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，以上层海洋中微小的浮游动物表面为食。浮游动物被洋流分散，因此霍乱弧菌可以通过这条路线传播到不同的地区，可能会传播感染。当海面温度升高时，弧菌的数量会增加，被称为浮游动物的微小动物数量也会增加。在人类中，如果食用受污染的海产品或在受污染的水中洗澡，霍乱弧菌会引起霍乱，这是一种腹泻病，还会引起皮肤感染、脑膜炎和败血症。霍乱弧菌也可以生活在淡水或半咸淡水中，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种。在气候较暖的地区，存在流行病O1和0139变体，并且是地方性的。这些会引起严重的胃肠道疾病，导致死亡。然而，在温带的北部海洋地区，如英国和加拿大，存在着大量的非严重变异体，这些变异体有更有利的结果。然而，非严重类型可以演变为致病性，因此监测菌株类型以更好地预测和提供潜在感染事件的早期预警非常重要。遗传方法是测量和跟踪大量不断变化的霍乱弧菌菌株的最佳方法，并且有几个数据库绘制了不同菌株的全球分布，其中包括由CEFAS主持的欧盟参考实验室（EURL），该实验室是英国政府实验室，负责测试英国水域的食品和水安全。2018年4月，加拿大西北海岸不列颠哥伦比亚省温哥华岛爆发霍乱弧菌疫情，导致4人在食用鱼卵后感染霍乱。这在温带海域是很罕见的。这一事件发生在2014-2016年间该地区最近一次不寻常的海洋热浪之后不久，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水域。连续浮游生物记录仪（CPR）定期对加拿大不列颠哥伦比亚省周围的水域进行采样，该调查还记录了浮游动物种类，此外，加拿大渔业和海洋部（DFO）也在非常接近感染地点的水域进行了采样。尽管CPR样本保存在福尔马林中，这使得基因检测变得困难，但我们仍然能够量化和检测CPR样本中弧菌的变体。我们建议进行一项试点研究，利用全基因组富集技术在这些样品中浓缩霍乱弧菌，以便使用高通量测序检测该细菌的所有变体。这将使我们能够检测到感染类型，并通过将它们与EURL的菌株进行比较，找出它们来自哪里，菌株是否开始具有传染性，以及它们是否在其他地方（如英国水域）被发现，以及它们最终到达温哥华岛的路线。我们还将查明海面温度升高的程度是否使人类传染性霍乱弧菌在当地水域和更广泛的海洋区域增加和持续存在。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窝藏这种病原体，并促进其在海洋中的传播和持久存在。这将使我们能够弄清楚这些人类传染性霍乱弧菌菌株是短暂的还是持续的威胁，以及它们茁壮成长的环境条件。我们将把这一信息传递给地方政府的监测机构，以便他们在再次发现这种细菌时建立早期预警系统。”

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.