Penicillin G

The HB0801 strain was isolated from the lesioned lung of an infected beef cattle from Yingcheng city in Hubei province, China by this laboratory [4] and stored at the China Center for Type Culture Collection (CCTCC # M2010040) at Wuhan University, Wuhan, China. HB1007 was isolated from milk of a dairy cow with mastitis in Hubei in 2010.
The strain was cultured on a pleuropneumonia-like organisms (PPLO) agar plate at 37°C, in a 5% CO₂ atmosphere for 3 days or in PPLO broth (2.5 g glucose, 10.5 g PPLO, 2.5 g yeast, 50 mL donor equine serum (Thermo Fisher Scientific, Waltham, MA, USA), 5 mL10% arginine, 5 mL 10×MEM, 5 mL of 80,000 IU/mL penicillin-G, and 500 µL 1% phenol red) at 37°C for 3 days on an orbital shaker.

MGIA were performed as previously described (22 (link)–24 (link)). Briefly, fifteen milliliters of peripheral blood were drawn from the tail vein of healthy Holstein cows into heparinized Vacutainer tubes (Becton, Dickinson and Company, Sparks, MD, USA) and diluted 1:2 in Hanks balanced salt solution (HBSS). Leucosep tubes were filled with 15 ml of Ficoll-Paque (1.084 g/cm³) (GE Healthcare, Uppsala, Sweden) and centrifuged at 1,000 rpm for 30 seconds at room temperature. Subsequently, the diluted blood was overlaid on the top of the Ficoll-Paque and centrifuged at 800 g for 15 minutes at room temperature. The plasma layer was removed and the cell interphase containing peripheral blood mononuclear cells (PBMCs) was collected and transferred to a clean tube. PBMCs were washed twice in HBSS and centrifuged at 400 g for 10 minutes to remove platelets. Supernatants were aspirated and the purified PBMCs were resuspended in RPMI-1640 supplemented with 20 mM L-glutamine, 10% heat-inactivated bovine serum (Lonza, Spain), 100 U ml-1 penicillin G, and 100 mg ml-1 streptomycin sulfate (Lonza, Spain). PBMCs were cultured at a concentration of 1 x 10⁶ cells/ml into 24-well plates and incubated at 37°C in a humidified 5% CO₂ incubator for 2 h. Non-adherent cells were removed by washing, and adherent cells were incubated in fresh medium for 7 days at 37°C to allow differentiation to MDMs. Differentiated MDMs were inoculated in triplicate with a single-cell suspension of MAP K10 strain at a multiplicity of infection (MOI) of 10:1 (bacteria:cells). After 2 h, the supernatant was removed, and the cells were washed twice with HBSS to remove extracellular bacteria. Infected MDMs were lysed at this time (2 h p. i.) or were cultured at 37°C for 7 days in fresh medium. At each time point, the supernatant was aspirated and infected MDMs were lysed by vigorous pipetting with 0.5 ml of 0.1% Triton X-100 (Sigma-Aldrich) in sterile water for 10 min.

The human keratinocyte cell line HaCaT was obtained from the China Center for Type Culture Collection (0106) and the mouse keratinocyte cell line PAM212 was purchased from the Mingzhoubio (MZ-2610). HaCaT and PAM212 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific, C11995500BT) and Roswell Park Memorial Institute 1640 medium (RPMI-1640; Gibco, 11875119), respectively. And both mediums were supplemented with 10% (v:v) fetal bovine serum (FBS; Thermo Fisher Scientific, 10099141), 100 U/mL penicillin G, and 0.1 mg/mL streptomycin sulfate (Thermo Fisher Scientific, 15140122). The cell culture conditions were 37 °C and 5% CO₂. All cells were found to be free from mycoplasma contamination.