Ureaplasmas isolated from the human genital tract and from the genital and respiratory tracts of cattle were grown in association with organ cultures of bovine oviduct (uterine tube). All strains of unreaplasmas multiplied in organ cultures, stopped ciliary activity, and caused histological lesions. Most strains grew well, and 10(8) to 10(9) color-changing units were determined 18 to 144 h after inoculation. Twenty-four to 144 h after inoculation with unreaplasmas, ciliostasis was complete. Ciliostasis was also caused by additions of nonviable cultures at pH 8.8 (or adjusted to 7.4) or washed disrupted cells (100 mug of protein/ml); it occurred in 48 to 96 h. The cilia-stopping effect of nonviable cultures was diminished by heating (56 C for 30 min) and was abolished by boiling. When added to fresh medium in amounts exceeding 25%, nonviable unreaplasmal cultures completely inhibited ureaplasmal growth. By light, scanning, and transmission electron microscopy, cilia-stopping effect was correlated with collapse and sloughing of the cilia (the initial lesion was "bent" cilia), with bulging and vacuolization of secretory and ciliated cells, and finally with disorganization of the epithelium, necrosis, and desquamation.
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.
No abstract
Stalheim , O. H. V. (University of Wisconsin, Madison) and J. B. Wilson . Leptospiral colonial morphology. J. Bacteriol. 86: 482–489. 1963.—A sequence of apparent colonial types was observed with colonies of Leptospira pomona, L. canicola, L. icterohaemorrhagiae , and L. grippotyphosa in agar medium. Although some colonies of these serotypes had a different appearance initially, they eventually developed the mature or final appearance characteristic of the serotype. Colonies of freshly isolated, virulent cultures of L. pomona, L. canicola , and L. icterohaemorrhagiae were similar in appearance to colonies of avirulent strains of the same serotype. Additional studies of three stable and distinct colonial types of a laboratory strain of L. autumnalis revealed no differences in antigenicity, catalase activity, or mouse infectivity; however, differences in susceptibility to lysis by oleic acid were found. Although the colonial variants were stable during several in vitro variations, including growth in the presence of homologous antiserum and mutation to growth in a chemically characterized medium, rapid dissociation in vivo was found.
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