Mycoplasmas.

The UAB CT strain of M. pulmonis was used in all experiments (70). Stock cultures were grown as previously described (71). On the day of infection, an aliquot of M. pulmonis stock was diluted in prewarmed Hayflick's broth to achieve a final concentration of 2 × 10⁸ to 3 × 10⁸ CFU. Hayflick’s medium was prepared for both broth and agar as follows: ultrapure water (196.25 ml), 1% (wt/vol) phenol red (Sigma-Aldrich Co., Buchs, Switzerland), 5.63 g PPLO broth base (BD Biosciences, San Jose, CA), 0.05 g equine sperm DNA (Sigma-Aldrich Co.), and (for agar only) 2.5 g noble agar (Sigma-Aldrich). Medium was autoclaved on a liquid cycle for 20 min, after which 65 μl Cefobid, 250 mg/ml (Sigma-Aldrich Co.), 2.5 ml Bacto-dextrose, 50% (wt/vol) (BD Biosciences), and 50 ml heat-inactivated equine serum (Thermo-Fisher Scientific, Waltham, MA) were added aseptically. Hayflick’s agar plates were prepared, and both agar and liquid broth were cooled to room temperature prior to being stored at 4°C. The diluted mycoplasmas were incubated at 37°C for 1 h before we intranasally (i.n.) infected the anesthetized mice with 20 μl of diluted mycoplasmas containing 2 × 10⁵ to 3 × 10⁵ CFU.