2.2. Treponema spp. Inoculum

Treponema spp. isolated from bovine DD cases were used to inoculate mice. Glycerol stocks were grown at 37 °C for 7 days in anaerobic novel oral spirochete media (NOS) containing 10% bovine fetal serum and 10% rabbit serum, supplemented with rifampicin and enrofloxacin at 5 μg/mL, as previously described [36]. All cultures were incubated in an anaerobic chamber (85% N₂, 5% CO₂ and 10% H₂). Bacterial concentration, active motility and the absence of spherical bodies were determined by counting spiral forms under a dark-field microscope, using a Petroff-Hausser chamber. For inoculum preparation, cultures were washed with pre-reduced PBS, and final concentrations of 1 × 10⁹ or 2 × 10⁹ spirochetes/mL were obtained. The inoculum (always 500 μL) was injected subcutaneously along the dorsal midline, between the scapulae. To ensure cell viability, all inoculations were performed within 15 min after inoculum preparation.