The use of PrP transgenic Drosophila to replace and reduce mice in the bioassay of mammalian prions

Prion病包括牛疯牛病、宫颈慢性萎缩性疾病、羊瘙痒病和人类CJD。这些疾病是传染性的，可以在同一物种或不同物种的个体之间传播。动物Prion病可以传播给人类，因此对公共健康构成威胁，典型的疯牛病在牛身上暴发，随后在人类中出现变异型CJD(VCJD)。导致这些传染性疾病的病原体是在神经元中发现的一种正常蛋白质的聚集流氓形式，被称为普恩。疯牛病的传播被认为是通过饮食摄入受疯牛病污染的食物而发生的。现在，严格的控制措施可以保护人类免受疯牛病的侵袭，包括在屠宰牛时移除最有可能含有传染性普恩病毒的牛组织。这些牛组织被称为特定危险物质(SRM)，不进入人类食物链。唯一可靠的检测疯牛病病毒传染性的方法是对实验动物进行生物检测，传统上实验动物是啮齿动物，如老鼠。小鼠的Prion生物检测包括将疑似BSE感染的样本注射到实验小鼠中，并等待这些动物是否会患上Prion疾病。这些分析是缓慢和繁琐的，因为在小鼠中，蛋白病毒病的潜伏期可能需要1-2年才能出现明显的临床症状，并达到预定的终点。总的来说，疯牛病病毒传染性研究在很长一段时间内使用了大量的小鼠，并使这些动物中的高比例经历了实验性神经疾病的晚期临床症状。自从经典的疯牛病出现以来，对这种疾病的加强监测导致了对新形式的疾病的识别，这种疾病对人类食物链构成了新的威胁，因为我们不知道受影响牛的哪些组织含有这些新形式的疯牛病病毒。因此，至关重要的是核实目前的SRM控制措施是否足以防止新形式的疯牛病Prion进入人类食物链。更多的小鼠将被用于拟议的食品安全研究计划，以测量感染新形式疯牛病的牛的广泛样本中的普恩传染性水平。总的来说，这些鼠普恩生物检测将使用超过10万只小鼠。在我们的实验室中，我们在果蝇黑腹果蝇身上模拟普恩病毒疾病，因为它们相对容易和经济地使用，并且是被广泛接受的对包括老鼠在内的高等生物的伦理替代。我们已经开发了一种基于果蝇的Prion生物检测方法，它可以检测疯牛病Prion的感染性，以便提供一种替代小鼠的牛Prion生物检测方法，并减少使用小鼠和其他脊椎动物物种来测量Pron感染性的一般方法。为了做到这一点，我们将一种基因引入果蝇中，使它们能够产生在患有普恩病毒疾病的动物的大脑中聚集的蛋白质。我们已经知道，当转基因果蝇被喂食感染的普恩时，这种哺乳动物蛋白质会聚集在一起，并导致普恩疾病。同样重要的是，转基因果蝇在接触普恩病毒材料后的几周内，对感染了普恩病毒的材料的反应是明显的。我们现在可以开发一种比目前存在的更快、更通用和更灵敏的生物检测方法来检测疯牛病病毒的传染性。我们的目标是将这项技术转让给APHA，APHA是小鼠Pron生物检测的主要用户和国际公认的参考实验室。我们的技能和知识转移到APHA将提供最好的机会，以确保我们新的基于果蝇的Prion生物检测在世界范围内产生影响。

Prion diseases include BSE of cattle, chronic wasting disease of cervids, scrapie of sheep and CJD of humans. These conditions are infectious and can spread between individuals of the same or different species. Animal prion diseases can be transmitted to humans and are therefore a threat to public health, evidenced by the outbreak of classical BSE in cattle followed by the emergence of variant CJD (vCJD) in humans. The pathogen that causes these transmissible diseases is an aggregated rogue form of a normal protein found in neurons and is referred to as a prion. Transmission of BSE to humans is believed to have occurred by dietary intake of BSE-contaminated food. Strict controls now protect humans from BSE, including removal of cattle tissues most likely to contain infectious prions when the animal is slaughtered. These cattle tissues are called specified risk material (SRM) and do not enter the human food chain.The only reliable way to detect BSE prion infectivity is by bioassay in experimental animals, which have traditionally been rodents, such as mice. The mouse prion bioassay involves injecting suspected BSE-infected samples into experimental mice and waiting to see if these animals develop prion disease. These assays are slow and cumbersome since the incubation time for prion disease in mice may be 1-2 years before clinical signs become evident and a predetermined end-point is reached. Collectively, BSE prion infectivity studies have utilised large numbers of mice over a long time period, and have subjected a high proportion of these animals to experience terminal clinical signs of experimental neurological disease. Since the emergence of classical BSE, the increased surveillance for the condition has led to identification of novel forms of the disease, which are a new threat to the human food chain as we do not know which tissues in affected cattle contain these new forms of BSE prion infectivity. It is essential therefore, to verify if the current SRM control measures are sufficient to prevent the new forms of BSE prions from entering the human food chain. Many more mice will be used in proposed food safety research programmes in order to measure prion infectivity levels in an extensive range of samples from cattle infected with new forms of BSE. In their entirety, these mouse prion bioassays will use more than 100,000 mice. It is vital to apply the 3Rs principles to this intended research programme by reducing and replacing, where possible, this large number of experimental mice with a prion bioassay that uses a less sentient host.In our laboratory we model prion disease in the fruit fly Drosophila melanogaster because they are relatively easy and economical to work with, and are a widely accepted ethical alternative to higher organisms including mice. We have developed a Drosophila-based prion bioassay that can detect BSE prion infectivity in order to provide an alternative to mice for the bioassay of bovine prions, and reduce the use of mice, and other vertebrate animal species, to measure prion infectivity in general. To do so, we have introduced a gene into the flies that allows them to produce the protein that aggregates in the brain of animals with prion disease. We already know that this mammalian protein will aggregate and cause prion disease in transgenic Drosophila when flies are fed infectious prions. What is equally significant is that the response by the transgenic flies to prion-infected material is evident within a few weeks following exposure to prion material. We can now develop a faster, more versatile and more sensitive bioassay to detect BSE prion infectivity than currently exists. We aim to transfer this know-how to the APHA, a main user and internationally recognised reference laboratory of the mouse prion bioassay. Our Skills and Knowledge Transfer to the APHA will provide the best opportunity to ensure world-wide impact of our new Drosophila-based prion bioassay.