A bovine alveolus model to replace cattle in the study of host-pathogen interactions in bovine tuberculosis

牛分枝杆菌（M. bovis）是牛结核病（BTB）的病原体，感染牲畜会造成严重的社会经济后果，并对动物健康产生影响。除了对养牛业社区和政府造成经济和情感上的影响外，这种疾病也是发展中国家人类和牲畜健康的主要风险。事实证明，在英国和爱尔兰，对结核病的控制存在问题。在没有改进控制的情况下，预计未来十年英国的经济负担将达到10亿英镑。应对结核分枝杆菌病需要更深入地了解宿主-病原体相互作用，否则不太可能在开发有效的疾病干预工具方面取得任何重大突破。牛支原体感染的主要途径是通过吸入传染性气溶胶。吸入后，牛分枝杆菌到达肺组织，尤其是肺泡。接下来发生的事情决定了宿主是否继续获得结核病，或者宿主是否应对这种威胁。对人类结核病的研究表明，肺泡内的上皮细胞远不止是对病原体的简单物理屏障。事实上，结核分枝杆菌能够穿透这些细胞，进入更深的组织，同时避免被免疫系统消灭。反过来，上皮细胞检测到分枝杆菌的存在，通过产生参与抗菌活性、炎症和病理的分子来做出反应。由于肺上皮对结核病的早期反应至关重要，因此需要更好地了解当毒力分枝杆菌到达牛肺泡时发生的早期事件和相互作用。目前还没有模型来进行这些研究，并且感染早期阶段的关键事件难以在活体动物中进行研究。如果我们要获得关于如何增强宿主对感染的抵抗力（例如通过接种疫苗）的新见解，了解在肺泡内发生的早期事件至关重要。为了满足这一需求，我们将开发一种牛肺泡组织培养模型来研究牛分枝杆菌与牛宿主的相互作用。该模型代表了一种非动物替代方法，通过不需要用分枝杆菌感染牛来回答结核病发病机制的基本问题，从而研究BTB的发病机制，并为没有动物设施的研究人员提供了牛的有效替代品。该模型的简单性使其优于使用整个动物，并且适合回答需要在感染后几分钟内收集数据或需要进行时间过程研究的问题。这将成为科学界可用的新工具。它的使用不仅局限于BTB，而且将适用于一般牛呼吸道感染的研究，许多具有全球重要性的牛呼吸道感染，例如牛呼吸道疾病（BRD）。针对结核分枝杆菌开发的疫苗可产生特定宿主反应，这将是目前情况的重大进展，因为疫苗必须在牛身上进行经验试验，以评估其效力。该项目的一个具体目标是确定模型中卡介苗/牛乳杆菌和宿主细胞的行为是否与我们储存血细胞进行评估的牛攻击研究中看到的保护功效相关。在我们的模型中确定与整个动物的疫苗效力相关的读出可能是筛选候选疫苗的基础，而不需要用牛分枝杆菌挑战牛。这将减少动物实验的严重度和持续时间，并显著降低其成本。我们假设疫苗介导的抗BTB保护的一个重要方面是在肺泡内宿主-病原体相互作用水平上表达的。

Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis (BTB) and infects livestock with severe socio-economic consequences and an impact on animal health. As well as the financial and emotional impact BTB has on the cattle farming community and government, the disease is a major risk to human and livestock health in developing countries. The control of BTB has proved problematic in Great Britain and Ireland. In the absence of improved control the projected economic burden to GB over the next decade is predicted to be £1 billion.Tackling BTB requires deeper insights into host-pathogen interactions otherwise it is unlikely any major breakthroughs in developing effective tools for disease intervention will occur. The principle route of infection with M. bovis is via inhalation of infectious aerosols. On inhalation, M. bovis reaches the lung tissues, especially the alveolus. What happens next determines whether the host goes on to acquire TB, or whether the host deals with the threat. Research in human TB shows that epithelial cells lining the alveolus are far more than a simple physical barrier to pathogens. Indeed, M. tuberculosis is able to penetrate these cells and gain access to the deeper tissues whilst evading elimination by the immune system. In turn, the epithelium detects the presence of mycobacteria, responding by producing molecules involved in antimicrobial activity, inflammation, and pathology. As the lung epithelium is central to early response to TB, better understanding of the early events and interactions that occur when virulent mycobacteria arrive in the bovine alveolus are needed. No model currently exists with which to conduct these studies and the crucial events in the earlier stages of infection are intractable for study in the live animal. Understanding early events that are played out within the alveolus is critical if we are to gain new insights in how to enhance host resistance to infection, e.g. through vaccination. In response to this need, we shall develop a tissue culture model of the bovine alveolus with which to study the interaction of M. bovis with the bovine host. The model represents a non-animal alternative with which to study of the pathogenesis of BTB by removing the need to infect cattle with mycobacteria to answer fundamental questions in TB pathogenesis and provide a valid substitute for cattle that can be used by researchers without access to animal facilities. The simplicity of the model make it preferable over the use of the whole animal, and for answering questions that require data to be gathered within minutes of infection or where time course studies are required. This will be a new tool available to the scientific community. Its use is not confined to BTB, but would be applicable to the study of respiratory infections of cattle in general, many of global importance, such as bovine respiratory disease (BRD). Vaccines against BTB developed to generate a specific host response would be a significant advance on the current state of affairs where vaccines must be tested empirically in cattle to evaluate their efficacy. A specific objective of this project will determine whether the behaviour of BCG / M. bovis and host cells in the model correlates with protective efficacy seen in cattle challenge studies from which we have stored blood cells to evaluate. Identifying a read-out in our model that is related to vaccine efficacy in the whole animal could be a basis of screening vaccine candidates without the need to challenge cattle with M. bovis. This would reduce the severity and duration of animal experiments, as well as significantly reduce their cost. We hypothesise that a significant aspect of vaccine-mediated protection against BTB is expressed at the level of host-pathogen interactions within the alveolus.