A 7?5 kbp cryptic plasmid is found in almost all isolates of Chlamydia trachomatis. Real-time PCR assays, using TaqMan chemistry, were set up to quantify accurately both the chlamydial plasmid and the single copy, chromosomal omcB gene in the infectious, elementary bodies (EBs) of C. trachomatis L1 440. Plasmid copy number was also determined in the EBs of six other lymphogranuloma venereum (LGV) isolates (serovars L1-L3), ten trachoma isolates (serovars A-C) and nine urogenital isolates (serovars D-J). The results indicated an average plasmid copy number of 4?0±0?8 (mean±95 % confidence interval) plasmids per chromosome. During the chlamydial developmental cycle, up to 7?6 plasmids per chromosome were detected, indicating an increased plasmid copy number in the actively replicating reticulate bodies. Attempts to eliminate the plasmid from strain L1 440 using the plasmid-curing agents ethidium bromide, acridine orange or imipramine/novobiocin led to a paradoxical increase in plasmid copy number. It is speculated that the stress induced by chemical curing agents may stimulate the activity of plasmid-encoded replication (Rep) proteins. In contrast to C. trachomatis, only a single isolate of Chlamydophila pneumoniae bears a plasmid. C. pneumoniae strain N16 supports a 7?4 kbp plasmid in which ORF1, encoding one of the putative Rep proteins, is disrupted by a deletion and split into two smaller ORFs. Similar assay techniques revealed 1?3±0?2 plasmids per chromosome (mean±95 % confidence interval) in EBs of this strain. These findings are in agreement with the hypothesis that the ORF1-encoded protein is involved in, but not essential for, plasmid replication and control of copy number. INTRODUCTIONMembers of the Chlamydiaceae are obligately intracellular, Gram-negative bacteria of significant importance in both human and animal pathogenesis. These organisms exhibit a unique developmental cycle, in which a metabolically active reticulate body (RB) gives rise to a dense, infectious elementary body (EB) (Rockey & Matsumoto, 2000). The family comprises two genera, Chlamydophila and Chlamydia (Everett et al., 1999), although this classification remains controversial (Schachter et al., 2001).Chlamydophila pneumoniae is an important human pathogen implicated in arterial disease (Saikku et al., 1992); infection of the respiratory tract causes an atypical pneumonia (Hahn et al., 2002). Although predominantly a human pathogen, C. pneumoniae strains have been isolated from animal species, e.g. equines (Everett et al., 1999;Storey et al., 1993; Wills et al., 1990) and koala (Wardrop et al., 1999).Chlamydia trachomatis is not only the major infectious agent of preventable blindness in the developing world (Thylefors et al., 1995), but also the commonest cause of non-specific urethritis in developed countries (Burstein & Zenilman, 1999). Sexually transmitted disease caused by C. trachomatis is frequently undiagnosed in women, whereupon an ascending infection may lead to pelvic inflammatory disease, salpingitis and consequent infe...

Chlamydia trachomatis L2 was used to infect BGMK cells at an m.o.i. of 1.0, and the developmental cycle was followed by transmission electron microscopy and quantitative PCR (QPCR) for both chromosomal and plasmid DNA. Samples were taken at sequential 6 h time points. Subsequent analysis by QPCR showed that there was an initial slow replication period (0–18 h), followed by a rapid phase (18–36 h) coinciding with exponential division when the DNA doubling time was 4.6 h. Chromosomal DNA was amplified 100–200-fold corresponding to 7–8 generations for the complete developmental cycle. Penicillin (10 and 100 units ml−1) was added to cultures at 20 h post-infection (p.i.). This blocked binary fission and also prevented reticulate body (RB) to elementary body transition. However, exposure to penicillin did not prevent chromosomal or plasmid DNA replication. After a short lag period, following the addition of penicillin, chlamydial chromosomal DNA replication resumed at the same rate as in control C. trachomatis-infected cells. C. trachomatis-infected host cells exposed to penicillin did not lyse, but instead harboured large, aberrant RBs in massive inclusions that completely filled the cell cytoplasm. In these RBs, the DNA continued to replicate well beyond the end of the normal developmental cycle. At 60 h p.i. each aberrant RB contained a minimum of 16 chromosomal copies.

Human rhinoviruses (HRVs) cause the common cold and often induce lower airway symptoms such as cough and wheezing. Although HRV infection is presumed to involve primarily ciliated epithelial cells, this has not been confirmed in vivo, and the cellular distribution and spread of infection as well as the pathogenesis of cold related nasal and chest symptoms remain speculative. We have developed in situ hybridization (ISH) to explore localization of the virus to airway tissues, employing HRV 16-derived oligonucleotide probes after sequencing part of the genome of this serotype. A reverse transcription-polymerase chain reaction was used to generate DNA from HRV 16 for sequencing; this yielded 305 nucleotide bases that showed considerable homology to other HRVs. The HRV 16 sequence was used to design oligonucleotides functioning as antisense and sense probes. These probes as well as random sequence and pathogen control oligonucleotides were applied to HRV-infected cell-clot complexes and finally to sections from six paired nasal biopsies obtained before, during, or after HRV-proven colds. Specificity of hybrids was established by the absence of signal in uninfected tissue, in cells infected with other viruses, after RNase pretreatment, and with application of control probes. Hybridization signals were observed in epithelial cells in three of six biopsies obtained during a cold, using probes to viral (+) strand; intermediate (-) strand, implying viral replication, was present in one biopsy. Evidence for infection of nonepithelial cells was inconclusive. HRVs cause productive infection of nasal epithelium during a cold and their intracellular localization may produce perturbation of inflammatory mediators and cytokine profiles.(ABSTRACT TRUNCATED AT 250 WORDS)