Figure 9.

Recovered yeasts induces looser granulomas, is more virulent and disseminate more than control yeasts. Groups of four C57BL/6 mice were infected by i.t. route with 1 × 10⁶ control P. brasiliensis yeasts or recovered yeasts contained in 50 µL of PBS. After eight and weeks of infection, the animals were euthanized and the lungs removed, which were stored in 10% formaldehyde at 4 °C. Sections of 5 µm were stained with Hematoxylin-Eosin (H&E; pink) for analysis of lesions and stained with Grocott (green) for fungal evaluation (A). The anatomy of the lesion was analyzed according to size, morphology, and presence of fungal cells. The image and the lesion area calculation were taken using the Leica DM750 microscope and the Leica Application Suite V4.8 software. Statistical analysis was performed using one-way ANOVA. Bars represent means ± standard error of lesion area (µm²) of groups of three mice (B). For the survival analysis (C) groups of six C57Bl/6 WT mice were infected via i.t. with 1x10⁶ yeasts extracted from granulomatous lesions or control yeasts to verify the virulence of freshly extracted granuloma yeasts. Deaths were recorded daily. The survival curve used the Log-rank test (Mantel-Cox) to compare the two groups. For the CFU assay (D) groups of three to four C57BL/6 mice were infected by the i.t. route with 1 × 10⁶ P. brasiliensis yeasts contained in 50 µL of PBS. After eight weeks of infection, the lungs, liver, and spleen were removed and macerated. After that, the samples were centrifuged to obtain the fungus and inoculated in plates containing BHI medium for subsequent counting of CFU. Bars represent means ± standard deviation of log10 or total yeast number. Values were considered significant when **p < 0.01; ***p<0.001; ****p<0.0001.