M. bovis BCG preparation, infection and CFU

M. bovis BCG Pasteur strain 1172 P2 (Pasteur Institute, Paris, France) was used and grown to the log phase in 7H9 middlebrook medium supplemented with oleic albumin dextrose catalase (OADC). The bacteria were then harvested, washed, and frozen at −80 °C in PBS plus 10% of glycerol. Bacterial load was determined by plating serial 10-fold dilutions on 7H10 middlebrook agar (supplemented with OADC) and counting colonies after incubation for at least 3 weeks³².

Mice were infected intravenously (i.v) with 1 × 10⁷ living M. bovis BCG strain Pasteur in 100 µL of saline, as previously reported¹⁸. Mice were monitored twice a week and sacrified 15 days after infection. Groups of uninfected or naïve mice of the different genotypes were killed and liver cells analyzed similarly to infected mice.

Colony forming units (CFU) were performed in liver tissues at 2-weeks post-infection as previously reported¹⁶.