Ultrastructural study of mode of entry of Chlamydia psittaci into L-929 cells.

The entry of Chlamydia psittaci into L-929 cells was studied morphologically by transmission electron microscopy and quantitatively by a method that discriminates between attachment and uptake. Upon adsorption of 3H-labeled elementary bodies (EBs) to host cells at 4 degrees C, the EBs bound efficiently to the L-cell surface. Binding reached an equilibrium level of 55% in 3 h. Ultrastructural analysis revealed that EBs were bound preferentially to the tips and sides of microvilli at this temperature. The EBs were also observed in coated pits located at the bases of microvilli and along smooth surfaces of the host cell. No internalization was observed at 4 degrees C. When cells with prebound 3H-labeled EBs were warmed to 37 degrees C, the EBs rapidly became resistant to proteinase K removal (half time = 5 min), indicating ingested chlamydiae. At 37 degrees C, the EBs were internalized within tightly bound vesicles surrounded by an electron-dense coat of fibrillar material. EBs were also present in smooth-surfaced pits and vesicles of the host cell. Using alpha 2-macroglobulin coupled to colloidal gold (a known marker for receptor-mediated endocytosis), we observed that the entry of EBs into cells via coated pits was identical in appearance to the internalization of alpha 2-macroglobulin. Also, when the two ligands were mixed together, they could be seen within the same coated pits and were cointernalized within endocytic vesicles of the host cell. These results suggest that C. psittaci can enter nonprofessional phagocytic cells by a pathway which is similar to that of receptor-mediated endocytosis of many physiologically important macromolecules, bacterial toxins, and viruses.     

Cytotoxic cells induced after Chlamydia psittaci infection in mice.

The ability of spleen cells from Chlamydia psittaci-infected mice to lyse C. psittaci-infected and uninfected target cell monolayers was studied. The cytotoxicity assay used was a terminal label method in which the number of adherent target cells surviving the interaction with effector cells was determined by measuring the uptake of [3H]uridine by such cells. It was observed that in the first few days postinfection (3 to 5), spleens contained cells that lysed infected and uninfected targets with equal efficiency. Subsequently, infected targets were killed primarily. The activity of effector spleen cells for infected targets continued, although at a reduced level, beyond 21 days postinfection. Intact effector cells were required since a disruption by sonication resulted in a loss of cytotoxicity. The enhanced killing observed with infected targets was also observed when target cells were sensitized with heat- or UV-inactivated C. psittaci. This study suggests that the induction of cytotoxic cells after C. psittaci infection may contribute to the ability of the host to control multiplication of the microorganism.     

Monoclonal antibody typing of Chlamydia psittaci strains derived from avian and mammalian species.

A total of 77 Chlamydia psittaci strains of avian, human, and mammalian origin were grouped into four serovars with 11 monoclonal antibodies recognizing the lipopolysaccharide and the major outer membrane protein antigens. The avian and human strains, which were closely related to each other, were distinct from the mammalian strains. Immunological typing of C. psittaci with monoclonal antibodies seems practical.     

Parasite-specified phagocytosis of Chlamydia psittaci and Chlamydia trachomatis by L and HeLa cells.

Phagocytosis of the 6BC strain of Chlamydia psittaci and the lymphogranuloma venereum 440L strain of Chlamydia trachomatis by L cells and HeLa 229 cells occurred at rates and to extents that were 10 to 100 times greater than those observed for the phagocytosis of Escherichia coli and polystyrene latex spheres. Both species of Chlamydia were efficiently taken up by host cells of a type they had not previously encountered. Phagocytosis of chlamydiae was brought about by the interaction of parasite surface ligands with elements of the host cell surface. The chlamydial ligands were readily denatured by heat, were masked by antibody, and were resistant to proteases and detergents. The host cell components were reversibly removed by proteases. Chlamydial phagocytosis was inhibited when host cells were incubated for many hours with cycloheximide. It was suggested that the presence on the chlamydial cell surface of ligands with high affinity for normal, ubiquitously occurring structures on the surface of host cells is an evolutionary adaptation to intracellular existence. The term parasite-specified phagocytosis was used to describe the efficient phagocytosis of chlamydiae by nonprofessional phagocytes and to distinguish it from the host-specified immunological and non-immunological phagocytosis carried out by professional phagocytes.     

Isolation of Chlamydia psittaci from avian sources using growth in cell culture

A method is described for the isolation of Chlamydia psittaci using cell culture treated with 5-iodo-2-deoxyuridine, and subsequent identification by direct fluorescent antibody staining. The method was applied to 110 sets of tissues from a variety of avian specimens submitted for diagnosis. Chlamydiae were isolated and identified in 24 specimens: 13 from parrots, 7 from turkeys and 4 from pigeons.     

Comparison of avian Chlamydia psittaci isolates by restriction endonuclease analysis and serovar-specific monoclonal antibodies.

Avian Chlamydia psittaci isolates were examined by restriction endonuclease analysis and serovar-specific monoclonal antibodies and compared with ovine abortion and polyarthritis isolates. The avian isolates were divided into four serovars (turkey, psittacine, pigeon, and duck) based on their reactivity to the monoclonal antibodies. The DNA digest patterns were similar across the four avian serovars; most bands were identical when the isolates were tested with PstI, BamHI, and EcoRI restriction endonuclease enzymes. The turkey group restriction endonuclease analysis patterns were distinguished from those of the other avian strains by three to four band differences with all enzymes. The duck and pigeon isolates showed only minor DNA pattern differences when compared with the psittacine isolates. Four psittacine isolates from various locations in Texas had an extra band with the EcoRI restriction enzyme, suggesting that they were from a common source; however, they were indistinguishable from the other psittacine isolates when examined with the monoclonal antibodies. The avian isolates were distinctly different from either abortion or polyarthritis isolates by both restriction endonuclease analysis and monoclonal antibody analysis. The data demonstrate that the avian isolates form a distinct group or separate biovar with at least four serovars.     

Pathogenicity for turkeys of Chlamydia psittaci strains belonging to the avian serovars A, B and D.

Four groups of 20 specific pathogen-free turkeys kept in isolators were exposed to aerosols of one of four Chlamydia psittaci strains. These were strain 92/1293 (avian serovar D), strain 84/55 (avian serovar A), the Texas Turkey strain (avian serovar D) and strain 89/1326 (avian serovar B). A further group of four specific pathogen-free turkeys consisted of sham-infected controls. The birds were observed daily for clinical signs. At daily intervals for 10 days and then twice weekly up to 34 days post-infection, one bird in each group was killed and examined for gross and microscopic lesions. Clinical signs, macroscopic and microscopic lesions were most severe in turkeys infected with strains 92/1293 and 84/55, and there was some mortality. Signs and lesions were less severe in the group infected with the Texas Turkey strain and there was no mortality. The turkeys infected with strain 89/1326 developed only mild clinical disease and lesions. Thus, differences in pathogenicity for turkeys were shown not only between strains belonging to different serovars, but also between strains within a single serovar.     

Ultrastructural cytochemical evidence for the activation of lysosomes in the cytocidal effect of Chlamydia psittaci.

The cytopathic effect of the polyarthritis strain of Chlamydia psittaci was studied in cultured bovine fetal spleen cells and found to be mediated by the release of lysosomal enzymes into the host cytoplasm during the late stages of chlamydial development. Ultrastructural cytochemical analysis and cell fractionation studies of infected cells revealed a close relationship between the stage of chlamydial development, fine structural features of the host, and localization of lysosomal enzyme activities. After adsorption, chlamydiae entered the host cells by endocytosis. The endocytic vacuoles containing individual chlamydiae and later the inclusion vacuoles containing the different chlamydial developmental forms were always free from lysosomal enzyme activity. Even after extensive multiplication of chlamydiae, lysosomal enzymes remained localized within lysosomes or their precursors in the host cell. Coincident with the process of chlamydial maturation, lysosomal enzymes were released into the host cytoplasm and were always associated with disintegration of host cell constituents and lysis. The chlamydiae appeared to be protected from this lysosomal enzyme activity by the inclusion membrane. After release from the inclusion, elementary bodies maintained their fine structural features, whereas all other chlamydial developmental forms lost their ultrasturctural integrity.     

Isolation and characterization of phagosomes containing Chlamydia psittaci from L cells.

The obligate intracellular procaryote Chlamydia psittaci enters host cells by a mechanism similar to, but distinct from, conventional phagocytosis. To better understand chlamydial uptake, L-cell phagosomes containing a single chlamydial cell were isolated and studied. Two rounds of dextran rate-zonal gradient centrifugation of L cells homogenized 1 h after infection with C. psittaci yielded phagosomes relatively free of other membranous structures. In double-label experiments, the phagosomes were enriched over 40-fold for radioactivity derived from chlamydiae as compared with the initial homogenate. Several lines of evidence showed that the structures isolated on dextran gradients were chlamydial phagosomes. These structures and free chlamydiae banded at different positions on discontinuous sucrose gradients. The difference was destroyed by the nonionic detergent Nonidet P-40, which disrupts plasma membranes but has no effect on C. psittaci. Material labeled on the surface of the L-cell plasma membrane cosedimented with the phagosome fractions. Electron microscopy of these fractions revealed structures having the appearance of a chlamydial elementary body surrounded by a unit membrane. Sodium dodecyl sulfate-polyacrylamide gels of the phagosome membranes displayed 10 major protein bands, less than the total number of surface-labeled proteins in the L-cell plasma membrane. Seven of the proteins of phagosome membranes had electrophoretic mobilities corresponding to those of proteins exposed on the surface of L cells. Two of them were cleaved by both trypsin and chymotrypsin, enzymes that decrease the susceptibility of L cells to infection with C. psittaci. These proteins may therefore be involved in the attachment and ingestion of C. psittaci by L cells.     

Detection of Chlamydia pneumoniae and Chlamydia psittaci in sputum samples by PCR.

AIMS--To use the polymerase chain reaction (PCR) to detect Chlamydia pneumoniae and Chlamydia psittaci in sputum samples. METHODS--A nested PCR was developed, the first stage of which amplified DNA from both C pneumoniae and C psittaci while the second stage targeted specifically at C pneumoniae, allowing the two species to be differentiated. The primers were designed not to amplify sequences from C trachomatis. A panel of 26 sputum samples from patients with community acquired pneumonia evaluated previously by enzyme linked immunosorbent assay (ELISA), direct immunofluorescence (DIF), and culture was tested blind by PCR. Most of these specimens also had accompanying serial serum samples which were tested for species specific antibodies using microimmunofluorescence (micro-IF). RESULTS--PCR detected C pneumoniae DNA in 10 of the 26 samples and C psittaci DNA in four. There was good concordance between ELISA, DIF, micro-IF and PCR in the C pneumoniae group. Two of the C psittaci identified by PCR were labelled C pneumoniae by DIF but the PCR results were supported by serology or a history of bird contact. Of the PCR negative group: six were true negative results; two contained C trachomatis. There were four discrepant results. CONCLUSIONS--The data suggest that PCR is effective in the detection of C pneumoniae. The sensitivity for C psittaci is inevitably lower due to the strategy taken but specificity seemed to be good.     

PCR-based diagnosis,molecular characterization and detection of atypical strains of avian Chlamydia psittaci in companion and wild birds

Chlamydiosis is one of the most important infectious diseases of birds. In this study, 253 clinical samples were taken from 27 bird species belonging to seven orders. Thirty-two (12.6%) samples were positive for Chlamydia psittaci major outer membrane gene (ompA) DNA by a nested polymerase chain reaction (PCR). Twelve nested PCR-positive specimens were typed by ompA gene-based PCR-restricted fragment length polymorphism, using CTU/CTL primers and AluI restriction enzyme. Four restriction patterns were identified, including genotype A (two specimens from an African grey parrot [Psittacus erithacus] and a lorikeet [Trichoglossus haematodus]), genotype B (two specimens from a rock dove [Columbia livia] and a canary [Serinus canaria]), a third new restriction pattern (six specimens from African grey parrots), and a fourth new restriction pattern (two specimens from a ring-necked parakeet [Psittacula krameri] and an Alexandrine parakeet [Psittacula eupatria]). The third and the fourth restriction patterns are suggested to be provisional genotypes I and J, respectively. Partial sequencing of the ompA gene of seven specimens completely correlated with the results of PCR-restricted fragment length polymorphism and confirmed the presence of genotypes A and B and the two new provisional genotypes I and J. The two new genotypes have the closest identity with C. psittaci genotype F and Chlamydia abortus, respectively. From an evolutionary perspective, both new genotypes, particularly genotype J, are intermediate between the two species, C. psittaci and C. abortus.     

Isolation of Chlamydia using McCoy cells and Buffalo green monkey cells

Unselected eye and genital specimens from 233 patients were cultured for Chlamydia. The isolation rates were compared using McCoy cells and Buffalo green monkey cells, both procedures being performed with and without the addition of cycloheximide and centrifugation. No significant difference was found in the isolation rates using the four methods. The characteristics of the two cell lines and the advantages of omitting cycloheximide treatment and centrifugation are discussed.     

Murine granulated metrial gland cells are susceptible to Chlamydia psittaci infection in vivo.

Granulated metrial gland (GMG) cells are the most numerous lymphoid cells in the uteroplacental unit in rodent pregnancy. In an experimental murine model of abortion-causing infection, we have studied the responses of GMG cells to Chlamydia psittaci. Chlamydial inclusions have been found within GMG cells, both in apparently healthy cells and in cells with degenerative changes. Establishing the existence of GMG cells infected by C. psittaci opens a new and interesting chapter in the study of these cells.     

Protection against Chlamydia psittaci in mice conferred by Lyt-2+ T cells.

A murine model was used to study the respective roles of L3T4+ and Lyt-2+ T cells in protection against Chlamydia psittaci. Donor mice were intravenously (i.v.) infected with 1 x 10(5) plaque-forming units (PFU) per mice of live C. psittaci. One month after inoculation, splenic cells from donors were transferred into syngenic recipients (5 x 10(7) cells/mouse). As measured by splenic colonization on Day 6 after i.v. challenge (1 x 10(5) PFU/mouse), transfer with primed (untreated) cells conferred a 3 log protection in this model. In vitro treatment, before transfer, of splenic cells with anti-Lyt-2 monoclonal antibody (mAb) and complement, markedly impaired the protection in comparison with control mice transferred with primed untreated cells, whereas treatment with anti-L3T4 mAb did not reduce the transferred protection. Resistance to a reinfection with C. psittaci was also studied after selective in vivo depletion of L3T4+ and Lyt-2+ T cells. One month after primary infection, mice were treated with anti-L3T4 or anti-Lyt-2 mAb and challenged thereafter (i.v., 1 x 10(5) PFU). The splenic colonization on Day 6 after challenge demonstrated that treatment with anti-Lyt-2 mAb impaired resistance against a subsequent infection with C. psittaci. Treatment with anti-L3T4 mAb in vivo had no effect on protection, as previously described in vitro. The mechanisms by which Lyt-2+ T cells could participate in the elimination of bacteria were discussed.     

Chemical, biological, and immunochemical properties of the Chlamydia psittaci lipopolysaccharide.

The lipopolysaccharide (LPS) of Chlamydia psittaci was extracted from yolk sac-grown elementary bodies, purified, and characterized chemically, immunochemically, and biologically. The LPS contained D-galactosamine, D-glucosamine, phosphorus, long-chain fatty acids, and 3-deoxy-D-manno-2-octulosonic acid in the molar ratio of approximately 1:2:2:6:5. The antigenic properties of the isolated LPS were compared with those of the LPS from Chlamydia trachomatis and Salmonella minnesota Re by the passive hemolysis and passive hemolysis inhibition tests, absorption, hydrolysis kinetics, and Western blot analysis with rabbit polyclonal antisera against chlamydiae and with a mouse monoclonal antibody recognizing a genus-specific epitope of chlamydial LPS. Two antigenic determinants were identified, one of which was chlamydia specific and the other of which was cross-reactive with Re LPS. Both determinants were destroyed during acid hydrolysis, whereby a third antigen specificity was exposed which was indistinguishable from the lipid A antigenicity. In rabbit polyclonal antisera prepared against Formalin-killed elementary bodies or detergent-solubilized membranes, two antibody specificities were differentiated. One of these was chlamydia specific, and the other was cross-reactive with Re LPS. The LPS of C. psittaci was inactive within typical endotoxin parameters (lethal toxicity, pyrogenicity, local Shwartzman reactivity); it was, however, active in some in vitro assays, such as those testing for mouse B-cell mitogenicity and the induction of prostaglandin E2 in mouse peritoneal macrophages.     

A rapid method for staining inclusions of Chlamydia psittaci and Chlamydia trachomatis.

A new staining method was developed for the detection of inclusions of Chlamydia psittaci and Chlamydia trachomatis inclusions in cell cultures. Using a combination of methyl green and neutral red stains and washing at pH 5.0, inclusions were stained red while cell cytoplasm was pale pink and cell nuclei were pale green. The method was significantly better than Giemsa staining and comparable to immunofluorescence for detecting C psittaci inclusions. Its sensitivity for detection C trachomatis inclusions by dark field microscopy was similar to that of Giemsa staining.     

Attachment defect in mouse fibroblasts (L cells) persistently infected with Chlamydia psittaci.

Antibiotic-associated pseudomembranous colitis has been linked with Clostridium difficile toxin. We examined the effect of toxins from four strains of C. difficile isolated from patients with pseudomembranous colitis on colonic adenylate (EC 4.6.1.1) and guanylate cyclase (EC 4.6.1.2) activities. Partially purified toxins had a cytotoxic effect on hamster fibroblasts in culture at a concentration of 10 ng/ml. Likewise, these toxins enhanced colonic guanylate cyclase activity two- to threefold, with the maximal stimulation being at 10 ng/ml. These toxins also enhanced guanylate cyclase activity in ileum, cecum, and duodenum. Both the cytotoxic activity on hamster fibroblasts and the enhancement of hamster guanylate cyclase activity were inhibited by antiserum to C. difficile toxin. These same toxins inhibited adenylate cyclase activity at a 100-ng/ml concentration, but had no effect at 10 ng/ml. They also had no effect at any concentration on colonic Na+-K+ adenosine triphosphatase. To be sure that the findings were not due to a contaminant, a purified C. difficile cytotoxin was used, and the same findings were found with the pure cytotoxin (at a 100-fold-lower concentration). The data suggest that activation of guanylate cyclase may be a factor in the pathogenesis of antimicrobial-associated pseudomembranous colitis.     

Interaction between turkey monocytes and avian Chlamydia psittaci in the presence of Mycoplasma sp.: the importance of nitric oxide

The interaction between Chlamydia psittaci and turkey monocytes was studied in vitro. Purified monocytes were inoculated with C. psittaci, in the presence or absence of Mycoplasma hyorhinis. Whereas turkey monocytes produced high amounts of nitric oxide (NO) following the inoculation with M. hyorhinis, inoculation with C. psittaci did not induce NO production in these phagocytes. The monocytes strongly supported chlamydial growth, as demonstrated by the presence of inclusion forming units, the positive direct immunofluorescence staining and transmission electron microscopy. In contrast, upon co-inoculation of the monocytes with C. psittaci and M. hyorhinis, a reduced replication rate of C. psittaci was observed. N(G)-monomethyl-L-Arginine, a competitive inhibitor for the enzyme NO-synthase, inhibited the NO production and reversed the antichlamydial activity of the M. hyorhinis co-inoculated turkey monocytes. These results imply two considerations. First, as chlamydiae are obligate intracellular bacteria, special care should be taken to guard chlamydial cultures from mycoplasmal contamination, in order to prevent false results when investigating the response of immunomodulating cells to chlamydial infection. Secondly, as a mycoplasmal co-infection in vitro has the capacity of inducing antichlamydial activity in turkey monocytes, through the action of NO, it could be suggested that a similar interaction might take place in vivo. Moreover, it was shown that avian M. gallisepticum strains were also able to induce NO in turkey monocytes. Considering the high prevalence of both C. psittaci and Mycoplasma sp. in turkeys, this interaction, through the pivotal role of NO, might influence the outcome of respiratory diseases in turkeys.     

Protein synthesis early in the developmental cycle of Chlamydia psittaci.

The incorporation of [35S]methionine into protein by intracellular and host-free Chlamydia psittaci 6BC was analyzed at intervals between 15 min and 28 h postinfection by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. The profiles of proteins synthesized in the two systems were similar at all times, indicating that the host-free system can be used to monitor the temporal expression of genes in chlamydiae. The host-free system permitted detection of synthesis of chlamydial proteins as early as 15 min postinfection. Some of the proteins synthesized during the initial phases of reorganization of elementary bodies to reticulate bodies either were not synthesized or were synthesized in greatly reduced amounts during the other phases of the developmental cycle. The effects of rifampin and actinomycin D indicated that host-free protein synthesis was at least partially dependent on the initiation and continuation of RNA synthesis in the isolated organisms.