2.2. Preparation of Mycoplasma Capricolum Subsp. Capripneumoniae (Mccp) Antigen

The Mycoplasma capricolum subspecies capripneumoniae (Mccp) antigen was produced from the Mccp F-38 vaccine strain [12] from the AU-PANVAC (Debrezeit, Ethiopia) vaccine seed repository as per the protocol previously described [13] with slight modifications. Briefly, the CCPP vaccine seed was reconstituted in 5 mL of pleuropneumonia-like organisms (PPLO) broth (Difco, Sparks, MD, USA) without serum and filtered through a 0.45-µm syringe filter. The flow-through was inoculated in PPLO media supplemented with 20% heat inactivated horse serum (GIBCO, Waltham, MA, USA) and 10% yeast extract (Difco, Sparks, MD, USA). The inoculated media was incubated at 37 °C for 10–14 days without shaking and with continuous pH monitoring. When the pH reached between 6.65 and 6.90, the culture was again inoculated into fresh PPLO medium at a ratio of 1/10 culture to fresh PPLO medium and incubated at 37 °C for up to 10 days until the desired turbidity and pH were observed. The CCPP antigen was prepared by centrifuging the final culture at 10,000× g for 30 min at 4 °C. The supernatant was discarded and the pellet washed three times with sterile phosphate buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA) by centrifuging at the same speed for 30 min to remove non-specific proteins from the culture media. The pellet was re-suspended in an adequate volume of sterile PBS (approximately 30 mL) and lysed with 0.1% Triton-X (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4 °C. The antigen was finally titrated by chessboard titration as previously described [14,15,16,17] with the Mccp25-horse radish peroxidase (HRP) conjugate (AU-PANVAC, Debre-Zeit, Ethiopia) to determine the optimal dilutions of both the antigen and conjugate for use in setting up CCPP b-ELISA.