Mucorales Virulence Assay in Mice

The murine infection assays for Mucorales virulence were performed using OF-1 male mice weighing 30g (Charles River, Barcelona, Spain) [13 (link),18 (link),27 (link)]. The mice were immunosuppressed with the administration of cyclophosphamide (200 mg/kg of body weight) via intraperitoneal injection, 2 days prior to infection and once every 5 days thereafter. Groups of 10 mice were challenged with 1x10⁶ spores of the strains R7B, NRRL3631, MU419, and MU412. The infections were performed intravenously via retroorbital injection following the protocol described by Chang et al. 2019 [5 (link)]. Before the injection, mice were anesthetized by inhalation of isoflurane, and then the animals were visually monitored while recovering from the anesthesia. Mice were housed under established conditions with free food and autoclaved water. The animal welfare was checked twice daily for 20 days, and those following the criteria for discomfort were euthanized by CO₂ inhalation. The significance of survival rates was quantified using the Kaplan-Meier estimator (GraphPad Prism). Differences were considered statistically significant at a P ≤ 0.05 in a Mantel-Cox test.