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PubMed2005

Conjunctival swab cytology from a guinea pig: it's elementary!

Three 3-month-old guinea pigs (Cavia porcellus) were evaluated for purulent ocular discharge. Conjunctival swabs were obtained for cytologic evaluation of Wright's-Giemsa-stained preparations. The specimen from the most severely affected guinea pig consisted primarily of karyolytic neutrophils and small lymphocytes. Epithelial cells occasionally were observed that contained intracytoplasmic coccoid basophilic organisms, 0.5-1.5 microm in diameter. The intraepithelial inclusions were most consistent with Chlamydia sp elementary and reticulate bodies. Specimens from the other 2 guinea pigs had a similar inflammatory response, but organisms were not observed. Polymerase chain reaction (PCR) analysis of a conjunctival swab from the most severely affected guinea pig was positive for C psittaci, which also is referred to as Chlamydophila caviae, immunotype 8, formerly known as the guinea pig inclusion conjunctivitis strain of C psittaci. Chlamydial conjunctivitis is a common problem in guinea pig populations, with C caviae being specific for this species. Cytologic identification of elementary or reticulate bodies within epithelial cells is diagnostic for the organism in Giemsa-stained preparations. However, PCR is an important complementary tool when organisms are not observed and for accurate classification of the Chlamydia species.

Animals↗
PubMed1989

Laboratory diagnosis of human chlamydial infections.

Chlamydia trachomatis is a human pathogen that causes ocular disease (trachoma and inclusion conjunctivitis), genital disease (cervicitis, urethritis, salpingitis, and lymphogranuloma venereum), and respiratory disease (infant pneumonitis). Respiratory chlamydioses also occur with infection by avian strains of C. psittaci or infection by the newly described TWAR agent. Diagnosis of most acute C. trachomatis infections relies on detection of the infecting agent by cell culture, fluorescent antibody, immunoassay, cytopathologic, or nucleic acid hybridization methods. Individual non-culture tests for C. trachomatis are less sensitive and specific than the best chlamydial cell culture system but offer the advantages of reduced technology and simple transport of clinical specimens. Currently available nonculture tests for C. trachomatis perform adequately as screening tests in populations in which the prevalence of infection is greater than 10%. A negative culture or nonculture test for C. trachomatis does not, however, exclude infection. The predictive value of a positive nonculture test may be unsatisfactory when populations of low infection prevalence are tested. Tests that detect antibody responses to chlamydial infection have limited utility in diagnosis of acute chlamydial infection because of the high prevalence of persistent antibody in healthy adults and the cross-reactivity due to infection by the highly prevalent C. trachomatis and TWAR agents. Assays for changes in antibody titer to the chlamydial genus antigen are used for the diagnosis of respiratory chlamydioses. A single serum sample that is negative for chlamydial antibody excludes the diagnosis of lymphogranuloma venereum.

Chlamydia Infections↗
PubMed1970

Plaque formation by Chlamydia in L cells.

Chlamydiae were found capable of producing plaques in several cell lines. Mouse fibroblast cells, L-929, proved the most sensitive to infection and yielded plaques of the highest clarity. Assay of chlamydial infectivity by plaque titration was at least as sensitive as egg ld(50) determination. Among chlamydial isolates of avian, mammalian, and human origin, only slow-growing trachoma-inclusion-conjunctivitis agents did not produce plaques. The plaque assay is highly sensitive, reproducible, and offers a potential tool for investigations requiring accurate measurement of small changes in chlamydial infectivity.

Journal Article↗
PubMed1970

Plaque reduction technique for demonstrating neutralizing antibodies for Chlamydia.

Some members of the genus Chlamydia (Bedsonia or psittacosis-lymphogranuloma venereum-trachoma-inclusion conjunctivitis group of microorganisms) produce plaques in monolayers of mouse fibroblast (L-929) cells. Hyperimmune chicken antisera may be capable of specifically reducing plaque counts. When the test was applied to chlamydiae isolated from avian species, the results indicated that different isolates with a common source had similar antigenic reactivity. The plaque reduction test is a potentially useful method for serotyping chlamydiae. The difficult aspect of the method appears to be a readily reproducible means of producing neutralizing antiserum.

Journal Article↗
PubMed1981

Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis.

Elementary bodies (EB) of Chlamydia trachomatis serotypes C, E, and L2 were extrinsically radioiodinated, and whole-cell lysates of these serotypes were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiography of the polypeptide profiles identified a major surface protein with an apparent subunit molecular weight of 39,500 that was common to each C. trachomatis serotype. The abilities of nonionic (Triton X-100), dipolar ionic (Zwittergent TM-314), mild (sodium deoxycholate and sodium N-lauroyl sarcosine), and strongly anionic (SDS) detergents to extract this protein from intact EB of the L2 serotype were investigated by SDS-PAGE analysis of the soluble and insoluble fractions obtained after each detergent treatment. Only SDS readily extracted this protein from intact EB. Sarkosyl treatment selectively solubilized the majority of other EB proteins, leaving the 39,500-dalton protein associated with the Sarkosyl-insoluble fraction. Ultrastructural studies of the Sarkosyl-insoluble EB pellet showed it to consist of empty EB particles possessing an apparently intact outer membrane. No structural evidence for a peptidoglycan-like cell wall was found. Morphologically these chlamydial outer membrane complexes (COMC) resembled intact chlamydial EB outer membranes. The 39,500-dalton outer membrane protein was quantitatively extracted from COMC by treating them with 2% SDS at 60 degrees C. This protein accounted for 61% of the total COMC-associated protein, and its extraction resulted in a concomitant loss of the COMC membrane structure and morphology. The soluble extract obtained from SDS-treated COMC was adsorbed to a hydroxylapatite column and eluted with a linear sodium phosphate gradient. The 39,500-dalton protein was eluted from the column as a single peak at a phosphate concentration of approximately 0.3 M. The eluted protein was nearly homogeneous by SDS-PAGE and appeared free of contaminating carbohydrate, glycolipid, and nucleic acid. Hyperimmune mouse antiserum prepared against the 39,500-dalton protein from serotype L2 reacted with C. trachomatis serotypes Ba, E, D, K, L1, L2, and L3 by indirect immunofluorescence with EB but failed to react with serotypes A, B, C, F, G, H, I, and J, with the C. trachomatis mouse pneumonitis strain, or with the C. psittaci feline pneumonitis, guinea pig inclusion conjunctivitis, or 6BC strains. Thus, the 39,500-dalton major outer membrane protein is a serogroup antigen of C. trachomatis organisms.

Antigens, Bacterial↗
PubMed1987

Analysis of the humoral immune response to chlamydial genital infection in guinea pigs.

Studies using the guinea pig model of chlamydial genital infection with the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis (GPIC) have shown that serum and local antibodies play a role both in the resolution of infection and in protection against reinfection. Thus, this model is suited for further exploration of immune mechanisms and for vaccine studies with chlamydial macromolecules. We have further characterized the model by assessing the antigen-specific antibody response to experimental genital infection by using immunoblotting to assay both genital secretions and serum. The GPIC agent was characterized by analysis of outer membrane proteins, which indicated that the GPIC agent possessed a major outer membrane protein (MOMP), with a molecular mass of 39 kilodaltons (kDa), and a 61-kDa protein, analogous to cysteine-rich 60-kDa proteins or doublets of Chlamydia trachomatis strains. As indicated by immunoblotting, most infected animals produced serum immunoglobulin G antibodies to MOMP, the 61-kDa proteins, an 84-kDa outer membrane protein, and lipopolysaccharide. Such serum antibodies persisted for at least 813 days after primary genital infection. Immunoglobulin A antibodies against the 61-kDa proteins, lipopolysaccharide, and MOMP, but not the 84-kDa protein, were detected in secretions. Animals challenged with GPIC 825 days after primary infection became infected again despite the presence of serum antibodies, but the period of chlamydial shedding was significantly shorter and less intense than in primary infections. Although the specific mechanism is not known, these data suggest that a long-lasting immune effect is capable of altering the course of infection late after primary infection. Correlation of the antigen-specific antibody response and other immune parameters with the duration and degree of protective immunity induced by infection or vaccination may be helpful in further understanding the nature of such protective immunity.

Animals↗
PubMed1989

Cloning and sequence analysis of the major outer membrane protein genes of two Chlamydia psittaci strains.

We cloned and sequenced the gene encoding the major outer membrane protein (MOMP) of two Chlamydia psittaci strains, guinea pig inclusion conjunctivitis (GPIC) strain 1, and meningopneumonitis (Mn) strain Cal-10. Intraspecies alignment of the two C. psittaci MOMP genes revealed 80.6% similarity, and interspecies comparison of C. trachomatis and C. psittaci MOMP genes yielded about 68% similarity. As found previously for C. trachomatis MOMP sequences, stretches of predominantly conserved sequences of GPIC and Mn MOMPs were interrupted by four variable domains whose locations were identical to those of C. trachomatis MOMPs. Seven of eight cysteine residues were found at precisely the same positions in GPIC, Mn, and C. trachomatis MOMPs, emphasizing their importance in structure and function of the protein. Collectively, these results indicate that C. psittaci and C. trachomatis MOMP genes diverged from a common ancestor.

Amino Acid Sequence↗
PubMed1990

Characterization of the cytochalasin D-resistant (pinocytic) mechanisms of endocytosis utilized by chlamydiae.

The cytochalasin D-resistant (pinocytic) portion of the entry of two chlamydia strains (Chlamydia trachomatis L2/434/Bu and Chlamydia psittaci GPIC [guinea pig inclusion conjunctivitis]) was examined. By ultrastructural criteria, few organisms of either strain were observed in association with coated host-cell plasma membrane during entry into McCoy cells; this argues against a coated-pit mechanism of entry. When association with a coated membrane was seen, coat material appeared to pinch off ahead of internalizing chlamydiae. However, entry of both strains was substantially reduced by cytosol acidification, a procedure shown to prevent coated-pit vesiculation (K. Sandvig, S. Olsnes, O. W. Petersen, and B. van Deurs, J. Cell Biol. 105:679-689, 1987). No conclusive evidence of displacement of the fluid-phase marker [3H]sucrose from constitutively forming endocytic vesicles was found. Indeed the entry of strain 434 (but not strain GPIC) was accompanied by the influx of a large volume of fluid, suggesting an inducible mechanism. Additionally, entry of strain 434 (but not strain GPIC) was partially inhibitable by amiloride, yet the drug had no effect on the entry of transferrin, a ligand known to enter solely via coated pits. Our findings endorse the view that chlamydial entry can occur via a pathway involving coated pits. However, the unusual morphology of entry and lack of fluid exclusion are consistent with a process whereby although chlamydiae are not fully enclosed by coat material, their entry is dependent on the vesiculation of coated pits. Furthermore, the data support the proposition that a significant proportion of the entry of strain 434 occurs via an inducible pathway independent of coated-pit uptake.

Ammonium Chloride↗
PubMed1990

Immunization against chlamydial genital infection in guinea pigs with UV-inactivated and viable chlamydiae administered by different routes.

Female guinea pigs were immunized with viable or UV light-inactivated chlamydiae (agent of guinea pig inclusion conjunctivitis), belonging to the species Chlamydia psittaci, by intravenous, subcutaneous, oral, or ocular routes. All animals were then inoculated vaginally with viable chlamydiae to determine the extent of protection against challenge infection induced by the various regimens. The course of genital infection was significantly reduced in intensity in all groups of animals except the unimmunized controls and those animals immunized orally with inactivated antigen. Guinea pigs immunized with viable antigen were more likely to develop resistance to challenge infection and, in general, had a significantly greater degree of protection than animals immunized with inactivated antigen. No one route seemed superior in producing a protective response. Animals in all groups demonstrating protection developed serum and secretion immunoglobulin G antibody responses to chlamydiae. Lymphocyte proliferative reactions to chlamydial antigen were variable among groups. Immunoblot analysis of serum and secretions indicated a wide range of antibody specificities, but most protected animals produced antibodies to the major outer membrane protein, lipopolysaccharide, and the 61-kilodalton protein. No definitive associations could be made between the increased ability of immunization with viable organisms to produce resistance to challenge infection and a particular immune parameter. These data indicate that viable chlamydiae given by various routes are able to induce a strong immune response which can provide resistance against reinfection in some cases or at least reduce the degree of infection to a greater degree than inactivated antigen. However, complete resistance to genital tract infection may be difficult to obtain and alternate immunizations strategies may have to be developed.

Animals↗
PubMed1990

Multiple tandem promoters of the major outer membrane protein gene (omp1) of Chlamydia psittaci.

The transcription of omp1, the gene encoding the major outer membrane protein, was studied for two strains of Chlamydia psittaci, guinea pig inclusion conjunctivitis (GPIC) and mouse pneumonitis (Mn). The transcriptional initiation sites for the omp1 of each strain were mapped by S1 nuclease and primer extension analyses. Three different sizes of omp1 transcripts were observed for GPIC and four were observed for Mn. The production of these transcripts appeared to be the consequence of multiple tandem promoters. The order in which the omp1 RNA transcripts appeared during the growth cycle of the C. psittaci strains was found to differ from that of C. trachomatis.

Animals↗
PubMed1991

Endocytic mechanisms utilized by chlamydiae and their influence on induction of productive infection.

The microfilament-disrupting drug cytochalasin D and, initially, inoculation at 20 degrees C were used to differentiate between phagocytosis (sensitive to both treatments) and pinocytosis (resistant to both treatments) to assess whether chlamydial uptake into McCoy cells occurred by one or both mechanisms and whether each could contribute to productive infection. Both treatments suppressed the infectivity of Chlamydia trachomatis L2/434/Bu and C. psittaci GPIC (the guinea pig inclusion conjunctivitis strain) following static inoculation by only 50%, indicating that there was simultaneous operation of both phagocytosis and pinocytosis during uptake that led to productive infection. Measurement of the entry of organisms by two separate assays established that both strains predominantly used a cytochalasin D-resistant (pinocytic) mechanism, implying that phagocytic uptake was coupled to a higher frequency of productive infection. Integration of the data on infectivity and entry allowed the potential for an organism to infect a host cell to be quantified. This synthesis revealed that for both strains the infectivity potential following phagocytic entry was ca. 10-fold greater than that following pinocytic entry. However, both entry mechanisms were exploited more efficiently by strain L2/434/Bu than by strain GPIC (unless the latter was inoculated with centrifugation), indicating that intrinsic strain properties are more important for infectivity potential than the endocytic mechanism utilized.

Cell Line↗
PubMed1994

Role for CD8+ T cells in antichlamydial immunity defined by Chlamydia-specific T-lymphocyte clones.

The role of CD8+ T cells in antichlamydial immunity was investigated in a murine model of chlamydial genital infection by using T-cell clones generated against the Chlamydia trachomatis agent of mouse pneumonitis (MoPn). Two CD8+ T-cell clones tested (2.1F and 2.14-9) were chlamydia antigen specific and MHC restricted and reacted against MoPn as well as the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis and C. trachomatis serovar E, suggesting the recognition of a genus-specific antigen. Upon adoptive transfer into persistently MoPn-infected nu/nu mice, 55.6% of the recipients of clone 2.1F (15 of 27) resolved the infection but recipients of clone 2.14-9 did not. The ability to resolve the MoPn infection correlated with the capacity of clone 2.1F to elaborate a combination of gamma interferon and tumor necrosis factor alpha. The results suggested that in addition to CD4+ T cells, CD8+ T cells may also contribute to antichlamydial T-cell immunity in vivo.

Animals↗
PubMed1995

Tumor necrosis factor alpha activity in genital tract secretions of guinea pigs infected with chlamydiae.

Previous studies using the guinea pig model of chlamydial genital infection demonstrated that primary infection is associated with a marked acute inflammatory response early on, while chronic inflammation appears later, at a time when the level of infection is reduced. Challenge infections result primarily in a chronic inflammatory response. The stimuli that initiate inflammation and lead to tissue damage have not been defined. We investigated the possibility that tumor necrosis factors (TNFs) play a role in the inflammatory response to chlamydial genital tract infection. Cytotoxicity assays for TNF were performed on genital tract secretions collected from female guinea pigs during infection with the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis. During the early days of primary infection, high levels of TNF-alpha were detected in genital tract secretions from inbred S2 strain and outbred Hartley strain guinea pigs. Significantly lower levels of TNF-alpha were detected in secretions from both strains during challenge infection. In general, the intensity of the TNF-alpha response was proportional to the intensity of infection. High TNF-alpha levels were present during primary infection at a time of marked neutrophil influx. Thus, TNF-alpha may play an important role in the response to primary chlamydial genital tract infection.

Animals↗
PubMed1995

Deviation of immune response to Chlamydia psittaci outer membrane protein in lipopolysaccharide-hyporesponsive mice.

The outcome of infection is determined by both the quantity and the quality of an induced immune response. In particular, it has been demonstrated for selected pathogens that induction of TH1 or TH2 type helper T-cell subsets determines whether an immune response gives rise to protective immunity or disease-associated immunopathology. The nature of the antigen and the type of antigen-presenting cells recruited in the induction of a response are critical factors that influence the quality of the immune response. Of particular interest in this respect is the immune response to bacterial particles and the impact of cell wall-associated lipopolysaccharide (LPS) on that response. Nonspecific activation of macrophages and B lymphocytes by LPS could skew the phenotype of activated antigen-presenting cells and selectively alter the immunoglobulin isotypes and helper T-cell subsets that are induced following infection. In an initial attempt to detect immune deviation associated with LPS stimulation, we have compared the immunoglobulin isotypes of antibodies specific for the cysteine-rich outer membrane protein Omp2 induced in normal and LPS-hyporesponsive mice following immunization with Chlamydia psittaci strain guinea pig inclusion conjunctivitis whole elementary bodies. We report that there is a dramatic shift of Omp2-specific antibody from predominantly immunoglobulin G2a (IgG2a) isotype in LPS-hyporesponsive mice to high levels of IgG1 isotype in LPS-responder strains. The dependence of the IgG1 isotype shift on the LPS responder status is linked to the structure of the antigen and its natural processing pathway since LPS-hyporesponsive mice are not, in general, deficient in IgG1 antibody production. In particular, the antibody response to purified recombinant Omp2 is predominantly of the IgG1 isotype even in LPS-hyporesponsive mice.

Animals↗
PubMed1995

Interaction of outer envelope proteins of Chlamydia psittaci GPIC with the HeLa cell surface.

The chlamydial life cycle involves the intimate interaction of components of the infectious elementary body (EB) surface with receptors on the susceptible eukaryotic cell plasma membrane. We have developed an in vitro ligand binding assay system for the identification and characterization of detergent-extracted EB envelope proteins capable of binding to glutaraldehyde-fixed HeLa cell surfaces. With this assay, the developmentally regulated cysteine-rich envelope protein Omp2 of Chlamydia psittaci strain guinea pig inclusion conjunctivitis was shown to bind specifically to HeLa cells. HeLa cells bound Omp2 selectively over other cell wall-associated proteins, including the major outer membrane protein, and the binding of Omp2 was abolished under conditions which alter its conformation. Furthermore, trypsin treatment, which reduces EB adherence, resulted in the proteolytic removal of a small terminal peptide of Omp2 at the EB surface and inactivated Omp2 in the ligand binding assay, while having a negligible effect on the major outer membrane protein. Collectively, our results suggest that Omp2 possesses the capacity to engage in a specific interaction with the host eukaryotic cell. We speculate that, since Omp2 is present only in the infectious EB form, the observed in vitro interaction may be representative of a determining step of the chlamydial pathogenic process.

Animals↗
PubMed1998

High-level expression of Chlamydia psittaci major outer membrane protein in COS cells and in skeletal muscles of turkeys.

The omp1 genes encoding the major outer membrane proteins (MOMPs) of avian Chlamydia psittaci serovar A and D strains were cloned and sequenced. The nucleotide sequences of the avian C. psittaci serovar A and D MOMP genes were found to be 98.9 and 87.8% identical, respectively, to that of the avian C. psittaci serovar A strain 6BC, 84.6 and 99.8% identical to that of the avian C. psittaci serovar D strain NJ1, 79.1 and 81.1% identical to that of the C. psittaci guinea pig inclusion conjunctivitis strain, 60.9 and 62.5% identical to that of the Chlamydia trachomatis L2 strain, and 57.5 and 60.4% identical to that of the Chlamydia pneumoniae IOL-207 strain. The serovar A or D MOMPs were cloned in the mammalian expression plasmid pcDNA1. When pcDNA1/MOMP A or pcDNA1/MOMP D was introduced into COS7 cells, a 40-kDa protein that was identical in size, antigenicity, and electrophoretic mobility to native MOMP was produced. Recombinant MOMP (rMOMP) was located in the cytoplasm of transfected COS7 cells as well as in the plasma membrane and was immunoaccessible. Intramuscular administration of pcDNA1/MOMP in specific-pathogen-free turkeys resulted in local expression of rMOMP in its native conformation, after which anti-MOMP antibodies appeared in the serum.

Amino Acid Sequence↗
PubMed2000

Characterization of lymphocyte response in the female genital tract during ascending Chlamydial genital infection in the guinea pig model.

It is well known that pathology caused by chlamydial infection is associated closely with the host response to the organism and that both innate and adaptive host responses contribute to tissue damage. While it is likely that the organism itself initiates the acute inflammatory response by eliciting cytokine and chemokine production from the host cell, the adaptive response is the result of activation of the cell-mediated immune response. While there are several studies describing the nature of the pathologic response in primate, guinea pig, and murine models, there is less information on the kinetics of the CD4 and CD8 response following primary and challenge infections. In this study, we have quantified by flow cytometry the mononuclear cell response to genital infection with the agent of guinea pig inclusion conjunctivitis in the cervix, endometrium, and oviducts at various times following a primary intravaginal infection and after a challenge infection. Tissues from individual animals were assessed for cells expressing CD4, CD8, or Mac-1 and for B cells. Peak responses of each subset occurred 10 to 14 days after a primary infection. The number of Mac-1-expressing cells in each tissue site was found to be dependent on the size of the inoculating dose of chlamydiae. The responses of each cell type were generally stronger in the cervix than in the upper genital tract. In contrast to the murine model but consistent with the primate models, there were equal numbers of CD4 and CD8 cells present in the infiltrates. Twenty-one days after challenge infection, which was performed 50 days after the primary infection, there was a significant increase in the number of CD4, CD8, and B cells in the oviduct compared to the number of these cells at the same time after a primary infection, providing clear cellular evidence for a cell-mediated immune pathologic response.

Animals↗