In vitro cultivation of Treponema pallidum: independent confirmation

Norris, Steven J.

doi:10.1128/iai.36.1.437-439.1982

Cited by 54 publications

(23 citation statements)

References 11 publications

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“…pallidum, is a chronic infection with many diverse clinical manifestations that occur in distinct stages. Over the years, because T. pallidum cannot be readily cultured (51,147) or stained with simple laboratory stains (197,213), other laboratory methods to identify infection in the various stages of syphilis have been developed. These tests for syphilis fall into four categories: (i) direct microscopic examination, used when lesions are present; (ii) nontreponemal tests, used for screening; (iii) treponemal tests that are confirmatory; and (iv) direct antigen detection tests currently used in research settings and as gold standards for test evaluation.…”

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Laboratory diagnosis and interpretation of tests for syphilis

1995

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The lack of a method for demonstrating the presence of Treponema pallidum by growth necessitates the use of alternative methods. Traditionally, these methods are divided into direct detection methods (animal inoculation, dark-field microscopy, etc.) and serologic tests for the presence of patient antibody against T. pallidum. Serologic methods are further divided into two classes. One class, the nontreponemal tests, detects antibodies to lipoidal antigens present in either the host or T. pallidum; examples are the Venereal Disease Research Laboratory and rapid plasma reagin and tests. Reactivity in these tests generally indicates host tissue damage that may not be specific for syphilis. Because these tests are easy and inexpensive to perform, they are commonly used for screening, and with proper clinical signs they are suggestive of syphilis. The other class of test, the treponemal tests, uses specific treponemal antigens. Confirmation of infection requires a reactive treponemal test. Examples of the treponemal tests are the microhemagglutination assay for antibodies to T. pallidum and the fluorescent treponemal antibody absorption test. These tests are more expensive and complicated to perform than the nontreponemal tests. On the horizon are a number of direct antigen, enzyme-linked immunosorbent assay, and PCR techniques. Several of these techniques have shown promise in clinical trials for the diagnosis of congenital syphilis and neurosyphilis that are presently difficult to diagnose.

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Laboratory diagnosis and interpretation of tests for syphilis

1995

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“…Antigenic characterization of Treponema pallidum has been hampered by the inability to obtain large quantities of pure, motile, and virulent organisms (1,9,10,21,22,(28)(29)(30). Nonpathogenic treponemes, however, can be grown in vitro in pure culture.…”

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Molecular characterization of common treponemal antigens

Hanff¹,

Miller²,

Lovett³

1983

Infect Immun

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A molecular characterization of cross-reactive antigens of Treponema pallidum Nichols and Treponema phagedenis biotype Reiter that are reactive with normal and syphilitic human sera is described. At least 8 common polypeptides, 14 T. pallidum-specific antigens, and 2 T. phagedenis biotype Reiter-specific antigens were identified.Antigenic characterization of Treponema pallidum has been hampered by the inability to obtain large quantities of pure, motile, and virulent organisms (1,9,10,21,22,(28)(29)(30). Nonpathogenic treponemes, however, can be grown in vitro in pure culture. Thus, in an attempt to obtain information about treponemal antigenic interrelationships, investigators have concentrated their efforts toward the examination of cross-reactive antigens obtained from the nonpathogens. Most of the studies have identified antigens common to both T. pallidum Nichols and the host-indigenous, nonpathogenic Treponema phagedenis biotype Reiter (referred to here as Reiter). Complement fixation (3-5, 7, 8, 12, 18, 20), gel diffusion (12,18,20), fluorescentantibody (6,13,17,27), crossed immunoelectrophoresis (20,(23)(24)(25)(26) and Western blot (15) techniques have revealed the presence of shared protein, polysaccharide, and/or protein-lipopolysaccharide complex antigens. Furthermore, absorption studies have shown that the presence of treponemicidal activity in normal human serum against T. pallidum and Reiter is stimulated by common T. pallidum and Reiter immunogens (P. A. Hanif and J. N. Miller, submitted for publication). However, with the exception of axial filament proteins (12, 21), the common treponemal antigens have not been well characterized in terms of their cellular location or molecular weight. As a prelude to isolation and purification of these particular cell components, we have characterized the common polypeptide antigens of T. pallidum and Reiter with normal and syphilitic human sera by using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by electrophoretic transfer of the t Present address:

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“…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). In this system, T. pallidum extracted from infected rabbit testes is added to SflEp cottontail rabbit epithelial cell cultures containing a modified tissue culture medium with 20% fetal bovine serum (FBS) and 0.63 mM dithiothreitol (DTT).…”

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

Norris¹,

Edmondson²

1986

Infect Immun

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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.

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In vitro cultivation of Treponema pallidum: independent confirmation

Cited by 54 publications

References 11 publications

Laboratory diagnosis and interpretation of tests for syphilis

Laboratory diagnosis and interpretation of tests for syphilis

Molecular characterization of common treponemal antigens

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

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