Quantitative M. ovipneumoniae Inoculum Preparation

M. ovipneumoniae inoculum was grown from a previously described nasal wash isolate (MSU NW-4) and expanded exactly as described in the original publication [11 (link)]. Briefly, inoculum was grown at 37 °C in Mycoplasma broth under microaerophilic conditions. On the day of inoculation, media was removed (10,000× g, 10 min, 4 °C) and culture was resuspended in sterile FACS buffer (2% fetal bovine serum and 0.1% sodium azide in DPBS) for counting by flow cytometry [67 (link)]. Cells were subsequently stained with SYBR Safe DNA Gel Stain (Invitrogen, Carlsbad, CA, USA) at 4 °C for 30 min and Absolute Counting Beads (Invitrogen, Carlsbad, CA, USA) were added to solution immediately prior to analysis using an Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).