Murine models of cryptococcosis.

Female C57BL/6 mice (20 to 22 g) were obtained from the Animal Resource Centre, Floreat Park, Western Australia, Australia. Cryptococcus strains were grown overnight in YPD broth, washed twice with phosphate-buffered saline (PBS), and resuspended in PBS. Prior to both intranasal and intravenous infection, mice were transiently anesthetized by inhalation of 3% isoflurane. For intravenous infection, fungal suspensions were injected via the retro-orbital plexus (5,000 cells in 200 μl PBS; seven mice in each infection group). For intranasal infection, 5 × 10⁴ (survival study with organ burden at the time of death) or 1 × 10⁵ (assessment of immune cell infiltrate) cells in 20 µl of PBS were delivered into the nares using a pipette as described previously (41). For the survival assays, 10 mice in each group were monitored daily and euthanized by CO₂ asphyxiation when they had lost in excess of 20% of their preinfection weight or if showing debilitating symptoms of infection, i.e., loss of appetite, moribund appearance, or labored breathing. Differences in survival were determined using a Kaplan-Meier log rank test. For blood sampling, mice were anesthetized by inhalation of 3% isoflurane, and blood was collected from the retro-orbital plexus. For organ burden analysis, mice were sacrificed by CO₂ inhalation on designated days postinfection or when they had lost 20% of the preinfection weight. Lungs and brain were then removed, weighed, and mechanically disrupted in 2 ml sterile PBS using a BeadBug (Benchmark Scientific). Serial dilutions of the organ samples were plated onto SDA agar plates and incubated at 30°C for 2 days. Colony counts were performed and adjusted to reflect the total number of CFU per gram of tissue.