Transcription of seven genes in a model of interferon­Î³-induced persistent Chlamydia psittaci infection.

The obligate intracellular bacterium Chlamydia psittaci is the causative agent of psittacosis in birds and humans. The capability of this zoonotic pathogen to develop a persistent phase may serve a role in the chronicity of infections, in addition to the failure of antibiotic therapy or immunoprophylaxis. In the present study, a C. psittaci strain 6BC persistent infection cell model was induced using interferon (IFN)­Î³, alterations in the infectivity and morphology of the pathogen were analyzed, and the transcript profile of seven selected genes was analyzed. Following treatment with IFN­Î³, the infectivity of C. psittaci 6BC was decreased, the inclusion bodies appeared to be smaller, reticulate bodies were larger and the number of infectious elementary bodies was decreased compared with acute infection. In IFN­Î³­induced persistently infected cells, the relative mRNA expression levels of the genes CPSIT­0208, CPSIT­0310, CPSIT­0846, CPSIT­0844 and CPSIT­0594 were upregulated at 2­48 h post­infection (p.i.). The genes CPSIT­0959 and CPSIT­0057 were downregulated at 2­36 h p.i. The results of the present study advanced the understanding of C. psittaci persistent infection and demonstrated a number of previously unknown alterations in chlamydial gene expression, which may provide novel targets to further analyze this particular host­pathogen interaction.

The LPS localization might explain the lack of protection of LPS-specific antibodies in abortion-causing Chlamydia psittaci infections.

Four monoclonal antibodies against chlamydial lipopolysaccharide (LPS) were used to study their localization and distribution in the Chlamydia psittaci AB7 abortion-causing strain by immunoelectron microscopy. A non-embedding technique on whole chlamydiae, together with a post-embedding technique on McCoy cells infected with the strain, were performed. Immunogold labelling was observed on the surface of reticular bodies (RB), but not on elementary bodies (EB). Immunolabelling was observed in ultrathin sections on both sides of the external chlamydial membrane, mainly on the inner side of EB and on the outer side of RB. Immunogold density was higher in EB than in RB; however, the absolute number of gold particles was higher in RB than EB, suggesting a loss of immunolabelling during the transformation of RB into EB. Specific labelling of LPS was also found in electrodense and adielectronic vacuoles near the surface of the cytoplasmic membrane of infected McCoy cells. These results suggest that the lack of protection against some chlamydial strains, despite the presence of anti-LPS specific antibodies, is due to the localization of LPS on the inner side of the external membrane of EB.

Common surface epitope of Bartonella bacilliformis and Chlamydia psittaci.

A serosurvey revealed intense cross-reactivity between Bartonella bacilliformis and Chlamydia psittaci. One of the cross-reacting Bartonella antigens was identified as lipopolysaccharide which reacted with Bartonella as well as with Chlamydia serum antibodies. A monoclonal Bartonella antibody bound to Bartonella lipopolysaccharide as well as to the surfaces of Bartonella bacilliformis and Chlamydia psittaci. It was thus demonstrated that Chlamydia psittaci carries a surface epitope identical to an epitope of Bartonella lipopolysaccharide. The lipopolysaccharide was preliminarily characterized by polyacrylamide gel electrophoresis and by a lectin-binding assay. The lipopolysaccharides of Bartonella bacilliformis and Chlamydia psittaci are not identical.

The morphology of Chlamydia pneumoniae.

The morphology of a recently isolated strain of Chlamydia pneumoniae, YK-41, was compared by electronmicroscopy with C. pneumoniae TWAR, Chlamydia trachomatis L2/434/Bu and Chlamydia psittaci Cal 10. The results showed that "pear-shaped" morphology was not typical of C. pneumoniae. Basic morphological features, such as surface projections and hexagonally arrayed, regular structures in the inside layer of the outer membrane of elementary bodies, were very similar in these strains. The structure of strain YK-41 was identical with that of C. trachomatis and C. psittaci, but the profiles of elementary bodies were different from those of C. pneumoniae TWAR strains.

A comparative ultrastructural and molecular biological study on Chlamydia psittaci infection in alpha-1 antitrypsin deficiency and non-alpha-1 antitrypsin deficiency emphysema versus lung tissue of patients with hamartochondroma.

BACKGROUND: Chlamydiales are familiar causes of acute and chronic infections in humans and animals. Human pulmonary emphysema is a component of chronic obstructive pulmonary disease (COPD) and a condition in which chronic inflammation manifested as bronchiolitis and intra-alveolar accumulation of macrophages is common. It is generally presumed to be of infectious origin. Previous investigations based on serology and immunohistochemistry indicated Chlamydophila pneumoniae infection in cases of COPD. Furthermore, immunofluorescence with genus-specific antibodies and electron microscopy suggested involvement of chlamydial infection in most cases of pulmonary emphysema, but these findings could not be verified by PCR. Therefore, we examined the possibility of other chlamydial species being present in these patients. METHODS: Tissue samples from patients having undergone lung volume reduction surgery for advanced alpha-1 antitrypsin deficiency (AATD, n = 6) or non-alpha-1 antitrypsin deficiency emphysema (n = 34) or wedge resection for hamartochondroma (n = 14) were examined by transmission electron microscopy and PCR. RESULTS: In all cases of AATD and 79.4% of non-AATD, persistent chlamydial infection was detected by ultrastructural examination. Intra-alveolar accumulation of macrophages and acute as well as chronic bronchiolitis were seen in all positive cases. The presence of Chlamydia psittaci was demonstrated by PCR in lung tissue of 66.7% AATD vs. 29.0% non-AATD emphysema patients. Partial DNA sequencing of four positive samples confirmed the identity of the agent as Chlamydophila psittaci. In contrast, Chlamydophila pneumoniae was detected only in one AATD patient. Lung tissue of the control group of non-smokers with hamartochondroma was completely negative for chlamydial bodies by TEM or chlamydial DNA by PCR. CONCLUSIONS: These data indicate a role of Chlamydophila psittaci in pulmonary emphysema by linking this chronic inflammatory process to a chronic infectious condition. This raises interesting questions on pathogenesis and source of infection.

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.

Comparison of ovine abortion and non-abortion isolates of Chlamydia psittaci using inclusion morphology, polyacrylamide gel electrophoresis, restriction endonuclease analysis and reactivity with monoclonal antibodies.

Twelve reference and four Northern Ireland ovine Chlamydia psittaci isolates including ovine abortion, faecal, conjunctivitis and arthritis isolates were compared. Inclusion morphology was shown to provide a useful means of differentiating the abortion and the non-abortion isolates studied. Identical SDS-PAGE polypeptide profiles were produced by the ovine abortion isolates. The polypeptide profiles of the non-abortion isolates were similar to one another and clearly distinct from the abortion isolate profiles. The restriction endonuclease profiles of the abortion isolates were remarkably similar whereas different profiles were produced by most of the non-abortion isolates. Monoclonal antibodies were prepared and characterized. A number of these reacted with all the isolates of chlamydia tested. Three mAbs reacted exclusively with the ovine abortion isolates while four mAbs reacted exclusively with a number of the faecal isolates.

Abortion in Japanese cows caused by Chlamydia psittaci.

An outbreak of abortion in cows occurring in Niigata Prefecture was shown to be caused by Chlamydia psittaci. Elementary bodies characteristic of Chlamydia were found in the liver of aborted fetuses and C. psittaci antigen was demonstrated by indirect immunofluorescence. Chlamydia was isolated from the liver of aborted fetuses by the yolk sac inoculation of developing chick embryos and by the intraperitoneal inoculation of guinea pigs. Abortion occurred mostly in middle or late pregnancy. Aborted fetuses showed subcutaneous edema and gelatinous infiltration, enlarged liver and spleen, and dark red pleural and ascitic fluid. Focal necrosis was shown in the liver, spleen and lymph nodes. Serological findings and isolation of Chlamydia from fecal specimens indicated a wide dissemination of C. psittaci among cows in the area.

Electron microscopic observations concerning the in vivo uptake and release of the agent of guinea-pig inclusion conjunctivitis (Chlamydia psittaci) in guinea-pig exocervix.

This report details electron-microscopical observations concerning C. psittaci infection in vivo. The model employed was that of the guinea-pig infected at the exocervical region with the agent of guinea-pig inclusion conjunctivitis (GPIC). Our observations indicate that chlamydial particles gain access to their target cells by the mechanism of endocytosis. Single GPIC elementary bodies were seen to be positioned within individual endosomes. The observations reported here provide evidence that chlamydial particles that had undergone their developmental cycle within the exocervical epithelial cells may leave the epithelium in 2 ways; within entire infected cells that had been shed into the lumen of the cervix and by means of the liberation of chlamydial particles from disrupted cells. The mechanism of cell disruption and shedding is thought to involve the large number of PMNs observed to be present within the enlarged intercellular spaces of the infected epithelium.

[Avian Chlamydia psittaci phyla and their pathogenic significance for turkeys]. / Aviaire Chlamydia psittaci stammen en hun pathogene betekenis voor kalkoenen.

The present study demonstrates that Chlamydia psittaci is an important cause of respiratory disease in turkeys in Europe. The serotyping of isolates revealed that European turkeys frequently become infected with Chlamydia psittaci serovar D strains, but also can become infected with serovar A and B strains. In turkeys, differences in pathogenicity were observed not only between strains belonging to the serovars A (strain 84/55), serovar B (strain 89/1326) and serovar D (Texas Turkey strain and strain 92/1293), but also between two strains belonging to the same serovar D (Texas Turkey strain and strain 92/1293). In order to find an explanation for these differences in pathogenicity, the pathogenesis of the infection was studied in specific pathogen free turkeys. However, for all three serovars examined a similar pathogenetic sequence of events was deduced. In order to try and elucidate the observed differences in pathogenicity between different Chlamydia psittaci strains, the bacterium-host cell interaction was studied in BGM cell culture using transmission electron microscopy and immuno electron microscopy. Strains most pathogenic for turkeys, namely the serovar A strain and strain 92/1293 (serovar D) produced significantly larger inclusions with more numerous infectious organisms, produced the most severe degenerative changes in the host cell an also replicated freely in the cytoplasm of the host cell.

Kinetic studies on the appearance of antigens of Chlamydia psittaci during its developmental cycle.

The kinetics of the antigen production of Chlamydia psittaci strains Izawa-1 and Pigeon-1041 (P-1041) was examined every 6 hr after infection up to 48 hr, by the indirect immunofluorescent antibody technique using monoclonal antibodies (MAbs). All three genus-specific antigenic determinants on lipopolysaccharide (LPS) appeared during the whole growth cycle. Antigenic determinants on proteins were, on the other hand, detected at various time periods from the early to the late stages of infection. However, a cross-reactive antigenic determinant on protein recognized by a MAb 3E9 was also detected during the whole growth cycle, similar to that on LPS. The time of appearance of common antigenic determinants on proteins of Izawa-1 and P-1041 was examined using cross-reactive MAbs, and it varied depending on heterologous and homologous MAbs. From the relationship between the detection of antigenic determinants and the morphological changes of chlamydial particles revealed by electron microscopy during the growth cycle, the antigenic determinants on proteins of Chlamydia psittaci were divided into two groups; one was specific to the elementary body and the other was coexisting in both the elementary body and the reticulate body.

Biological properties and genetic analysis of the ompA locus in chlamydiae isolated from swine.

Eight strains of Chlamydia psittaci isolated from swine with pneumonia, pleuritis, pericarditis, and enteritis were characterized through analysis of the major outer membrane protein gene ompA by a two-step polymerase chain reaction, by their interactions with cells in culture, and by the morphologic features and ultrastructure of intracellular inclusions. Amplified chlamydial ompA DNA fragments were differentiated by restriction endonuclease digestion. Chlamydial isolates were separated into 2 types on the basis of ompA restriction fragment length polymorphism. Strains of type L71 had finely granular inclusions, whereas those of type 1710S contained pleomorphic reticulate bodies (RB) in the inclusions, which are characteristic of aberrant chlamydial developmental forms. Chlamydial types L71 and 1710S required centrifuge-assisted inoculation for efficient infection of cell cultures. Cultivation in cell culture medium containing cycloheximide increased the numbers of chlamydial inclusions about 1.5-fold. These strains formed few elementary bodies in yolk sac cells of chicken embryos. Ultrastructurally, unique doublet RB were observed, particularly in strains of the ompA type L71. These doublets consisted of 2 RB, bounded by a cytoplasmic membrane, contained within a common cell wall and an extended periplasmic space. Ultrastructural examination of strains of the ompA type 1710S confirmed the aberrant chlamydial developmental forms, but evidence of viral infection of the RB as a cause of these aberrant forms was not found. The strain S45 isolated from intestinal sites of swine was a trachoma restriction fragment length polymorphism type. With the mouse biotype, it represented the second isolate from animals of Chlamydia trachomatis.

Experimentally induced feline chlamydial infection (feline pneumonitis).

Cats exposed to aerosols of feline Chlamydia psittaci developed a disease characterized principally by conjunctivitis. Signs of conjunctivitis appeared between postexposure days (PED) 5 and 10, were often unilateral initially, and persisted for 22 to 45 days. Fever followed the onset of conjunctivitis (PED 11 to 15) and persisted for 3 to 8 days. Signs of mild rhinitis (occasional sneezing and mild serous nasal discharge) occurred in some cats between PED 8 and 37. Neither signs of lower respiratory tract disease nor significant pulmonary lesions were produced by the feline pneumonitis agent. Small foci of pneumonia were detected microscopically in 3 of 6 cats examined between PED 7 and 14. Chlamydiae were identified between PED 7 and 14 in the cytoplasm of epithelial cells in stained conjunctival smears. Conjunctivitis persisted for at least 18 days after chlamydiae no longer were detectable in conjunctival smears. Low levels of chlamydial infectivity, however, still were present in conjunctiva and lung on PED 45.

Serological detection of phage infection in Chlamydia psittaci recovered from ducks.

Antiserum prepared against a phage which infects a Chlamydia psittaci isolate recovered from domestic ducks was used to screen other recent avian C psittaci isolates by indirect immunofluorescence. Two more phage infected strains from ducks were discovered. However, phage was not detected in every isolate examined from common source ducks, although such birds are likely to be infected with the same C psittaci strain. Moreover, phage could not always be demonstrated by indirect immunofluorescence in McCoy cell monolayers infected with the phage-containing strain. The results suggest that phage infection is probably an integral part of duck chlamydiosis in the United Kingdom at present, but that the infection is often cryptic.

[A study of antichlamydial effect of rokitamycin].

MICs of a new macrolide antibiotic, rokitamycin (RKM), for Chlamydia psittaci and Chlamydia trachomatis were determined. Meanwhile, the organisms were observed under the electron microscope for morphologic changes with the addition of RKM. 1. MICs of RKM for C. psittaci MP and 3 strains of C. psittaci isolated from budgerigars kept by patients ranged from 0.05 to 0.10 microgram/ml, and those for 3 strains of C. trachomatis B, E and L2 ranged from 0.20 to 0.39 microgram/ml. These MICs were higher than MICs of minocycline (MINO), doxycycline and rifampicin, but lower than MICs of erythromycin and midecamycin against these organisms. 2. The addition of MINO or RKM to C. psittaci Izawa and C. trachomatis L2 at concentrations twice as high as MICs resulted in no formation of elementary body or intermediate form inside the inclusion body, and abnormal enlargement of reticulate body containing irregularly distributed cytoplasmic components.

[Use of a cryoultramicrotomy method for the purpose of studying chlamydial ultrastructure]. / Primenenie metoda krioul'tramikrotomii s tsel'iu izucheniia ul'trastruktury khlamidii.

The method of cryoultramicrotomy was adapted for the study of the ultrastructure of HeLa and McCoy cells in monolayer cultures infected with Chlamydia, obligatory intracellular procaryotic parasites, the causative agents of ornithosis (strain Loth) and paratrachoma (strain LB 1). The cryosections were obtained by the fixation of the monolayer with 2.5% glutaraldehyde, by the gradual infiltration of precipitated cells with sucrose (0.6--1.2--1.8--2.3 M) prior to freezing in liquid nitrogen, and by the treatment of sections with 1% aqueous methyl cellulose solution before drying. This method ensured good preservation of both Chlamydia, in intracytoplasmic inclusions and host cells, as well as regular reproducibility of the results. Ultrathin sections showed a considerable polymorphism in the vegetative forms of Chlamydia, which was probably due to the structure of their cell walls. Chlamydia, were found to form small vesicle-like structures in the cavities of inclusions. The cell walls and granules inside the elementary bodies of the causative agent of ornithosis were stained with the use of phosphotungstic acid--HCl, pH 0.5.

[Pathomorphology of experimental ornithosis pneumonia]. / Patomorfologiia éksperimental'noi ornitoznoi pnevmonii.

Model experiments in white mice reproduced chronic ornithosis pneumonia morphological features of which included early outgrowth of connective tissue cells of the type of carnification, considerable accumulation of tropoglycogen, and disturbed process of collagen fibers formation at a minimal number of leukocytes. Such foci occur in the lungs very early, before the onset of the clinical signs of the disease which appears to be a typical feature of ornithosis pneumonia and may explain causes and mechanisms of frequent occurrence of chronic forms of this disease. In cases of significant multiplication of the ornithosis agent there is mass migration of leukocytes into the focus of lesion followed by the development of acute inflammation. Being an obligatory intracellular parasite, the ornithosis agent affects both large and small alveolar cells. Multiplying by binary division, it stays free in the cytoplasm without forming a common vacuole. Ultrastructurally typical various forms of the ornithosis agent reflecting the stages of its complicated life cycle have been detected.