Psittaci Elementary Bodies Research Articles

Intranasal immunization with inactivated chlamydial elementary bodies formulated in VCG-chitosan nanoparticles induces robust immunity against intranasal Chlamydia psittaci challenge

Vaccines based on live attenuated Chlamydia elementary bodies (EBs) can cause disease in vaccinated animals and the comparably safer inactivated whole EBs are only marginally protective. Recent studies show that a vaccine formulation comprising UV-inactivated EBs (EB) and appropriate mucosal delivery systems and/or adjuvants induced significant protective immunity. We tested the hypothesis that intranasal delivery of UV-inactivated C. psittaci EB formulated in Vibrio cholerae ghosts (VCG)-chitosan nanoparticles will induce protective immunity against intranasal challenge in SPF chickens. We first compared the impact of VCG and CpG adjuvants on protective immunity following IN mucosal and IM systemic delivery of EB formulated in chitosan hydrogel/microspheres. Immunologic analysis revealed that IN immunization in the presence of VCG induced higher levels of IFN-γ response than IM delivery or the CpG adjuvanted groups. Also, vaccine efficacy evaluation showed enhanced pharyngeal bacterial clearance and protection against lung lesions with the VCG adjuvanted vaccine formulation, thereby establishing the superior adjuvanticity of VCG over CpG. We next evaluated the impact of different concentrations of VCG on protective immunity following IN mucosal immunization. Interestingly, the adjuvanticity of VCG was concentration-dependent, since protective immunity induced following IN mucosal immunization showed dose-dependent immune responses and protection. These studies reveal that formulation of inactivated chlamydial antigens with adjuvants, such as VCG and chitosan increases their ability to induce protective immune responses against challenge.

Seroprevalence of Chlamydophila Psittaci among Employees of Two German Duck Farms.

Psittacosis is a zoonotic infectious disease that is caused by Chlamydophila psittaci. To determine the occupational risk of getting the infection, we investigated the seroprevalence of C. psittaci among employees of two German duck farms and two slaughterhouses according to their level of exposure to the pathogen during the years 2010, 2007, and 2004. In summary, we found low seroprevalence (≈ 8%) throughout the study population almost irrespective of the duty of a given worker. Surprisingly, in 2010, the anti-C. psittaci-specific antibody prevalence in the group of slaughterer (38.9%) was significantly increased in comparison to the non-exposed employees (p = 0.00578). This indicates that individuals in the surrounding of slaughterhouses exposed especially to aerosols containing C. psittaci elementary bodies bear a greater occupational risk of getting infected.

Analysis of cross-reactive and specific anti-carbohydrate antibodies against lipopolysaccharide from Chlamydophila psittaci

Chlamydiae contain a rough-type lipopolysaccharide (LPS) of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid residues (Kdo). Two Kdo trisaccharides, 2.8/2.4- and 2.4/2.4-linked, and a branched 2.4[2.8]2.4-linked Kdo tetrasaccharide occur in Chlamydiaceae. While the 2.8/2.4-linked trisaccharide contains a family-specific epitope, the branched Kdo oligosaccharide occurs only in Chlamydophila psittaci and antibodies against it will be useful in human and veterinarian diagnostics. To overcome the generation of cross-reactive antibodies that bind with high affinity to a dominant epitope formed by 2.4/2.4-linked Kdo, we immunized mice with a synthetic 2.4[2.8]-linked branched Kdo trisaccharide and used phage display of scFv to isolate recombinant antibody fragments (NH2240-31 and SAG506-01) that recognize the branched Kdo oligosaccharide with a K(D) of less than 10 nM. Importantly, although these antibodies used germline genes coding for an inherited Kdo recognition site, they were able clearly to distinguish between 2.4[2.8]2.4- and 2.4/2.4-linked Kdo. Sequence determination, binding data, and X-ray structural analysis revealed the basis for the improved discrimination between similar Kdo ligands and indicated that the alteration of a stacking interaction from a phenylalanine residue in the center of the combining site to a tyrosine residue facing away from the center favors recognition of branched 2.4[2.8]2.4-linked Kdo residues. Immunofluorescence tests of infected cell monolayers using this antibody show specific staining of C. psittaci elementary bodies that allow it to be distinguished from other pathogenic chlamydiae.

ELISA test to detect Chlamydophila pneumoniae IgG

A new ELISA test (Chlamydophila pneumoniae IgG, Vircell, Spain) to detect Chlamydophila pneumoniae IgG was evaluated. The micro-immunofluorescence (MIF) test was used as reference method. Chlamydia trachomatis and Chlamydophila psittaci elementary bodies were also assayed. Two hundred and sixteen sera were included in the study: 66 from patients with peripheral arterial occlusive disease (Panel 1), 68 from adults with pneumonia (Panel 2), 44 from healthy adults (Panel 3) and 38 from patients with a sexuality transmitted disease by C. trachomatis (Panel 4). In Panel 1, 51 sera (77%) had antibody titres between 32 and 128; 4 out of 15 sera with IgG titres < 32 were positive by ELISA test and 2 sera with 32 IgG titres were uncertain by ELISA; the remaining 60 sera were correctly classified, giving a 91% concordance between the techniques. In Panel 2, 55 sera (81%) had IgG titres between 32 and 512; 2 out of 13 sera with IgG titres < 32 were positive by ELISA and 2 sera with 32 titres were uncertain by ELISA; the remaining 64 sera were correctly classified, giving a 97% concordance. In Panel 3, 22 sera (50%) had IgG titres between 32 and 64; only 1 out of 22 sera with IgG titres < 32 was positive by ELISA, giving a 97% concordance between the techniques. In Panel 4, there were 24 (63%) negative, 10 (26%) uncertain and 4 (10%) positive results by ELISA, giving an 86% concordance. The C. pneumoniae ELISA test demonstrated 100% sensitivity and 85% specificity. The IgG ELISA test demonstrated a good concordance with the MIF test without the drawbacks associated with the latter assay. We conclude that the ELISA test could be an alternative to the MIF test.

Cloning and characterization of a Chlamydia psittaci gene coding for a protein localized in the inclusion membrane of infected cells.

Chlamydiae are obligate intracellular bacteria which occupy a non-acidified vacuole (the inclusion) throughout their developmental cycle. Little is known about events leading to the establishment and maintenance of the chlamydial inclusion membrane. To identify chlamydial proteins which are unique to the intracellular phase of the life cycle, an expression library of Chlamydia psittaci DNA was screened with convalescent antisera from infected animals and hyperimmune antisera generated against formalin-killed purified chlamydiae. Overlapping genomic clones were identified which expressed a 39 kDa protein only recognized by the convalescent sera. Sequence analysis of the clones identified two open reading frames (ORFs), one of which (ORF1) coded for a predicted 39 kDa gene product. The ORF1 sequence was amplified and fused to the malE gene of Escherichia coli and antisera were raised against the resulting fusion protein. Immunoblotting with these antisera demonstrated that the 39 kDa protein was present in lysates of infected cells and in reticulate bodies (RBs), but was at the limit of detection in lysates of purified C. psittaci elementary bodies. Fluorescence microscopy experiments demonstrated that this protein was localized in the inclusion membrane of infected HeLa cells, but was not detected on the developmental forms within the inclusion. Because the protein produced by ORF1 is deposited on the inclusion membrane of infected cells, this gene has been designated incA, (inclusion membrane protein A) and its gene product, IncA. In addition to the inclusion membrane, these antisera labelled structures that extended from the inclusion over the nucleus or into the cytoplasm of infected cells. Immunoblotting also demonstrated that IncA, in lysates of infected cells, had a migration pattern that seemed indicative of post-translational modification. This pattern was not observed in immunoblots of RBs or in the E. coli expressing IncA. Collectively, these data identify a chlamydial gene which codes for a protein that is released from RB and is localized in the inclusion membrane of infected cells.

The development of proliferative responses of ovine peripheral blood mononuclear cells to Chlamydia psittaci during pregnancy

In vitro lymphoproliferative responses to Chlamydia psittaci elementary bodies were detected in the peripheral blood mononuclear cells of pregnant sheep infected with C. psittaci. These responses were suppressed in cells collected in the immediate peri-parturient period, but returned to levels significantly above background within 14 days. Infection also suppressed the lymphoproliferative response to the T-cell mitogen Con A until abortion or lambing, but did not affect the proliferative response to the B-cell mitogen LPS.

Differential modulation of lymphocyte proliferative responses and lymphokine secretion in mice during development of immunity to Chlamydia psittaci

A murine model was utilized to study immune responses occurring during the period of acquisition of immunity to chlamydial infection. C3H (H-2k) mice were immunized by intramuscular injection of 5 x 10(3) viable Chlamydia psittaci elementary bodies (EBs) by a protocol which permits animals to survive an otherwise lethal intraperitoneal challenge 10 days later with the homologous chlamydial strain. Spleen cells assayed during the 10-day period of development to immunity showed depressed proliferative responses in vitro to the T-cell mitogen, concanavalin A, and also exhibited suppressor cell activity. Spleen cell mitogen responses returned to normal levels by 30 days postimmunization, concomitant with the detectable development in vitro of responses to chlamydia-specific antigen. In marked contrast to the reduced proliferative responses, mitogen-stimulated production of the T-cell-derived lymphokines interleukin-2 and gamma interferon by spleen cells from immunized animals was within the normal range at 10 days postimmunization, and supernatant fluids containing these products had both microbicidal and microbistatic effects on chlamydial organisms in vitro. These results demonstrate that independent regulation of T-cell proliferation and lymphokine production occurs in vivo as part of the development of an antigen-specific protective immune response. These results also suggest that such differential modulation of T-cell responses may contribute to the development of protective immunity to chlamydiae in mice, perhaps through limited T-cell clonal expansion coupled with early or preferential maturation of cytokine-secreting helper T cells.

The effect of intracellular Chlamydia psittaci on lysosomal enzyme activities in mouse peritoneal macrophages.

Live or heat killed (30 min at 56 degrees C) Chlamydia psittaci elementary bodies (EB) were phagocytosed by mouse peritoneal macrophages. Inoculation with killed Chlamydia caused rises in three lysosomal enzyme activities tested, especially in acid phosphatase activity. In contrast, after infection with live Chlamydia, only a negligible increase was seen in acid phosphatase activity, and no change in the activities of beta-glucuronidase and cathepsin D was observed. It was concluded that regulation of lysosomal enzyme synthesis in macrophages may be linked to signals mediated by phago-lysosome fusions.

Surface projections of Chlamydia psittaci elementary bodies as revealed by freeze-deep-etching.

The morphology of the surface projections of Chlamydia psittaci elementary bodies in the intracytoplasmic inclusion was the same as that of the projections on the purified elementary bodies. Each projection emerged from the center of a flower structure, which was composed of nine leaves arranged radially.

Interaction of Chlamydia psittaci with mouse peritoneal macrophages

L-cell-grown Chlamydia psittaci elementary bodies (EB) were rapidly phagocytized by mouse peritoneal macrophages in vitro. However, the intracellular fate of chlamydiae in macrophages appeared to be dependent on the multiplicity of infection (MOI), i.e., the EB-to-macrophage ratio, and the treatment of the EB. At an MOI of 1:1 or less, survival is maximal, and growth and multiplication of live, untreated chlamydiae did occur. In contrast, at a high MOI (100:1), survival of chlamydiae is reduced, as confirmed by release of 3H-labeled nucleic acid into the supernatant. At the high MOI, macrophage damage occurred that resulted in significant release of the lactic dehydrogenase, beginning 2 h postinfection. This immediated macrophage cytotoxicity as abolished by pretreatment of EB with heat (5 min at 56 degrees C) and was reduced about 50% by coating EB with homologous antibody. Pretreatment of the chlamydia with heat or opsonizing antibody provides increased uptake of EB by macrophages but may contribute to increased destruction of these obligate intracellular pathogens in professional phagocytic cells.

Effect of alkali on the structure of cell envelopes of Chlamydia psittaci elementary bodies.

Suspensions of isolated cell envelopes of infectious elementary bodies (EB) of Chlamydia psittaci at alkaline pH showed a rapid, extensive decrease in absorbance, accompanied by the release of a cell envelope component in a sedimentable form. This phenomenon was observed both at 0 C and with envelopes which had been previously heated to 100 C. Monovalent and divalent cations effectively inhibited the turbidity loss, whereas ethylenediaminetetraacetate (EDTA) caused an accelerated decrease in turbidity. The turbidity loss observed after incubation of the envelopes at alkaline pH could be reversed to the level of the initial value by dialysis against distilled water containing Mg2+. Thin-section electron photomicrographs of purified EB exposed to alkaline buffer with EDTA revealed the loss of the internal contents of cells, but these cells still maintained their round shapes. The cell surface of treated EB appeared pitted in negatively stained preparations, whereas intact EB had a smooth surface. Electron microscopic studies on negatively stained preparations of the clear supernatant obtained after the treatment of the envelope with alkaline buffer containing EDTA demonstrated the presence of spherical particles, approximately 6 to 7 nm in diameter, and rodlike particles, which appeared to be made up of two or more spherical particles.