Further studies on replication of virulent Treponema pallidum in tissue cultures of Sf1Ep cells

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The in vitro interaction between Treponema pallidum and SflEp cells during treponemal replication was investigated by using transmission electron microscopy. The SflEp cells grown on Teflon-treated cover slips after 12 days of cocultivation were fixed in situ, overlaid with agar, embedded, and vertically sectioned. Large numbers of treponemes were found extracellularly not only at the upper cell surfaces but also in the narrow spaces between the cells and between the cells and the cover slips. These narrow spaces supported treponemal growth and survival, as did those at the upper cell surfaces. Although few in number, organisms were also seen in cell vacuoles either surrounded by a membrane or free in the cytoplasm. Some extracellular treponemes attached to host cells by body spirals or the terminal end and formed electron-dense layers at attachment sites. Some treponemes were often surrounded with amorphous, extracellular material which appeared to "connect" them to host cell surface. After 12 days of cocultivation, host cells showed excessive vacuolation and appeared to be damaged. This did not seem to be due to treponemal infection alone, because cells from uninfected cultures also showed similar vacuolation.

mentioning

confidence: 99%

“…Passage levels utilized in these experiments ranged from 73 to 85. The tissue cultures were infected with T. pallidum as previously described (6).…”

mentioning

confidence: 99%

Electron microscopy of Treponema pallidum (Nichols) cultivated in tissue cultures of Sf1Ep cells

Konishi¹,

Yoshii²,

Cox³

1986

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“…The organisms are unicellular bacteria which exhibit a characteristic spiral shape and corkscrew motility (15). Fieldsteel et al have achieved replication of T. pallidum in vitro (3), but continuous cultivation of these pathogens outside the human host or appropriate animal model has met with failure (6). These organisms are morphologically indistinguishable and cannot be identified as to species on the basis of conventional serological tests for syphilis (19).…”

mentioning

confidence: 99%

Molecular basis of immunological cross-reactivity between Treponema pallidum and Treponema pertenue

Baker‐Zander¹,

Lukehart²

1983

Protein antigens of Treponema pallidum, Nichols strain, and Treponema pertenue, Gauthier strain, were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. Treponemal proteins were solubilized in 1% sodium dodecyl sulfate, electrophoresed on 12.5% polyacrylamide gels, and either stained with Coomassie brilliant blue or electrophoretically transferred to nitrocellulose paper. These antigen blots were incubated with sera from rabbits infected with either T. pallidum or T. pertenue and 125I-labeled staphylococcal protein A and exposed to X-ray film for visualization of antigenic molecules. Protein profiles of each organism separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue showed no distinguishable differences. Antigenic profiles as determined by Western blots were similar with two exceptions. A 39,500-dalton band was present on T. pertenue but absent from T. pallidum, and a 19,000-dalton band was present on T. pallidum but absent from T. pertenue (although two additional antigenic bands at 21,000 and 18,000 daltons were seen on T. pertenue). Because these differences were detected by using antisera raised against either T. pallidum or T. pertenue, these molecules must contain some antigenic determinants in common despite their differences in molecular weight.

“…Treponema pallidum, the causative agent of syphilis, is one of the few bacteria pathogenic to humans which has not yet been cultivated continuously in vitro, necessitating the use of organisms cultured in rabbits for research and diagnostic purposes. In 1981, a major barrier was overcome when consistent multiplication was obtained in a tissue culture system developed by Fieldsteel et al (2,3). In contrast to previous reports of T. pallidum cultivation, the results obtained by this group were highly reproducible and have been corroborated by other laboratories (5,6).…”

mentioning

confidence: 61%

Factors affecting the multiplication and subculture of Treponema pallidum subsp. pallidum in a tissue culture system

Norris¹,

Edmondson²

1986

Limited multiplication of Treponema pallidum subsp. pallidum (Nichols strain) can be obtained in the presence of SflEp rabbit epithelial cell cultures, but continuous culture has not yet been achieved. In the system currently employed, growth is exponential for the first 10 to 15 days of culturing, after which multiplication and the percentage of motile organisms decrease. In an effort to identify culture conditions which may adversely affect treponemal viability and growth, eight culture parameters were monitored over a 12-day period of incubation. Several of these parameters, including pH, redox potential, dissolved oxygen concentration, and glucose levels were found to change dramatically during the course of incubation, indicating that they may be responsible for the cessation of treponemal multiplication. The feasibility of extending the period of growth by subculturing was also investigated. In preparation for planned serial subcultivation experiments, several subculture procedures were tested and found to be effective in allowing the transfer of T. pallidum from 3-day-old primary cultures to secondary cultures without loss of motility or growth potential. Increases of up to 55-fold were observed in secondary cultures, but increased growth due to subculturing was not a consistent finding. Use of subculture intervals of .6 days resulted in a progressive decrease in treponemal multiplication in secondary cultures, although retention of motility was extended in the subcultures compared with motility in the primary cultures. These results indicate that the lack of continued multiplication of T. pallidum in subcultures is not due to damage to the treponemes during subculture. Prolonged multiplication of T. pallidum may be obtained through the stabilization of culture conditions by either performing subcultures at regular intervals or by medium replacement techniques. It was also found that primary T. pallidum cultures could be established by using as the inoculum treponemes that had been stored at -70°C in a medium containing 15% glycerol.