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PubMed1990

Muramic acid is not detectable in Chlamydia psittaci or Chlamydia trachomatis by gas chromatography-mass spectrometry.

By using the powerful separation technique of capillary gas chromatography combined with the selectivity of mass spectrometric detection, muramic acid was not detectable in purified elementary bodies of Chlamydia psittaci Cal 10 (less than or equal to 0.006%) or C. trachomatis serovar E (less than or equal to 0.02%). This confirms previous reports which suggested the absence of a typical peptidoglycan in Chlamydia spp.

Chlamydia trachomatis↗
PubMed1986

Synthesis of disulfide-bonded outer membrane proteins during the developmental cycle of Chlamydia psittaci and Chlamydia trachomatis.

The disulfide bond cross-linked major outer membrane protein (MOMP) of the extracellular elementary bodies (EBs) of Chlamydia psittaci was reduced to its monomeric form within 1 h of entry of EBs into host cells by a process which was inhibited by chloramphenicol, while monomeric forms of three cross-linked cysteine-rich proteins could not be detected in Sarkosyl outer membrane complexes at any time in either extracellular or intracellular forms of C. psittaci. Synthesis and incorporation of the MOMP into outer membrane complexes were detected early in the infection cycle (12 h postinfection), while synthesis and incorporation of the cysteine-rich proteins were not observed until reticulate bodies had begun to reorganize into EBs at 20 to 22 h postinfection. By 46 h postinfection, the intracellular population of C. psittaci consisted mainly of EBs, the outer membrane complexes of which were replete with monomeric MOMP and cross-linked cysteine-rich proteins. Upon lysis of infected cells at 46 h, the MOMP was rapidly cross-linked, and infectious EBs were released. The status of the MOMP of intracellular Chlamydia trachomatis was similar to the status of the MOMP of C. psittaci in that the MOMP was largely uncross-linked at 24 and 48 h postinfection, but formed interpeptide disulfide bonds when it was exposed to an extracellular environment late in the developmental cycle. In contrast to C. psittaci, only a fraction of the cross-linked MOMP of infecting EBs of C. trachomatis was reduced by 4 h postinfection, and reduction of the MOMP was not inhibited by chloramphenicol. Exposure of extracellular EBs of C. trachomatis and C. psittaci to dithiothreitol reduced the MOMP but failed to stimulate metabolic activities normally associated with reticulate bodies.

Bacterial Outer Membrane Proteins↗
PubMed1995

Evaluation of Sanofi Diagnostics Pasteur Chlamydia Microplate EIA shortened assay and comparison with cell culture and Syva Chlamydia MicroTrak II EIA in high- and low-risk populations.

Seven hundred thirty-two female urogenital samples were collected for Chlamydia trachomatis testing by both the Sanofi Diagnostics Pasteur (Chaska, Minn.) Chlamydia Microplate EIA by the shortened protocol and the Syva (San Jose, Calif.) MicroTrak II EIA, and the results were compared with those obtained by cell culture. For the analysis of samples from female patients, the patients were divided into high- and low-risk categories. An additional 121 male urethral samples were collected and tested by the Sanofi Microplate EIA and cell culture; for the analysis of samples from male patients, the patients were divided into asymptomatic and symptomatic categories. All specimens positive by enzyme immunoassay (EIA) were confirmed by a blocking assay following the respective manufacturer's instructions. Specimens negative by EIA that fell within a gray zone 30% below the cutoff and negative cultures with one or more corresponding positive EIA results were tested further by cytocentrifugation and direct immunofluorescent assay. The overall sensitivity, specificity, positive predictive value, and negative predictive value for Syva versus culture were 94, 98.8, 85.5 and 99.6%, respectively. After resolution, the results were 94.5, 99.6, 94.5, and 99.6%, respectively. The parallel results for the Sanofi Microplate EIA versus culture were 94.0, 98.7, and 83.9, and 99.6%, respectively, and after being resolved, the results were 94.9, 100, 100, and 99.6%, respectively. In the small male population tested, the resolved results of the Sanofi Microplate EIA versus culture demonstrated sensitivity, specificity, positive predictive value, and negative predictive value of 100, 100, 100, and 100%, respectively. The present study demonstrated that the Sanofi Microplate EIA shortened protocol is highly sensitive and specific in comparison with cell culture and the Syva MicroTrak II EIA.

Age Factors↗
PubMed1982

A rapid method for staining inclusions of Chlamydia psittaci and Chlamydia trachomatis.

A new staining method was developed for the detection of inclusions of Chlamydia psittaci and Chlamydia trachomatis inclusions in cell cultures. Using a combination of methyl green and neutral red stains and washing at pH 5.0, inclusions were stained red while cell cytoplasm was pale pink and cell nuclei were pale green. The method was significantly better than Giemsa staining and comparable to immunofluorescence for detecting C psittaci inclusions. Its sensitivity for detection C trachomatis inclusions by dark field microscopy was similar to that of Giemsa staining.

Animals↗
PubMed1993

Detection of Chlamydia pneumoniae and Chlamydia psittaci in sputum samples by PCR.

AIMS: To use the polymerase chain reaction (PCR) to detect Chlamydia pneumoniae and Chlamydia psittaci in sputum samples. METHODS: A nested PCR was developed, the first stage of which amplified DNA from both C pneumoniae and C psittaci while the second stage targeted specifically at C pneumoniae, allowing the two species to be differentiated. The primers were designed not to amplify sequences from C trachomatis. A panel of 26 sputum samples from patients with community acquired pneumonia evaluated previously by enzyme linked immunosorbent assay (ELISA), direct immunofluorescence (DIF), and culture was tested blind by PCR. Most of these specimens also had accompanying serial serum samples which were tested for species specific antibodies using microimmunofluorescence (micro-IF). RESULTS: PCR detected C pneumoniae DNA in 10 of the 26 samples and C psittaci DNA in four. There was good concordance between ELISA, DIF, micro-IF and PCR in the C pneumoniae group. Two of the C psittaci identified by PCR were labelled C pneumoniae by DIF but the PCR results were supported by serology or a history of bird contact. Of the PCR negative group: six were true negative results; two contained C trachomatis. There were four discrepant results. CONCLUSIONS: The data suggest that PCR is effective in the detection of C pneumoniae. The sensitivity for C psittaci is inevitably lower due to the strategy taken but specificity seemed to be good.

Bacterial Outer Membrane Proteins↗
PubMed1999

Multiplex polymerase chain reaction for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples.

AIMS: To develop a multiplex polymerase chain reaction (PCR) for the simultaneous detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Chlamydia psittaci in respiratory samples. METHODS: Oligonucleotide primers for the amplification of the DNA of these three organisms were optimised for use in combination in the same reaction. PCR products were detected by hybridisation with pooled internal probes using an enzyme linked immunosorbent assay. Those with positive signals were further differentiated using species specific probes. Quality of DNA extraction and PCR inhibition were controlled by amplification of a human mitochondrial gene. A panel of 53 respiratory samples with known results was evaluated blindly. This was followed by a retrospective study on sputa collected from 244 patients with suspected community acquired pneumonia. RESULTS: The multiplex assay had a lower sensitivity than PCR with individual primers by about one log. The resultant sensitivity was considered acceptable for diagnostic use. Of the panel of 53 samples, nine of 11 M pneumoniae, 11 of 11 C pneumoniae, six of seven C psittaci, and 24 of 24 negative samples were correctly identified. Of the 244 patients with pneumonia, seven (2.9%) had detectable M pneumoniae, six (2.5%) had C pneumoniae, and one (0.4%) had C psittaci. The case notes from 11 patients were studied. The PCR finding was of possible significance in at least eight of these patients. CONCLUSIONS: This multiplex PCR assay has the potential to be used as a diagnostic and epidemiological tool. Further prospective studies are needed to establish its clinical value.

Adolescent↗
PubMed1996

Isolation of Chlamydia pneumoniae from the coronary artery of a patient with coronary atherosclerosis. The Chlamydia pneumoniae/Atherosclerosis Study Group.

BACKGROUND: Atherosclerosis is pathologically similar to a chronic inflammatory response. Recent reports have suggested that Chlamydia pneumoniae may play a role in the pathogenesis of atherosclerosis. OBJECTIVE: To determine, by using various detection methods, whether C. pneumoniae is present in the coronary arteries of patients with coronary atherosclerosis. DESIGN: Multicenter investigation. SETTING: The Jewish Hospital Heart and Lung Institute in Louisville, Kentucky, and several laboratories. PATIENTS: 12 patients seeking heart transplantation. MEASUREMENTS: Culture for C. pneumoniae was done in HEp-2 cell monolayers. Other methods of detection included polymerase chain reaction (PCR) assay, immunocytochemistry, transmission electron microscopy, and in situ hybridization. RESULTS: Chlamydia pneumoniae was cultured from atherosclerotic plaques in one patient with severe coronary artery disease. The organism was found in the atheromas of this patient by PCR assay, immunocytochemistry, electron microscopy, and in situ hybridization. In addition, at least one testing method showed C. pneumoniae in coronary artery tissue in six of nine additional patients with coronary atherosclerosis. CONCLUSIONS: This study provides direct evidence of the presence of viable C. pneumoniae in atheromatous lesions. A chronic inflammatory response caused by a persistent infection of the coronary arteries may explain the link between C. pneumoniae and atherosclerosis.

Chlamydia Infections↗
PubMed2005

Differences in growth characteristics and elementary body associated cytotoxicity between Chlamydia trachomatis oculogenital serovars D and H and Chlamydia muridarum.

AIM: In vitro growth and elementary body (EB) associated cytotoxicity of two Chlamydia trachomatis strains belonging to serovars D and H and C muridarum were compared to identify difference(s) that correlate with virulence variations between these strains in the mouse model of human female genital tract infection, and phenotypic characteristics that could explain human epidemiological data on serovar prevalence and levels of shedding during serovar D and H infection. METHODS: Replication cycle kinetics, inclusion characteristics, and EB associated cytotoxicity were assessed in McCoy cell monolayers using culture, light microscopy, and lactate dehydrogenase release. RESULTS: Over 72 hours, more rapid production and release of inclusion forming units (ifu) allowed C muridarum to initiate two replication rounds, resulting in 4-8 times more ifu/input unit of infection than with serovars D and H. Although C muridarum EBs were significantly more cytotoxic to McCoy cell monolayers than serovar D at moderate and high multiplicity of infection ratios (MOI), serovar H EBs were significantly more cytotoxic than C muridarum, even at the lowest MOI tested. CONCLUSIONS: These phenotypic differences are consistent with the more invasive course and severe pathological outcome of infection in mice infected with C muridarum, providing an objective basis for questioning the appropriateness of C muridarum as a surrogate for the human biovar of C trachomatis in the murine model of female genital tract infection. The differences seen between the human strains could help explain human epidemiological data relating to differences in prevalence and level of shedding that occurs during infection with oculogenital serovars D and H.

Cell Cycle↗
PubMed1994

Remarkable sequence relatedness in the DNA encoding the major outer membrane protein of Chlamydia psittaci (koala type I) and Chlamydia pneumoniae.

DNA encoding the major outer membrane protein (MOMP) of the koala type-I strain of Chlamydia psittaci (pathogen responsible for blindness and infertility in koalas) was cloned and sequenced. Comparison with momp gene sequences from other chlamydial species revealed a remarkable degree of homology (> 97%) with that of the human pathogen, Chlamydia pneumoniae. In comparison, the sequence only shared 75% DNA sequence homology with other C. psittaci members and 69% homology with C. trachomatis. The open reading frame consisted of 1167 bp encoding a 389-amino acid (aa) pre-MOMP including a leader sequence of 23 aa, similar to the C. pneumoniae gene. These genes were closely related even within the four variable domains (86-100% homology). Specific antibodies were capable of distinguishing between koala type I and C. pneumoniae. This very high degree of relatedness between C. pneumoniae, a human pathogen, and an individual strain of C. psittaci in the momp gene raises further questions on the host specificity, classification and evolutionary relationships of the different chlamydial species.

Amino Acid Sequence↗
PubMed1996

Homologs of Escherichia coli recJ, gltX and of a putative 'early' gene of avian Chlamydia psittaci are located upstream of the 'late' omp2 locus of Chlamydia psittaci strain guinea pig inclusion conjunctivitis.

The nucleotide sequence of nearly 6 kb of genomic DNA located immediately upstream of the omp3-omp2 operon of Chlamydia psittaci strain GPIC was obtained, revealing four significant open reading frames (ORFs), named ORF1, ORF2, ORF4 and ORF5. Searches for homologous sequences in the GenBank/EMBL databases have revealed that: (a) the open-ended ORF1 putatively encodes an homolog of RecJ of Escherichia coli, thought to be required for RecBCD-independent and conjugational recombination, and for UV repair; (b) the predicted translation product of ORF4 is highly homologous to the putative product of EUO, a previously described ORF of avian C. psittaci strain 6BC which is preferentially transcribed early during the life cycle; and (c) ORF5 putatively encodes an homolog of bacterial glutamyl-tRNA synthetases. This analysis establishes the genetic linkage of late (omp3-omp2) and of a proposed early (EUO) genes in Chlamydia.

Amino Acid Sequence↗
PubMed1999

Single channel analysis of recombinant major outer membrane protein porins from Chlamydia psittaci and Chlamydia pneumoniae.

We recently demonstrated that the major outer membrane protein of Chlamydia psittaci, the primary vaccine candidate for combating chlamydial infections, functions as a porin-like ion channel. In this study, we have cloned, expressed and functionally reconstituted recombinant major outer membrane proteins from C. psittaci and Chlamydia pneumoniae and analysed them at the single channel level. Both form porin-like ion channels that are functionally similar to those formed by native C. psittaci major outer membrane protein. Also, like the native channels, recombinant C. psittaci channels are modified by a native major outer membrane protein-specific monoclonal antibody. This is the first time that native function has been demonstrated for recombinant chlamydial major outer membrane proteins. Future bilayer reconstitution will provide a strategy for detailed structure/function studies of this new subclass of bacterial porins and the work also has important implications for successful protein refolding and the development of improved subunit vaccines.

Antibodies, Monoclonal↗
PubMed1996

Association of Positive Chlamydia trachomatis and Chlamydia pneumoniae Immunoglobulin G Titers with Increasing Age

Due to previously reported associations of ovarian cancer with pelvic inflammatory disease, tubal infertility, and positive Chlamydia trachomatis titers, women undergoing follow-up for ovarian cancer were screened with a C. trachomatis IgG titer. Positive titers were seen in 15 (79%) of 19 women with ovarian cancer, 9 (90%) of 10 age-matched controls, and 14 (67%) of 21 patients with infertility and pain. When analyzed by age, 4 (40%) of 10 women under 30 years of age and 34 (85%) of 40, 30 years of age or older had positive titers (p = 0.007). Of 21 women with positive Chlamydia pneumoniae titers, 17 (81%) had positive C. trachomatis titers, whereas 17 (85%) of 20 with positive C. trachomatis titers had positive C. pneumoniae titers. The high rate of positive C. trachomatis titers in older women may be due to cross-reaction with C. pneumoniae titers. Although C. trachomatis IgG titers may be useful as an immunologic screening marker in infertile women, the results of these tests should be interpreted with caution. A positive test may not be evidence of C. trachomatis infection and is not an indication for specific therapy.

Journal Article↗
PubMed2002

Foam cell formation inhibits growth of Chlamydia pneumoniae but does not attenuate Chlamydia pneumoniae-induced secretion of proinflammatory cytokines.

BACKGROUND: It has not yet been determined whether lipid-loaded macrophages (foam cells), a major cellular component of atherosclerotic lesions, have the capacity to support growth of Chlamydia pneumoniae and be activated to secrete proinflammatory cytokines in response to C pneumoniae infection. METHODS AND RESULTS: Lipid loading of RAW 264.7 cells and mouse peritoneal macrophages with either oxidized or acetylated LDL significantly inhibits the growth of C pneumoniae. Modified forms of LDL are not directly toxic to C pneumoniae and do not inhibit either the initial binding or internalization of C pneumoniae by macrophages. Lipid loading does not reduce infection of macrophages with Chlamydia trachomatis. Treatment of lipid-loaded macrophages with live, heat-killed, or UV-inactivated C pneumoniae stimulates secretion of cytokines. C pneumoniae also induces expression of the mRNA for tumor necrosis factor-alpha in foam cells despite inhibition of nuclear factor-kappaB binding to DNA by prior treatment with oxidized LDL. CONCLUSIONS: Foam cell formation is not conducive to growth of C pneumoniae but does not inhibit the C pneumoniae-induced secretion of proinflammatory cytokines.

Animals↗
PubMed1992

[The ultrastructure of a new species of Chlamydia--Chlamydia pneumoniae].

The dynamic study of a new Chlamydia species, C. pneumoniae (strain TWAR, isolate TW-480), inoculated into the monolayer culture of cells L-929 was made 24, 48, 72 and 96 hours after inoculation. When compared with C. trachomatis and C. psittaci, C. pneumoniae were found to stand between these two species with respect to the morphology of their intracytoplasmic microcolonies (inclusions): they were round, almost bubble-like, but more densely packed with chlamydiae, surrounded by an undulate membrane, preserving its integrity until the late stages of their development cycle. In cells L-929 C. pneumoniae had a typical development cycle accompanied by the formation of vegetative and spore-like cells, reticular and elementary bodies, as well as intermediate cells, though this process was slower than in C. trachomatis and C. psittaci. Besides normal elementary bodies, many altered ones were formed in the process of the development of C. pneumoniae in cells L-929. Most of these alterations were similar to the process of bacterial L-transformation and could be regarded as the manifestation of chlamydial pathology related to the adaptation to new host cells.

Animals↗
PubMedDate not supplied

Immunoblots of outer-membrane proteins of chlamydiae with sera from turkeys experimentally exposed to avian and mammalian Chlamydia psittaci.

Outer-membrane protein (OMP)-enriched preparations from avian (turkey/TT3 and parrot/VS1) and mammalian (sheep abortion/B577) strains of Chlamydia psittaci were compared by immunoblotting using sera from turkeys exposed to these strains. Turkeys inoculated with avian chlamydiae became infected and developed strong serological responses, but turkeys inoculated with B577 failed to develop detectable serological responses. Sera from turkeys exposed to either of the two avian strains could be differentiated on the basis of immunoreaction patterns with OMPs of homologous and heterologous strains. Fewer bands and often weaker reactions were detected using sera from TT3- and VS1-inoculated birds with the heterologous avian strain. Sera from turkeys inoculated with either avian strain reacted with the 97,400-molecular weight (MW) protein. The sera reacted with the major outer-membrane protein (MOMP) of the homologous strains but not consistently with the MOMP of the heterologous strain. Results suggest that the 97,400-MW protein is highly immunogenic for turkeys and antigenically more complex than the MOMP.

Animals↗
PubMed1999

Different humoral immune response to Chlamydia trachomatis major outer membrane protein variable domains I and IV in Chlamydia-infected patients with or without reactive arthritis.

OBJECTIVE: The possibility that some bacterial-specific factor(s) may play a role in triggering Chlamydia trachomatis reactive arthritis was investigated. METHODS: Since the variable domains of the major outer membrane protein (MOMP) contain the serovar-determining epitopes of C. trachomatis, the ability of serum IgG to recognize peptides mimicking these epitopes was determined in 2 groups of infected patients, one with and the other without reactive arthritis. Because asymptomatic C. trachomatis infections are frequent, and nonspecific reactions due to inflammation could be observed, this study was also performed with samples from healthy blood donors and from patients with inflammatory arthritis unrelated to C. trachomatis infection. RESULTS: A predominant reactivity against peptides duplicating the J serovar-specific epitopes was only observed in the group of patients with reactive arthritis. For positive samples, differences between the two groups of C. trachomatis-infected patients were clearly observed. The mean numbers of positive responses obtained for each of the 7 peptides of the MOMP domain I or each of the 8 peptides of the MOMP domain IV were significantly higher for samples from patients with reactive arthritis (4.7 and 6) than for those from patients with only C. trachomatis urogenital infection (1.3 and 2.9). CONCLUSION: Patients with reactive arthritis had a pattern of reactivities that was compatible with infection by several serotypes of bacteria. Repeated exposures to C. trachomatis might therefore be involved in the development of the disease.

Adolescent↗
PubMed1999

Lower prevalence of Chlamydia pneumoniae DNA compared with Chlamydia trachomatis DNA in synovial tissue of arthritis patients.

OBJECTIVE: To assess the presence of Chlamydia pneumoniae DNA in the joints of patients with reactive arthritis (ReA) and other arthritides. METHODS: DNA was prepared from synovial tissue (ST) and several synovial fluid (SF) samples from 188 patients with either ReA, undifferentiated oligoarthritis, or other forms of arthritis, and from 24 normal (non-arthritis) individuals. Preparations were screened using polymerase chain reaction (PCR) assays that independently targeted the C. pneumoniae 16S ribosomal RNA and major outer membrane protein genes. RESULTS: Twenty-seven of 212 ST samples (12.7%) were PCR positive for C. pneumoniae DNA; 10 SF samples from these 27 patients were similarly positive. Among the PCR-positive patients, 3 had ReA, 2 had Reiter's syndrome, 7 had undifferentiated oligoarthritis, 4 had undifferentiated monarthritis, 6 had rheumatoid arthritis, and 5 had other forms of arthritis. No samples from normal control individuals were PCR positive. CONCLUSION: DNA of C pneumoniae is present in synovial specimens from some arthritis patients. The prevalence of this organism in the joints was lower than that of C trachomatis, and synovial presence of the organism was not associated with any distinct clinical syndrome. Widely disseminated nucleic acids such as those of C. pneumoniae might have some role in the pathogenesis of several arthritides, since the organism was not found in the ST from normal control individuals.

Arthritis, Reactive↗
PubMed1996

The nPCR detection of Chlamydia pneumoniae and Chlamydia trachomatis in children hospitalized for bronchiolitis.

Nasopharyngeal aspirates from children admitted with the diagnosis of bronchiolitis, were screened for Chlamydia pneumoniae, and C. trachomatis. The nested PCR was found to be more sensitive that amplified the DNA extracts of up to two elementary bodies of the test strains. Using this technique, C. pneumoniae was detected in two (1.3%), and C. trachomatis in 26 17.1%) of the 152 samples tested. This study indicated C. pneumoniae infections to be unusual in children with bronchiolitis.

Antibodies, Bacterial↗