Structures of and allelic diversity and relationships among the major outer membrane protein (ompA) genes of the four chlamydial species.

DNA sequences coding for 81% of the ompA gene from 24 chlamydial strains, representing all chlamydial species, were determined from DNA amplified by polymerase chain reactions. Chlamydial strains of serovars and strains with similar chromosomal restriction fragment length polymorphism had identical ompA DNA sequences. The ompA sequences were segregated into 23 different ompA alleles and aligned with each other, and phylogenetic relationships among them were inferred by neighbor-joining and maximum parsimony analyses. The neighbor-joining method produced a single phylogram which was rooted at the branch between two major clusters. One cluster included all Chlamydia trachomatis ompA alleles (trachoma group). The second cluster was composed of three major groups of ompA alleles: psittacosis group (alleles MN, 6BC, A22/M, B577, LW508, FEPN, and GPIC), pneumonia group (Chlamydia pneumoniae AR388 with the allele KOALA), and polyarthritis group (ruminant and porcine chlamydial alleles LW613, 66P130, L71, and 1710S with propensity for polyarthritis). These groups were distinguished through specific DNA sequence signatures. Maximum parsimony analysis yielded two equally most parsimonious phylograms with topologies similar to the ompA tree of neighbor joining. Two phylograms constructed from chlamydial genomic DNA distances had topologies identical to that of the ompA phylogram with respect to branching of the chlamydial species. Human serovars of C. trachomatis with essentially identical genomes represented a single taxonomic unit, while they were divergent in the ompA tree. Consistent with the ompA phylogeny, the porcine isolate S45, previously considered to be Chlamydia psittaci, was identified as C. trachomatis through biochemical characteristics. These data demonstrate that chlamydial ompA allelic relationships, except for human serovars of C. trachomatis, are cognate with chromosomal phylogenies.     

Detection of Chlamydia psittaci using DNA probes and the polymerase chain reaction

Fewer than 10(5) elementary bodies of Chlamydia psittaci could be detected by using DNA hybridisation with a plasmid probe specific for avian chlamydial strains. PCR amplification of chlamydial DNA using primers specific for conserved regions of the major outer membrane protein gene enabled the detection of fewer than 10 elementary bodies. DNA could be amplified from 22 of the 24 chlamydial strains tested including avian, feline, ovine, caprine, koala and lymphogranuloma venereum strains.     

High-yield culture and purification of Chlamydiaceae bacteria

Research on intracellular bacteria of the family Chlamydiaceae, and the diseases they cause, requires large amounts of infectious elementary bodies (EB). We describe an approach that maximizes the generation of Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia abortus, or Chlamydia pecorum EBs in several replication cycles over approximately 10 days or more in a saturated equilibrium monolayer cell culture system. Buffalo Green Monkey Kidney (BGMK) cells, Human Epidermoid Carcinoma-2 (HEp-2) cells, or mouse McCoy cells were tested. BGMK cells best supported C. pneumoniae replication when cultivated in Iscove's Modified Dulbecco's Medium. From day 1 to day 9 after inoculation, C. pneumoniae genomes per ml culture medium increased from 10(5.1) to 10(8.6) in BGMK, from 10(5.6) to 10(8.1) in HEp-2, and remained at 10(5.2) in McCoy cell cultures. Three-month pre-inoculation maintenance of BGMK cells in different culture media did not influence C. pneumoniae yields. Inoculation at multiplicities of infection (MOI) of 10 or higher and supplementation of the cell culture medium on day 7 after inoculation with 0.1% glucose enhanced C. pneumoniae EB yields in harvested cell culture medium. For purification, EBs in medium were concentrated by sedimentation, followed by low-speed centrifugation for removal of host cell nuclei, and by step-gradient centrifugation of the supernatant in a 30% RenoCal-76-50% sucrose step-gradient. Extensive sonication increased yield and infectivity of chlamydial EB. The combined method typically produced from 1000 ml infected BGMK culture medium 10 ml homogeneous, single-cell, highly infectious EB stock containing approximately 5x10(11) C. pneumoniae genomes equivalent to 4-5x10(11) inclusion forming units.     

Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis

Following the initial report of the use of SYBR Green I for real-time polymerase chain reaction (PCR) in 1997, little attention has been given to the development of alternative intercalating dyes for this application. This is surprising considering the reported limitations of SYBR Green I, which include limited dye stability, dye-dependent PCR inhibition, and selective detection of amplicons during DNA melting curve analysis of multiplex PCRs. We have tested an alternative to SYBR Green I and report the first detailed evaluation of the intercalating dye SYTO9. Our findings demonstrate that SYTO9 produces highly reproducible DNA melting curves over a broader range of dye concentrations than does SYBR Green I, is far less inhibitory to PCR than SYBR Green I, and does not appear to selectively detect particular amplicons. The low inhibition and high melting curve reproducibility of SYTO9 means that it can be readily incorporated into a conventional PCR at a broad range of concentrations, allowing closed tube analysis by DNA melting curve analysis. These features simplify the use of intercalating dyes in real-time PCR and the improved reproducibility of DNA melting curve analysis will make SYTO9 useful in a diagnostic context.     

Prevalence of chlamydiae in semen and genital tracts of bulls, rams and bucks

Chlamydiae infect male genital organs of ruminants. However, little is known about their prevalence. Hence, we investigated fresh and cryopreserved semen (bulls: n=304; rams: n=78; bucks: n=44) by polymerase chain reaction (PCR), as well as genital organs (bulls: n=13; rams: n=10; bucks: n=6) by immunohistochemistry (IHC) and PCR. Sera from bulls (n=104) and small ruminants (n=61) were tested by LPS and rMOMP (recombinant major outer membrane protein) ELISA and competitive ELISA (cELISA), respectively. Three PCR assays were compared in this study for detection of chlamydial DNA in semen: 16S rRNA, IGS-S (intergenic spacer 16S/23S-short), and IGS-L (intergenic spacer 16S/23S-long) PCRs. PCR sensitivity and inhibitory effects were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. In bull semen, detection limits of the 16S, IGS-S and IGS-L PCRs were 10, 10, 100 templates, respectively. However, PCR sensitivity was reduced in ram and buck semen suggesting the presence of potential PCR inhibitors. Of 304 bull semen samples, the 16S PCR revealed DNA of chlamydiae in 20 samples (6.6%), including Cp. abortus (n=2), Cp. psittaci (n=1), Chlamydia suis (n=2), and Chlamydia-like organisms (n=15). In rams, one semen sample was positive for Chlamydia-like organism. All investigated male genital organs were negative for Chlamydia. Serology revealed 47.1% (49/104) positive bulls by LPS ELISA. Of these, 30 samples were positive by rMOMP ELISA, predominantly for Cp. pecorum. In small ruminants, cELISA displayed 34.8% (16/46) and 60% (9/15) positivity for Cp. abortus in rams and bucks, respectively. There was no correlation between serology and PCR of semen. The presence of chlamydiae in semen suggests the possibility of venereal transmission, although risk may be low in Switzerland.     

Determination of chlamydial load and immune parameters in asymptomatic, symptomatic and infertile women

The regulation of immune response and chlamydial infectious load in the cervix of human females is largely unknown. Infectious load in terms of inclusion-forming units (IFUs) was determined by quantitative cultures in Chlamydia -positive women, in asymptomatic women, women with mucopurulent cervicitis (MPC) and women with fertility disorders (FD). CD4⁺, CD8⁺, CD14⁺ cells, myeloid and plasmacytoid dendritic cells (mDCs and pDCs) in the cervix were quantified by flow cytometry. Cervical cytokines, levels of β-estradiol and C-reactive protein (CRP) in serum and cervical immunoglobulin A antibody to chlamydial major outer membrane protein antigen, chlamydial heat shock protein 60 and 10 antigens were measured by an enzyme-linked immunosorbent assay. In asymptomatic women, chlamydial load showed significant positive correlations with CD4, mDCs, interleukin-12 (IL-12) and IL-2; however, negative correlations were found with CD8 and IL-8 levels. In women with MPC, chlamydial IFUs correlated positively with CD8, pDC number, IL-8, CRP and interferon-γ (IFN-γ). In women with FD, chlamydial load showed a significant positive correlation with the pDC number, IL-10 and estradiol level and a negative correlation with CD4 and IFN-γ. Overall, these results suggest that the interplay between chlamydial infectious load and host immune responses may be the deciding factor for the clinical condition presented during Chlamydia trachomatis infection.     

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.     

Molecular characterization of atypical Chlamydia and evidence of their dissemination in different European and Asian chicken flocks by specific real-time PCR

Chlamydia psittaci is a zoonotic pathogen associated primarily with avian chlamydiosis. New chlamydial agents with suspected zoonotic potential were recently detected from domestic poultry in Germany and France indicating that the spectrum of Chlamydiaceae encountered in birds is not confined to a single chlamydial species. For further characterization, a specific real-time PCR targeting the conserved 16S rRNA gene was developed and validated for a specific detection of these atypical Chlamydiaceae. In order to address the epidemiological importance of the new chlamydial agents and their distribution, Chlamydiaceae-positive chicken samples collected from flocks from five different countries were examined. The results confirmed that C.psittaci is not the predominant chlamydial species among chickens examined and suggested that the new chlamydial agents could putatively be widespread in poultry flocks (France, Greece, Croatia, Slovenia and China at least) justifying their systematic investigation when poultry samples are submitted to laboratories for avian chlamydiosis diagnosis. Besides, 16S rRNA-based dendrogram, including sequences from both isolates of the new chlamydial agents or positive samples as well as representative sequences from species belonging to the order Chlamydiales, showed the new chlamydial agents to form a distinct line of descent separated from those of other chlamydial species, but clearly grouped within the family Chlamydiaceae. Finally, the phylogenetic tree inferred from the multi-locus sequence typing based on four housekeeping fragments (gatA, gidA, enoA and hflX) and the ompA-based dendrogram showed an almost identical topology of the new chlamydial agents with that recovered by 16S rRNA-based dendrogram. Interestingly, partial ompA gene sequences displayed considerable diversity among isolates.     

Effect of Ionizing Irradiation on Susceptibility of McCoy Cell Cultures to Chlamydia trachomatis

The effect of graded doses of irradiation (cobalt-60) on the morphology of McCoy cells was analyzed, and 4,000 to 5,000 r was selected as a satisfactory dose for production of giant cells. The susceptibility of radiation-induced giant cells to chlamydial infection was compared with that of nonirradiated cells by using three strains of Chlamydia trachomatis and one of C. psittaci. Monolayers of giant cells were more susceptible than normal McCoy cells as indicated by (i) greater numbers of inclusions (four- to eightfold) per unit area of monolayer, (ii) larger inclusions (fourfold greater in area), (iii) higher infective titers (1 log or more greater) of harvested cells, and (iv) greater ease of promoting a second cycle of growth. Graded doses of irradiation were applied also to mouse fibroblast (L) cells, and a similar increase in susceptibility to chlamydial infection was noted. It is concluded that giant cells produced by irradiation possess advantages over nonirradiated cells in culture for growth of Chlamydia.     

Oxygen-independent inhibition of intracellular Chlamydia psittaci growth by human monocytes and interferon-gamma-activated macrophages

We have demonstrated previously that Chlamydia psittaci grows well in human monocyte-derived macrophages, but to a limited extent in lymphokine-or interferon-gamma (IFN-gamma)-activated macrophages. In this investigation, freshly explanted human monocytes inhibited chlamydial inclusion formation by 85% as compared to macrophages, and the level of inhibition was similar to that exhibited by lymphokine-activated macrophages (79%). To determine whether the oxygen-dependent antimicrobial mechanisms of the mononuclear phagocyte were involved in the inhibition, cells were infected with C. psittaci in the presence of agents that either inhibit the respiratory burst (glucose deprivation) or diminish the effect of H2O2 (catalase). These treatments had no effect on the capacity of monocytes and lymphokine-activated macrophages to restrict chlamydial growth. In addition, monocytes and activated macrophages from an individual with chronic granulomatous disease suppressed chlamydial growth as effectively as normal cells. Oxidatively deficient HeLa and endothelial cells, once stimulated by lymphokine, also displayed normal levels of antichlamydial activity. The induction of this apparently oxygen-independent antichlamydial effect by lymphokine was completely neutralized by a monoclonal anti-IFN-gamma antibody, and could be achieved by treatment with recombinant (r)IFN-gamma alone. These results indicate that the primary antimicrobial mechanism of the human monocyte against C. psittaci is oxygen-independent, and that this response can be effectively stimulated in the macrophage by lymphokine (IFN-gamma).     

Initial characterization of a chlamydial receptor on mammalian cells

We have examined characteristics of the binding of eukaryotic cells to chlamydial elementary body (EB)-specific proteins. A wide variety of eukaryotic cell lines bound to representatives of both Chlamydia trachomatis lymphogranuloma venereum (LGV) and trachoma biovars and a C. psittaci strain meningopneumonitis (Mn) suggesting the presence of a common host cell receptor. Neither tunicamycin nor neuraminidase treatment of HeLa cells impaired binding to C. trachomatis EB, implying that host cell N-linked carbohydrate domains and sialic acid moieties, respectively, are not involved in attachment. However, trypsinized HeLa cells do not bind to EB, suggestive of a proteinaceous host cell receptor. The trypsin sensitivity of two EB-specific binding proteins Mr = 18,000 and 31,000) was also examined, and the finding that 125I-labeled HeLa cells bind both the 18,000 and 31,000-dalton proteins after chlamydial trypsinization corroborates our earlier observation that these EB binding proteins mediate attachment.     

Endotoxic activity and chemical structure of lipopolysaccharides from Chlamydia trachomatis serotypes E and L2 and Chlamydophila psittaci 6BC.

The lipopolysaccharide (LPS) of Chlamydia trachomatis serotype E was isolated from tissue culture-grown elementary bodies and analyzed structurally by mass spectrometry and 1H, 13C and 31P nuclear magnetic resonance. The LPS is composed of the same pentasaccharide bisphosphate alphaKdo-(2-8)-alphaKdo-(2-4)-alphaKdo-(2-6)-betaGlcN-4P-(1-6)-alphaGlcN-1P (Kdo is 3-deoxy-alpha-d-manno-oct-2-ulosonic acid) as reported for C. trachomatis serotype L2[Rund, S., Lindner, B., Brade, H. and Holst, O. (1999) J. Biol. Chem. 274, 16819-16824]. The glucosamine disaccharide backbone is substituted with a complex mixture of fatty acids with ester or amide linkage whereby no ester-linked hydroxy fatty acids were found. The LPS was purified carefully (with contaminations by protein or nucleic acids below 0.3%) and tested for its ability to induce proinflammatory cytokines in several readout systems in comparison to LPS from C. trachomatis serotype L2 and Chlamydophila psittaci strain 6BC as well as enterobacterial smooth and rough LPS and synthetic hexaacyl lipid A. The chlamydial LPS were at least 10 times less active than typical endotoxins; specificity of the activities was confirmed by inhibition with the LPS antagonist, B1233, or with monoclonal antibodies against chlamydial LPS. Like other LPS, the chlamydial LPS used toll-like receptor TLR4 for signalling, but unlike other LPS activation was strictly CD14-dependent.     

Antichlamydial activity of ofloxacin

The in vitro activity of ofloxacin, a new pyridone-carboxylic acid, against 11 strains of Chlamydia trachomatis and six strains of Chlamydia psittaci was determined. All test strains of both species were inhibited by 0.39 micrograms of ofloxacin per ml. The antichlamydial activity of ofloxacin was comparable to that of doxycycline and four- to eightfold less than that of minocycline. The results of this susceptibility test, coupled with those of previous pharmacokinetic analyses of ofloxacin, warrant further evaluation of its clinical usefulness against chlamydial infections.     

Cloning and antigenic expression of two genes from Chlamydia psittaci avian strain

Abstract: Genes from Chlamydia psittaci P-1041 were cloned into the Bam HI site of pUC19 and were transformed to host Escherichia coli JM109. Two recombinant plasmids that expressed protein antigens of Chlamydia were isolated. The sizes of the DNA fragments were 1350 and 1710 bp, and encoded for polypeptides of M _r 25 and 42 kilodaltons (kDa), respectively. The 25-kDa protein had cross-reactivity with antisera to ten C. psittaci strains and two C. trachomatis strains, whereas the 42-kDa protein reacted only with homologous antiserum to the C. psittaci P-1041 strain. Furthermore, in Southern hybridization analysis these two fragments as probes hybridized with DNA of ten C. psittaci strains and four C. trachomatis strains. These results indicated that the two fragments shared a DNA sequence common to the chlamydial genus.     

The application of real-time PCR to the identification, detection and quantification of Pyrenophora species in barley seed

A real-time quantitative PCR technique has been used to develop a rapid and sensitive seed health test for Pyrenophora spp. on barley seed. Using the fluorescent reporter dye SYBR Green I for real-time detection of PCR amplification, pathogen DNA extracted from infected seed can be quantified to the picogram level. The amount of Pyrenophora DNA extracted from seed samples of an artificial infection level gradient, constructed by mixing infected and uninfected seed, correlated with good agreement ( r  = 0.931) to percentage infection levels of the same samples measured by agar plate testing. In addition, a correlation of r  = 0.883 was obtained between the two testing methods for naturally infected seed, ranging from 0% to 89% infection. Samples could be quantified to below the 2% voluntary threshold required for deciding on seed treatment. The proposed test was performed in three parts: (i) quantification of Pyrenophora spp. infection using Pyrenophora -specific PCR primers; (ii) test of any negative samples from (i) with barley-specific PCR primers to check the DNA extraction process; (iii) test of positive samples from (i) for the presence of Pyrenophora graminea using P. graminea -specific PCR primers. All PCRs were performed in the LightCycler™ instrument allowing each PCR run and analysis to be completed within 30 min. With the current daily receipt of samples (batches up to 16) the test can be completed in 8 h, compared to 7 days for the traditional agar plate test.     

Chlamydia parasitism: ultrastructural characterization of the interaction between the chlamydial cell envelope and the host cell.

Ultrastructural analysis of the growth cycles of Chlamydia trachomatis and Chlamydia psittaci showed that the chlamydial cell envelope became rigid and septated at the time of the reorganization from reticulate to elementary body. This process occurred in the immediacy of the inclusion membrane and in close proximity with the mitochondria or the endoplasmic reticulum of the host cell.     

Proteinase Produced by Chlamydia psittaci in L Cells

L cells (mouse fibroblasts) infected with Chlamydia psittaci (strain meningopneumonitis) produced a proteinase differing in solubility in ammonium sulfate from the proteinase of uninfected L cells. Synthesis of the enzyme was inhibited by chloramphenicol but not by cycloheximide, indicating that the new proteinase in infected L cells was synthesized by Chlamydia psittaci. The chlamydial proteinase had no demonstrable ion requirements and was not inhibited by a variety of inhibitors of proteinase activity. Gel filtration experiments suggested a molecular weight of approximately 250,000. The proteinase appeared in infected L cells at the time host cells began to die and the large chlamydial cells began to reorganize into small ones. Some possible functions for the chlamydial proteinase were proposed.     

Chlamydiosis: seroepidemiologic survey in a red deer (Cervus elaphus) population in Italy

Chlamydiae are obligate, intracellular, gram-negative bacteria that are responsible for important diseases in humans, other mammals, and birds. Studies have shown that chlamydiae could be present in wild ruminants, but the serodiagnostic method most commonly used did not allow identification of chlamydial species. We determined the prevalence of antibodies to Chlamydia pecorum, Chlamydia suis, Chlamydia abortus, and Chlamydia psittaci in 271 red deer (Cervus elaphus) of a central Italian population, by using the microimmunofluorescence test that shows antibody response against genus-specific and species-specific antigens. No sera had detectable antibodies to C. pecorum and C. abortus. Antibodies were detected against C. psittaci (9.6%) and C. suis (3.3%). Antibody response could be related to contact of the red deer with birds and wild boars (Sus scrofa), respectively, and confirm an extended host range of individual Chlamydia species. In view of the potential zoonotic risk related to exposition of C. psittaci, our findings suggest surveillance of wild ruminants as potential reservoirs for chlamydiae.     

Comparison of the SYBR Green and the hybridization probe format for real-time PCR detection of HHV-6

A comparative study was conducted of a novel real-time quantitative PCR test (LightCycler System) with FastStart DNA Master(PLUS) SYBR Green I dye to detect DNA of human herpes virus 6 (HHV-6). Results were compared with those of a real-time quantitative PCR with hybridization probe (HP) formats using the fluorescence resonance energy transfer method, and with those of a single qualitative PCR test. The detection limit of the test with SYBR Green I dye was 20 copies of the virus, similar to that of the other two tests. The reproducibility was satisfactory. The new test has the same advantages as real-time PCR with HP formats and offers a greater versatility at lower cost.     

Analysis of synonymous codon usage bias in Chlamydia

Chlamydiae are obligate intracellular bacterial pathogens that cause ocular and sexuallytransmitted diseases,and are associated with cardiovascular diseases.The analysis of codon usage mayimprove our understanding of the evolution and pathogenesis of Chlamydia and allow reengineering of targetgenes to improve their expression for gene therapy.Here,we analyzed the codon usage of C.muridarum,C.trachomatis(here indicating biovar trachoma and LGV),C.pneumoniae,and C.psittaci using the codonusage database and the CUSP(Create a codon usage table)program of EMBOSS(The European MolecularBiology Open Software Suite).The results show that the four genomes have similar codon usage patterns,with a strong bias towards the codons with A and T at the third codon position.Compared with Homosapiens,the four chlamydial species show discordant seven or eight preferred codons.The ENC(effectivenumber of codons used in a gene)-plot reveals that the genetic heterogeneity in Chlamydia is constrained bythe G+C content,while translational selection and gene length exert relatively weaker influences.Moreover,mutational pressure appears to be the major determinant of the codon usage variation among the chlamydialgenes.In addition,we compared the codon preferences of C.trachomatis with those of E.coli,yeast,adenovirus and Homo sapiens.There are 23 codons showing distinct usage differences between C.trachomatisand E.coli,24 between C.trachomatis and adenovirus,21 between C.trachomatis and Homo sapiens,butonly six codons between C.trachomatis and yeast.Therefore,the yeast system may be more suitable for theexpression of chlamydial genes.Finally,we compared the codon preferences of C.trachomatis with those ofsix eukaryotes,eight prokaryotes and 23 viruses.There is a strong positive correlation between the differ-ences in coding GC content and the variations in codon bias(r=0.905,P<0,001).We conclude that thevariation of codon bias between C.trachomatis and other organisms is much less influenced by phylogeneticlineage and primarily determined by the extent of disparities in GC content.