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

隐孢子虫是一种通过水传播的病原体，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，疫苗开发也没有立竿见影的前景。乳品业和养牛业的新生小牛中的局部感染很普遍；受感染的动物会出现严重腹泻，失去病情，在极端情况下可能会死于这种疾病。隐孢子虫也是撒哈拉以南非洲地区儿童腹泻的第二大病原体，即使在发达国家，人类疫情也可能涉及数万人。这种疾病仍然受到慈善组织、研究机构和制药公司的关注，但越来越多的关注集中在研发药物上，以消除隐孢子虫这一人类病原体。由于这种病原体的维持依赖于小牛或小鼠的培养，最近增加的资金将极大地增加动物在这一研究领域的使用。隐孢子虫是一种单细胞微生物，与导致疟疾的病原体有关。它会感染哺乳动物的肠道内层。它在肠道细胞内繁殖，杀死它们，并将大量具有抵抗力的感染阶段(卵囊)释放到肠腔内，这些阶段通过粪便排出，在环境中休眠，直到被新生的牛犊等动物吃掉。对这种疾病的研究步伐不断加快，需要越来越多的隐孢子虫卵囊用于实验，这种供应完全来自新生牛犊的感染，因为到目前为止，还没有一种不使用活动物的有效培养方法。奈杰尔·亚利特教授于2016年开发并发表了一种前景看好的不使用牛犊培育隐孢子虫的新方法。这种方法在使用新技术培养的哺乳动物肠道细胞中培养寄生虫阶段。该方法声称每天能产生多达108个隐孢子虫卵囊。如果能将这一方法应用到常规方法中，卵囊的产出将满足研究团体的需求，而不需要使用小牛或其他活动物。这项提议寻求资金，以访问纽约的Yarlett教授的实验室，学习制造大量感染期的新方法，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的卵囊生产设施。我们的长期目标是取代小牛生产卵囊，首先专注于取代我们在加的夫自己的实验室的供应，然后将这些供应提供给其他欧洲隐孢子虫实验室。关键是，在Yarlett的方法可以被用作隐孢子虫卵囊供应的选择方法之前，我们必须(A)证明该方法对于不同隐孢子虫种类和变种(包括感染人类的形式)生长的可靠性，以及(B)确保在该系统中生长了许多代的寄生虫相对于在牛犊中饲养的寄生虫的遗传稳定性。因此，我们需要证明这种新方法在产生感染阶段的数量和质量方面都好于使用动物。这项工作将传播给英国和欧洲的感兴趣的研究单位，解释该方法，并展示与动物模型相比的优势。将向相关学会成员提供培训，并将通过科学出版物和在线沉积公开来自两个系统的隐孢子虫的比较数据(体外培养和牛犊培养)。

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.