Sterile defibrinated sheep blood

Trueperella pyogenes isolates were obtained from cows suffering from endometritis and preserved in our laboratory. Isolates were classified as msrA-positive or msrA-negative strains by PCR amplification of the msrA gene in our previous study. Eight msrA-positive T. pyogenes isolates (HC03-1, HC-H03-3, HC-H02-2, BM-07-1, BM-H06-3, BM-H11-1, BM-H01-1 and RY04-2) and two msrA-negative T. pyogenes isolates (HC-H10 and RY14-3) were included in this study. T. pyogenes isolates were grown on Mueller–Hinton agar (MH(A), AOBOX, Beijing, China) supplemented with 5% sterile defibrinated sheep blood (Solarbio, Beijing, China) for 48 h at 37 °C in 5% CO₂. Before assays, 3–5 colonies were inoculated into nutrient broth (NB, AOBOX, Beijing, China) containing 8% foetal bovine serum (FBS, Gibco, USA) and cultured in tubes for 24 h at 37 °C with shaking at 180 rpm to obtain a bacterial suspension that reached the logarithmic phase.

Fn (Fn standard strain ATCC 25586, donated by the University of Louisville, USA) was cultured in a brain–heart infusion medium (Solarbio) containing 5% sterile defibrinated sheep blood (Solarbio), 1% haem chloride (Solarbio), and 0.1% vitamin K1 (Solarbio) at 37 °C in an anaerobic workstation (COY) containing 85% N₂, 10% H₂, and 5% CO₂. The purity of Fn was detected by gram staining and culture on Columbia blood agar plates [12 (link)]. The Fn bacterial solution (OD600 = 1–2) with better viability was selected. When the cell confluence was 60–70%, Fn was added into the cell culture medium at an MOI of 10. Different times of infection (24 or 48 h) were used. A follow-up experiment was performed.