2.2. Bacterial strains and growth conditions
B. canis strain RM6/66 (ATCC 23365) was grown in glycerol-dextrose agar and incubated in aerobic atmosphere for 48-72 h. Bacteria were collected, resuspended in sterile deionized water, heat-inactivated at 60 °C for 2 h and centrifuged at 3500 g for 30 min. The pellet was then washed 3 times with deionized water, dissolved in 0.2 M Tris–maleate, pH 9.0, at ratio 1:5, mixed for 2 h at room temperature (RT) with stirring and then stored at 5 ± 3 °C for 90 days before use. The antigen was then titrated and further diluted with 0.2 M Tris–maleate, pH 9.0, to obtain a ready-to-use antigen.