Lipopolysaccharide in cells infected by Chlamydia trachomatis.

In view of the controversy concerning the expression of chlamydial lipopolysaccharide (LPS) in the eukaryotic host cell, the presence of this molecule was examined in three cell types which were experimentally infected with Chlamydia trachomatis serotype E. LPS was detected in the McCoy cell line, human endometrial epithelium and human amniotic epithelium with two monoclonal antibodies. The appearance and distribution of LPS at the host cell surface during the chlamydial developmental cycle and its transmission to neighbouring cells were examined by immunofluorescence microscopy after air drying of the host cells. LPS distribution was not uniform; it was first observed on regions of the cell surface in close proximity to the chlamydial endosome (inclusion). Soon after, the antigen was also detected at points of contact with neighbouring uninfected cells. Immunofluorescent plaques of host cells contaminated with LPS were thus formed in the vicinity of infected cells. These plaques increased in size over 2 d before becoming smaller as host cell lysis occurred. The major outer-membrane protein (MOMP) was not visualized on the host cell surface after air drying. No cell-surface LPS antigen was observed in live cells or those fixed in formaldehyde without air drying. Conventional methanol fixation and immunolocalization of LPS and MOMP in parallel infected cultures stained these antigens within inclusions in the expected fashion. Radio-immunoassays were used to quantify LPS in confluent McCoy cell monolayers during the chlamydial developmental cycle. Cell-surface-associated and inclusion-associated LPS, measured by direct binding of 125I-labelled anti-LPS monoclonal antibodies to air-dried or methanol-fixed monolayers respectively, increased for up to 3 d then declined.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlamydia trachomatis infection of cultured motile cells after uptake of chlamydiae from the substratum.

The ability of motile cells to remove small inanimate particles from solid substrata is well documented. We show here that motile cells will pick up and internalize infectious particles of the obligate intracellular parasite Chlamydia trachomatis when they are adherent to the substratum over which the host cells move. Two cell types were used to assess chlamydial uptake; a feeder independent human squamous cell carcinoma variant (AC3A cells) and the McCoy cell line. Purified chlamydia elementary bodies were attached to glass or collagen-coated glass by centrifugation. Suspensions of cells were then allowed to sediment on to the substrata to which chlamydiae had attached. Both types of cell picked up chlamydiae and transported them over their surface during the course of attachment and spreading. Stereoscopic images obtained by confocal microscopy demonstrated that chlamydiae were found mainly on the surface of non-spread cells. After the cells had spread on the substratum they began to move around forming tracks where the chlamydiae had been removed. Some cell-surface-attached chlamydiae were endocytosed and a proportion of these proliferated during the 48 h after plating. However, chlamydiae attached to the substratum lost infectivity by a simple exponential decay process within a few hours of incubation in the extracellular environment. Therefore, increasing numbers of non-viable organisms were probably endocytosed as the time of extracellular incubation increased. This mode of infection may be relevant to in vivo situations where cell migration occurs after damage to mucosal surfaces.

Genome analysis of adenovirus 4 isolated over a six year period.

Genome analysis was carried out on 74 adenovirus 4 (Ad4) isolates from patients in Manchester between 1984 and 1989. Most of the isolates were associated with conjunctivitis. Of the 74 isolates studied, 51 were Ad4a and 10 were Ad4p (the prototype strain). The remaining isolates consisted of two new genome types we have designated Ad4a2 (10 isolates) and Ad4a3 (3 isolates). Most of the genome types co-circulated during the period of study. The Bst E II and Xho I restriction maps of the new variants are presented and compared with those of Ad4p. We are unable to associate genome types with particular clinical presentations.

Purification of Chlamydia trachomatis by a simple and rapid filtration method.

A simple method for filter purification of Chlamydia trachomatis from cell culture is described. Crude homogenates of chlamydiae-infected cells were passed through a glass prefilter and a 0.6 microns pore diameter polycarbonate filter. The filtrate was then passed through a 0.2 microns pore diameter filter on which the chlamydiae were trapped. This filter was then back-washed to collect the organisms. These procedures removed cell debris and soluble protein, and yielded particles with a narrow size distribution. The mean yield of viable chlamydiae purified by filtration was 64% when the filters were washed at each stage of the process.

Ofloxacin in the treatment of uncomplicated gonorrhea and chlamydial genital infection.

The subjects were 149 patients (96 men, 53 women) with gonorrhea only (n = 66), chlamydial infection only (n = 48), or both gonorrhea and chlamydial infection (n = 35). All patients with gonorrhea were culture positive; all isolates cultured before treatment were sensitive to ofloxacin. Chlamydial infection was diagnosed by culture, inclusions being identified by indirect immunofluorescence. The patients with gonorrhea received a single dose of 400 mg of ofloxacin. Clinical and microbiologic cure was evident in 86 of 88 patients evaluated at seven days after treatment and in 71 of 72 patients at 14 days. Three patients developed postgonococcal urethritis; the cause was chlamydial in two. The patients with chlamydial infection received 200 mg of ofloxacin twice daily for seven days. Clinical and microbiologic cure was evident in all 78 patients evaluated one day after treatment and in 73 of 74 patients at 14 days. Side effects were reported by 11 patients. It is concluded that ofloxacin is a safe and effective treatment for uncomplicated gonorrhea in patients with and without concurrent chlamydial infections.

An immune dot-blot test for the diagnosis of ocular infection with Chlamydia trachomatis.

We report a new simple non culture technique for the diagnosis of chlamydial eye disease. The immune dot-blot test (IDBT) detects chlamydial lipopolysaccharide (LPS) antigen which, after being trapped on nitrocellulose membrane, is detected by autoradiography with 125I-labelled genus specific monoclonal antibody. This test was evaluated over a two year period in adults and neonates, by comparing it to culture, serological detection of chlamydial antibodies and clinical features. We demonstrate that the IDBT is more than twice as sensitive as culture, and suggest that in order to achieve a reliable diagnosis of chlamydial eye infection an immunological test for chlamydial antigen should be used in preference to tests which detect the organisms themselves.

Comparison of cotton wool swab and cytobrush for detection of chlamydial infection in women attending a genitourinary medicine clinic.

Endocervical specimens obtained by cytobrush and conventional cotton wool swabs from 90 women attending a genitourinary medicine clinic were compared for their efficiency in detecting chlamydial infection. Isolation of Chlamydia trachomatis and detection of the chlamydial lipopolysaccharide antigen were attempted on each specimen. Antigen was detected in 18% of cytobrush and 17% of swab specimens. The cytobrush proved less suitable than swabs for isolation because 8 cytobrush specimens (9%) were toxic to the McCoy cells. Toxicity was significantly associated with an infected endocervix (2P = 0.004). Cytobrush therefore appeared to have little advantage over the much cheaper alternative, the cotton wool swab.

An immune dot-blot technique for the diagnosis of ocular adenovirus infection.

The development of a monoclonal antibody based radio-immune dot-blot technique (IDBT) for the rapid detection of adenovirus is described. 718 conjunctival swabs from patients with acute keratoconjunctivitis were examined by conventional cell culture isolation techniques and IDBT. IDBT identified adenovirus in 64 of 75 culture positive samples and also in a further 34 culture negative samples [Sensitivity (IDBT versus culture) 85.3%; Specificity 92.2%]. IDBT is considered to be a simple, clinically relevant, technique for the rapid identification of adenovirus infection of the eye.

Characterisation of Chlamydia psittaci isolated from a horse.

This paper describes the isolation and characterisation of a strain of Chlamydia psittaci obtained from a nasal swab taken from a horse with serous nasal discharge. Initial isolation was achieved in cycloheximide-treated McCoy cell monolayers. Chlamydial inclusions stained by immunofluorescence either with a rabbit antiserum raised against C. psittaci or with a monoclonal antibody directed against the genus-specific lipopolysaccharide antigen were single and compact. They did not stain with iodine or with a monoclonal antibody reactive against Chlamydia trachomatis. The agent was re-isolated in the yolk sacs of embryonated hens eggs and designated N16. Identification of the agent was confirmed by electron microscopy. Unique plasmid DNA was prepared from a purified suspension of chlamydial elementary bodies (EBs), and analysed by electrophoresis through 1.0% agarose gels stained by ethidium bromide. This strain of C. psittaci grew relatively slowly in cycloheximide-treated McCoy cells, and the yield of elementary bodies during the course of one growth cycle was relatively low.

Analysis of the complete nucleotide sequence of Chp1, a phage which infects avian Chlamydia psittaci.

We report the complete nucleotide sequence of bacteriophage Chp1. The genome was found to be 4877 bases long and it potentially codes for 11 proteins. Open reading frames (ORFs) 6 and 7 lie within ORFs 2 and 1 respectively but are in a second reading frame. No significant DNA homology was found when Chp1 was compared to the EMBL database. The N-terminal amino acid sequences of the three structural proteins VP1, VP2 and VP3 were determined and it was found that they were encoded by ORFs 1, 2 and 3 respectively. Amino acid homology studies revealed that VP1 has homology with the major structural protein of bacteriophages phi X174 and S13, and that the protein inferred from ORF 4 shows homology to the A proteins of phi X174, S13 and G4. The genome of Chp1 has an organization similar to that of phi X174 although it is 509 bases smaller. We propose that Chp1 is a member of the Microviridae but that it is sufficiently different to warrant its own subfamily which we have called the Chlamydiavirinae.

The effect of Chlamydia trachomatis infection on the host cell cytoskeleton and membrane compartments.

Human epithelial cells and the McCoy cell line were infected with Chlamydia trachomatis, serotype E. The organization of the cytoplasm was then studied with probes which stained cytoskeletal components and membrane compartments. The major actin-containing stress fibre bundles were not associated with inclusions due to the peri-basal and peri-apical location of these bundles within the host cell. The cytokeratin network was distorted by the presence of inclusions so that a common basket of these intermediate filaments surrounded both nucleus and peri-nuclear inclusions. The microtubule network was similarly distorted, but the nucleus and inclusion were surrounded by separate rather than joint baskets of tubules. After reversible depolymerization by nocadazole the microtubules in amniotic epithelial cells began to reassemble at the peri-nuclear microtubule-organizing centre, so that independent microtubule networks were rapidly regenerated around the nucleus and inclusion. Mitochondria of amniotic epithelial cells were vitally stained with the fluorescent probe DiOC6 (3,3'-dihexyloxacarbocyanine iodide) after 48 h of infection and found to be widely distributed throughout the host cytoplasm. When the morphology of the Golgi complex was examined with C6-NBD-ceramide (N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)] aminocaproyl sphingosine) the main cisternae were retained in a juxta-nuclear position, although scattered stained structures were also present close to the cytoplasmic surface of the inclusion. These results demonstrate that the peri-nuclear position of inclusions is determined by the configuration of the cytoskeleton, and that normal host-cell architecture is maintained during infection, albeit in a distorted form.

Further characterization of a bacteriophage recovered from an avian strain of Chlamydia psittaci.

The genome of a 22 nm icosahedral phage which infects some avian Chlamydia psittaci strains recovered from domestic ducks has been characterized as a ss circular DNA molecule of about 4850 bases. The replicative form of this genome was isolated from purified chlamydial organisms. A restriction endonuclease cleavage site map of the genome was constructed from dsDNA synthesized in vitro from ss phage DNA and EcoRI fragments were then cloned into pUC9. The phage genome was detected only by Southern blot hybridization in C. psittaci which was productively infected with phage; no evidence was found for the integration of phage DNA into the chlamydial chromosome. Three viral polypeptides, of approximate Mr values 75K, 30K and 16.5K were identified when phage was analysed by SDS-PAGE. This virus, which we have designated Chp 1, is either an aberrant member of the Microviridae or the first member of a new bacteriophage family.

The development of Chlamydia trachomatis inclusions within the host eukaryotic cell during interphase and mitosis.

The dynamic nature of Chlamydia trachomatis inclusions was studied by video and 35 mm time-lapse photomicrography of live cells, and by immunolocalization of inclusions in fixed cells. A serotype E isolate was used to infect the MCCoy cell line and endometrial epithelia. Then resulting inclusions were observed over 4 d. They appeared as slowly expanding fluid-filled membrane vesicles whose growth varied considerably, and which were subject to great physical distortion by the host cell during interphase and mitosis. When this distortion became extreme the inclusion was observed to divide. However, as inclusions were mobile within the cytoplasm and thus able to come into contact with each other, there was a net tendency for the opposite process of inclusion fusion to occur when cells contained more than one inclusion. The proportion of infected cells decreased with time as a result of host cell proliferation, despite transmission of inclusions to progeny at the time of mitosis. Inclusion growth physically disrupted karyokinesis and cytokinesis so that host cell division became distorted or blocked on the second or third day of infection. Cell death eventually occurred by a very rapid lysis event.

Plasmid diversity within the genus Chlamydia.

Examination of 12 Chlamydia psittaci strains recovered from nine different host species (three avian and six mammalian) revealed the presence of a 7.5 kb plasmid in all isolates except two ovine abortion strains, the human strain IOL207 and the Cal 10 strain. Restriction mapping analysis distinguished four different plasmids that were associated with avian, feline, equine and guinea-pig C. psittaci isolates, respectively. The restriction maps of these four C. psittaci plasmid types all differed from that of the plasmid recovered from C. trachomatis L2/434. Despite this plasmid diversity, which is likely to be of taxonomic importance, all four plasmids identified within the species C. psittaci were found to share some sequence homology, which was mapped to two separate regions in the plasmid molecules. One region, which showed a high degree of homology between C. psittaci plasmids and also detectable homology with the C. trachomatis plasmid, may represent a common replication control region for plasmids of this genus.

Expression of a secretory product by microvillous and ciliated cells of the human endometrial epithelium in vivo and in vitro.

A monoclonal antibody which identifies a component of post-ovulatory endometrial secretions is now shown to be expressed within the cytoplasm and on the cell surface of both microvillous and ciliated epithelial cells. A glandular explantation model was developed in order to study the 'carry over' of this secretion to the regenerative phase endometrium. A loss of cytoplasmic antigen was observed in vitro. However, it was retained on the cell surface in a fashion consistent with its expression at the time of explantation. Mosaicism of expression of this secretory component occurs throughout the secretory-phase and is particularly pronounced at the time of transition from proliferative to secretory phase. It is concluded that both ciliated and microvillous epithelial cells produce a post-ovulatory secretory component which may be retained on the cell surface in the absence of hormonal stimulation.

Sensitive immune dot blot test for diagnosis of Chlamydia trachomatis infection.

The sensitivity and specificity of an immune dot blot test (IDBT), which relies on a 125I-labeled genus-specific monoclonal antibody to detect the Chlamydia lipopolysaccharide (LPS) antigen, were improved by pretreatment of specimens with proteinase K. This enzyme destroys protein A and therefore eliminates false-positive reactions caused by the presence of Staphylococcus aureus. Proteinase K treatment also improved the ability of the assay to detect the Chlamydia LPS antigen. When the improved IDBT was compared with culture for detection of C. trachomatis in 1,394 urogenital specimens obtained from a genitourinary medicine clinic, the overall sensitivity was 96%, and LPS antigen was detected in 76 of 83 (92%) specimens that yielded less than 10 inclusions in culture. The specificity and positive and negative predictive values of the test were 97, 81.5, and 99%, respectively. Of 123 conjunctival swabs, 7 were positive by both tests and 4 swabs were positive only by IDBT. This improved IDBT provides a simple, reliable alternative to culture for the detection of C. trachomatis in urogenital and conjunctival specimens.

The efficacy of ofloxacin against infection caused by Neisseria gonorrhoeae and Chlamydia trachomatis.

Ofloxacin was used to treat patients with gonorrhoea and/or Chlamydia trachomatis infection. Gonorrhoea was treated with a single 400 mg dose and chlamydial infection with a seven day course (200 mg bd). Fifty of 84 patients with gonorrhoea (60 men and 24 women) returned for two follow-up examinations 7 and 14 days after treatment, 17 patients returned for only one follow-up examination and 17 defaulted. Neisseria gonorrhoeae was re-isolated from three patients who had probably become re-infected. Treatment was successful in 64 patients, 58 of whom were assessed after a single 400 mg dose of ofloxacin. Chlamydial infection was identified in 30 patients with gonorrhoea (13 men and 17 women) and in 35 other patients (17 men and 18 women). C. trachomatis was not recovered from any of the 49 patients who returned for two follow-up examinations, or from the ten patients who attended for one follow-up visit only. Sixteen patients did not return for follow-up examination. Our results suggest a seven day course of ofloxacin would eradicate N. gonorrhoeae and C. trachomatis in patients infected with both organisms. Such treatment may be advisable in patients with gonorrhoea if microbiological tests for C. trachomatis are not available.

An in vitro model of Chlamydia trachomatis infection in the regenerative phase of the human endometrial cycle.

An in vitro model of the regenerative phase of the human endometrial cycle was developed in order to study the growth of Chlamydia trachomatis during the period following menses. Glandular epithelial fragments were prepared from curettings of endometria and explanted onto coated substrata. Epithelial cells migrated rapidly from the explant in a fashion which closely mimicked the regeneration of the surface epithelium after menses. The cultures were then experimentally infected with C. trachomatis serotype E at various times during formation of the outgrowth. Chlamydial inclusions developed both within the explants and in the outgrowing epithelial sheets. They were also found in isolated epithelial and non-epithelial cells. However, the most striking feature of chlamydial inclusion development within these cultures was the tendency for inclusions to be located in cells at the periphery of the epithelial sheets. This was partly due to the failure of the cells within the sheets to bind chlamydiae after centrifugation of the organisms onto the culture and partly due to a phenomenon similar to phagokinesis. During this process infectious chlamydial particles were cleared from the substratum by migrating cells with free motile edges, which occasionally led to internalization and inclusion development within these cells.

Recent respiratory and enteric adenovirus infection in children in the Manchester area.

Seventy-three group B adenoviruses (29 type 3 and 44 type 7) identified in a recent community outbreak were analysed with restriction endonucleases. Considerable genetic heterogeneity was identified, particularly amongst the type 3 isolates, but this genome variation could not be correlated with either clinical or epidemiological findings. Group F adenoviruses were found in 132 (4.1%) of 3202 stool specimens from children with gastroenteritis and, after rotaviruses, they were the most common viruses identified. Unlike rotaviruses, these enteric adenoviruses were endemic throughout the 3-year study period and the greatest proportion of infections (47.6%) were found in babies under 6 months old.