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Two cases are presented of simultaneous infection with Treponema pallidum and herpes simplex virus. The occasional concurrence of these diseases suggests that all cases of herpes genitalis, including those with negative darkfield examinations and initial nonreactive serologic tests for syphilis, should be followed in two to four weeks by a repeat serologic test for syphilis.
Virulent Treponema pallidum has been shown to survive in KCN-containing artificial medium. Oxygen uptake being sensitive to cyanide, the observation indicates that treponemes do not have a cytochrome oxidase system. KCN had a killing effect only at a 15--30 mM final concentration. The data show that the Budapest strain of T. pallidum is an anaerobic organism.
A modification of the in vitro immobilization assay together with freeze-fracture analysis was used to determine the factors responsible for the prolonged time required in vitro to achieve killing of Treponema pallidum subsp. pallidum. The modified immobilization assay permitted separate determination of the time required for binding of antibody to the surface of T. pallidum and for C activation. Treponemes were preincubated in heat-inactivated immune rabbit serum (IRS) followed by washing the organisms in 2.5% BSA/PBS to remove unbound IRS antibody before the addition of C. The results showed that a comparable degree of C-dependent killing occurred when treponemes were preincubated in heat-inactivated IRS for either 30 min or 16 h, indicating that treponemicidal antibody rapidly binds to the surface of T. pallidum. Preincubation of treponemes for 17 h in heat-inactivated IRS followed by a 1-h incubation in C resulted in the loss of 80% treponemal motility, indicating that C activation results in rapid killing of T. pallidum. Treponemes preincubated in IRS for 1 h, then incubated for 8 h and 16 h in heat-inactivated normal serum also lost a significant level of motility after the addition of C; in contrast, motility was unaffected after 30 min and 4 h of incubation in heat-inactivated normal serum under similar conditions. These results demonstrate that, whereas antibody binding to and C-mediated killing of treponemes can proceed rapidly, the prolonged time to C activation limits the rate at which treponemicidal activity occurs in vitro. In addition, treponemicidal activity using the modified immobilization assay could not be demonstrated with antiserum against T. pallidum endoflagella, antiserum against proteins solubilized from T. pallidum using the detergent Triton X-114, and a mAb to the T. pallidum r190-kDa "4D" protein, suggesting that these molecules are not accessible to surface binding antibody. Freeze-fracture analysis, recently used in our laboratory to demonstrate that the outer membrane of T. pallidum has rare constituent protein, was utilized to demonstrate outer membrane target Ag of IRS antibody. T. pallidum rare outer membrane protein (TROMP) molecules were shown in freeze-fracture electron micrographs to be consistently aggregated following a 16-h incubation of treponemes in IRS. In contrast, no aggregation of TROMP was present in treponemes incubated in normal rabbit serum for 16 h or in treponemes incubated in IRS for 2 h. These findings suggest that the rate of C activation leading to in vitro treponemicidal activity is limited by the time required for aggregation of antibody-bound TROMP molecules.
The ultrastructure of treponema pallidum obtained directly from human chancres by biopsy was studied by electron microscopy. The treponemes were enveloped by a trilaminar cytoplasmic membrane and a trilaminar periplastic membrane. The central part of the periplastic membrane corresponds to the protective mucoid layer. In undamaged organisms bunches of axial filaments were seen to entwine the whole cytoplasmic body without any disruption or overlapping. The number of axial filaments varied between three and four. Identical nose pieces were demonstrable in both ends of the treponemes. Axial filaments and nose pieces seem to differ from those of Nichols' strain.
In biopsies obtained from syphilitic chancres of varying ages in 10 patients, a total of 766 ultrathin sections of Treponema pallidum were studied by electron microscopy. The course and number of axial filaments observed reveal that one bunch of 3-4 filaments without interruption entwine the whole cytoplasmic body. In 9.2% of the sections a trilaminar or a fragmentary trilaminar periplastic membrane was observed outside the cytoplasmic membrane and the axial filaments. The occurrence of the periplastic membrane decreased with advancing ages of the chancres. A protective function of the membrane is discussed. A peritreponemal fine reticular halo demonstrable in most fragments is supposed to be due to fixation induced shrinkage of treponemal hyaluronidase-influenced semifluid glycosaminoglycans. Peritreponemal reticular halos were also observed in collagen tissue. A destructive effect of the treponemes on collagen fibres could explain how the organisms penetrate through the collagen rich meninges into the central nervous system. A surface associated narrow border of electron dense amorphous substance, probably originating from the host organism, yields to tangentially cut treponemes a spiny caterpillar-like appearance.
Treponema pallidum was isolated from various types of secondary syphilitic skin lesions. From moist genital papules and from condylomata lata several treponemes were isolated whereas few were isolated from dry papules of the trunk. One third of the observed treponemes were morphologically different from treponemes isolated from human chancres. Especially the nose-piece structures of the terminal parts of the treponemes were deviating. Some nose-pieces were coated by a fuzzily outlined electron dense substance, whereas others were degenerated or nearly separated from the cytoplasmic body. Other treponemes were missing their nose-piece as avirulent saprophytic treponemes. Recent studies have indicated that the nose-pieces are essential for the tissue attachment of treponemes and the treponemal virulence. The significance of the altered nose-piece structure observed is discussed.
Rabbits immunized with the protease resistant recombinant Treponema pallidum surface-associated Ag 4D showed an altered course of experimental syphilis after intradermal challenge with virulent T. pallidum. Vaccination trials using three different protocols were examined. In one experiment, a combined i.m.-i.v. immunization protocol was compared with an i.m. injection schedule. Four of five rabbits immunized by the i.m.-i.v. protocol developed morphologically atypical lesions at each of 16 sites, whereas the i.m. injected animals showed no evidence of attenuated disease. Moreover, immunization by both protocols elicited equally high ELISA titers of 4D-specific IgG antibody suggesting that cellular immune mechanisms may have been involved. In an effort to augment cell-mediated responses to 4D, seven rabbits received i.v. immunizations with an emulsion of 4D, purified BCG cell wall skeletons, and trehalose dimycolate in an oil-microdroplet form (4D-cell wall skeleton-trehalose dimycolate). Splenic lymphocytes from three representative immunized animals had a strong in vitro proliferative response to 4D as compared to controls. Further, all animals developed high anti-4D titers in response to immunization. After challenge with virulent T. pallidum, six of the seven rabbits developed an early cutaneous response (3-8 days) at the sites of inoculation consistent with a hypersensitivity reaction followed by morphologically atypical lesions. Aspirates from each of three representative atypical lesions were devoid of treponemes by darkfield examination at day 14, whereas motile T. pallidum were observed in aspirates from four of five control sites. Between days 20 and 24, lesions in the 4D-cell wall skeleton-trehalose dimycolate immunized rabbits began to enlarge and become more typical in appearance; aspirates from six of seven representative lesions were darkfield positive. We conclude that 1) immunization with the r4D Ag alters the course of experimental syphilis in a manner consistent with previously defined parameters of partial protection and 2) immunizations protocols containing an i.v. component are more effective than protocols using the i.m. route alone.
The contribution of individual specific molecules of Treponema pallidum subspecies pallidum to cellular immunity in experimental syphilis was evaluated by combining the techniques of Ag identification and purification with the lymphocyte proliferation assay. Proliferative responses of splenic lymphocytes from syphilitic rabbits to complex treponemal Ag and Con A were vigorous throughout the course of intratesticular infection (6, 10, 17, 30, and 210 days). Normal rabbits did not respond to any treponemal preparations and all rabbits failed to respond to normal rabbit testicular Ag (NRT). Seven defined treponemal Ag (47 kDa, 37 kDa, 35, 33-kDa, 30-kDa, 14 kDa, and 12 kDa) stimulated lymphocytes from infected rabbits. Cellular responses to the 37-kDa and 30-kDa fractions were evident by day 6 of infection and responses to the 35, 33-kDa and 14-kDa Ag were first detected on day 10; responsiveness to these Ag continued throughout the observation period. Cellular responses to the 47-kDa molecule were detectable but lower when compared with other individual Ag. Responsiveness to the 12-kDa Ag was not evident until 7 mo postinfection. Specific immunoblot reactivity of serum from rabbits used in this study generally correlated with the development of cellular reactivity to individual Ag of T. pallidum.
In this research 113 sera have been analysed with three methods for IgM treponema-specific determination: IgM-SPHA, IgM-EIA and 19S IgM FTA-ABS. Among these sera, 33 samples related to non-treated patients at different stages of infection, and 80 samples to treated patients. The results point out a light sensibility of the IgM-SPHA in primary lues. The IgM-EIA test has performed a good specificity, but displayed a sensibility lower to the 19S IgM FTA-ABS, which proved the best of tests. About the cases of treated lues at different stages, in 23 samples with VDRL negative has been found no positivity at three tests used, while in 49 samples with VDRL positive 8 are resulted positive at 19S IgM FTA-ABS.
The authors proceed with their studies of magnetic microfields detected round T. pallidum and of the interactions of these fields with the biologic objects round the treponemas; these studies will prompt approaches to physiotherapeutic management of infectious diseases, making use of a new (for medicine) source of physical fields. They have revealed that T. pallidum fluorescence intensity and the width of the 'fan' of T. pallidum on the domain structures of ferrite/garnet films indicates the degree of T. pallidum pathogeneity.
The kinetics of the humoral response to pathogen-specific polypeptides was examined in Treponema pallidum-infected young (3-5 months old) and old (10-20 months old) inbred strain-2 guinea pigs. Sera collected before and at various times after infection were pooled and examined by immunoblotting and two serologic tests (ELISA and FTA) before and after sequential adsorption with CNBr-activated sepharose coupled to normal rabbit proteins and antigens from five nonpathogenic treponemal species. Prior to adsorption the kinetics of the humoral response to T. pallidum antigens did not seem to differ significantly between the two groups. After adsorption, however, a delay in the appearance of detectable antibodies and a milder response to various pathogen-specific polypeptides was observed in the older group. After adsorption, a sharp drop in ELISA-TP, ELISA-TR and FTA titres occurred in both groups. Six months post-infection, between 9 and 10 pathogen-specific polypeptides (97, 57, 47, 45, 43, 39, 37, 33, 17 and 15 kD) were recognized by both groups. The effect of age and levels of natural treponemal antibodies on the clinical symptoms of primary lesions and humoral response to pathogen-specific polypeptides is discussed.
Plasmid DNA has been isolated by soft alkaline and hard alkaline lysis from a pathogenic strain (Nichols) and two cultural strains (Reiter and VIII) of Treponema pallidum. Plasmid DNA was identified in all three strains. The molecular mass of identified plasmid DNA is 7 x 10(6) daltons according to the data of electrophoretic analysis in the agarose gel.
When 47 pigs were dosed orally with cultures of Treponema hyodysenteriae, 44 (94 per cent) developed swine dysentery. Of those which recovered and were rechallenged, nine of 21 (43 per cent) showed clinical signs, as did one of 10 (10 per cent) challenged on a third occasion. Clinical disease was associated with development of specific IgG, IgA and IgM antibodies in serum and the local production of IgA in gut mucosal tissues. The appearance of antibody was not directly related to protection but rather indicated either prolonged exposure (in the case of serum IgG) or recent exposure to T hyodysenteriae (for secretory IgA). Infection also resulted in the appearance of IgG and IgA memory cells in gut-associated lymphoid tissue. However, these studies indicated that humoral immunity alone is not responsible for the onset of a protective response to T hyodysenteriae in the colon.
Properties of the attachment of Treponema hyodysenteriae to Henle intestinal epithelial (HIE 407) cells were examined. The frequency of attachment depended on the motility and viability of the spirochetes. Rabbit hyperimmune and swine convalescent antisera inhibited attachment. Treatment of HIE cells with neuraminidase had no effect on attachment; however, treatment of spirochetes with the enzyme decreased adherence significantly (P = 0.01). Attachment was inhibited by N-acetylneuraminic acid, D-glucuronic acid, and fetuin. Adherence was increased following coincubation with N-acetylglucosamine or yeast mannan. Surface antigens of T hyodysenteriae, isolated by chemical extraction, competitively inhibited adherence. Concentrated T hyodysenteriae culture supernatant fractions inhibited adherence, but concentrated phosphate buffered-saline washings of the spirochete and concentrated uninoculated media did not inhibit adherence. Sialic acid was detected in unwashed T hyodysenteriae and spent culture supernatant fractions in higher concentrations than from washed spirochetes and uninoculated media. It was concluded that the binding adhesins on T hyodysenteriae for cultured HIE cells may contain sialic acid residues.
Treponema pallidum has not been yet cultivated. Hence any in vitro investigation is excluded, and it is owing to the experimental animal model, the rabbit, that we have studied the susceptibility of that germ to ofloxacin. This quinolone, owing to its pharmacokinetic and therapeutic properties, can specially be indicated in the treatment of Sexually Transmitted Diseases. Thus, its appeared to be of the utmost importance to know if the suggested schedule of treatment for STD, might not be susceptible to modify the course of a co-existing incubating syphilis by either delaying or inhibiting the apparition of the clinical features of primary syphilis. This study was undertaken at the incubation period, in syphilitic rabbits, using kinetic data obtained in man, after a given dosage of ofloxacin. Results were appraised upon converging data: lesions, bacteriology, and serology of the tested lot compared with two control batches of infected rabbits, the first one being untreated, the other having received the reference antibiotic treatment. From the data obtained and in the experimental settled conditions where this study was done, it results that ofloxacin has no effect on the course of the experimental syphilitic infection.
The relationship between Treponema pallidum infection and the synthesis of proteoglycans by organ cultures of rabbit testes was investigated. Two proteoglycan fractions, large (CL-6B Kav 0.05) and small (Kav 0.25), that were not synthesized at detectable levels by cultures from rabbits infected with T. pallidum for 0 or 5 days, were produced by cultures from 10-, 15- and 20-day infected rabbits. The small proteoglycan appeared to be synthesized first because greater amounts of this fraction were detected in extracts of cultures from 10-day infected animals. The large proteoglycan fraction may have been induced directly by T. pallidum because increased synthesis correlated with maximal treponemal numbers, 15 days after infection. In contrast, the induction of the smaller proteoglycan did not appear to be directly related to numbers of elutable organisms. The proteoglycans synthesized between 10 and 20 days after infection were analyzed for glycosaminoglycan (GAG) size and composition. The size of GAGs beta-eliminated from the proteoglycans generally increased over the 10-20-day infection period. Whereas the composition of the small proteoglycan fraction was largely unchanged during the 10-20-day period (45% chondroitin sulphate (CS), 55% dermatan sulphate (DS)), the amount of DS in the high molecular weight proteoglycan fraction decreased from 50-20% during this period (CS 50-80%). Autoradiography studies revealed that increased proteoglycan synthesis in T. pallidum-infected testes was localized to cells lining the seminiferous tubules and to fibroblasts infiltrating peritubular spaces.
Experimental studies have indicated that macrophages are involved in the pathogenesis of syphilis. Whether macrophages alone or with immune serum are ultimately responsible for killing of treponemes is disputed. We have demonstrated that BCG-vaccinated hamsters administered normal serum contained fewer treponemes in the inguinal and popliteal lymph nodes than did the nonvaccinated controls. When BCG-vaccinated hamsters were injected with syphilitic immune serum and challenged with Treponema pallidum ssp. endemicum, treponemicidal activity was enhanced. Treponemicidal activity was also detected in BCG-vaccinated hamsters challenged with treponemes treated in vitro with immune serum and its immunoglobulin fractions, especially IgG2. The immune IgG2 fraction had more treponemicidal activity than did the immune IgG1 fraction and the unfractionated immune serum. Our observations indicate an important synergistic role for macrophages and immune serum, especially IgG2, for elimination of T. pallidum ssp. endemicum from the host.
In a study to determine the reliability of specific direct fluorescent antibody staining of Treponema pallidum (DFATP) in lesion exudate, and to evaluate it as a potential diagnostic test to be used in lieu of or as an adjunct to the darkfield and other laboratory tests as an aid in the diagnosis of early syphilis, two types of comparisons were made: Study A-Replicate specimens from each of 350 lesions were examined in the state laboratory by DFATP and in the field by darkfield microscopy and the results were compared; Study B--95 specimens of lesion exudate were tested by the DFATP test and compared with the Fluorescent Treponemal Antibody Absorption (FTA-ABS) test on patients' sera. The tests in each study were evaluated as to sensitivity, specificity, and agreement with each other. The DFATP proved to be as reliable as the darkfield, was advantageous in some instances, and can be valuable when a darkfield examination is not feasible. Much of the responsibility of an adequate DFATP or darkfield test rests on the one who takes the specimen.