Infection of zebrafish with natural pathogen Mycobacterium marinum is a useful surrogate for studying the human granulomatous inflammatory response to infection by Mycobacterium tuberculosis. The adaptive immune system of the adult stage zebrafish offers an advance on the commonly used embryo infection model as adult zebrafish form granulomas with striking similarities to human-M. tuberculosis granulomas. Here, we present workflows to perform high content analyses of granulomas in adult zebrafish infected with M. marinum by cryosectioning to take advantage of strong endogenous transgenic fluorescence adapted from common zebrafish embryo infection tools. Specific guides to classifying granuloma necrosis and organisation, quantifying bacterial burden and leukocyte infiltration of granulomas, visualizing foam cell formation, analysing extracellular matrix remodelling and granuloma fibrosis are also provided. We use these methods to characterize neutrophil recruitment to M. marinum granulomas across time and find an inverse relation to granuloma necrosis suggesting granuloma necrosis is not a marker of immunopathology in the natural infection system of the adult zebrafish-M. marinum pairing. The methods can be easily translated to studying the zebrafish adaptive immune response to other chronic and granuloma-forming pathogens.

斑马鱼感染天然病原的海洋分支杆菌是研究人类肉芽肿性炎症反应对结核分枝杆菌感染的一个有用的替代品。成年斑马鱼的适应性免疫系统在常用的胚胎感染模型上取得了进展，因为成年斑马鱼形成肉芽肿与人类结核分枝杆菌肉芽肿具有惊人的相似性。在这里，我们提出了工作流程，以对感染了海藻支原体的成年斑马鱼进行肉芽肿的高含量分析。通过冷冻切片，利用从常见斑马鱼胚胎感染工具改编而成的强大的内源性转基因荧光。还提供了对肉芽肿坏死和组织进行分类，定量肉芽肿的细菌负荷和白细胞浸润，可视化泡沫细胞形成，分析细胞外基质重塑和肉芽肿纤维化的具体指南。我们使用这些方法来表征嗜中性白细胞跨时间募集到M. marinum肉芽肿，并发现与肉芽肿坏死成反比关系，这表明肉芽肿坏死不是成年斑马鱼-M. marinum自然感染系统中免疫病理学的标志。配对。该方法可以轻松转换为研究斑马鱼对其他慢性和肉芽肿病原体的适应性免疫反应。