。然而，在温带北方海洋地区，如英国和加拿大，存在大量非严重变异，这些变异具有更有利的结果。然而，非严重类型可以演变成致病性，因此监测菌株类型以更好地预测潜在的感染事件并提供早期预警是重要的。遗传学方法是测量和跟踪众多不断变化的霍乱弧菌菌株的最佳方法，目前有几个数据库绘制了不同菌株的全球分布图，包括CEFAS主办的欧盟参考实验室（EURL），CEFAS是英国政府实验室，负责测试英国沃茨的食品和水安全。2018年4月，加拿大西北海岸不列颠哥伦比亚省的温哥华岛爆发霍乱，导致4人在食用鱼卵后感染霍乱。这在温带海洋沃茨中是罕见的。这一事件发生在2014-2016年该地区最近一次异常海洋热浪之后不久，我们有兴趣确定较高的海面温度是否改变了浮游动物群落，使致病性霍乱弧菌能够茁壮成长。这样的事件可能发生在英国水域，因为英吉利海峡和北海是英国周围变暖最快的沃茨。加拿大不列颠哥伦比亚省周围的沃茨由连续浮游生物记录仪（CPR）调查定期取样，该调查还记录浮游动物物种，此外，加拿大渔业和海洋部（DFO）在感染地点附近捕获了水。虽然CPR样本保存在福尔马林中，这使得遗传检测困难，但我们仍然能够从CPR样本中定量和检测弧菌的变体。我们提出了一个试点研究，集中了霍乱弧菌在这些样品中使用全基因组富集，使所有的变种，这种细菌被检测使用高通量测序。这将使我们能够检测感染类型，并通过将它们与EURL的菌株进行比较，找出它们的来源，病毒是否一开始就具有传染性，以及是否在其他地方发现（如英国沃茨）以及它们经过什么路线到达温哥华岛。我们亦会研究海水温度上升，会否令人类传染性霍乱弧菌在当地增加及持续存在沃茨和更广泛的海洋区域。由于浮游动物是霍乱弧菌的宿主，我们将确定是否有某些浮游动物物种或浮游动物群体窝藏这种病原体，并促进其在海洋沃茨的扩散和持久性。这将使我们能够弄清楚这些人类传染性霍乱弧菌菌株是暂时的还是持续的威胁，以及它们茁壮成长的环境条件。我们将把这些信息传递给当地政府监测机构，以便他们在再次发现这种细菌时建立预警系统。

Vibrio cholerae is a marine bacteria living and feeding on the surfaces of tiny microscopic animals called zooplankton in the upper oceans. Zooplankton are dispersed by ocean currents and so Vibrio cholerae bacteria can spread to different regions via this route potentially spreading infections. Vibrio bacteria increase in numbers when sea surface temperatures have increased and tiny microscopic animals called zooplankton are at high abundance. In humans, V. cholerae causes Cholera, a diarrhoeal disease along with skin infections, meningitis and septicaemia if contaminated seafood is consumed or by bathing in contaminated waters. V. cholerae can also live in fresh or brackish water and so can infect people drinking contaminated freshwater too. There are many different variant forms of V. cholerae. In warmer climates, epidemic O1 and 0139 variants exist and are endemic. These cause severe gastrointestinal disease leading to fatalities. However, a multitude of non-severe variants exist in temperate Northern oceanic regions, such as UK and Canada, and these have a more favourable outcome. However non-severe types can evolve to become pathogenic thus it is important to monitor strain types to better predict and provide early warning for potential infectious events. Genetic methods are the best way to measure and track the multitude of ever changing Vibrio cholerae strains and several databases exist mapping the global distribution of different strains, including the European Union Reference Laboratory (EURL) hosted by CEFAS, the UK government lab that tests for food and water safety in UK waters. A recent outbreak of V. cholera occured in April 2018 in Vancouver Island, British Columbia on the Northwest coast of Canada causing four people to suffer cholera infection after consuming fish eggs. This is a rare occurence in temperate oceanic waters. This event happened soon after a recent unusal marine heatwave in this region between 2014-2016 and we are interested in determining whether the higher sea surface temperatures had altered zooplankton communities to enable pathogenic V. cholerae to thrive. Such events may happen in UK water as the English Channel and North sea are the fastest warming waters surrounding the UK. The waters surrounding BC Canada are regularly sampled by the Continuous Plankton Recorder (CPR) survey that also records zooplankton species and additionally by the Department of Fisheries and Oceans (DFO) in Canada that have captured water very near the site of infection. Although CPR samples are preserved in formalin which makes genetic detection difficult, we have nevertheless been able to quantify and detect variants of Vibrio from CPR samples. We propse a pilot study to concentrate up Vibrio cholerae using Whole Genome Enrichment in these samples to allow all of the variants of this bacteria to be detected using high-throughput sequencing. This will allow us to detect the infectious types and, by comparing them with strains from EURL, find out where they came from, whether the strain started out as infectious and if they are found elsewhere (such as UK waters) and the route they travelled to end up in Vancouver Island.We will also find out if the extent that increased sea surface temperatures allow human infectious Vibrio cholerae to increase and persist in local waters and in wider oceanic regions. As zooplankton act as hosts to Vibrio cholerae, we will determine if there are certain zooplankton species or groups of zooplankton that harbour this pathogen and facilitate its dispersal and persistence in oceanic waters. This will allow us to work out if this is these human infectious Vibrio cholerae strains are a transient or persistent threat and the environmental conditions in which they thrive. We will trasnmit this information to local governmental monitoring agencies to allow them to set up an early warning system if they find this bacteria again.