High content analysis of granuloma histology and neutrophilic inflammation in adult zebrafish infected with Mycobacterium marinum.

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.