A murine model for the study of Chlamydia trachomatis genital infections during pregnancy

Pregnant BALB/c mice were inoculated intravaginally on day 5 of gestation with the Chlamydia trachomatis mouse pneumonitis biovar. Animals that received 10(5), 10(6), or 10(7) inclusion-forming units (IFU) of C. trachomatis delivered prematurely on days 15 to 16 of gestation. A focal inflammatory infiltrate was observed in the wall of the uterus on the day 14 of gestation in animals inoculated with 10(5) IFU. In this group of mice, immunohistochemical analysis showed chlamydial inclusions in the endometrium and fetal membranes.     

Effect of Chlamydia trachomatis infection and subsequent tumor necrosis factor alpha secretion on apoptosis in the murine genital tract

The pathology observed during Chlamydia infection is due initially to localized tissue damage caused by the infection itself, followed by deleterious host inflammatory responses that lead to permanent scarring. We have recently reported that the infection by Chlamydia in vitro results in apoptosis of epithelial cells and macrophages and that infected monocytes secrete the proinflammatory cytokine interleukin-1beta. At the same time, proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) can also trigger apoptosis of susceptible cells. To study the possible relationship between Chlamydia trachomatis infection and apoptosis in vivo, we used the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling technique to determine whether infection may cause apoptosis in the genital tract of mice and, conversely, whether cytokines produced during the inflammatory response may modulate the level of apoptosis. Our results demonstrate that infected cells in the endocervix at day 2 or 7 after infection are sometimes apoptotic, although there was not a statistically significant change in the number of apoptotic cells in the endocervix. However, large clumps of apoptotic infected cells were observed in the lumen, suggesting that apoptotic cells may be shed from the endocervix. Moreover, there was a large increase in the number of apoptotic cells in the uterine horns and oviducts after 2 or 7 days of infection, which was accompanied by obvious signs of upper tract pathology. Interestingly, depletion of TNF-alpha led to a decrease in the level of apoptosis in the uterine horns and oviducts of animals infected for 7 days, suggesting that the inflammatory cytokines may exert part of their pathological effect via apoptosis in infected tissues.     

Effect on Chlamydia trachomatis infection of the murine genital tract of adoptive transfer of congenic immune cells or specific antibody

Groups of progesterone-treated female CBA/nu mice were adoptively transferred with immune spleen cells or pooled antisera from congenic immunocompetent CBA donors that had been infected with a 'fast', human strain (SA-2f) of Chlamydia trachomatis. The spleen cells were given either intravenously (6.3 X 10(7) cells) or intraperitoneally (9.5 X 10(7) cells), and the antiserum (antibody titre 1:4096) was given intravenously. Strain SA-2f was introduced into the uterine cavity of these mice approximately 3 h after cell or antiserum transfer; antiserum was given also at intervals up to 23 days later. Untreated mice serving as controls were inoculated with chlamydiae in the same way. Subsequent recovery of chlamydiae from mice in the various groups indicated that transfer of cells or antiserum had not abrogated the chlamydial infections, despite high titres of chlamydial IgG antibody in the sera of all the recipient mice. These results confirm our earlier findings but are unlike those of some other investigators working with different mouse model systems. It seems that there are differences between systemic/respiratory immune mechanisms and those which operate locally in the uterus, which may be regarded as an immunologically privileged site.     

Prevalence of Chlamydia trachomatis in genital samples from Abidjan]

A study of direct genital swabs achieved in Abidjan, on 116 men and 131 women consulting for urogenital complaints, has revealed that the men show a prevalence of 28.4% Chlamydia trachomatis, and of 18.1% Neisseria gonorrhoeae. Concerning the women the prevalence of the same germs are 13.7% for Chlamydia trachomatis, and 4.6% for Neisseria gonorrhoeae. These results show the importance of Chlamydia trachomatis as a sexually transmitted disease in Abidjan (C?te-d'Ivoire). No differences were observed according to age in the two groups.     

A Chlamydia trachomatis-specific Th2 clone does not provide protection against a genital infection and displays reduced trafficking to the infected genital mucosa

A T helper type 1 (Th1) response is essential for resolving genital infections with the mouse pneumonitis biovar of Chlamydia trachomatis (MoPn). However, T-cell-dependent anti-chlamydial antibody is produced and may also contribute to protective immunity. We produced a MoPn-specific CD4 Th2 clone (Th2-MoPn) to study the role of a Th2 response during infection. We found that Th2-MoPn was unable to eradicate chlamydiae from the genital tract (GT) when it was transferred into MoPn-infected nude mice. Mice that received Th2-MoPn produced greater titers of MoPn-specific serum immunoglobulin G (IgG) antibody than mice that received a MoPn-specific Th1 clone (Th1-MoPn) (log(10) titers, 1.89 +/- 0.84 and 0.58 +/- 0.76 [mean +/- standard deviation], respectively [P < 0.01]). Also, the IgG isotypes were different for the two groups; whereas IgG1 was associated with Th2-MoPn, IgG2a was associated with Th1-MoPn. Also, infected nude mice that received Th2-MoPn produced higher levels of IgA in vaginal secretions. Although clone Th2-MoPn was detected in the GT, it was less efficient at migrating (112 +/- 35.6 labeled Th2 clone cells/10(5) GT cells) than Th1-MoPn (505 +/- 51.6 Th1 clone cells/10(5) GT cells) (P < 0.001, as determined by a t test). This may have been due to reduced expression of alpha4beta7 and P-selectin ligand 1 on Th2-MoPn. However, Th2-MoPn cells were retained in the GT during chronic infection and comprised 10 to 15% of the total GT cells 80 days after transfer. The data show that the MoPn-specific Th2 cells are important for serum and vaginal antibody production and may accumulate in the GT during chronic infection.     

Prior genital tract infection with a murine or human biovar of Chlamydia trachomatis protects mice against heterotypic challenge infection

We sought to assess the degree of cross-protective immunity in a mouse model of chlamydial genital tract infection. Following resolution of genital infection with the mouse pneumonitis (MoPn) biovar of Chlamydia trachomatis, mice were challenged intravaginally with either MoPn or human serovar E or L2. The majority of animals previously infected with MoPn were solidly immune to challenge with either of the two human biovars. Surprisingly, approximately 50% of animals became reinfected when homologously challenged with MoPn, although the secondary infection yielded significantly lower numbers of the organism isolated over a shorter duration than in the primary infection. Primary infection with serovar E also protected against challenge with MoPn or serovar L2, although the degree of immune protection was lower than that resulting from primary infection with MoPn. Blast transformation and assessment of delayed-type hypersensitivity indicated that mice previously infected with either human or murine biovars produced broadly cross-reactive T cells that recognized epitopes of either murine or human biovars of C. trachomatis. Immunoblotting demonstrated that primary MoPn infection produced immunoglobulin G (IgG) antibody to antigens of MoPn as well as at least three distinct antigenic components of human serovar E, one of which was identical in molecular weight to the major outer membrane protein (MOMP). Primary infection with serovar E produced IgG antibody reactive against serovar E but not MoPn MOMP and against at least one ca. 60-kDa protein of both chlamydial strains. Our results indicate that primary genital infection of mice with murine C. trachomatis induces immunity against challenge with either of two human biovars.     

Effect on Chlamydia trachomatis infection of the murine genital tract of adoptive transfer of congenic immune cells or specific antibody.

Groups of progesterone-treated female CBA/nu mice were adoptively transferred with immune spleen cells or pooled antisera from congenic immunocompetent CBA donors that had been infected with a ''fast'', human strain (SA-2f) of Chlamydia trachomatis. The spleen cells were given either intravenously (6.3 X 10(7) cells) or intraperitoneally (9.5 X 10(7) cells), and the antiserum (antibody titre 1:4096) was given intravenously. Strain SA-2f was introduced into the uterine cavity of these mice approximately 3 h after cell or antiserum transfer; antiserum was given also at intervals up to 23 days later. Untreated mice serving as controls were inoculated with chlamydiae in the same way. Subsequent recovery of chlamydiae from mice in the various groups indicated that transfer of cells or antiserum had not abrogated the chlamydial infections, despite high titres of chlamydial IgG antibody in the sera of all the recipient mice. These results confirm our earlier findings but are unlike those of some other investigators working with different mouse model systems. It seems that there are differences between systemic/respiratory immune mechanisms and those which operate locally in the uterus, which may be regarded as an immunologically privileged site.     

HLA-B27 expression does not modulate intracellular Chlamydia trachomatis infection of cell lines

Chlamydia trachomatis is an obligate intracellular pathogen. Infection of susceptible individuals with this bacterium can trigger the development of reactive arthritis, an acute inflammation that is associated with the expression of the class I major histocompatibility antigen, HLA-B27. Other facultative intracellular pathogens, such as Yersinia and Salmonella spp., are also known triggers of reactive arthritis. Previous studies report conflicting results concerning whether the presence of HLA-B27 modulates the infection of cells with these enteric pathogens. In the present study, we have examined whether the expression of HLA-B27 can influence the infection of cell lines with C. trachomatis and also whether the replication of these bacteria is altered in HLA-B27-expressing cell lines. To do this, we have used a sensitive flow cytometric approach. We fixed and permeabilized cells and used fluorescein isothiocyanate-conjugated monoclonal antibody specific for chlamydia lipopolysaccharide to detect intracellular bacteria. The staining pattern obtained closely resembled the intracellular life cycle of chlamydia, with the appearance of brightly staining cells correlating to the microscopic detection of mature inclusion bodies. Moreover, since the percentage of cells that stained with the antibody was proportional to the infectious inoculum used, we were able to use the technique to quantitate the number of infectious organisms recoverable from infected cell lines. An important component of our study was the use of heparin to prevent reinfection of cells and thus enable the infection to be followed from a discrete time point. Our results suggest that HLA-B27 influences neither the infection nor replication of C. trachomatis serovar L2 within cell lines. Consequently, the role of HLA-B27 in the pathogenesis of reactive arthritis may lie downstream of the invasion and replication stages of the triggering pathogenic infection.     

In situ analysis of the evolution of the primary immune response in murine Chlamydia trachomatis genital tract infection

Adaptive immune responses contribute to the resolution of Chlamydia trachomatis genital tract infection and protect against reinfection, but our understanding of the mechanisms of those protective responses is incomplete. In this study, we analyzed by in situ immunohistochemistry the progression of the inflammatory and cytokine responses in the genital tracts of mice vaginally infected with C. trachomatis strain mouse pneumonitis. The cellular inflammatory response was characterized by an initial elevation in myeloid cells in the vagina (day 3) and uterine horns (day 7), followed by a marked rise in the number of T cells, predominantly CD4(+) cells. CD8(+) T cells and CD45R(+) B cells were also detected but were much less numerous. Perivascular clusters of CD4(+) T cells, which resembled clusters of T cells seen in delayed-type hypersensitivity responses, were evident by 2 weeks postinfection. Following the resolution of infection, few CD8(+) T cells and CD45R(+) B cells remained, whereas numerous CD4(+) T cells and perivascular clusters of CD4(+) T cells persisted in genital tract tissues. Interleukin-12 (IL-12)- and tumor necrosis factor alpha (TNF-alpha)-producing cells were observed in vaginal tissue by day 3 of infection and in uterine tissues by day 7. Cells producing IL-4 or IL-10 were absent from vaginal tissues at day 3 of infection but were present in uterine tissues by day 7 and were consistently more numerous than IL-12- and TNF-alpha-producing cells. Thus, the evolution of the local inflammatory response was characterized by the accumulation of CD4(+) T cells into perivascular clusters and the presence of cells secreting both Th1- and Th2-type cytokines. The persistence of CD4(+)-T-cell clusters long after infection had resolved (day 70) may provide for a readily mobilizable T-cell response by which previously infected animals can quickly respond to and control a secondary infectious challenge.     

Experimental infection of the marmoset genital tract with Chlamydia trachomatis.

A strain of Chlamydia trachomatis isolated in McCoy cells from the urethra of a patient suffering from non-gonococcal urethritis was inoculated into the vagina of 8 female marmosets. Chlamydiae were isolated repeatedly for 10-42 days from the lower genital tract of 7 of the marmosets. Six of the infected animals developed an acute inflammatory reaction in the genital tract and chlamydial inclusions in epithelial cells were seen in smears from 2 of them. In addition, each of 6 infected marmosets examined developed humoral antibodies to C. trachomatis. In contrast, 3 control animals inoculated intravaginally with chlamydia-free McCoy cells showed no evidence of chlamydial infection. Since the marmoset is small and easily bred in captivity, it should provide a useful model for studying the mechanisms of chlamydial pathogenicity.     

Multicenter evaluation of the AntigEnz Chlamydia enzyme immunoassay for diagnosis of Chlamydia trachomatis genital infection.

To evaluate the AntigEnz Chlamydia enzyme immunoassay (EIA) (Baxter/Bartels, Issaquah, Wash.), we studied 320 men and 1,209 women attending clinics for sexually transmitted diseases in Baltimore, San Francisco, and Seattle. At examination, two separate swabs were obtained from each patient, one for chlamydial culture and one for EIA. Cervical samples were collected from women, and urethral samples were collected from men. The prevalence of chlamydial infection by culture was 9% in Baltimore (n = 532), 11% in Seattle (n = 500), and 9% in San Francisco (n = 497). To resolve specimens with discrepant culture and EIA results, the EIA transport buffer was centrifuged and the resuspended pellet was stained by direct immunofluorescence to determine whether elementary bodies were present. Overall sensitivity of the AntigEnz Chlamydia assay compared with culture was 87% in men and 86% in women, and overall specificities were 94 and 97%, respectively. Differences between centers were seen, with sensitivities ranging from 76% among men and 79% among women in Seattle to 100% among men and 95% among women in Baltimore. With a true positive considered to be either a culture-positive or an EIA- and direct immunofluorescence-positive specimen, the revised sensitivity was 91% in men and 88% in women. Overall revised specificity was 99% in both men and women. We conclude that in this high-prevalence population, the sensitivity and specificity of this assay compare favorably with those of other noncultural antigen detection tests for the diagnosis of chlamydial genital infection.     

Detection of Chlamydia trachomatis in genital specimens by the Chlamydiazyme test.

Cotton swabs were used to collect two specimens each from 416 patients (206 males, 210 females) attending a sexually transmitted disease clinic. The first swab was transported in Specimen Storage Reagent and extracted in Specimen Dilution Buffer for enzyme immunoassay by Chlamydiazyme (Abbott Laboratories); the second swab was extracted into 2SP and inoculated into McCoy cell cultures. In the first phase of the study (215 patients: 111 males, 114 females) enzyme immunoassay results were positive (optical density greater than or equal to 0.1) in 30 of 35 instances in which Chlamydia trachomatis was isolated (sensitivity, 86%). Of 18 false-positive enzyme immunoassay results, 15 (83%) were cervical swabs (specificity, 90%). In a phase II study, using a modified Chlamydiazyme kit, 201 patients were tested (95 males, 106 females). Of 41 chlamydial isolates, 8 were not detected by the Chlamydiazyme test (sensitivity, 81%). Only three positive Chlamydiazyme test results could not be confirmed by culture (specificity, 98%). Overall, Chlamydiazyme assay provided a rapid (4 h), sensitive, and specific assay for the detection of chlamydial antigens.     

Immunity to murine Chlamydia trachomatis genital tract reinfection involves B cells and CD4(+) T cells but not CD8(+) T cells

CD4(+) T-helper type 1 (Th1) responses are essential for the resolution of a primary Chlamydia trachomatis genital tract infection; however, elements of the immune response that function in resistance to reinfection are poorly understood. Defining the mechanisms of immune resistance to reinfection is important because the elements of protective adaptive immunity are distinguished by immunological memory and high-affinity antigen recognition, both of which are crucial to the development of efficacious vaccines. Using in vivo antibody depletion of CD4(+) and CD8(+) T cells prior to secondary intravaginal challenge, we identified lymphocyte populations that functioned in resistance to secondary chlamydial infection of the genital tract. Depletion of either CD4(+) or CD8(+) T cells in immune wild-type C57BL/6 mice had a limited effect on resistance to reinfection. However, depletion of CD4(+) T cells, but not CD8(+) T cells, in immune B-cell-deficient mice profoundly altered the course of secondary infection. CD4-depleted B-cell-deficient mice were unable to resolve a secondary infection, shed high levels of infectious chlamydiae, and did not resolve the infection until 3 to 4 weeks following the discontinuation of anti-CD4 treatment. These findings substantiated a predominant role for CD4(+) T cells in host resistance to chlamydial reinfection of the female genital tract and demonstrated that CD8(+) T cells are unnecessary for adaptive immune resistance. More importantly, however, this study establishes a previously unrecognized but very significant role for B cells in resistance to chlamydial reinfection and suggests that B cells and CD4(+) T cells may function synergistically in providing immunity in this model of chlamydial infection. Whether CD4(+) T cells and B cells function independently or dependently is unknown, but definition of those mechanisms is fundamental to understanding optimum protective immunity and to the development of highly efficacious immunotherapies against chlamydial urogenital infections.     

Immunity to reinfection of the genital tract of marmosets with Chlamydia trachomatis.

Eleven marmosets inoculated intra-vaginally with either of 2 serotypes (D/E and H) of Chlamydia trachomatis developed a self-limited infection which persisted usually for 10-42 days. Animals re-inoculated on one or more occasions were, however, infected generally for a shorter duration, usually 3-7 days. Curtailed infections were observed after re-inoculation with either the same or a different serotype, indicating that immunity was not serotype specific but cross-protective. IgM and/or IgG chlamydial antibody, measured by micro-immunofluorescence, developed in most of the marmosets on primary infection and was not serotype specific. The antibody titres were boosted on re-infection and there was a correlation between pre-existing high antibody titres and infections of short duration. Chlamydial infection of the genital tract was accompanied by acute inflammation which persisted in about half of the immune animals for up to several weeks despite rapid clearance of the organisms. These features of the experimental infection should help to provide a greater understanding of the immunobiology and pathogenesis of chlamydial genital-tract infections of humans.     

Interrelationship of Chlamydia trachomatis and other pathogens in the female genital tract.

The isolation of Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, and Candida albicans in the female genital tract was studied in 1323 patients attending a venereal disease clinic. Disruption of the cell monolayers use for the isolation of C. trachomatis was significantly associated with the presence of T. vaginalis; this effect was markedly reduced by the addition of vancomycin to gentamicin and amphotericin B in the transport and growth media. The only significant positive association was the more frequent isolation of C. trachomatis in the presence of N. gonorrhoeae. There was a negative association between N. gonorrhoeae and C. albicans and between T. vaginalis and C. albicans, the fungus being isolated significantly less frequently when these microorganisms were present.     

Detection of Chlamydia trachomatis in genital specimens by the Microtrak direct specimen test

The conventional cell culture method for detection of Chlamydia trachomatis requires two to six days and is technically difficult to perform. The authors evaluated a new, relatively simple, non-culture method (MicroTrak, Syva Co., Palo Alto, CA) that requires less than one hour to complete. Two hundred fifty-one cervical and 209 male urethral specimens from three Richmond health clinics were read by direct immunofluorescence staining and compared with cell culture technics using iodine staining. Patient specimens were applied directly onto microscope slides (8 mm well) and stained with a fluorescein-labeled monoclonal antibody. Slides were examined for 10-15 minutes at X1,000 using an AO epifluorescent microscope and were considered positive if five or more typical elementary bodies were seen. The sensitivity, specificity, positive and negative predictive values for the direct smear were 89%, 97%, 85%, and 98% for males, and 93%, 96%, 85%, and 98% for females, respectively. The rapid direct specimen test appears to be a satisfactory method for detecting chlamydia in male and female genital specimens.     

Growth of Neisseria gonorrhoeae in the female mouse genital tract does not require the gonococcal transferrin or hemoglobin receptors and may be enhanced by commensal lactobacilli

Neisseria gonorrhoeae is capable of utilizing a variety of iron sources in vitro, including human transferrin, human lactoferrin, hemoglobin, hemoglobin-haptoglobin complexes, heme, and heterologous siderophores. Transferrin has been implicated as a critical iron store for N. gonorrhoeae in the human male urethra. The demonstration that gonococci can infect the lower genital tracts of estradiol-treated BALB/c mice in the absence of human transferrin, however, suggests that other usable iron sources are present in the murine genital tract. Here we demonstrate that gonococcal transferrin and hemoglobin receptor mutants are not attenuated in mice, thereby ruling out transferrin and hemoglobin as essential for murine infection. An increased frequency of phase variants with the hemoglobin receptor "on" (Hg(+)) occurred in ca. 50% of infected mice; this increase was temporally associated with an influx of neutrophils and detectable levels of hemoglobin in the vagina, suggesting that the presence of hemoglobin in inflammatory exudates selects for Hg(+) phase variants during infection. We also demonstrate that commensal lactobacilli support the growth of N. gonorrhoeae in vitro unless an iron chelator is added to the medium. We hypothesize that commensal lactobacilli may enhance growth of gonococci in vivo by promoting the solubilization of iron on mucosal surfaces through the production of metabolic intermediates. Finally, transferrin-binding lipoprotein (TbpB) was detected on gonococci in vaginal smears, suggesting that although gonococci replicate within the genital tracts of mice, they may be sufficiently iron-stressed to express iron-repressible proteins. In summary, these studies support the potential role of nontransferrin, nonhemoglobin iron sources during gonococcal infection of the female genital tract.     

Evaluation of chlamydiazyme for the detection of genital infections caused by Chlamydia trachomatis.

Chlamydiazyme is a 4-h enzyme-linked immunoassay that detects an antigen of Chlamydia trachomatis directly in clinical specimens. This immunoassay was compared with cell culture for the diagnosis of chlamydial infections of the genital tract. The assay was evaluated at five clinics with a total of 1,277 cervical specimens of which 239 were culture positive. At three of these clinics where urethral samples were taken from males, 99 of 363 samples were culture positive. The sensitivity of the assay averaged 89.5% for detecting cervical infections and 78.8% for detecting male urethral infections. Specificity was 97.0% when samples from either males or females were tested. Some patients who were culture negative were infected with chlamydiae according to both Chlamydiazyme and a monoclonal antibody test that detected a chlamydial antigen distinct from the antigen detected by Chlamydiazyme. If the 15 females and 2 males who were positive by both immunoassays but culture negative were considered positive for chlamydial infection, the specificity of the assay was 98.4% in females and 97.7% in males. Chlamydiazyme is a simple and relatively rapid immunoassay that has sufficient sensitivity and specificity to supplant culture in the detection of genital chlamydial infections.     

Disease outcome subsequent to primary and secondary urogenital infection with murine or human biovars of Chlamydia trachomatis

A susceptible strain of mice infected intravaginally with the mouse pneumonitis biovar of Chlamydia trachomatis became infertile and sustained high rates of hydrosalpinx formation regardless of prior infection with a human serovar. Conversely, susceptible mice infected with human serovars remained fertile unless challenged with a homologous human serovar.