Konrad Wicher

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.

Treponema pallidum subsp. pertenue Displays Pathogenic Properties Different from Those of T. pallidum subsp. pallidum

ABSTRACT 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.

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).

Extensive Cross Reactivity between Treponema pallidum and Cultivable Treponemes Demonstrated by Sequential Immunoadsorption

Extensive cross reactivity between Treponema pallidum and three nonpathogenic species of treponemes was demonstrated by the Western blot technique. Rabbit antiserum produced by adjuvant immunization with solubilized T. pallidum antigens reacted with 34 T. pallidum antigens and with approximately 30 antigens each of T. phagedenis biotype Reiter, T. noguchii and T. vincentii. Adsorption of the antiserum with T. phagedenis Reiter removed only about half of the cross-reacting antibodies. Sequential adsorption with all three nonpathogenic treponemes removed antibodies to all but three polypeptides of 36,000, 34,000 and 27,000 daltons.

Differences in susceptibility to infection with Treponema pallidum (Nichols) between five strains of guinea pig.

Groups of 10 young male guinea pigs of inbred strains 2 and 13 and outbred strains Hartley A, Hartley B, and one deficient in the fourth component of complement (C4D) were infected intradermally with 80 X 10(6) Treponema pallidum (Nichols). The course of infection and production of antitreponemal antibody were examined. Strain C4D guinea pigs were the most susceptible to infection (100%); inbred strains 2 and 13 and outbred strain Hartley B showed 80-90% symptomatic infection; and the Hartley A strain was the least susceptible to infection (10%). Strain 13 animals responded with the highest antitreponemal antibody activity, and the Hartley A strain with the lowest. The results suggest that genetic factors or complement, or both, may influence the degree of susceptibility to infection with T pallidum in guinea pigs.

Experimental neonatal syphilis in a susceptible (C4D) and a resistant (Albany) strain of guinea pig

Despite similar levels of natural antibodies and treponemicidal activity, 83% of fourth complement component-deficient (C4D) mother guinea pigs developed ulcerative lesions to a challenge of 5 x 10(7) Treponema pallidum, whereas 75% of offspring 1 to 5 days old were temporarily (2-3 months) resistant to development of dermal lesions. In contrast, only 17% of Albany-strain mothers developed small papular lesions, while 68% of 1- to 5-day-old newborns developed large papular or ulcerative lesions within 9-15 days postinfection. These findings, together with the late development of both dermal lesions and antibodies in C4D neonates, preclude the concept of an antibody-associated natural resistance. T. pallidum infection in either C4D or Albany neonates was not associated with depletion of any particular cell population in lymphoid tissue. However, marked age- and strain-dependent histologic differences were noted. Histologic examination of lymph nodes and spleens from 17-day-old and 3- to 4-month-old animals showed that maturation of lymphoid tissues in C4D animals lagged behind the Albany strain at either age. Moreover, 75% of C4D newborns contained significantly higher levels of immunomodulatory alpha 1 fetoprotein than Albany neonates. The possibility that differences in susceptibility to T. pallidum infection between C4D and Albany guinea pigs as neonates and again as adults is the result of genetically associated changes in immunologic recognition is discussed.

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.

Kinetics of Antibody Response to Polypeptides of Pathogenic and Nonpathogenic Treponemes in Experimental Syphilis

Antigenic cross-reactivity between Treponema pallidum subspecies pallidum (TP) and nonpathogenic Treponema phagedenis biotype Reiter (TR), Treponema refringens strain Noguchi (TN), and Treponema vincentii (TV) was examined by the Western immunoblotting technique in pooled sera from five rabbits infected intratesticularly with T. pallidum. Sera were obtained before infection and on days 6, 12, 20, 30, 60, and 120 after infection. The pooled preinfection sera reacted with nine polypeptides of TV, nine of TS, and five of TR. The pooled sera did not show any clear-cut reactions with TP, but some individual rabbit sera did demonstrate visible reaction with three to five polypeptides of TP. Twelve days after infection, multiple serum antibodies reacting with polypeptides of all treponemes were detected. The number of antibodies reacting with polypeptides and the intensity of reaction increased with the duration of infection; for the nonpathogenic treponemes (TV, TS, TR) 21-26 polypeptides were identified on day 30, and by day 60 a total of 21 were detected for TP. By day 120 the reaction had become less pronounced, and fewer reactive polypeptides were seen. The extent of the cross-reactivity with the three nonpathogenic treponemes reflects the complex structure of T. pallidum, which should be viewed as a mosaic of more cross-reacting than strain- or species-specific antigens.

Increased Production of Antibodies to Spermatozoa and Seminal Fluid in Rabbits Used as Semen Donors

Sera of 20 male rabbits that were used as frequent donors of semen and age-matched normal male and female rabbit controls were examined by an enzyme-linked immunosorbent assay (ELISA), with sperm and seminal fluid as antigens. Five of the 20 semen donors developed an especially high humoral response to seminal fluid and spermatozoa, similar to that observed in some female breeders. The antibodies increased gradually during the 9 months of semen collection. The specificity of the antibodies was demonstrated by total or partial absorption with seminal fluid or spermatozoa. The antibodies were virtually all IgM and were directed against the acrosomal cap region of the spermatozoa, but variable fluorescence was also observed in the postnuclear region and tail.

Factors Affecting the Clinical Course of Treponema pallidum Infection in Guinea Pigs

The clinical course of infection with Treponema pallidum (Nichols) in inbred strain 2 guinea pigs was shown to be affected by age, sex and site of inoculation.