Inoculation and isolation of C. muridarum from genital tract swabs

To regulate estrous cycles, all mice were injected subcutaneously with 2.5 mg of progesterone in 100 μL of phosphate-buffered saline (PBS) on day 17 of the 24-day stressing period. On day 24, stressed and non-stressed mice were inoculated intravaginally with 10⁵ IFU of C. muridarum in a volume of 30 μL of PBS while under Ketamine-Xylazine induced anesthesia to minimize discomfort. The course of infection was monitored by cervicovaginal swabbing at 3-day intervals for the first 42 days of the primary course of infection. Chlamydia muridarum was recovered from swabs by staining infected monolayers of McCoy tissue culture with fluorescein isothiocyanate labeled genus-specific anti-chlamydial antibodies purchased from Fisher Scientific (Pittsburgh, PA). Inclusion bodies in 10 fields/well were visualized, imaged, and counted under a fluorescent Mitic AE31E microscope (Carlsbad, CA) and inclusion-forming units per mL (IFU/mL) was determined as previously described in our lab [49].