Experimental protothecosis in a murine model.

Prototheca spp. are unicellular, yeast-like microalgae, and the only plant lineage known to cause opportunistic infections in vertebrates, including humans. The aim of the study was to establish a comprehensive murine model of protothecosis to systematically investigate the influence of Prototheca species, inoculum dose, infection route, and host immune status on disease development and severity. Three pathogenic (P. wickerhamii, P. bovis, P. ciferrii) and one saprophytic (P. stagnora) species were used to infect immunocompetent or athymic mice. A total of 324 animals were split into 54 groups according to inoculum size (10⁶ or 10⁷) and infection route (subcutaneous, intramammary, or intraperitoneal). Six weeks post-infection, mice were euthanized, and organs were collected for microbiological and histopathological analyses. All four Prototheca species produced infection in mice, yet the infection potential differed considerably between the species. P. ciferrii exhibited the highest infection rate (61.1%), followed by P. bovis (45.8%) and P. wickerhamii (31.9%), whereas P. stagnora was the least virulent (11.1%). Athymic mice were markedly more susceptible compared to wt mice (45.1% vs. 29.9%) and more prone to develop multifocal infections. Higher inocula (10⁷) increased infection yield, while the inoculation route influenced the infection site but not its severity. In the cytokine profile, IL-10 and TNF-α were most prominently elevated, with significantly higher levels in wt than in athymic mice. This study highlights three hallmarks of protothecal disease: a species-specific infection pattern, chronic, asymptomatic infection as the clinical manifestation, and the essential role of host immunity in determining disease trajectory and severity.