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.