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.

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.

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.

ABSTRACTThe present study described the susceptibility of C4D guinea pigs to cutaneous infection withTreponema pallidumsubsp.pertenueHaiti B strain. The general manifestations of the disease in adults and neonates differ, to a certain degree, from those induced byT. pallidumsubsp.pallidumNichols 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 toT. pallidumsubsp.pallidum,T. pallidumsubsp.pertenuedoes 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.

This chapter describes the various technical aspects of the guinea-pig model of acquired and congenital syphilis. This model has been employed since 1907 by various investigators for studies of syphilis. Of all small laboratory mammals, the guinea-pig reproductive system resembles the humans most, as it has a long cycle, ovulates spontaneously, and has an actively secreting corpus luteum. Infected animals are kept in air-conditioned rooms at 15–16° C to enhance the spirochetal growth. The animals are fed with antibiotic-free Purina 5025 guinea-pig diet reinforced with vitamin C and water ad libitum. Guinea-pigs are anesthetized by an intramuscular (i.m.) injection of ketamine for treatments such as clipping, shaving, bleeding, and infection. The animal returns to normal behavior within 1 hour. For skin biopsy and tattooing the i.m. injection of ketamine is supplemented with a local injection of 0.05 ml oflidocaine 2%. Clipping and depilation is done prior to intradermal infection and then periodically to keep the infected area cool. This also facilitates the observation and follow-up of lesion development. T. pallidum infection in guinea-pigs is affected by the factor of age, sex, and breed. In the C4D guinea-pigs, the onset of primary lesions at the site of inoculation is approximately 5–6 days after infection. It starts as an induration, which evolves into severe ulcerative lesions of 8–18 mm diameter between 9 and 13 days. Infection with T.pallidurn does not cause the animals discomfort or increase of body temperature as the pattern of behavior and intake of food and water resemble those of normal animals. Transplacental infection in guinea-pigs occurs regardless of the maternal susceptibility to T. pallidurn infection, which is facilitated by the animal's relatively long gestation period.

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.

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.

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.

ABSTRACT 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 withTreponema 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 ofT. pallidum from primary sites of infection is prolonged and incomplete.

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).

Using a semi‐quantitative multiplex reverse transcription–polymerase chain reaction assay, we examined cytokine mRNA expression for interleukin‐1α (IL‐1α), IL‐2, IL‐10, IL‐12p40, tumour necrosis factor‐α (TNF‐α) and transforming growth factor‐β (TGF‐β) 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‐1α, IL‐12p40, and TNF‐α 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.

Spleen, lymph node, and peripheral blood lymphocytes from healthy guinea pigs (gp) were examined for their ability to produce polyreactive autoantibodies to a battery of self-antigens and to cryptic determinants (phosphatidylcholine) on bromelain-treated mouse red blood cells (Br-MRBC). The mouse monoclonal antibody (Mab) 8BE6 anti-gp pan-T (CD5) marker was used for identification of CD5+ B1 cells by the plaque-forming assay (PFC), immunofluorescence, complement-mediated cytotoxicity, and immunocytochemistry. The detection of CD5+ cells by the 8BE6 Mab depended on the method used. They were better demonstrated by cytolysis and immunocytochemistry than by FACS analysis. By the latter method, the level of the CD5+ B cell subpopulation was associated neither with the age of the gp nor with the organ examined. Similarly wide ranges of PFC were detected in untreated or LPS-treated animals regardless of age and organ. The vast majority of the LPS-stimulated IgM antibody-secreting B lymphocytes reacting with the Br-MRBC, and those producing natural autoantibodies, did not bind the 8BE6 Mab.

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°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 (μ-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.

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.