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Objective: To study the localisation of treponemes and to analyse the inflammatory infiltrate in biopsy specimens from patients with primary or secondary syphilis, or early infectious yaws., Materials and Methods: Skin biopsies originating from human lesions of primary (29x) or secondary (15x) syphilis (Rotterdam), or early yaws (18x) (West Sumatra) were studied. Different histochemical and immunohistochemical detection methods were used in this study., Results and Conclusion: The histochemical silver staining method according to Steiner revealed the presence of T. pallidum in all cases of primary syphilis studied. In 10 out of 14 cases of secondary syphilis, treponemes were demonstrated. With an immunofluorescence staining technique (IF) using anti-T. pallidum antiserum raised in rabbits (a-Tp), T. pallidum was demonstrated in 28 out of 29 cases of primary syphilis, and in 14 out of 14 studied cases of secondary syphilis. The silver staining method and IF showed identical localisations of T. pallidum (mainly in the dermal-epidermal junction zone or throughout the dermis). Using a-Tp antiserum in the indirect immunofluorescence technique, T. pertenue could be demonstrated in the dermis more often than with Steiner silver staining. However, epidermotropism of T. pertenue in yaws specimens was remarkable, compared with more mesodermotropism of T. pallidum; numbers of T. pertenue in the dermis were limited in all specimens. The dermal inflammatory infiltrate in primary and secondary syphilis was composed mainly of lymphocytes and plasma cells. In most cases more T (CD3 positive) cells than B (CD22 positive) cells were present. Regarding T cell subpopulations, in primary syphilis, T helper/inducer (CD4 positive) cells predominated in 86% of cases. In secondary syphilitic lesions, numbers of T helper/inducer cells were less frequent than or equal to T-suppressor/cytotoxic (CD8 positive) cells in 60% of cases. Remarkably, in yaws specimens the inflammatory infiltrate consisted mainly of IgG, but also IgA and IgM producing plasma cells. T or B lymphocytes were scarce, which is in sharp contrast with findings in syphilitic lesions.

Objectives: Investigation of sera, especially rabbit serum, in preventing in vitro immobilisation of Percoll purified T. pallidum., Materials and Methods: The immobilisation of Percoll purified T. pallidum (Nichols) was studied after pre-incubations with basal reduced medium (BRM), heat-inactivated serum of seven different species of animals, heat-inactivated normal human serum (NHS) and rabbit sera containing a different level of antitreponemal antibodies. Also increasing percentages of heat-inactivated normal rabbit serum (NRS) were studied., Results: The rapid immobilisation of purified treponemes by NHS is delayed by pre-incubation with NRS in a dose-dependent manner. The treponemes from 5-day infections were immobilised significantly more slowly than treponemes from 7- and 8-day infections. Compared with NRS, pre-incubations with a high-titred, low-titred and "autologous" serum resulted in significantly more rapid immobilisation of the treponemes. With most other animal sera resistance to immobilisation was slight compared with that produced by NRS. Immunofluorescent studies revealed that the treponemes were covered with a layer of the human third complement factor (C3b), within an hour of incubation. With two sequential pre-incubations, a delay of the immobilisation was only noted in those test mixtures in which NRS had been present in both preincubations., Conclusion: Rabbit serum delays the rapid in vitro immobilisation of Percoll purified treponemes by normal human serum. There was no evidence that this was caused by preventing access of antibodies (in vivo as well as in vitro) to, or preventing the activation of complement on, the treponemal surface. The evidence points to a mechanism in the fluid phase, suggesting participation of a third factor in the immobilisation process, for instance an enzyme, which can be partially inhibited by rabbit serum component(s).

Objective: To study ultrastructural aspects of infection with Treponema pertenue (Pariaman strain), originating from West Sumatra, Indonesia., Materials and Methods: Biopsy material originating from skin lesions in ten young children suffering from early infectious yaws in Indonesia, and rabbit testicular tissue inoculated with T pertenue. Human skin as well as rabbit testicular tissue was examined by means of conventional electron microscopy., Results: In human skin, treponemes were found in interepidermal spaces in 5 out of 10 specimens. In two of five positive specimens, treponemes were also seen in the dermis. In one out of five specimens from rabbit testicular tissue a profusion of treponemes was found lying in the interstitial myxomatous tissue. Microorganisms showed no adhesion to fibroblasts., Conclusion: This ultrastructural study of T pertenue demonstrated the scarcity and focal distribution of treponemes in tissue and did not reveal any morphological differences from the Gauthier strain of T pertenue. No differences from the ultrastructure of T pallidum were observed either.

The results of a yaws survey on the island of Sumatra in Indonesia are presented. The prevalence of yaws in the investigated region was found to be very high, a minimum of 300 cases per 100,000 individuals, which indicates that yaws is far from being eradicated and that campaigns for treatment are necessary. Patients suffering from early infectious yaws showed florid skin lesions. Of 101 serum samples from such patients, 100 had a positive reaction in one or more treponemal tests. The Treponema pallidum haemagglutination assay was found to be the most sensitive test (97% positive) in detecting antibodies against Treponema pallidum subsp. pertenue, followed by the fluorescent treponemal antibody absorption test (94%), the Venereal Disease Research Laboratory test and the TmpA enzyme immunoassay (91%), and analysis by Western blot using Treponema pallidum antigens (88%). Of 42 asymptomatic contacts of yaws patients 32 showed positive reactions in one or more tests, indicating that many people in the investigated region have been infected with treponemes. Eight new Treponema pallidum subsp. pertenue strains were isolated from yaws skin lesions. In vitro amplification of treponemal DNA and hybridisation with specific DNA probes showed that all eight strains were identical with Treponema pallidum subsp. pertenue CDC 2575, with regard to the subsp. pertenue specific tyfl gene.

The use of Percoll-purified treponemes in an assay similar to the Treponema pallidum Immobilisation test demonstrated that immobilisation of purified treponemes by seronegative normal human serum proceeded at a much higher rate than that of unpurified treponemes. This suggests that the removal of the testicular extract makes the treponemes more vulnerable to this action. A preincubation of the purified treponemes with the testicular extract from infected or uninfected testes delayed their rate of immobilisation to that demonstrated by the unpurified treponemes. This showed that substances produced during the infection are probably not responsible for the delay in immobilisation. Discrimination between the classical and the alternative pathway of complement activation, studied by the ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) method, showed that the classical pathway was responsible for the rapid immobilisation of the purified treponemes. However, the slow immobilisation in the EGTA-serum samples suggested a minor role of the alternative pathway in the immobilisation of the purified treponemes. Since the testicular extracts exerted an anti-complement activity, it needs to be investigated whether the protection offered to the purified treponemes by the testicular extracts is based on their deteriorating effect on the classical complement pathway or is due to a re-establishment of the protective cover around the treponemes.

Fresh and aged adherent T pallidum were used to study the accessibility of their outer membrane to antibodies by means of an indirect immunofluorescent technique. The integrity of the outer membrane was demonstrated by the non-reactivity with a monoclonal antibody directed against the axial filaments. Using the sera from patients with sero-positive primary and secondary syphilis no binding of IgG and IgM antibodies was observed. However, IgG and IgM antibody fractions isolated from the sera of patients with secondary syphilis, gave with the fresh fibroblast-adhering treponemes a mean of 14.5% IgG- and of 43.2% IgM positive treponemes. These means were 32.1% and 87.3% respectively for aged treponemes. Lower percentages were observed when fibronectin adhering treponemes were used. This demonstrates the inability of the outer membrane to bind antibodies in a majority of the fresh treponemes. This is partly lost on in vitro aging. Absence of IgG- and IgM fluorescence was also observed when sequential incubations with the antibody fractions and control sera were used. This was accompanied by the deposition of the third complement factor (C3) around the treponemes. Incubations of IgG- or IgM pre-coated adherent treponemes with heat-inactivated control sera or a C3 deficient serum did not result in the deposition of C3, and partially restored the detection of human antibodies. The most likely explanation for the absence of fluorescence is that antibodies become buried in an extra-cellular layer of serum proteins. The deposition of C3 from control sera alone most probably points to the classical pathway of complement activation and suggests that antibodies of rabbit origin constitute a part of the extracellular layer of treponemes.

Synthetic DNA probes specific for either the tpf-1 gene of Treponema pallidum subsp. pallidum Nichols or the tyf-1 gene of Treponema pallidum subsp. pertenue CDC 2575 were used for hybridization with in vitro-amplified chromosomal DNAs of 10 different Treponema isolates. tpf-1 and tyf-1 differ only in one nucleotide at residue 123, and three of four syphilis strains were of the Nichols type, whereas five of six yaws strains were of the CDC 2575 type, indicating that the nucleotide at position 123 of the tpf-1 or tyf-1 gene is not a definitive trait for either T. pallidum subspecies.

A new rapid technique for detection of serum treponemal antibodies is described which is based on an enzyme-linked immunoassay using nitrocellulose as solid phase. With this technique antigen-antibody binding is accelerated by the filtration of the antibody solution through the antigen-coated nitrocellulose filter instead of its remaining over the solid phase for incubation. Test results are available in less than 15 min. Serum specimens from 255 syphilitics and 829 non-infected subjects were investigated. The sensitivity and specificity of the Treponema pallidum enzyme-linked immunofiltration assay were comparable to those of the Treponema pallidum haemagglutination assay and the fluorescent treponemal antibody absorption test.

In an attempt to identify antigenic differences between Treponema pallidum subsp. pallidum (T. pallidum) and Treponema pallidum subsp. pertenue (T. pertenue) a gene bank of T. pertenue was constructed in lambda vector EMBL3. Clones carrying the T. pertenue gene encoding a 190 kDa protein, TyF1, were selected and the DNA was expressed in E. coli. TyF1 was shown to be closely related, but slightly different from the previously cloned T. pallidum antigen TpF1. TyF1 and TpF1 are high molecular weight antigens of about 190 kDa, which dissociate into 19 kDa subunits after heat treatment in presence of SDS. The difference between the two proteins is most obvious after treatment with proteinase K, which yields a 115 kDa component from TyF1 and a 95 kDa component from TpF1, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The structural genes encoding TyF1 and TpF1 were sequenced and the predicted amino acid sequences differed in a single amino acid residue at position 40, which is arginine in TyF1 and glutamine in TpF1. Similarities TyF1 and TpF1 with the previously described 4D antigen are discussed. The antibody response to TyF1 and TpF1 seems higher in syphilis patients than in yaws patients. The possibility of using the difference between these T. pallidum and the T. Pertenue antigens for serological discrimination of syphilis and yaws is discussed.

The mucopolysaccharide nature of the material deposited in rabbit testes infected with Treponema pallidum was confirmed by histochemical staining with alcian blue. Differential staining of mucopolysaccharides showed the presence of sulphated mucopolysaccharides as an almost constant feature, whereas in little more than half of the orchitic testes studied variable deposits of hyaluronic acid were seen. The treponemes were almost exclusively present in the areas rich in mucopolysaccharide. A combination staining with the Warthin-Starry method and alcian blue showed treponemes in close association with pre-existing fibrils and cells contained in these fibrils. The latter findings were confirmed by electron microscopy, and the fibroblasts to which treponemes adhered displayed the characteristics of activated cells. The close parallel between the histopathological changes observed here and their descriptions in published reports shows that our specific strain still behaves the same as the original Nichols pathogenic strain of T pallidum.

A new IgM capture enzyme linked immunosorbent assay (ELISA) was compared with the 19S(IgM) fluorescent treponemal antibody absorption (19S(IgM)FTA-ABS) test for detecting IgM antibodies to treponemes. Serum samples from 180 people, 109 with various stages of untreated syphilis, 45 with treated syphilis, and 26 non-infected, were investigated. In all diagnostic groups of syphilis the reactivity of the IgM capture ELISA was similar to that of the 19S(IgM)FTA-ABS test except in untreated neurosyphilis, for which the IgM capture ELISA was significantly less sensitive. The IgM capture ELISA was very sensitive in congenital (100%, 5/5) and primary (82%, 18/22) syphilis, but less sensitive in secondary (60%, 12/20), latent (53%, 16/30), neurosyphilis (34%, 11/32), and treated (11%, 5/45) syphilis. False positive IgM capture ELISA results were not found in five people who gave false positive Venereal Disease Research Laboratory (VDRL) reactions or in 21 neonates born to mothers adequately treated for syphilis before or during pregnancy. This indicated that the IgM capture ELISA was very specific. The course of antitreponemal IgM reactivity after treatment of early infectious syphilis was followed up in six patients. The quantity of IgM antibody declined in nearly all patients after treatment, but still remained detectable in five patients up to six months after treatment. In contrast, non-treponemal antibodies measured by the VDRL test disappeared in four out of six patients within five months from starting treatment. In conclusion, the IgM capture ELISA may be useful for easy and sensitive detection of IgM antibodies to treponemes in patients with congenital and primary syphilis. A positive test result in these cases indicates that patients should receive treatment if they have not been treated recently. The test is not, however, recommended to replace the VDRL test to monitor patients treated for syphilis.

The amount and nature of mucopolysaccharides present in extraction fluids routinely obtained in the isolation procedure of Treponema pallidum from infected rabbit testes was investigated. The mean quantity of mucopolysaccharides extracted from both testes of groups of 10 rabbits was 3.09 mg after infection for seven days and 26.88 mg after infection for 12 days, while from the testes of uninfected rabbits a mean of 0.42 mg was obtained. On electrophoresis the isolated mucopolysaccharides showed only one single band with the migration characteristics of hyaluronic acid. This band disappeared completely after pretreatment with hyaluronidase from bovine testes, which showed that during infection with T pallidum increasing amounts of hyaluronic acid accumulate. They can, at least in part, be extracted by a gentle extraction procedure, suggesting that this material binds loosely. The amount of hyaluronic acid isolated 12 days after infection showed positive correlations with the wet weight of testes as well as the number of treponemes isolated; seven days after infection such correlations were not present.

The hypothesis that experimentally determined survival times of Treponema pallidum in stored donor blood could be related to the number of treponemes initially present in the treponeme-blood mixtures was investigated by inoculating rabbits with three graded doses of treponemes suspended in donor blood and stored at 4 degrees C for various periods of time. The storage periods up to which living treponemes could be detected in the testes of the inoculated rabbits as well as those storage periods up to which seroconversion occurred are related to the number of treponemes present in the treponeme-blood mixtures. Increasing numbers of treponemes present in the donor blood resulted in longer periods up to which positive results in both parameters were found. All available evidence suggests that the upper limit of seroconversion coincides with the upper limit of treponemal survival. Inoculation with 5 X 10(4) treponemes per ml of donor blood resulted in a treponemal survival time of 48 h, inoculation with 1.25 X 10(6) treponemes per ml in a treponemal survival time of 72 h and inoculation with 2.5 X 10(7) treponemes per ml donor blood in a survival time of 120 h.

Serum samples from 25 patients at five different stages of syphilis were investigated for their ability to inhibit the adherence of pathogenic Nichols treponemes to cultured human fibroblasts. Serum taken from patients at the end of the primary stage showed an appreciable inhibition of treponemal adherence, and maximum inhibition of adherence was produced by serum from patients with secondary syphilis. Some freshly harvested treponemal suspensions were resistant to the adherence inhibition factors in serum from patients with syphilis; after incubation in vitro for 24 hours this resistance was lost. In vitro incubation almost doubled the number of adherent treponemes/fibroblast. These phenomena are discussed in terms of loss and reconstruction of the treponemal outer envelope. This leads to the suggestion that adherence occurs not only at the tips of the treponemes, but that surface components are also implicated, possibly as an initial contact mechanism. The composition of the outer envelope may in this way determine localisation versus dissemination of the treponemes.

The development of immunoglobulin G (IgG) subclass-specific anti-Treponema pallidum antibodies during the course of syphilis in humans was studied with sera from 50 untreated male patients. The patients were divided into five diagnosis groups. In the fluorescent treponemal antibody test, which delineates the presence of cross-reacting antibodies, as well as specific antitreponema antibodies, IgG1, IgG2, and IgG3 subclass antibodies were already present during the seronegative primary stage. Specific antibodies, which were detected by the fluorescent treponemal antibody absorption test, were first present during the serotype-variable primary stage. These antibodies were almost exclusively of the IgG1 and IgG3 subclasses. In later stages, antibodies of other subclasses were detectable. Titration of IgG1 antitreponema antibodies in three electrophoretically different IgG fractions revealed an asymmetric distribution in these fractions during primary syphilis. The antibodies were largely confined to the most basic fraction during primary syphilis. A sudden change in the distribution was noted between the end of the primary stage and the secondary stage; an even distribution of IgG1 antitreponema antibodies existed in the late latent stage. These findings confirm and extend previous results from our laboratory. The development of antibodies detected by both tests is discussed in terms of a sequential stimulation of the immune system due to the presence of an extracellular layer covering the treponemas or, alternatively, in terms of a suppression of the immune response during early syphilis.

The influence of storage at 4 degrees C on the survival of Treponema pallidum in donor blood, artificially infected with treponemes of the virulent Nichols strain was studied. Rabbits were inoculated in each testis with 0.5 ml of the blood-treponeme mixture, containing 5 X 10(5) microorganisms/ml, which was stored for up to 336 h. Under these circumstances, the blood-treponeme mixture appeared to be infectious for up to 96 h of storage, based on the demonstration of T. pallidum and serological findings, using FTA-ABS, TPHA and VDRL tests. The two former tests were found to be equally sensitive in detecting incubating syphilis in the rabbits, the VDRL test was significantly slower in this respect. From the absence of orchitis as well as seroconversion in rabbits, inoculated with blood-treponeme mixtures stored for 120 h or longer periods of time, it is concluded that after 120 h of storage infectivity is lost. The presently found survival time of treponemes in blood between 96 and 120 h of storage is discussed in relation with survival times found by other investigators.