Virulence Assay of Fungal Mutants in Immunosuppressed Mice

Male OF-1 mice weighing 30 g and two months old (Charles River, Barcelona, Spain) [30 (link),32 (link),33 (link)] were used as animal models for virulence assays. The mice were immunosuppressed by intraperitoneal administration of cyclophosphamide (200 mg/kg of body weight), 2 days prior to infection and then once every 5 days. Eight-mouse groups were challenged intravenously by retroorbital injection of 1 × 10⁶ spores [32 (link)]. The assay was repeated twice using two independent mutants of mcwc-1a (MU242 and MU243). During this procedure, the mice were anesthetized using isoflurane via inhalation and monitored until they recovered from the anesthesia. Mice were housed under established conditions with free food and autoclaved water. The animal welfare was checked twice a day for 20 days, and those mice following the criteria for discomfort were euthanized by CO₂ inhalation. Survival rates during the time were plotted in a Kaplan–Meier curve (Graph Pad Prism), and differences were considered statistically significant with a p-value ≤ 0.05 in a Mantel–Cox test.