Using Zebrafish To Study Candida albicans-Mediated Systemic Candidiasis in vivo.

Candida albicans is a common oral opportunistic pathogen that causes thrush and rarely systemic candidiasis, a nosocomial infection which involves the haematogenous spread of Candida to multiple organs and is associated with high mortality. To cause tissue damage C.albicans must bind to the endothelium of blood vessels, leave the circulation and invade tissues. However, little is known about the mechanisms involved in these processes. Objectives: To use transparent embryonic zebrafish (Danio rerio) as an in vivo infection model in conjunction with an in vitro flow adhesion assay to study how C.albicans bind to the endothelium, leave the circulation and invade tissues. Methods: Suspensions of viable or treated C.ablicans were flowed over monolayers of endothelial cells and adherent Candida counted. Zebrafish were injected with C.albicans and mortality determined after 18h. Time-lapse confocal microscopy using fluorescent C.albicans and blood vessels was performed to image dissemination. Results: Viable C.albicans bound to endothelium significantly (p<0.05) more than heat-killed Candida but at a similar frequency to formalin-fixed Candida under conditions of flow in vitro. In vivo, only viable Candida caused disseminated infection with a maximal dose-dependent mortality of around 40% after 18h. Wild-type C.albicans (CAF-2) was significantly (p<0.01) more lethal compared to a mutant strain (SSY50-B) locked in the yeast form. Real-time microscopy showed that adherence to the endothelium in vivo is rapid. Some yeast forms of C.albicans were observed binding to and transmigrating across the endothelium into tissues where they remained as yeast, whereas others bound to the endothelium and underwent intravascular hyphal transformation before ramifying the tissues causing organ damage. Conclusion: Zebrafish are a useful tool to study systemic candidiasis in vivo. Viability and transition from yeast to hyphae is crucial for virulence. Hyphal formation predominately occurs at the site of adhesion to the endothelium before invading tissues and causing damage.