High-throughput in vitro culture system for Cryptosporidium oocysts: replacing animals in research

隐孢子虫卵囊高通量体外培养系统：替代研究中的动物

• 批准号: NC/R000913/1
• 负责人: Joanne Cable
• 金额: $ 9.63万
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FR000913%2F1
• 关键词: throughput; vitro; culture; system; Cryptosporidium

Cryptosporidium is a water-borne pathogen infecting farm animals and humans. It poses a major threat to animal health and welfare, and to public health, because there is no proper treatment and no immediate prospect of vaccine development. Focal infections in new-born calves within the dairy and beef industries are widespread; infected animals experience severe diarrhoea, lose condition and in extreme cases can die of the disease. Cryptosporidium is also the second most important diarrhoeal pathogen of small children in sub-Saharan Africa, and even in developed countries human outbreaks can involve tens of thousands of people. The disease has remained under the radar of charity organisations, research institutions and pharma companies, but increasing attention has focussed substantially more funding to develop drugs to eliminate Cryptosporidium as a human pathogen. As maintenance of the pathogen depends on cultures kept in calves or mice, recent increase in funding will dramatically increase animal use in this research field.Cryptosporidium is a single-celled microbe, related to the pathogen which causes malaria. It infects the gut lining of mammals. It multiplies within the cells of the gut, killing them and releasing into the gut lumen large numbers of resistant infective stages (oocysts), which are passed out in faeces and remain dormant in the environment until eaten by an animal such as a new-born calf. The accelerating pace of research into the disease requires an increasing supply of Cryptosporidium oocysts for experimentation, a supply that is met entirely from infections of neonatal calves, because to date there has never been an effective culture method which does not use live animals. A promising new method of rearing Cryptosporidium without using calves was developed and published in 2016 by Prof Nigel Yarlett. This method grows parasite stages in mammalian gut cells grown in culture using new technology. The method claims to generate up to 108 Cryptosporidium oocysts per day. If this can be adapted into a routine methodology, the output of oocysts would meet the needs of the research community without using calves or other live animals.This proposal seeks funds to visit the laboratory of Prof Yarlett in New York to learn the new methodology for producing large numbers of infective stages, and to return this technology to Cardiff University where a facility to produce oocysts without animal use will be built. Our long-term ambition is to replace calves for the production of oocysts, focussing first on replacing supply in our own laboratory in Cardiff, and then making this supply available to other European Cryptosporidium laboratories. Critically, before Yarlett's method can be adopted as the method of choice for Cryptosporidium oocyst supply, it is imperative that we (a) demonstrate the reliability of the method for the growth of different Cryptosporidium species and variants, including the human-infecting forms, and (b) assure the genetic stability of parasites grown in this system for many generations, relative to parasites reared in calves. Thus, we need to show that this new method is better than using animals both in terms of quantity and quality of infective stages produced.The work will be disseminated to interested research units in the UK and Europe explaining the method, and demonstrating advantages compared to the animal model. Training will be offered to relevant Society members, and data comparing Cryptosporidium derived from the two systems (in vitro culture vs. calves) will be made publicly available through scientific publication and deposition on line.

隐孢子虫是一种水媒病原体，可感染农场动物和人类。它对动物健康和福利以及公共卫生构成重大威胁，因为没有适当的治疗方法，也没有立即开发疫苗的前景。乳业和牛肉行业新生小牛的局灶性感染很普遍；受感染的动物会出现严重腹泻，失去健康，在极端情况下可能死于该病。隐孢子虫也是撒哈拉以南非洲地区幼儿腹泻的第二大重要病原体，即使在发达国家，人类暴发也可能涉及数万人。这种疾病一直在慈善组织、研究机构和制药公司的关注之下，但是越来越多的注意力集中在开发消除隐孢子虫作为人类病原体的药物上。由于病原体的维持依赖于小牛或小鼠的培养物，最近资金的增加将大大增加该研究领域的动物使用。隐孢子虫是一种单细胞微生物，与引起疟疾的病原体有关。它会感染哺乳动物的肠壁。它在肠道细胞内繁殖，杀死它们，并向肠道内释放大量具有耐药性的感染阶段（卵囊），这些卵囊随粪便排出，在环境中保持休眠状态，直到被新生的小牛等动物吃掉。对这种疾病的研究步伐加快，需要越来越多的隐孢子虫卵囊用于实验，这一供应完全来自新生牛犊的感染，因为迄今为止还没有一种不使用活体动物的有效培养方法。奈杰尔·耶利特教授于2016年开发并发表了一种不使用小牛饲养隐孢子虫的新方法。该方法利用新技术在哺乳动物肠道细胞中培养寄生虫阶段。该方法声称每天可产生多达108个隐孢子虫卵囊。如果这一方法可以应用到常规方法中，卵囊的产出将满足研究界的需求，而无需使用小牛或其他活体动物。该提案寻求资金，以访问Yarlett教授在纽约的实验室，学习生产大量感染阶段的新方法，并将这项技术返回卡迪夫大学，在那里将建立一个不使用动物生产卵囊的设施。我们的长期目标是取代小牛来生产卵囊，首先重点是取代我们在卡迪夫的实验室的供应，然后将这种供应提供给其他欧洲隐孢子虫实验室。至关重要的是，在Yarlett的方法可以被采用作为隐孢子虫卵囊供应的选择方法之前，我们必须(a)证明该方法在不同隐孢子虫种类和变体（包括人类感染形式）生长的可靠性，以及(b)确保在该系统中生长的寄生虫的遗传稳定性，相对于在小牛中饲养的寄生虫。因此，我们需要证明这种新方法在产生的感染阶段的数量和质量方面都优于使用动物。这项工作将传播给英国和欧洲感兴趣的研究单位，解释该方法，并展示与动物模型相比的优势。将为相关协会成员提供培训，并通过科学出版物和在线沉积公开两种系统（体外培养与小牛）衍生隐孢子虫的比较数据。