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PubMed1996

A model of genital Chlamydia trachomatis infection using human xenografts in severe combined immunodeficiency mice.

We developed a new model of human genital Chlamydia trachomatis infection in order to characterize the pathogen-host relationship in a clinically relevant system using a human strain of C. trachomatis instead of the commonly employed mouse biovar (MoPn). Human endometrial tissue was xenografted into the skin of mice homozygous for the mutation severe combined immunodeficiency and inoculated with C. trachomatis serovar K. C. trachomatis efficiently infected the endometrium as shown by cell culture and immunofluorescence microscopy and persisted for more than 6 weeks. Chlamydial inclusions detected by direct immunofluorescence and electron microscopy appeared to be smaller than those produced by in vitro cell culture-grown chlamydiae. A pattern of localized mild infection prevailed, and infiltrative uncontrolled spread of chlamydiae was observed in only 1 of 10 infected grafts. This might correspond to the well-known tendency of the agent to cause asymptomatic infections. This model allows the study of a human genital infection resembling the clinical situation and offers the possibility to better characterize the host-parasite relationship with respect to pathogenicity and therapy.

Animals↗
PubMed1997

In vitro infection of smooth muscle cells by Chlamydia pneumoniae.

Recent observations have shown that both Chlamydia pneumoniae antigens and DNA may be found within atherosclerotic lesions. In this study, we evaluated the ability of C. pneumoniae to infect cells that make up atherosclerotic lesions, including endothelial cells, smooth muscle cells, and cholesterol-loaded smooth muscle cells. The organism readily infected rabbit, bovine, and human aortic smooth muscle cells. Cholesterol-loaded smooth muscle cells were even more susceptible to C. pneumoniae infection. Chlamydia trachomatis inefficiently infected smooth muscle cells, demonstrating that this is not a characteristic of all members of the genus Chlamydia. C. pneumoniae infected bovine endothelial cells poorly. This study demonstrates that C. pneumoniae readily infects one of the important types of cells found within atherosclerotic lesions, i.e., smooth muscle cells with and without cholesterol loading.

Animals↗
PubMed1997

Characterization of the Chlamydia trachomatis vacuole and its interaction with the host endocytic pathway in HeLa cells.

Chlamydia trachomatis, an obligate intracellular parasite and a major human pathogen, invades eukaryotic host cells and replicates within a membrane-bound compartment (termed the vacuole or inclusion) in the cytoplasm of the host cell. In this report, we describe in detail the characteristics of the vacuole throughout the chlamydial life cycle in terms of the endocytic pathway, as determined by epifluorescent and confocal immunofluorescence microscopy. By indirect immunofluorescence, the transferrin receptor (TfR), a component of early endosomes, and the cation-independent mannose-6-phosphate receptor (CI-M6PR), a component of late endosomes, were found in close association with the chlamydial vacuole as early as 4 h postinfection (hpi) and as late as 20 hpi. Fluorescein isothiocyanate (FITC)-labeled Tf was also found to colocalize with the vacuole at 4, 12, and 20 hpi, indicating that exogenously added ligands can be transported to the region of the vacuole. Antibodies to several different lysosomal proteins failed to label the chlamydial vacuole at any time point during the life cycle. Indirect immunofluorescence of cells infected with chlamydiae stained with an antibody to the trans-Golgi network (TGN) protein TGN38 demonstrated that in infected cells, the integrity and structure of the TGN was altered. The rates of Tf recycling in infected and uninfected cells were compared by fluorescence microscopy and quantitated with 125I-Tf. While the rate of FITC-Tf recycling from endocytic compartments in chlamydia-infected cells did not appear different from that of uninfected cells, a small pool of FITC-Tf that had accumulated adjacent to the chlamydial vacuole recycled at a slower rate. Quantitation of Tf recycling with 125I-Tf showed that Tf was recycled more slowly in infected cells than in uninfected cells. The altered distribution of several endocytic pathway markers and the slowed Tf recycling are consistent with the hypothesis that the chlamydial vacuole interacts with the endocytic pathway of the host. These results furthermore suggest that the chlamydial vacuole does not correspond to a canonical endocytic compartment but that it is a unique and dynamic organelle that shares several characteristics with recycling endosomes of the host cell. Interactions with the early and/or late endosomal compartments, in addition to the Golgi apparatus, may provide a source of membrane or nutrients for the replicating organisms.

Biomarkers↗
PubMed1997

Genital tract infection with Chlamydia trachomatis fails to induce protective immunity in gamma interferon receptor-deficient mice despite a strong local immunoglobulin A response.

CD4+ T cells have been found to play a critical role in immune protection against Chlamydia trachomatis infection. Since both humoral and cell-mediated antichlamydial immunity have been implicated in host protection, the crucial effector functions provided by the CD4+ T cells may rely on Th1 or Th2 functions or both. In the present study, we evaluated the development of natural immunity following vaginal infection with C. trachomatis serovar D in female gamma interferon receptor-deficient (IFN-gammaR-/-) mice with a disrupted Th1 effector system. We found that in comparison with wild-type mice, the IFN-gammaR-/- mice exhibited a severe ascending primary infection of prolonged duration which stimulated almost 10-fold-stronger specific local immunoglobulin A (IgA) and IgG responses in the genital tract. Following resolution of the primary infection and despite the augmented antibody responses to chlamydiae, the IFN-gammaR-/- mice were completely unprotected against reinfection, suggesting that local antibodies play a subordinate role in host protection against chlamydial infection. Immunohistochemical analysis of frozen sections of the genital tract revealed many CD4+ T cells in the IFN-gammaR-/- mice, with a dominance of interleukin 4-containing cells in mice following resolution of the secondary infection. However, in contrast to the findings with wild-type mice, the typical clusters of CD4+ T cells were not found in the IFN-gammaR-/- mice. Few and similarly distributed CD8+ T cells were observed in IFN-gammaR-/- and wild-type mice. Whereas chlamydia-infected macrophages from wild-type mice had no inclusion bodies (IB) and produced significant amounts of nitric oxide (NO) in the presence of IFN-gamma, macrophages from IFN-gammaR-/- mice contained many IB but no NO. These results indicate that CD4+ Th1 cells and IFN-gamma, rather than local antibodies, are critical elements in host immune protection stimulated by a natural ascending C. trachomatis infection in the female genital tract.

Animals↗
PubMed1998

Chlamydia trachomatis infection in the female reproductive tract of the rat: influence of progesterone on infectivity and immune response.

As the most common cause of sexually transmitted disease in women, chlamydial infections can lead to pelvic inflammatory disease, infertility, and ectopic pregnancy. To better understand the role played by sex hormones in modulating the immune response of the genital tract to microbial infections, we have developed a rat model to study Chlamydia trachomatis infection. Inbred female Lewis rats were primed with progesterone and inoculated by intrauterine instillation of C. trachomatis (mouse pneumonitis strain MoPn) into each uterine horn. When infected animals were examined for the presence of chlamydial antigens 14 days postinfection, both the uterus and vagina were found to be positive compared to those of saline-treated animals, which did not show specific staining. The involvement of local and systemic immune systems following chlamydial infection was determined by analyzing major histocompatibility complex (MHC) class II expression in the reproductive tract and lymphocyte proliferation in response to mitogenic and chlamydia-specific stimulation of cells from the spleen and lymph nodes (LN) draining the reproductive tract. Enhanced proliferation was observed in LN following mitogenic but not antigenic (MOMP [major outer membrane protein]) stimulation. In contrast, spleen cell proliferation was lower in chlamydia-infected rats than in saline-treated controls. MHC class II expression, an indicator of inflammatory responses, was upregulated in the uterus, on glandular epithelial cells, and adjacent to glands in response to chlamydial infection. In other experiments, when rats were infected at estrus and diestrus without prior progesterone priming, chlamydial inclusions were not detected in either the uterus or vagina. However, enhanced lymphocyte proliferation was observed in response to mitogenic and MOMP stimulation in the reproductive tract-draining LN from estrous and diestrous animals. These findings indicate that under appropriate endocrine conditions, the rat uterus is susceptible to C. trachomatis infection and that immune responses to this pathogen can be detected locally and systemically. Further, they suggest that clearance of the infection from the reproductive tract involves immune cells from the LN draining the reproductive tract.

Animals↗
PubMed1998

Human mannose-binding protein inhibits infection of HeLa cells by Chlamydia trachomatis.

The role that collectin (mannose-binding protein) may play in the host's defense against chlamydial infection was investigated. Recombinant human mannose-binding protein was used in the inhibition of cell culture infection by Chlamydia trachomatis (C/TW-3/OT, E/UW-5/Cx, and L2/434/Bu), Chlamydia pneumoniae (AR-39), and Chlamydia psittaci (6BC). Mannose-binding protein (MBP) inhibited infection of all chlamydial strains by at least 50% at 0.098 microg/ml for TW-3 and UW-5, and at 6.25 microg/ml for 434, AR-39, and 6BC. The ability of MBP to inhibit infection with strain L2 was not affected by supplementation with complement or addition of an L2-specific neutralizing monoclonal antibody. Enzyme-linked immunosorbent assay and dot blot analyses showed MBP bound to the surface of the organism to exert inhibition, which appeared to block the attachment of radiolabeled organisms to HeLa cells. Immunoblotting and affinity chromatography indicated that MBP binds to the 40-kDa glycoprotein (the major outer membrane protein) on the outer surface of the chlamydial elementary body. Hapten inhibition assays with monosaccharides and defined oligosaccharides showed that the inhibitory effects of MBP were abrogated by mannose or high-mannose type oligomannose-oligosaccharide. The latter carbohydrate is the ligand of the 40-kDa glycoprotein of C. trachomatis L2, which is known to mediate attachment, suggesting that the MBP binds to high mannose moieties on the surface of chlamydial organisms. These results suggest that MBP plays a role in first-line host defense against chlamydial infection in humans.

Bacterial Adhesion↗
PubMed2000

Expression of mucosal homing receptor alpha4beta7 is associated with enhanced migration to the Chlamydia-infected murine genital mucosa in vivo.

The CD4 T helper cell type 1 (Th1) response is essential for the resolution of chlamydial genital infection in mice. However, not all Th1 clones are equally protective in eradicating the infection. Since oral immunization regimens produce protective immunity, we evaluated the role of the mucosa-associated homing receptor, alpha4beta7, in trafficking to the genital mucosa. Using a panel of CD4, Th1 cell lines and clones, we compared the lymphocyte homing patterns of a Chlamydia-specific, protective clone (P-MoPn), a nonprotective clone (N-MoPn), and a keyhole limpet hemocyanin (KLH)-specific cell line (KLH-1). T cells were labeled with the fluorescent dye PKH-26, adoptively transferred into Chlamydia-infected mice, and monitored at different time points throughout the course of a genital infection. We found that clones P-MoPn and N-MoPn migrated to similar extents to the genital tract and in significantly greater numbers than the KLH-specific T-cell line. Both clones and the KLH-1 line expressed similar levels of the adhesion molecules alpha4, beta1, CD44, and CD11a. However, clones P-MoPn and N-MoPn expressed higher levels of the mucosal homing receptor, alpha4beta7. Also, clones P-MoPn and N-MoPn but not the KLH-1 line migrated to the mesenteric lymph node, suggesting a mucosal recirculation pattern. Moreover, blocking alpha4beta7 adhesion interaction in vivo significantly reduced the recruitment of P-MoPn but not KLH-1 to the genital tract. These findings show that the mucosal homing receptor alpha4beta7 is utilized by a subset of CD4 cells during migration to the Chlamydia-infected genital tract.

Animals↗
PubMed2000

Induction of protective immunity against Chlamydia trachomatis genital infection by a vaccine based on major outer membrane protein-lipophilic immune response-stimulating complexes.

The significance of delivery systems in modern vaccine design strategies is underscored by the fact that a promising vaccine formulation may fail in vivo due to an inappropriate delivery method. We evaluated the immunogenicity and efficacy of a candidate vaccine comprising the major outer membrane protein (MOMP) of Chlamydia trachomatis delivered with the lipophilic immune response-stimulating complexes (ISCOMs) as a vehicle with adjuvant properties, in a murine model of chlamydial genital infection. Immunocompetent BALB/c mice were immunized intranasally (IN) or intramuscularly (IM) with MOMP, MOMP-ISCOMs, and live or heat-inactivated C. trachomatis serovar D. The level of local genital mucosal Th1 response was measured by assaying for antigen-specific Th1 cell induction and recruitment into the genital mucosa at different times after immunization. Immunization with MOMP-ISCOMs by the IM route induced the greatest and fastest local genital mucosal Th1 response, first detectable 2 weeks after exposure. Among the other routes and regimens tested, only IN immunization with MOMP-ISCOMs induced detectable and statistically significant levels of local genital mucosal Th1 response during the 8-week test period (P < 0.001). In addition, when T cells from immunized mice were adoptively transferred into syngeneic naive animals and challenged intravaginally with Chlamydia, recipients of IM immunization of MOMP-ISCOMs cleared their infection within 1 week and were resistant to reinfection. Animals that received IN immunization of MOMP-ISCOMs were partially protected, shedding fewer chlamydiae than did control mice. Altogether, the results suggested that IM delivery of MOMP-ISCOMs may be a suitable vaccine regimen potentially capable of inducing protective mucosal immunity against C. trachomatis infection.

Adjuvants, Immunologic↗
PubMed2000

Effect of Chlamydia trachomatis infection on atherosclerosis in apolipoprotein E-deficient mice.

We have previously demonstrated that Chlamydia pneumoniae accelerates plaque formation in apolipoprotein E-deficient (ApoE(-/-)) mice following intranasal inoculations. In this study, we evaluated the effect of respiratory tract infection with Chlamydia trachomatis on the progression of atherosclerosis in ApoE(-/-) mice. The study showed that in contrast to infection with Chlamydia pneumoniae, infection of the lung and aorta with C. trachomatis was mild and transient and did not significantly accelerate plaque development.

Animals↗
PubMed2000

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.

Animals↗
PubMed2000

Effects of estradiol and progesterone on susceptibility and early immune responses to Chlamydia trachomatis infection in the female reproductive tract.

We have used a previously described rodent model to examine the influence of hormonal environment on susceptibility and immune responses to genital Chlamydia infection. Ovariectomized rats were administered estradiol, progesterone, or a combination of both, infected with Chlamydia trachomatis via the intrauterine route, and sacrificed 5 days later. Histopathological examination showed severe inflammation in the uteri and vaginae of progesterone-treated animals, whereas animals receiving estradiol or a combination of both hormones showed no inflammation. Large numbers of chlamydiae were found in vaginal secretions of progesterone-treated and combination-treated animals, while estradiol-treated animals had none. Tissue localization showed that numerous chlamydial inclusions were present in the uterine epithelium of the progesterone group and the cervicovaginal epithelium of the combination group. Examination of the acute immune responses of the infected animals showed that maximum activation was present in the draining lymph node cells from the progesterone-treated group, and these cells were producing large amounts of interleukin-10 and gamma interferon compared to other hormone-treated groups. In contrast, spleen cell proliferation was suppressed in progesterone-treated animals compared to other hormone-treated groups. We conclude that progesterone increases and estradiol decreases susceptibility to intrauterine chlamydial infection in this rat model. Our data demonstrate that hormone environment, at the time of infection, has a profound effect on the outcome of microbial infection in the female reproductive tract.

Animals↗
PubMed2001

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.

Cell Line↗
PubMed2002

Chemokine expression patterns differ within anatomically distinct regions of the genital tract during Chlamydia trachomatis infection.

Untreated infections with Chlamydia trachomatis commonly result in ascending infection to fallopian tubes and subsequent immune-mediated tubal pathology in females. The proposed immune-mediated injury may be associated with the increased recruitment of CD4 cells to the upper genital tract (GT) (oviducts) in comparison to the lower GT (cervix) during infection, as shown in animal models. To understand the mechanisms responsible for this biased recruitment of CD4 cells within the GT, we characterized chemokine expression patterns in the upper and lower GTs in mice during infection with the murine pneumonitis biovar of Chlamydia trachomatis. Enzyme-linked immunosorbent assays of supernatants from GT homogenates revealed that the levels of the Th1-associated chemokines CXCL9 (monokine induced by gamma interferon), CXCL10 (interferon-inducible protein 10), and CCL5 (RANTES) were significantly higher in the upper GT than in the lower GT after infection, while the CCL3 (macrophage inflammatory protein 1 alpha) level was not increased. In contrast, the level of chemokine CCL11 (eotaxin) was significantly elevated in the lower GT later in the course of infection. Increased levels of mRNA confirmed the selective differences in chemokine expression within the upper and lower GTs. The increased levels of Th1-inducible chemokines in the upper GT were not due to differences in the magnitude of infection or progesterone pretreatment. These data demonstrate that the upper and lower regions of the GT respond differently to Chlamydia infection.

Animals↗
PubMed2004

The infecting dose of Chlamydia muridarum modulates the innate immune response and ascending infection.

Murine vaginal infection with the obligate intracellular bacterium Chlamydia muridarum is commonly used as a model for ascending Chlamydia infections of the human female genital tract. Gamma interferon-producing Th1 cells, in concert with other mononuclear infiltrates, primarily mediate antichlamydial immunity. However, many factors modify this response, including the bacterial load. To investigate the manner in which the inoculating dose of C. muridarum modulates a genital infection, we measured innate and adaptive cell numbers, CD4+ lymphocyte cytokine profile, chemokine expression, course of infection, and pathological sequelae in genital tracts of BALB/c mice infected with doses of C. muridarum ranging from 10(4) to 10(7) inclusion-forming units. We found that the influx of both innate and adaptive immune cells responded similarly in the lower genital tract (cervical-vaginal tissues) and upper genital tract (oviduct tissues) to increasing inoculating doses. However, cells expressing the innate markers Gr-1 and CD11c were affected to a greater degree by increasing dose than lymphocytes of the adaptive immune response (Th1, CD4+, CD8+, CD19+), resulting in a change in the balance of innate and adaptive cell numbers to favor innate cells at higher infecting doses. Surprisingly, we detected greater numbers of viable chlamydiae in the oviducts at lower inoculating doses, and the number of organisms appeared to directly correlate with hydrosalpinx formation after both primary infection and repeat infection. Taken together, these data suggest that innate immune cells contribute to control of ascending infection.

Animals↗
PubMed2004

Human antibody responses to a Chlamydia-secreted protease factor.

We have previously identified a chlamydia-secreted protein (designated chlamydial proteasome/protease-like activity factor, or CPAF) in the cytosol of chlamydia-infected cells. Although CPAF is known to degrade host transcription factors required for major histocompatibility complex antigen expression in cultured cells, it is not clear whether CPAF is produced and maintains similar functions in humans infected with chlamydial organisms. We now report that CPAF does not preexist in chlamydial organisms and that CPAF synthesis requires live organism replication in cultured cells. Mice inoculated with live, but not mice inoculated with dead, chlamydial organisms produced a strong antibody response to CPAF, correlating CPAF-specific antibody production with CPAF synthesis in animals. Sera from women diagnosed with Chlamydia trachomatis cervicitis displayed higher levels of antibodies to CPAF than to either chlamydial major outer membrane protein or heat shock protein 60, suggesting that CPAF is both produced and immunogenic during human chlamydial infection.

Animals↗
PubMed2025

A platform supporting generation and isolation of random transposon mutants in Chlamydia trachomatis.

Chlamydia species represent a paradigm for understanding successful obligate intracellular parasitism. Despite limited genetic malleability, development of genetic tools has facilitated the elucidation of molecular mechanisms governing infectivity. Random mutagenesis approaches provide one of the most powerful strategies available to accomplish untargeted elucidation of gene function. Unfortunately, initial progress in transposon-mediated mutagenesis of Chlamydia has been challenging. To increase efficiency, we developed a plasmid-based system that couples conditional plasmid maintenance with a previously described strategy leveraging inducible expression of the Himar1-derived C9 transposase. Our pOri-Tn(Q) construct was maintained in Chlamydia trachomatis cultivated with antibiotics but was rapidly cured in the absence of antibiotic selection. pOri-Tn(Q) supported transposition events when transposase expression was induced during infection. Induction was accompanied by loss of the plasmid backbone when penicillin G was used to select for only the transposable element. C9 induction during iterative passaging was used to increase the overall insertion frequency and accumulate an expanded pool of transposon mutants. The approach supported isolation of individual mutant strains from the mixed pool, and whole-genome sequencing confirmed that the recovered strains harbored single insertions.IMPORTANCEChlamydia trachomatis is a prevalent human pathogen exerting a tremendous negative impact on human health. A complete understanding of how these bacteria create and maintain an intracellular niche and avoid/subvert host defense mechanisms to cause disease is lacking. The utility of transposon-mediated, random mutagenesis in supporting forward genetic studies is well established in a multitude of genetically tractable systems. This study reports the development of a plasmid-based system capable of generating mutant pools and supporting subsequent isolation of individual transposon mutants. This step is an important advance in providing a mechanism capable of supporting downstream studies interrogating chlamydial biology.

Chlamydia trachomatis↗
PubMed1997

Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis.

Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore, the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster. The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine. Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C. trachomatis being the more recently evolved species within the genus Chlamydia.

Animals↗
PubMed1989

Molecular techniques for the detection of Chlamydia trachomatis.

A DNA probe assay (PACE; Gen-Probe, San Diego, Calif.) was compared with a culture reference method for the detection of Chlamydia trachomatis. Using stock isolates of each of the 15 serovars (A to K, Ba, L1, L2, and L3) of C. trachomatis, the lower limit of sensitivity for the DNA probe ranged between 1,086 inclusion-forming units (IFU) for serovar E (Bour) to 2,930 IFU for serovar L1 (440), with the only exception being serovar C (TW-3), with which 99 IFU was detected. There was no cross-reactivity with Chlamydia psittaci (Texas turkey) and Chlamydia pneumoniae (TWAR-183). Bacterial and fungal isolates representing 14 species of normal vaginal flora as well as Neisseria gonorrhoeae gave negative results with the DNA probe when tested at a level of 1.5 X 10(7) CFU/ml. In addition, the DNA probe, a direct fluorescent-antibody stain (DFA) (MicroTrak; Syva Corp., Palo Alto, Calif.), and an enzyme-linked immunosorbent assay (Chlamydiazyme; Abbott Laboratories, North Chicago, Ill.) were compared with culture for the detection of C. trachomatis, using 196 clinical cervical samples. Of the 196 samples, 20 (10%) were culture positive. Of the 176 culture-negative samples, 1 was not evaluated by DNA probe and 4, because of a lack of cellular material, were not evaluated by DFA. The sensitivities of the DNA probe, DFA, and enzyme-linked immunosorbent assay were 60, 75, and 85%, respectively, and specificities were 95, 99, and 97%, respectively. Of the false-positive direct results, there was only one specimen with which more than one direct method was positive, and with this specimen all three direct methods were positive. The majority of false-negative results by the direct methods were from specimens which by the culture method gave <100 IFU per culture.

Chlamydia Infections↗