Pathogenesis of maternal-fetal syphilis revisited.

Although congenital syphilis has been recognized for several centuries and an efficient treatment with penicillin became available more than a half-century ago, the disease is still with us. Inability to culture in vitro the causative agent, Treponema pallidum, and the lack of an adequate animal model have prevented exploration of the various immunopathological events affecting the natural course of congenital infection. The purpose of this review is to analyze the disease in the context of recent knowledge acquired from human and experimental animals, particularly from the guinea pig model of congenital and neonatal syphilis, and to describe how the infection interacts with the maternal-fetal unit and how it is further modulated by the conceptus' ontogenic development. We also attempt to elucidate several old immunologic concepts and misconceptions that have remained unchallenged for too long.

Treponema pallidum subsp. pertenue displays pathogenic properties different from those of T. pallidum subsp. pallidum.

The present study described the susceptibility of C4D guinea pigs to cutaneous infection with Treponema pallidum subsp. pertenue Haiti B strain. The general manifestations of the disease in adults and neonates differ, to a certain degree, from those induced by T. pallidum subsp. pallidum Nichols strain. Noticeable differences between the infections were reflected in the character of the skin lesions, their onset and persistence, and the kinetics of the humoral response. The incidence and dissemination of cutaneous yaws lesions in very young guinea pigs were remarkably different from the low frequency observed in a similar age group of syphilis infection, 100 versus 17%, respectively. Moreover, as opposed to T. pallidum subsp. pallidum, T. pallidum subsp. pertenue does not cross the placenta. Offspring born to yaws-infected mothers did not produce immunoglobulin M antibodies and their organs, examined by PCR and rabbit infectivity test (RIT), were all negative. Examination of a large number of tissues and organs in adult, neonate, and maternal yaws by PCR and RIT clearly demonstrated that, unlike syphilis, there was a low incidence and short persistence of the yaws pathogen in internal organs. These findings stress the dermotropic rather than the organotropic character of yaws and provide further evidence of distinctive biological and pathological differences between yaws and venereal syphilis.

The time-dependent clearance of virulent Treponema pallidum in susceptible and resistant strains of guinea pigs is significantly different.

The kinetics of clearance of Treponema pallidum spp. pallidum Nichols from skin and testes of susceptible C4-deficient (C4D) and -resistant Albany (Alb) strains of guinea pigs (gps) was evaluated using the polymerase chain reaction (PCR) and the rabbit infectivity test (RIT). For each strain there were two groups of animals, one infected with virulent T. pallidum (TP) and one control injected with heat-killed treponemes (HKTP). The kinetic studies and their statistical analysis showed that in the C4D strain the microbial clearance in both tissues was significantly slower (p < 0.005) and still incomplete at 3 months after infection. In the Alb strain the clearance was faster and apparently completed within a month. A greater permissiveness in bacterial growth in C4D compared to Alb appears to be one critical factor determining the different rate of local elimination after primary infection. In both strains there was some correlation between the severity and duration of cutaneous lesions and the local persistence of viable organisms. This correlation was not observed in testes. These studies suggest a genetic basis for the strain-specific susceptibility and resistance phenotypes in the pathogenesis of syphilis.

Vertical transmission of Treponema pallidum to various litters and generations of guinea pigs.

The transmission of congenital syphilis was studied in a 4-generation guinea pig family with 10 litters and 38 offspring. By use of one or all of the following tests (ELISA-IgM, polymerase chain reaction, and rabbit infectivity), transplacental infection was demonstrated through 5 litters and up to 4 generations. Twenty-eight (93%) of 30 animals were positive by >/=1 test, and 2 (7%) were negative by 1 or 3 tests. While transmission of the pathogen appeared to be unaffected by the maternal acquisition of immunity, signs of smoldering infection in the young was suggested by the decline in humoral responses in successive progeny and by unusual rabbit infectivity test results. With each pregnancy there was a remarkable booster in the maternal humoral response, which dropped significantly prior to term. These findings shed new light on the understanding and interpretation of serologic testing during pregnancy and the perinatal period.

Laboratory methods of diagnosis of syphilis for the beginning of the third millennium.

Despite that the whole genome of T. pallidum, the causative agent of syphilis, has been sequenced, syphilis is, and will remain for some time, diagnosed by direct clinical observation and by laboratory methods. This review presents comprehensively most of the practical techniques used for direct detection of T. pallidum and lists all practical methods for phospholipid and treponemal antibodies detection. It describes most novel tests for syphilis, discusses problems with sero-creossreactivity in Lyme disease, immune responses in HIV-syphilis coinfected patients, and reviews serologic responses to antibiotic treatment.

Cytokine gene expression in skin of susceptible guinea-pig infected with Treponema pallidum.

Using a semi-quantitative multiplex reverse transcription-polymerase chain reaction assay, we examined cytokine mRNA expression for interleukin-1alpha (IL-1alpha), IL-2, IL-10, IL-12p40, tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) in skin samples obtained from C4-deficient (C4D) guinea-pigs inoculated intradermally with virulent Treponema pallidum (VTP). Controls included unmanipulated animals, guinea-pigs injected with T. pallidum-free rabbit inflammatory testicular fluid (ITF) alone, or mixed with heat-killed organisms (HKTP). The expression of IL-1alpha, IL-12p40, and TNF-alpha mRNA [T helper type 1 (Th1)] remained within the normal range in both infected and control animals throughout the experimental period. However, a significant increase (P<0.05) in IL-10 mRNA (Th2) was found exclusively in the VTP-inoculated animals from 3 to 30 days post-infection. Another unique characteristic of the inflammatory response in infected guinea-pigs was the appearance, between 11 and 30 days post-inoculation, of a substantial number of eosinophils in addition to infiltrating mononuclear cells. The results showed a local Th2 response which is consistent with an inadequate immune response. This is reflected by the lengthy and incomplete clearance of the pathogen from the local site of entry and the chronic infection of distant organs.

Identification of persistent infection in experimental syphilis by PCR.

The studies described herein were designed to evaluate the usefulness of the PCR in detecting persistent syphilitic infection. Three groups of animals were used: a nonimmune group infected with Treponema pallidum (NI/TP), a nonimmune group injected with heat-killed treponemes (NI/HKTP), and an immune and reinfected group (I/TP). All animals were inoculated with similar numbers of organisms distributed at 10 sites on the clipped back and in both testes. The persistence of the treponemes was examined by PCR and the rabbit infectivity test (RIT). The kinetic studies and statistical analysis of their results demonstrated that the rate of bacterial clearance from the NI/TP group was very low and incomplete at 4 months after infection. It was significantly different from those of both the NI/HKTP (P < 0.001) and I/TP (P < 0.05) groups. No statistically significant differences in treponemal elimination were found between the NI/HKTP and I/TP groups. PCR can detect the DNA of dead organisms, but the latter are eliminated by the host relatively quickly (15 to 30 days) as compared to elimination of live treponemes (>120 days). PCR results correlated well with RIT results. These data suggest that PCR-positive specimens obtained from an untreated patient(s) or collected weeks after treatment indicate persistent infection. They also show that the process of elimination of T. pallidum from primary sites of infection is prolonged and incomplete.

Spontaneous cytokine gene expression in normal guinea pig blood and tissues.

The authors report, for the first time, the cloning, characterization and sequencing of guinea pig cDNAs for interleukin (IL)-2, IL-10, IL-12p40, and transforming growth factor beta (TGF-beta). Partial cDNAs for two additional cytokines, IL-1alpha and TNF-alpha, whose sequences are present in the GenEMBL database, were also cloned. The IL-10 clone is a full-length cDNA, while the remaining clones are partial cDNAs. The guinea pig cDNA sequences have high identity with their mouse and human counterparts. Northern blot analysis revealed that the guinea pig transcripts range in size from 1.0 kb to 2.2 kb. The constitutive expression of cytokines in two strains of guinea pig (C4D, Albany) that differ in susceptibility to infection with Treponema pallidum was examined. Since susceptibility to T. pallidum is also age dependent, both neonates and adults were examined. Spontaneous cytokine expression was examined in peripheral blood, skin, spleen, lymph node, brain, and peritoneal cells. In skin, lymph node, and peripheral blood, very low levels of IL-1alpha, IL-12p40, tumour necrosis factor alpha (TNF-alpha), and TGF-beta and moderate levels of IL-2 and IL-10 were observed. Cytokine gene expression was not observed in spleen and brain. Peritoneal cells expressed only TGF-beta. Age- and strain-associated differences were not observed, except for IL-12p40, which was elevated in guinea pigs resistant to T. pallidum infection (C4D neonates, Albany adults).

Immune abnormalities in guinea pigs with asymptomatic congenital syphilis.

Spleens from 1-20-wk-old guinea pigs infected in utero with Treponema pallidum and age-matched controls, born to normal and heat-killed (56 degrees C, 2 h.) T. pallidum-injected mothers, were examined for their in vitro lymphoproliferative response to phytohemagglutinin, concanavalin A, and lipopolysaccharide. Additionally, T cell surface markers (mu-chain, pan T, CD4, and CD8) were determined in spleen, lymph node, and peripheral blood from 10-wk infected and normal pups by single and dual parameter fluorescence-activated cell sorter analysis. Compared with control animals, congenitally infected animals showed a remarkable prolonged naive-type of immune response as reflected by the higher (p < 0.01) proliferative responses to both T cell mitogens (up to 20 wk of age), and the weaker response to the B cell mitogen, significantly different (p < 0.01) at 10 wk of age. As opposed to controls, in all organs examined the level of CD8+ (cytotoxic/suppressor) T cells was significantly diminished (p < 0.01); consequently, the CD4/CD8 ratio was significantly elevated (p < 0.05). The role of C4 complement component and the nature and potential role of the immature T and B lymphocyte responses in asymptomatic congenital syphilis is discussed.

Target organs of infection in guinea pigs with acquired congenital syphilis.

The target organs of infection in guinea pigs with asymptomatic acquired or congenital syphilis were identified by PCR and in some cases by rabbit infectivity test (RIT). The prevalence of Treponema pallidum DNA was examined in the following seven organs: the inguinal and mesenteric lymph nodes, spleen, liver, kidney, heart, and brain. Test samples consisted of 95 organs from two genetically different strains of female guinea pigs (C4-deficient and Albany) with different susceptibilities to cutaneous infection by T. pallidum and 195 organs from their asymptomatic offspring. Twenty organs from dams of both strains injected with heat-killed T. pallidum and 19 organs from their progeny served as negative controls. The infections of mothers and neonates were documented by PCR, RIT, and serology. Though any of the organs tested could be infected, there was a spirochetal predilection for some anatomical locations, such as the lymph nodes, heart, and brain, regardless of the strain, route of maternal infection, and age. None of the 49 organs collected from control animals were positive by PCR. In infected C4-deficient dams, one to four organs were positive by PCR, whereas the organs of 7 of their 27 (25%) asymptomatic offspring were treponemal DNA negative, despite evidence of immunoglobulin M treponemal antibodies. Comparative analysis done by both PCR and RIT on a limited number of samples showed 90% agreement between results. An examination of multiple samples obtained from single organs demonstrated that even within 24 h of spirochetemia, when most organs appeared to be infected, not all samples from an individual organ were positive by PCR. A specific immunological response in guinea pigs with congenital syphilis was a more consistent parameter of vertical transmission than was an analysis of T. pallidum DNA.

Congenital and neonatal syphilis in guinea-pigs show a different pattern of immune response.

C4-deficient (C4D) and Albany strains of guinea-pigs transplacentally and neonatally infected with Treponema pallidum showed distinctive patterns of humoral immune responses. Congenitally infected progeny of both strains originated from dams intradermally (i.d.) infected at mid-pregnancy with virulent T. pallidum. In the neonatal groups families of C4D and Albany strains consisting of 1-3-day-old offspring and their mothers were i.d. infected with a similar dose of T. pallidum. Regardless of the strain, asymptomatic congenitally infected guinea-pigs (n = 16) responded from the first day of life with high levels of IgM [T. pallidum (TP) ELISA] antitreponemal antibodies and up to 85% presented with IgM CIC (circulating immune complexes) and IgM RF (rheumatoid factor). Although relatively high levels of IgM antitreponemal antibodies persisted in these animals throughout the 4-month experimental period, significant levels of host IgG antitreponemal antibodies were detectable after 2-3 months of age. Neonatally infected guinea-pigs of both strains (n = 27) responded similar to the infected sow but with relatively lower levels of IgM and IgG antitreponemal antibodies at 1 and 4 weeks, respectively, both of which increased with the time of infection. Antibodies were also detected in these animals by fluorescent treponemal antibody adsorption test (FTA-ABS). Unlike congenital syphilis, neonatally infected animals developed IgG-CIC after 2-3 months of infection and none of them showed any RF. In neonatal syphilis, FTA-ABS antibody levels were closely associated with the onset of lesions, whereas those of TP ELISA were not. The distinctive immune responses observed in these experimental models have the potential to differentiate between congenitally and neonatally infected human infants, even though the current clinical management is the same.

Immunization of guinea pigs with Treponema pallidum recombinant antigens reveals the presence of novel epitopes.

The DNA technology employed in the construction and purification of recombinant antigens has the potential of creating epitopes with specificities other than those of native antigens. Such a phenomenon has been observed when guinea pigs were immunized with Treponema pallidum recombinant antigens, TmpA and TmpB, expressed in Escherichia coli K12. Adsorption of the immune sera with E. coli K12 and T. pallidum revealed the presence of antibodies directed against epitopes not present or exposed in the native antigens of the organisms from which the DNA has been cloned.

Hepatic lesions in rabbits infected with Encephalitozoon cuniculi administered per rectum.

Microsporidia have been recognized recently as opportunistic pathogens in acquired immunodeficiency syndrome patients. In an attempt to develop an animal model of enteric microsporidiosis, adult (5 to 6 months old) male Flemish Giant rabbits from a closed New York colony were administered 5 x 10(3), 5 x 10(5), and 5 x 10(7) Encephalitozoon cuniculi per rectum. Rabbits given 5 x 10(5) and 5 x 10(7) E. cuniculi had moderate granulomatous periportal infiltrates, characterized by the presence of numerous macrophages, epithelioid cells and a few multinucleated giant cells, lymphocytes, and plasma cells. Inflammatory cells also were seen infiltrating the tunica adventitia and tunica media of hepatic portal veins and branches of the hepatic artery. This study demonstrates that administration of E. cuniculi per rectum to rabbits results in infection that is characterized by high frequency and severity of hepatic lesions.

Strain- and age-associated differences in lymphocyte phenotypes and immune responsiveness in C4-deficient and Albany strains of guinea-pigs.

Spleen lymphocytes from C4-deficient (C4D) and Albany strains of guinea-pigs, 1-7 days, 3-6 and 12-16 months old, genetically related to inbred strains 13 and 2 respectively, were analysed in terms of their expression of cell surface markers, allogenic and T- and B-cell mitogenic responses, and interleukin-1 (IL-1) and IL-2 production. There were strain- and age-associated differences in phenotypic expression and immune responsiveness levels. In both strains a significant shift in immunocompetence apparently occurs postnatally before 3-6 months of age, with no further significant changes noticed in animals 12-16 months old. Phenotypic changes in cell surface markers did not always correlate with functional capability of lymphoid cells. H159+ (pan T) and H155+ (CD4) lymphocyte number and levels of T-cell responsiveness (mitogenic and allogenic responses, and IL-2 production) were higher in C4D neonates compared with age-matched Albany guinea-pigs or with young animals of the same strain. On the other hand, 31D2+ (B) lymphocytes in a significantly higher proportion in Albany neonates compared with similarly aged C4D, did not correlate at this age or at any other time with their proliferative response to lipopolysaccharide (LPS) or dextran sulphate (DS), two B-cell-specific mitogens.

Production of rheumatoid factor in adoptively immune guinea-pigs after challenge with Treponema pallidum.

Guinea-pigs of inbred strains 2 and C4D were infused with various concentrations (1 x 10(8) to 4 x 10(8) of syngeneic nylon wool-purified Treponema pallidum-immune T lymphocytes (TPI-T) and challenged 24 hr later with virulent T. pallidum (10(8) organisms). The degree of protection depended on the number of infused T cells and was associated with an accelerated production of IgM rheumatoid factor (RF). Fully protected animals (4 x 10(8) TPI-T) did not produce treponemal antibodies or circulating immune complexes (CIC) but produced IgM RF detectable 10 days after infection. Partially protected animals (< or = 2 x 10(8) TPI-T) produced, 30 days post-infection, relatively low levels of treponemal antibodies but high levels of CIC and RF. Control animals infused with 2 x 10(8) TPI-T lymphocytes but not infected with T. pallidum, when monitored for a period of 6 weeks, did not produce treponemal antibodies, CIC, or RF, excluding the possibility that IgM RF could be generated by the donor's B cells contaminating (circa 3%) the TPI-T lymphocytes. Moreover, unprotected syngeneic control animals infused, prior to infection, with T. phagedenis biotype Reiter-immune T cells or with T. pallidum-free testicular inflammatory fluid-immune T cells responded with increasing levels of treponemal antibodies; only a few animals produced RF and CIC 5 months after infection similarly to control guinea-pigs infected only. The production of RF in partially protected animals responding to infection with treponemal antibodies and CIC was apparently associated with the presence of the CIC; but the mechanism of RF production in fully protected animals in which no antibodies or CIC were detected is currently unknown.

Effect of passive immunization with purified specific or cross-reacting immunoglobulin G antibodies against Treponema pallidum on the course of infection in guinea pigs.

Whole immune serum or highly purified immunoglobulin G (IgG) antibodies to Treponema pallidum exhaustively adsorbed with three strains of nonpathogenic treponemes (TPI-IgG) were used for passive immunization of inbred strain 2 guinea pigs before and after intradermal challenge with 3.4 x 10(7) virulent T. pallidum Nichols organisms. Before challenge, control animals received a similarly purified IgG fraction containing either a cocktail of antibodies against three nonpathogenic treponemes (NPTI-IgG) or IgG prepared from normal guinea pig serum (NGPS-IgG). The purified fractions contained both IgG1 and IgG2 isotypes. The antibody levels (detected by fluorescent treponemal antibody test and enzyme-linked immunosorbent assay) and molecular specificities (immunoblot) of sera obtained from recipient animals before infection reflected those of the purified fractions used for immunization. Three protocols of passive immunization were used. Whole immune serum containing specific and cross-reacting antibodies afforded better protection than TPI-IgG even though asymptomatic animals were not fully protected. A single intradermal injection (0.1 ml) of TPI-IgG or NPTI-IgG into one hind leg 22 h before infection at the same site provided relatively higher protection than multiple intravenous injections (total, 15 ml) of the respective individual preparations. Since purified NGPS-IgG injected in the same animals, into the opposite hind leg, failed to protect against the challenging infection, it is reasonable to assume that specific and cross-reacting antitreponemal antibodies of the IgG1 subclass, which in guinea pigs are homocytotropic, play a relevant role in local protection.

Detection of Treponema pallidum in early syphilis by DNA amplification.

By using experimentally infected rabbits as a model for early syphilis, the applicability of in vitro DNA amplification was explored for detection of Treponema pallidum. It was determined that whole blood in heparin or EDTA (but not serum), lesion exudate, and punch biopsy as well as swabs of lesions are useful specimens for examination by the polymerase chain reaction. Swabs do not require special diluents, and the specimens, whether kept at room temperature or frozen, are well suited for use in the polymerase chain reaction.

Experimental congenital syphilis: guinea pig model.

Neonates born to female guinea pigs of either a highly susceptible (C4D) or a resistant (Albany) strain, infected prior to or during pregnancy with a single dose of Treponema pallidum, showed in their sera from the first day of life immunoglobulin M (IgM) antibodies to T. pallidum, circulating immune complexes consisting of IgM antibodies and treponemal antigens, and IgM rheumatoid factor. Although the animals were asymptomatic for a 6-month observation period, several lines of evidence indicated that they were infected in utero. Molecular analysis of whole sera, purified serum IgM fraction, or dissociated immune complexes demonstrated IgM reactivity against one (47 kDa) or more of several T. pallidum peptides (15, 17, 37, 42, 45, and 87 kDa) recognized as integral membrane components. Sequential analysis of the neonates' sera by immunoblot and enzyme-linked immunosorbent assay, using alcohol-treated T. pallidum, T. phagedenis biotype Reiter, and T. vincentii, demonstrated early IgM antibodies followed 3 to 4 months later by IgG2- and IgG1-specific antibodies to T. pallidum. Moreover, an infectivity test done in five rabbits with pooled tissue extracts prepared from liveborn or stillborn animals evoked a seroconversion in two rabbits (reactive Venereal Disease Research Laboratory and fluorescent treponemal antibody tests), suggesting the presence of T. pallidum in the organs. Sera from neonates born to either T. phagedenis biotype Reiter-injected mothers or three normal pregnant females were all serologically negative. The model offers new possibilities for exploration of factors responsible for asymptomatic infection often observed in human congenital syphilis.

Immunization of guinea pigs with recombinant TmpB antigen induces protection against challenge infection with Treponema pallidum Nichols.

Treponema pallidum-susceptible guinea pigs of strain C4D were immunized with recombinant T. pallidum antigens TmpA, TmpB, TmpC, and TmpA plus TmpB plus TmpC; with Escherichia coli membranes; or with adjuvant alone. Animals in groups of five received six immunizing injections, each of 100 micrograms of antigen incorporated in RIBI adjuvant. After the sixth immunization, all experimental and nonimmunized controls were intradermally challenged with 3 x 10(6) T. pallidum Nichols freshly extracted from infected rabbit testes. Although high titers of antitreponemal antibodies in the fluorescent-treponemal-antibody test or an enzyme-linked immunosorbent assay were evoked in all animals immunized with recombinant antigens, only guinea pigs receiving TmpB antigen demonstrated protection expressed by the development of significantly (P less than 0.01) smaller, atypical lesions of significantly (P less than 0.01) shorter duration and devoid of or containing fewer T. pallidum organisms than lesions in the remaining immunized and control animals.