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PubMed1990

A new attempt to distinguish serologically the subspecies of Treponema pallidum causing syphilis and yaws.

In an effort to serologically differentiate syphilis from yaws, 69 monoclonal antibody species raised against Treponema pallidum subsp. pallidum were tested by immunoblotting for their reactivity with Treponema pallidum subsp. pertenue. All monoclonal antibodies reacted with antigens with the same molecular weight of both subspecies. Furthermore, no differences in reactivity between sera from yaws patients and from syphilis patients were found by Western blot (immunoblot) analysis of cell lysates of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue. We tried to exploit the only known molecular difference between the subspecies. The subunits of the 190-kilodalton multimeric proteins TpF1 and TyF1 of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue, respectively, have previously been shown to differ in one amino acid residue at position 40. In this study, no difference was found in immunoreactivity of TpF1 or TyF1 with either syphilis sera or yaws sera. Synthetic peptides based on the sequence of TpF1 and of TyF1 were used in an enzyme-linked immunosorbent assay with syphilis sera and yaws sera. Again, no difference in reactivity between the T. pallidum subsp. pallidum- and T. pallidum subsp. pertenue-derived peptides was observed.

Amino Acid Sequence↗
PubMed1991

Sensitive detection of Treponema pallidum by using the polymerase chain reaction.

We have developed a sensitive assay for Treponema pallidum subsp. pallidum (T. pallidum), the agent of veneral syphilis, based upon the polymerase chain reaction (PCR). A 658-bp portion of the gene encoding the 47-kDa membrane immunogen was amplified, and the PCR products were probed by DNA-DNA hybridization with a 496-bp fragment internal to the amplitifed DNA. The assay detected approximately 0.01 pg of purified T. pallidum DNA, and positive results were obtained routinely from suspensions of treponemes calculated to contain 10 or more organism and from some suspensions calculated to contain a single organism. Specific PCR products were obtained for the closely related agent of yaws, Treponema pallidum subsp. pertenue, but not with human DNA or DNAs from other spirochetes (including Borrelia burgdoferi), skin microorganisms, sexually transmitted disease pathogens, and central nervous system pathogens. T. pallidum DNA was detected in serum, cerebrospinal fluids, and amniotic fluids from syphilis patients but not in in nonsyphilitic controls. T. pallidum DNA was also amplified from paraffin-embedded tissue. The diagnosis of syphillis by using PCR may become a significant addition to the diagnostic armamentarium and a valuable technique for the investigation of syphilis pathogenesis.

Animals↗
PubMed2000

rpoB gene analysis as a novel strategy for identification of spirochetes from the genera Borrelia, Treponema, and Leptospira.

Spirochetes are emerging pathogens for which culture and identification are partly unresolved. In fact, 16S rRNA-based sequencing is by far the most widely used PCR methodology that is able to detect such uncultivable pathogens. However, this assay actually has some limitations linked to potential problems of contamination, which hampers diagnosis. To circumvent this, we have devised a simple PCR strategy involving targeting of the gene encoding the RNA polymerase beta subunit (rpoB), a highly conserved enzyme. The complete sequence of the Leptospira biflexa (serovar patoc) rpoB gene was determined and compared with the published sequences for Borrelia burgdorferi and Treponema pallidum. From the resulting analysis, degenerate nucleotide primers were designed and tested for their ability to amplify a portion of the rpoB gene from various spirochetes. Using two different pairs of these primers, we succeeded in obtaining specific rpoB-amplified fragments for all members of the genera Leptospira, Treponema, and Borrelia tested and no other bacteria. Our findings may have significant implications for the development of a new tool for the identification of spirochetes, especially if clinical samples are contaminated or when the infecting strain is uncultivable.

Borrelia↗
PubMed2000

Comparison of the Serodia Treponema pallidum particle agglutination, Captia Syphilis-G, and SpiroTek Reagin II tests with standard test techniques for diagnosis of syphilis.

We compared the microhemagglutination assay for Treponema pallidum (MHA-TP), a treponemal test, with two other treponemal tests, the Serodia Treponema pallidum particle agglutination (TP-PA) assay and the Captia Syphilis-G enzyme immunoassay, using 390 clinical serum samples. We also compared two nontreponemal tests, the rapid plasma Reagin (RPR) card test and the SpiroTek Reagin II test. Agreements of the MHA-TP with the TP-PA test and the Syphilis-G test were 97.4 and 97.7%, respectively. There was 89.2% agreement between the RPR and Reagin II tests. The Reagin II test was more apt to be reactive if the treponemal test was also reactive. We conclude that either the Serodia TP-PA test or the Captia Syphilis-G test is an appropriate substitute for the MHA-TP and that the Spirotek Reagin II test could substitute for the RPR test as a screening test.

Antibodies, Bacterial↗
PubMed2003

Evaluation of an enzyme immunoassay technique for detection of antibodies against Treponema pallidum.

In the present study, the performance of an enzyme-linked immunosorbent assay (ELISA) technique (Eti-syphilis-G and Eti-syphilis-M; DiaSorin) for detection of Treponema pallidum immunoglobulin M (IgM) and IgG antibodies for the laboratory diagnosis of syphilis was evaluated. Four hundred forty-one samples were studied. The sensitivity and specificity of the ELISA were 100 and 93%, respectively, compared with the results of a microhemagglutination assay for Treponema pallidum (MHA-TP) and 99.4 and 100%, respectively, compared with the results of the fluorescent treponemal antibody absorption (FTA-Abs) test. The results of the ELISA technique were concordant with those of MHA-TP for 98% of the samples tested, while the rate of concordance with the FTA-Abs test was 99.5%. The sensitivities of the rapid plasma reagin (RPR) test, MHA-TP, and the ELISA in the different phases of syphilis compared with the results of the FTA-Abs test were 92, 88, and 100%, respectively, for patients with primary syphilis; 100% for all tests evaluated for patients with secondary syphilis; 97.2, 99.4, and 100%, respectively, for patients with latent syphilis; and 57.9, 92.6, and 97.9%, respectively, for patients with past treated syphilis. The RPR test was reactive with 12 samples that were negative by all the specific tests. IgM antibodies were most frequently detected by the ELISA for IgM antibodies (32.8%) than by the FTA-Abs for IgM antibodies (28.4%). Detection of these antibodies by the FTA-Abs test and the ELISA for IgM antibodies decreased with the stage of disease (72 and 88%, respectively, for patients with primary syphilis to 17 and 19%, respectively, for patients with early latent syphilis). The high sensitivity and specificity of this ELISA technique during all stages of syphilis, together with the fact that it is a simple, objective, and easily automated method, lead us to believe that it could be used as a screening test for syphilis.

Antibodies, Bacterial↗
PubMed2026

A new highly discriminatory typing scheme for Treponema pallidum reveals similar levels of genetic variability across lineages.

UNLABELLED: The global resurgence of treponematoses, particularly syphilis, poses a growing public health challenge. Despite advances in sequencing technologies, obtaining complete Treponema pallidum genome sequences for epidemiological studies remains challenging due to clinical sampling and methodological constraints. There is, therefore, a need for rapid, cost-effective, and accessible typing methods. Based on the analysis of 121 T. pallidum genomes spanning all three subspecies (TPA, TPE, and TEN) from diverse regions, we selected seven highly variable genes (tp0136, tp0326, tp0548, tp0705, tp0858, tp0865, and tp1031) to form a new typing system, combined with analysis of macrolide resistance mutations in the 23S rRNA gene. The scheme was validated on 542 global T. pallidum samples, using either Sanger reads or whole genome sequence data, obtaining 82 sequence types (STs) among the 415 fully typed samples. Macrolide resistance mutations were frequently detected, highlighting the need for ongoing epidemiological surveillance. Phylogenetic analyses based on concatenated multilocus typing (MLST) loci recovered the expected subspecies and lineage structure. Consistently, almost all sequence types formed monophyletic groups, indicating strong concordance between MLST-based classification and whole-genome phylogenies. In addition, population genetic analyses revealed comparable levels of within-lineage diversity across subspecies and lineages, despite pronounced differences in geographic distribution, and identified distinct regional genetic clusters consistent with localized transmission dynamics. Importantly, the scheme employs a single-step PCR for all seven targets, facilitating implementation in standard laboratories and is publicly accessible through PubMLST. Overall, our novel MLST scheme offers a rapid, cost-effective tool to advance molecular epidemiology of T. pallidum, facilitate transmission and resistance tracking, and support global surveillance to strengthen public health interventions for syphilis and endemic treponematoses control. IMPORTANCE: We have developed a new multilocus typing (MLST) scheme useful for all Treponema pallidum lineages after the analysis of 121 complete genome sequences of this species. The new scheme can be used directly with uncultured clinical samples, thus providing an excellent contribution to the molecular surveillance of syphilis and other treponematoses. The application of this MLST scheme to over 500 samples from all lineages and main geographical regions has revealed similar levels of genetic variation within them. Furthermore, the analyses show a complex pattern of spread, with global and local contributions to the observed distribution of genetic variation in the syphilis-producing sublineages. The new scheme represents a significant improvement over previous proposals and also reveals unsuspected levels of variability in T. pallidum lineages.

Treponema pallidum↗
PubMed1973

The Treponema pallidum haemagglutination (TPHA) test in biological false positive and leprosy sera.

The Treponema pallidum haemagglutination (TPHA) test was carried out on 274 sera known to show biological false positive reactions to reagin tests for syphilis. The Treponema pallidum immunization (TPI) and fluorescent treponemal antibody absorption (FTA-ABS) tests were non-reactive on all these sera. Thirty-one or 11.3% showed reactive results in the TPHA test. Sera from 267 people who had lepromatous leprosy were also tested in the TPHA test. Fourteen sera were reactive in the TPHA, TPI, and FTA-ABS tests and were from people who had both syphilis and leprosy. Biological false positive reactions were shown by 26 of the leprosy sera, of which three or 11.5% were also reactive in the TPHA test. A further four sera in the leprosy group were reactive only in the TPHA test. The possible cause of false reactive TPHA test results is discussed. It was concluded that where reagin and TPHA tests are reactive in a person who has no history or clinical signs of syphilis, the serum should be referred for TPI and FTA-ABS testing.

Antibodies, Bacterial↗
PubMed1978

Treponema pallidum immune adherence test for serodiagnosis of syphilis. 3: Clinical significance and evaluation of treatment.

Antibody titres were measured in patients with clinical syphilis, and the effect of treatment on the results of the Treponema pallidum immune adherence (TPIA) test is reported. In the Treponema pallidum haemagglutination (TPHA) test little change in antibody titre occurred after treatment while in the fluorescent treponemal antibody-absorbed (FTA-ABS) test only a slight decrease occurred. The decrease in the antibody titre in the TPIA test was similar to that in the glass plate test, but the findings were different. From the results of gel filtration of sera obtained from patients with syphilis at different intervals after treatment it was apparent that the decrease in antibody titre after treatment mainly concerned the IgM antibody; thus, because of the high sensitivity of the TPIA test to IgM antibody the test is useful in evaluating the effect of treatment.

Antibodies, Bacterial↗
PubMed1980

Lack of serological evidence for venereal spirochaetosis in wild Victorian rabbits and the susceptibility of laboratory rabbits to Treponema paraluis-cuniculi.

Sera from 608 wild rabbits were examined using serological tests for syphilis as an indicator of infection with Treponema paraluis-cuniculi. Only eight sera gave positive or weakly positive results in the rapid plasma reagin (RPR) test, and none of these eight sera gave positive results in the Treponema pallidum haemagglutination assay (TPHA). Thus, it appears that wild rabbit populations in Victoria, Australia, are not naturally infected with T paraluis-cuniculi. Normal Australian laboratory rabbits however were readily infected with T paraluis-cuniculi, either by intradermal or intratesticular inoculation or by the venereal route. In the latter case, treponeme-containing lesions developed after about five months' cohabitation with infected mates. The disease was successfully transmitted from male to female and from female to male rabbits by the venereal route. In most cases infected rabbits became RPR-positive (17/19 rabbits) and in all cases TPHA-positive (19/19), indicating that serological tests for syphilis can be used to screen rabbits for this disease.

Animals↗
PubMed1982

Mitogenic responses of hamsters infected with Treponema pertenue Lack of correlation with passive transfer of resistance.

Infection of the CB/Ss Lak hamster with Treponema pertenue is characterised by chronic cutaneous lesions and lymph nodes teeming with treponemes. Throughout the course of infection lymph node and spleen cells responded poorly to the mitogens concanavalin A, phytohaemagglutinin, and lipopolysaccharide. This impairment preceded clinical signs of infection and correlated well with the chronicity of framboesial infection. High concentrations of antigen from T pertenue, but not from the non-pathogenic Treponema phagedenis, depressed the mitogenic response of normal lymphoid cells. After framboesial hamsters were treated with penicillin the mitogenic activities of their lymph node and spleen cells were similar to or slightly raised above those of controls. No significant differences were detected among recipients of framboesial immune cells with or without mitogenic activity. Recipients of immune lymph node and spleen cells from penicillin-treated or non-penicillin-treated animals had no cutaneous lesions 21 days after infection and had significantly lower lymph node weights and fewer treponemes per node than recipients of cells from normal penicillin-treated or non-penicillin-treated animals. Since lymphocyte transformation in vitro does not correlate with in-vivo treponemicidal activity, it is not a valid approach to assess the protective immune capacity of the framboesial host.

Animals↗
PubMed1986

Enzyme linked immunosorbent assays with Treponema pallidum or axial filament of T phagedenis biotype Reiter as antigen: evaluation as screening tests for syphilis.

Enzyme linked immunosorbent assays with an ultrasonicate of Treponema pallidum (TP-ELISA) or axial filament of Treponema phagedenis biotype Reiter (AF-ELISA) were developed to detect treponemal antibody. TP-ELISA and AF-ELISA were compared with the T pallidum haemagglutination assay (TPHA), the fluorescent treponemal antibody-absorbed (FTA-ABS) test, and the Venereal Disease Research Laboratory (VDRL) test for sensitivity and specificity of serodiagnosis of syphilis. A total of 1423 serum samples, 253 from patients with various stages of syphilis, 500 from patients attending a sexually transmitted disease (STD) clinic, and 670 from people without syphilis, were investigated. At all stages of syphilis the sensitivity of the TP-ELISA, the AF-ELISA, the TPHA, and the FTA-ABS test did not differ significantly, except that the AF-ELISA was less sensitive than the TPHA (p less than 0.05) for treated syphilis. In primary syphilis, neurosyphilis, and treated syphilis the TP-ELISA and AF-ELISA were significantly more sensitive than the VDRL test (p less than 0.05). The specificity of all tests was comparable (p greater than 0.05). The TP-ELISA and AF-ELISA appear to be good alternatives to the TPHA as screening tests for syphilis. Because of the easy availability of a well defined antigen the AF-ELISA seems to be better suited for large scale testing.

Antigens, Bacterial↗
PubMed1984

Glucose metabolism of Treponema bryantii, an anaerobic rumen spirochete.

The pathway of glucose metabolism by Treponema bryantii, an obligately anaerobic spirochete isolated from bovine rumen contents, was studied. Washed cell suspensions of the spirochete consumed glucose and CO2 and produced equimolar amounts of acetate, formate, and succinate. Carbon dioxide was essential for glucose metabolism. Determination of radioactivity in products formed from 14C-labelled glucose and NaH14CO3 and assays of enzyme activities in cell-free extracts were used to determine the pathway of glucose metabolism. Treponema bryantii catabolized glucose to pyruvate via the Embden--Meyerhof--Parnas pathway. The spirochete used a coliform pyruvate-formate lyase to degrade pyruvate and produce formate and acetate. Succinate was formed by a pathway which involved the condensation of CO2 with pyruvate (or phospho(enol)pyruvate) formed from the breakdown of glucose.

Acetates↗
PubMed1984

Oxygen toxicity in Treponema pallidum: deoxyribonucleic acid single-stranded breakage induced by low doses of hydrogen peroxide.

The effect of hydrogen peroxide on Treponema pallidum was investigated. The in vitro loss of virulence (as measured by rabbit inoculation) of T. pallidum was accelerated by as low as 100 microM hydrogen peroxide in the complex maintenance medium used. Higher doses led to rapidly accelerated death with 500 microM hydrogen peroxide causing sterilization of the medium within 3 to 4 h. Since hydrogen peroxide is known to cause single-stranded breaks in DNA, the effect of hydrogen peroxide on the treponemal genome was examined. Extensive breakage was caused by 100 microM hydrogen peroxide as determined on alkaline sucrose gradients. A limit was reached at 250 microM and above. Single-stranded breaks could be demonstrated as early as 5-10 min after exposure to hydrogen peroxide when the treponemes were exposed to 250 microM hydrogen peroxide; accelerated death was evident by 2 h past exposure demonstrating that DNA breakage was preceding death. Treponemal death caused by penicillin did not result in DNA breakage. The repair-proficient bacterium Escherichia coli K-12 was compared with T. pallidum. It required 10-100 times more hydrogen peroxide to cause various levels of breakage. Escherichia coli K-12 rapidly repaired DNA breakage once hydrogen peroxide was removed by addition of catalase. Treponema pallidum, in comparison, showed little or no repair in vitro. Addition of catalase or dithiothreitol to the medium protected against all but a low level of breakage; this may reflect on the ability of catalase and reducing agents to protect T. pallidum against oxygen toxicity in vitro.

DNA Repair↗
PubMed1985

Lymphocyte transformation in inbred guinea pigs infected with Treponema pallidum nichols.

T lymphocytes were purified from peritoneal exudates from young male, inbred strain 2 guinea pigs infected with 8 X 10(7) Treponema pallidum Nichols and from control animals injected with normal rabbit testes extract. Groups of animals were sacrificed after 15, 30, 60, and 90 days of infection, and the cells were analyzed for their proliferative response to concanavalin A and phytohemagglutinin and to sonicated T. pallidum, Treponema phagedenis biotype Reiter, and normal rabbit testes extract. No significant differences were observed in the responses to mitogens. Cells from infected but not from control animals responded with significant proliferation to T. pallidum antigen, but neither infected nor control cells reacted to T. phagedenis Reiter antigen. Although cells from both infected and control animals responded to normal rabbit testes extract, this response in the infected animals was transient and much lower than the response to T. pallidum antigen.

Animals↗
PubMed1989

Immunogenicity of three recombinant Treponema pallidum antigens examined in guinea pigs.

The immunogenicity of recombinant treponemal antigens TmpA, TmpB and TmpC incorporated in RIBI adjuvant and injected into inbred strain 2 guinea pigs has been examined. The immune status of these animals has been challenged by infection with Treponema pallidum, Nichols. The immune response evaluated by the fluorescent-antibody test, microhemagglutination test and ELISA demonstrated high titers of antibodies to the T. pallidum antigens. The immunoblot analysis proved that the antibodies were directed to the 43-(Tmp A) 34- (Tmp B) and 35-kdalton (Tmp C) polypeptides. Antibodies cross-reacting with Treponema phagedenis biotype Reiter were, however, also detected. In spite of high titers of antibodies the animals were not protected against challenging infection with 10(8) organisms of T. pallidum.

Adjuvants, Immunologic↗
PubMed1984

Experimental neonatal syphilis. I. Evidence of resistance to symptomatic infection in neonatal rabbits following intradermal inoculation with Treponema pallidum (Nichols strain).

Resistance of 5- to 8-day-old neonatal rabbits to dermal lesion development after intradermal inoculation of Treponema pallidum was demonstrated. Clinical evidence of infection following inoculation of 1 X 10(6) Treponema pallidum at each of two sites was either minimal or absent. Atypical, nonprogressive, nonulcerative lesions occurred in 59% of the inoculated neonates and at 45% of inoculated sites. Differences in incubation periods, duration, and maximum diameters of lesions among adult controls versus neonatal rabbits were significant. The age of waning resistance was determined by inoculating groups of neonates ranging from 1 to 7 weeks of age. Five-week-old (31-36 days) neonates demonstrated waning resistance by the appearance of typically ulcerative, progressive lesions, though their parameters (duration, size) were not yet those of adult control lesions. The resistance demonstrated by neonates may be due in part to group housing (nesting) which could create unfavorable temperatures for T. pallidum survival; comparison of lesion development between nesting and individually housed neonates, 31 to 46 days of age, revealed a greater percentage of typical lesions developing among those individually housed (95 versus 52%). However, these differences may reflect the variability of typical lesion development found among animals of this age when resistance begins to wane. In both groups, the duration of typical lesions was significantly shorter than for adult controls. A heat-stable serum factor(s) was demonstrated in 19 of 20 basal sera from neonates 4 to 6 days of age; this presented another possible mechanism of resistance. The neutralizing serum factor(s) was not demonstrable in the sera of does either before mating, during gestation, or shortly after kindling.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1984

Experimental neonatal syphilis. II. Immunological responses of neonatal rabbits to intradermal inoculation with Treponema pallidum (Nichols strain).

The immunological competence of neonatal rabbits inoculated intradermally with Treponema pallidum was examined. Both cellular responses and the production of humoral antibody to specific T. pallidum antigens and to nonspecific antigens or mitogens were investigated. In blast transformation assays, splenic and popliteal lymph node lymphocytes from neonates inoculated with virulent T. pallidum responded to T. pallidum antigens in a manner similar to or greater than inoculated adult rabbits. Splenic and popliteal lymph node lymphocytes from both uninoculated and T. pallidum-inoculated neonate and adult animals showed consistent and similar responses to concanavalin A. Both neonate and adult animals inoculated with heat-killed T. pallidum also responded but to a significantly lesser degree. Immunofluorescent examination of skin sections from the site of inoculation of adult and neonatal animals revealed 1) that the early infiltrate was composed predominantly of T cells, 2) diffuse antibody staining with rare B cells, and 3) fewer treponemes with significant fragmentation in neonates as compared to adult controls. Antibody production by neonates inoculated with virulent T. pallidum was delayed 4 to 6 weeks postinoculation as measured by the fluorescent Treponemal antibody absorption and Venereal Disease Research Laboratory (VDRL) test procedures, respectively. Antibody was not detected among neonates inoculated with heat-killed treponemes during a 6-week observation period and only low levels of VDRL antibody were detected in a few adult control animals. Evidence for incomplete resistance of neonatal rabbits to the intradermal inoculation of Treponema pallidum was provided.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Adherence of Treponema pallidum subsp. pallidum in the rabbit placenta.

Congenital syphilis is the consequence of transplacental passage of Treponema pallidum. A system was developed to deliver virulent T. pallidum, Nichols strain, through an isolated uterine horn of a pregnant rabbit in order to investigate the mechanism by which T. pallidum is able to cross the placenta. While the pregnant rabbit was anesthetized, the ovarian artery and the uterine vein were cannulated and attached to a peristaltic pump. Treponema pallidum (2-5 x 10(8) in 10-15 ml RPMI-1640) were circulated via the peristaltic pump throughout the horn for 2 hr, after which the placentas were removed, fixed in formalin, and embedded in paraffin. This system was used to investigate treponemal binding to rabbit placenta at Day 20 and 26 during the gestation period of the rabbit (29-32 days). Examination of 5-microns Dieterle silver stained tissue sections revealed (i) a greater number of spirochetes in the later gestational stage placentas (Day 26) than in the earlier placentas (Day 20), (ii) organisms adhering to the trophoblastic tissue surrounding the maternal blood channels, and (iii) organisms appearing to be in the process of penetrating the trophoblastic tissue or that had completely penetrated from the channels into the trophoblastic elements. We suggest that T. pallidum may be adhering to placental components that are differentially expressed during gestation of the rabbit.

Animals↗