Use of sodium deoxycholate to extract cell wall components of virulent Mycobacterium bovis

Mycobacterium bovis ATCC No. 19210 was grown on Middlebrook 7H-10 medium with pyruvate. Cells were harvested, and extracts were prepared, using 2% sodium deoxycholate (DOC) and 0.003M EDTA (in 0.1M Tris-HCl, 0.15M NaCl with 0.02% sodium azide [pH 8.4]). Phenylmethylsulfonyl fluoride was added, and the cells were extracted for 48 hours at 4 C. Cells were removed by centrifugation at 10,000 X g for 30 minutes. The supernatant was filter sterilized and separated into 2 fractions (peak A and peak B) by size-exclusion chromatography. The nonfractionated DOC extract and DOC peak A elicited delayed-type hypersensitivity responses at each of the protein concentrations tested (0.5, 1.5, and 4.5 micrograms) in M bovis-sensitized guinea pigs; responses were not detected, using DOC peak B. Significant differences were detected for each of the antigens when enzyme-linked immunosorbent assay values were compared, using sera from cattle before and 10 months after they were exposed to M bovis (P less than 0.01).