Victoria 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.

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).

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.

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.

Effect of Irradiation and Depletion of C3-Complement Component on the Course of Treponema pallidum Infection in a Resistant Guinea Pig Strain

The role of complement and ionizing radiation in the natural resistance to Treponema pallidum infection of Albany guinea pigs was explored. Depletion of C3 by cobra venom factor for a period of 14 days affected neither the hosts susceptibility to infection nor the humoral response. Total body irradiation with 420 or 800 R was fatal within 20-30 days and there was no multiplication of treponemes in the infected host. Animals showing lethal signs were euthanized and tissues removed for examination. Exposure to a nonlethal dose of 300 R increased the susceptibility to infection (46% symptomatic lesions) and facilitated multiplication of treponemes at the site of inoculation and in the lymphoid organs, but the humoral response was not different from that of non-irradiated controls. The results seem to suggest a defect in antigen recognition by the immunocompetent cells in the resistant Albany guinea pigs.

Immunocompetence of inflammatory cells in rabbit testes infected with Treponema pallidum

Systematic studies were conducted in rabbits to delineate factors favoring the predilection for multiplication of T. pallidum in testes. The results strongly suggest that, in addition to the mucoid material produced during lesion development regardless of the site of infection, a whole array of testicular substances with immunomodulatory activity may largely contribute to the propagation and delayed clearance of the pathogen from the testicular environment and most likely from the host.

Lymphocyte Transformation in Inbred Guinea Pigs Infected with Treponema pallidum Nichols

T lymphocytes were purified from peritoneal exudates from young male, inbred strain 2 guinea pigs infected with 8 X 10(7) Treponema pallidum Nichols and from control animals injected with normal rabbit testes extract. Groups of animals were sacrificed after 15, 30, 60, and 90 days of infection, and the cells were analyzed for their proliferative response to concanavalin A and phytohemagglutinin and to sonicated T. pallidum, Treponema phagedenis biotype Reiter, and normal rabbit testes extract. No significant differences were observed in the responses to mitogens. Cells from infected but not from control animals responded with significant proliferation to T. pallidum antigen, but neither infected nor control cells reacted to T. phagedenis Reiter antigen. Although cells from both infected and control animals responded to normal rabbit testes extract, this response in the infected animals was transient and much lower than the response to T. pallidum antigen.

Chemotaxis in Experimental Syphilis

Peritoneal exudate leukocytes from normal and Treponema pallidum-infected rabbits were examined for their chemotactic response to various chemoattractants and treponemal preparations. Leukocytes from normal and infected animals responded well to bacterial and serum-derived chemoattractants. Suspensions of washed Treponema phagedenis biotype Reiter but not T. pallidum exerted significant chemotactic activity. Cells from rabbits infected for greater than or equal to 7 days demonstrated increased locomotion, suggesting the possibility of increased random motility.