Zebrafish embryos as a model to study bacterial virulence.

In recent years the zebrafish has gained enormous attention in infection biology, and many protocols have been developed to study interaction of both human and fish pathogens, including viruses, fungi, and bacteria, with the host. Especially the extraordinary possibilities for live imaging of disease processes in the transparent embryos using fluorescent bacteria and cell-specific reporter fish combined with gene knockdown, transcriptome, and genetic studies have dramatically advanced our understanding of disease mechanisms. The zebrafish embryo is amenable to study virulence of both extracellular and facultative intracellular pathogens introduced through the technique of microinjection. Several protocols have been published that address the different sites of injection, antisense strategies, imaging, and production of transgenic fish in detail. Here we describe a protocol to study the virulence profiles, ranging from acute fatal to persistent, of bacteria belonging to the Burkholderia cepacia complex. This standard operating protocol combines simple survival assays, analysis of bacterial kinetics, analysis of the early innate immune response with qRT-PCR, and the use of transgenic reporter fish to study interactions with host phagocytes, and is also applicable to other pathogens.