Leptospiral Selection, Growth, and Virulence in Synthetic Medium

Abstract

The need for protein in leptospiral cultural medium may be circumvented by the use of strains which tolerate the lytic activity of polyoxyethylene sorbitan monooleate (Tween 80), a relatively nonlytic source of essential fatty acids. In an otherwise adequate medium, the primary function of a serum protein (bovine albumin fraction V) in the cultivation of Leptospira pomona was detoxification of fatty acids. Treatment to destroy or block end groups (amino, sulfhydryl, or hydroxyl) did not impair this function, but, after treatment with trypsin, albumin was inactive. Synthetic and derived peptides or polyvinylpyrrolidone did not substitute for albumin. L. pomona grew in medium with surface tension values of 44 to 58 dynes/cm'; after growth, the values were increased slightly (5 to 8). The growth responses did not correlate with the surface tension of the medium, but they were in proportion to the concentration of Tween 80. Of six strains of L. pomona, five were transferred from medium containing rabbit serum and were subcultured in Tween synthetic medium (TSM) containing low, nonlytic concentrations (0.002%) of Tween 80. The poor antigenicity of L. pomona in carbon-limited TSM was associated with a deficiency of those carbonaceous cellular components which were extractable with 50% ethyl alcohol. After as few as four subcultures in TSM, L. pomona tolerated higher concentrations of Tween 80 (0.06% was optimal; MTSM). If grown on a shaker, the rate and amount of growth and the antigenicity of L. pomona in MTSM equaled that in medium supplemented with rabbit serum. After cultivation in MTSM, all of the five strains were avirulent when administered to hamsters, guinea pigs, and swine. They were still avirulent after three subcultures in complex media or after two serial passages in hamsters.

Growth of Pathogenic Leptospira in Chemically Defined Media

“…Growth of leptospires on synthetic media has obvious advantages over growth on protein-rich media, for preparation of vaccines and diagnostic antigens. However, the use of chemically defined media for these purposes should be carefully evaluated in view of recent reports on induced selective changes in antigenic composition by growth in serum-free media (18,19).…”

Growth Requirements of Pathogenic Leptospira

“…A mutant of L. pomona, resistant to high levels of free fatty acids, was employed to avoid the issue of fatty acid toxicity. Such mutants had been used by other authors for nutritional studies (25,26). Supernatant fluids from stationary phase cultures of both B-16 and the L. pomona mutant were adjusted to pH 7.4 with NaOH, heated to 56 C for 30 min, filter sterilized, and distributed into culture tubes.…”

Biological Effects of Leptospiral Lipids

“…Extraction and purification oflipids. Virulent Leptospira pomona strain DM-2 was grown to maximal cellular densities in medium supplemented with bovine albumin, whereas the avirulent variant was grown in a synthetic medium as previously described (10). When the cultures reached maximal turbidity (determined by nephelometry), they were harvested by centrifugation (10,000 X g for 30 min) at 5 to 7 C. After the leptospires were resuspended in 0.02 M phosphate buffer solution, pH 7.3 (PBS), they were sedimented again, lyophilized, and stored in an atmosphere of nitrogen at -70 C, until used.…”