KC from the conjunctiva of a koala. Monoclonal antibodies raised to C. pneumoniae IOL207 and shown to be C. pneumoniae-specific confirmed that N16 was more closely related to C. pneumoniae than to C. psittaci. Thus DNA homology and monoclonal antibody data both suggest that horse chlamydiae, as exemplified by N16, form a new second strain of C. pneumoniae. This species is probably more widespread and diverse than the current literature would suggest.

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.

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 a t 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 a t points of contact with neighbouring uninfected cells. lmmunofluorescent 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. Cellsurface-associated and inclusion-associated LPS, measured by direct binding of 1251-labelled anti-LPS monoclonal antibodies to air-dried or methanol-fixed monolayers respectively, increased for up to 3 d then declined. Cell-surfaceassociated LPS is not directly accessible to antibodies in the hydrated cell. This apparent masking of the antigen may have a significant advantage for persistence of the parasite in vivo, since such host-cell-associated antigen is unlikely to be a target for immune attack.

~ ~~ ~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 pm pore diameter polycarbonate filter. The filtrate was then passed through a 0.2 pm 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.

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. Chlamydia1 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.