Aspects of the epidemiology of Chlamydia psittaci infection in a population of koalas (Phascolarctos cinereus) in southeastern Queensland, Australia

A population of free-ranging koalas in southeastern Queensland was examined to determine the prevalence of Chlamydia psittaci infections. Although C. psittaci was isolated from 46 of 65 (71%) koalas studied, only six (9%) of these had clinical signs of disease. Most adult females (82%) had back or pouch young present even though 67% of them were infected. There were no significant correlations between age, sex or site of sampling (urogenital versus conjunctival tissues) and the isolation of C. psittaci. No other important bacterial or fungal pathogens were isolated. The complement fixation test had a sensitivity of 7% and a specificity of 94% in detecting chlamydial infections, suggesting that it is unsuitable for use as a screening test. Chlamydia psittaci infection within this population appeared to represent a generally well-balanced host-parasite relationship and few animals had clinical signs of disease. Only four of 27 (15%) healthy koalas infected with C. psittaci followed for 24 wk after sampling developed eye disease or "dirty tail." Two koalas with keratoconjunctivitis recovered without treatment during the study period. Additional factors, including the stresses imposed by loss of habitat, may act to produce overt disease in koalas with latent C. psittaci infections.     

Guineapig inclusion conjunctivitis (GPIC) in a commercial colony

Serological findings in a commercial colony of Hartley guineapigs revealed that about 70% had antibodies to Chlamydia psittaci as detected by the microimmunofluorescence method. Conjunctivitis was evident in 14% of 86 guineapigs examined. Chlamydial antigen was detected in conjunctival scrapings by a direct immunofluorescence test using Chlamydia-specific monoclonal antibody; however, C. psittaci was not demonstrated by other methods.     

Evaluation of an enzyme immunoassay test for the diagnosis of Chlamydia psittaci infection in free-ranging koalas (Phascolarctos cinereus) in southeastern Queensland, Australia

The IDEIA Chlamydia Test, a commercially available antigen-capture enzyme-linked immunosorbent assay (ELISA) test, based on a monoclonal antibody for the detection of chlamydia in clinical specimens, was evaluated in a population of 65 free-ranging koalas in southeastern Queensland determined to be infected with Chlamydia psittaci. Compared to isolation of the organism in tissue culture, the sensitivity of the IDEIA test ranged from 3 to 11%, and the specificity from 90 to 97%. The results indicated that the IDEIA test is unsuitable for use as a diagnostic screening test for C. psittaci in free-ranging koalas.     

Identification of a major envelope protein in Chlamydia spp.

A major cell envelope protein of Chlamydia psittaci with a molecular weight of approximately 43,000 was identified and partially characterized. It was present at all stages of the C. psittaci developmental cycle. A major protein with a similar molecular weight was also observed in two Chlamydia trachomatis strains.     

Monoclonal antibodies against Chlamydia psittaci

Five monoclonal antibodies were prepared against Chlamydia (C.) psittaci strain Pigeon-1041 isolated from a feral pigeon in Sapporo. Reactions of these antibodies to chlamydiae were examined using five strains of C. psittaci and two strains of C. trachomatis in an enzyme-linked immunosorbent assay, microimmunofluorescent test and complement fixation test. The antibodies were divided into two groups: three genus-specific (A2, D2, and I21) and two strain-specific (F2 and H9) antibodies. The antigenic determinant site of A2 was KIO4 sensitive, but those of D2, F2, and H9 were not affected greatly by KIO4 treatment. Nine C. psittaci strains from feral pigeons and 16 strains from budgerigars were classified into three groups and four groups, respectively, by reaction patterns against the monoclonal antibodies.     

Chlamydiosis: seroepidemiologic survey in a red deer (Cervus elaphus) population in Italy

Chlamydiae are obligate, intracellular, gram-negative bacteria that are responsible for important diseases in humans, other mammals, and birds. Studies have shown that chlamydiae could be present in wild ruminants, but the serodiagnostic method most commonly used did not allow identification of chlamydial species. We determined the prevalence of antibodies to Chlamydia pecorum, Chlamydia suis, Chlamydia abortus, and Chlamydia psittaci in 271 red deer (Cervus elaphus) of a central Italian population, by using the microimmunofluorescence test that shows antibody response against genus-specific and species-specific antigens. No sera had detectable antibodies to C. pecorum and C. abortus. Antibodies were detected against C. psittaci (9.6%) and C. suis (3.3%). Antibody response could be related to contact of the red deer with birds and wild boars (Sus scrofa), respectively, and confirm an extended host range of individual Chlamydia species. In view of the potential zoonotic risk related to exposition of C. psittaci, our findings suggest surveillance of wild ruminants as potential reservoirs for chlamydiae.     

Plasmids of Chlamydia psittaci: Cloning and comparison of isolates by Southern hybridisation

Abstract A chlamydial plasmid, 6.2 kb in size, was isolated from an avian strain of Chlamydia psittaci and cloned into the Eco RI site of pUC13. A restriction enzyme cleavage map of the resultant clone, pAP¹p, was very similar to the published map of the plasmid cloned from the C. psittaci meningopneumonitis strain Cal-10. Southern hybridisation analyses using pAP¹p as a probe, revealed the presence of plasmids with homologous DNA sequences in avian psittacosis, avian ornithosis, ovine polyarthritis and sporadic bovine encephalomyelitis strains of C. psittaci , as well as the LGV strain of Chlamydia trachomatis . Plasmid was not detected in koala conjunctivitis, ovine abortion or feline conjunctivitis isolates. The plasmid-containing isolates could be grouped according to size (6.2 or 7.2–7.3 kb) and restriction endonuclease pattern. These three plasmid categories correlate with previously reported C. psittaci biotypes, immunotypes and serotypes. The absence of plasmid from three infectious, pathogenic strains of C. psittaci suggests that, in this species at least, plasmid-encoded genes are not essential for survival, infectivity or virulence of the parasite.     

Glycogen Metabolism in Chlamydia-Infected HeLa-Cells

A difference in glycogen metabolism between two strains of Chlamydia agents was observed which can serve as a taxonomic marker to distinguish C. psittaci from C. trachomatis.     

Monoclonal antibodies to Chlamydia psittaci: their isolation, characteristics and use]

A panel of 22 hybridomas producing monoclonal antibodies (McAb) to C. psittaci was obtained. 15 hybridomas produced IgG1 antibodies, 4 hybridomas produced IgM antibodies and 3 hybridomas produced IgG2b, IgG3 or IgA antibodies. IgG1 antibodies and 2 IgM antibodies did not bind complement in the complement fixation test. All McAb were reactive in the enzyme immunoassay and the indirect immunofluorescence test and did not precipitate specific antigens. Peroxidase conjugates on the basis of McAb effectively detected Chlamydia antigen, prepared from the crude suspension of chick embryo yolk sack infected with different strains of C. psittaci and C. trachomatis, in different modifications of EIA.     

Proposal of Chlamydia pecorum sp. nov. for Chlamydia strains derived from ruminants.

Chlamydia pecorum sp. nov. is proposed as the fourth species of the genus Chlamydia on the basis of the results of a genetic analysis of Chlamydia strains that were isolated from cattle and sheep which had various diseases, including sporadic encephalitis, infectious polyarthritis, pneumonia, and diarrhea. The levels of DNA-DNA homology between C. pecorum and strains of C. psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis were less than 10%. Several DNA probes were used to identify C. pecorum. The C. pecorum strains were distinguished from C. psittaci strains by the results of immunological assays, including an immunofluorescence antibody assay performed with monoclonal antibodies and an immunoblot analysis of the immunological specificity of the major outer membrane protein. Species identification was based on results obtained from DNA analyses and serology. The type strain of C. pecorum is strain ATCC VR628.     

Comparative PCR-based restriction fragment length polymorphism analysis of the plasmid gene orf3 of Chlamydia trachomatis and Chlamydia psittaci

The BfaI digestion of PCR-based restriction fragment length polymorphism analysis of the plasmid orf3 of Chlamydia trachomatis and Chlamydia psittaci provided evidence for two distinct restriction patterns, respectively. The nucleotide sequences of orf3 genes confirmed these differences. Serum antibodies against recombinant C. psittaci protein (pgp3) encoded by orf3 were detected both in pigeons with C. psittaci infection and in a human patient with psittacosis.     

Immunotyping of Chlamydia psittaci by indirect immunofluorescence antibody test with monoclonal antibodies

Monoclonal antibodies against pigeon and budgerigar strains of Chlamydia psittaci were used to classify the immunotypes of C. psittaci strains by an indirect immunofluorescence antibody (IFA) test. Thirty-three C. psittaci strains from pigeons and 24 from budgerigars were divided into three immunotypes (P-I, P-II, and P-III) and (B-I, B-II, and B-III), respectively. Two strains from human psittacosis patients were identified as P-III and B-I, coinciding with the epidemiological evidence of each human infection. Two strains from psittacine birds, a parrot and a parakeet, were identical to the B-II immunotype. Other mammalian strains were quite distinct from avian strains in their IFA reaction with the monoclonal antibodies.     

Quantitative detection of Chlamydia spp. by fluorescent PCR in the LightCycler

Quantitative detection of intracellular bacteria of the genus Chlamydia by the standard cell culture method is cumbersome and operator dependent. As an alternative, we adapted hot-start PCR to the glass capillary quantitative PCR format of the LightCycler. The optimized PCR was consistently more efficient than commercially available pre-assembled PCRs. Detection by quantitative PCR of as few as single copies of DNA of Chlamydia spp. was accomplished by SYBR Green fluorescence of the dsDNA product and by fluorescence resonance energy transfer (FRET) hybridization probes. The PCRs were 15-fold more sensitive than the cell culture quantitative assay of C. psittaci B577 infectious stock. The number of chlamydial genomes detected by C. psittaci B577 FRET PCR correlated well with cell culture determination of inclusion forming units (IFUs) (r = 0.96, P < 0.0008). When infected tissue samples were analyzed by cell culture and PCR, the correlation coefficient between IFUs and chlamydial genomes was higher with C. psittaci B577 FRET PCR (r = 0.90, P < 0.0004) than with Chlamydia omp1 SYBR Green PCR (r = 0.85, P < 0.002).     

Cloning and antigenic expression of two genes from Chlamydia psittaci avian strain

Abstract: Genes from Chlamydia psittaci P-1041 were cloned into the Bam HI site of pUC19 and were transformed to host Escherichia coli JM109. Two recombinant plasmids that expressed protein antigens of Chlamydia were isolated. The sizes of the DNA fragments were 1350 and 1710 bp, and encoded for polypeptides of M _r 25 and 42 kilodaltons (kDa), respectively. The 25-kDa protein had cross-reactivity with antisera to ten C. psittaci strains and two C. trachomatis strains, whereas the 42-kDa protein reacted only with homologous antiserum to the C. psittaci P-1041 strain. Furthermore, in Southern hybridization analysis these two fragments as probes hybridized with DNA of ten C. psittaci strains and four C. trachomatis strains. These results indicated that the two fragments shared a DNA sequence common to the chlamydial genus.     

In vitro expression of factor-mediated cytotoxic activity generated during the immune response to Chlamydia in the mouse

Supernatant fluid (SF) prepared by mitogen incubation of spleen cells from A/J mice previously immunized against lethal challenge by the 6BC strain of Chlamydia psittaci was cytotoxic for mouse fibroblasts (L cells) infected with 6BC, as detected by the [3H]thymidine release assay and the trypan blue exclusion test. In contrast, SF prepared from spleen cells taken from unimmunized animals (controls) was not cytotoxic when added to infected L cells. No cytotoxicity was observed when SF was added to uninfected L cells. Maximal levels of cytotoxicity were observed only from cells infected with 6BC for at least 26 hr and exposed to SF for greater than 20 hr. Furthermore, the degree of cytotoxicity was dependent on both the dose of Chlamydia administered and the concentration of SF in the medium. We conclude that the capacity to secrete a spleen cell cytotoxic factor is an aspect of the immune response against the obligate intracellular prokaryotic pathogen Chlamydia. Our results indicate that SF-mediated cytotoxicity is induced subsequent to immunization with Chlamydia, and is significantly more pronounced against infected as opposed to uninfected L cells.     

Single channel analysis of recombinant major outer membrane protein porins from Chlamydia psittaci and Chlamydia pneumoniae

We recently demonstrated that the major outer membrane protein of Chlamydia psittaci, the primary vaccine candidate for combating chlamydial infections, functions as a porin-like ion channel. In this study, we have cloned, expressed and functionally reconstituted recombinant major outer membrane proteins from C. psittaci and Chlamydia pneumoniae and analysed them at the single channel level. Both form porin-like ion channels that are functionally similar to those formed by native C. psittaci major outer membrane protein. Also, like the native channels, recombinant C. psittaci channels are modified by a native major outer membrane protein-specific monoclonal antibody. This is the first time that native function has been demonstrated for recombinant chlamydial major outer membrane proteins. Future bilayer reconstitution will provide a strategy for detailed structure/function studies of this new subclass of bacterial porins and the work also has important implications for successful protein refolding and the development of improved subunit vaccines.     

Chlamydia psittaci ewe abortion agent: complete nucleotide sequence of the major outer membrane protein gene

The complete nucleotide sequence encoding the major outer membrane protein (MOMP) of Chlamydia psittaci strain A22/M, responsible for enzootic abortion of ewes (EAE), has been determined. An 800bp Eco RI/ Xba I fragment containing a portion of the MOMP coding sequence from C. trachomatis serovar L1 was used to probe a λL47.1 genomic library constructed from DNA obtained from C. psittaci EAE A22/M. The recombinant L47.1/EA1 was selected and contained the entire C. psittaci MOMP gene within a 7.5 kb Bam HI fragment. The DNA sequence revealed an open reading frame encoding 402 amino acids, including a 22 amino acid signal peptide, which exhibited 17/22 conservation with the signal peptide of C. trachomatis MOMP. The calculated molecular mass of the C. psittaci MOMP was 43 kDa. A comparison of the MOMP genes of C. psittaci and C. trachomatis revealed only 34% nucleotide sequence homology, but 65% amino acid homology.     

Synthesis of disulfide-bonded outer membrane proteins during the developmental cycle of Chlamydia psittaci and Chlamydia trachomatis.

The disulfide bond cross-linked major outer membrane protein (MOMP) of the extracellular elementary bodies (EBs) of Chlamydia psittaci was reduced to its monomeric form within 1 h of entry of EBs into host cells by a process which was inhibited by chloramphenicol, while monomeric forms of three cross-linked cysteine-rich proteins could not be detected in Sarkosyl outer membrane complexes at any time in either extracellular or intracellular forms of C. psittaci. Synthesis and incorporation of the MOMP into outer membrane complexes were detected early in the infection cycle (12 h postinfection), while synthesis and incorporation of the cysteine-rich proteins were not observed until reticulate bodies had begun to reorganize into EBs at 20 to 22 h postinfection. By 46 h postinfection, the intracellular population of C. psittaci consisted mainly of EBs, the outer membrane complexes of which were replete with monomeric MOMP and cross-linked cysteine-rich proteins. Upon lysis of infected cells at 46 h, the MOMP was rapidly cross-linked, and infectious EBs were released. The status of the MOMP of intracellular Chlamydia trachomatis was similar to the status of the MOMP of C. psittaci in that the MOMP was largely uncross-linked at 24 and 48 h postinfection, but formed interpeptide disulfide bonds when it was exposed to an extracellular environment late in the developmental cycle. In contrast to C. psittaci, only a fraction of the cross-linked MOMP of infecting EBs of C. trachomatis was reduced by 4 h postinfection, and reduction of the MOMP was not inhibited by chloramphenicol. Exposure of extracellular EBs of C. trachomatis and C. psittaci to dithiothreitol reduced the MOMP but failed to stimulate metabolic activities normally associated with reticulate bodies.     

Development of a simplified polymerase chain reactionenzyme immunoassay for the detection of Chlamydia pneumoniae

The 16S rRNA genes of two Chlamydia pneumoniae and two C. psittaci strains of different serovars were sequenced then compared to previously reported Chlamydia 16S rRNA gene sequences. Chlamydia pneumoniae -specific regions were identified and specific primers for nested PCR were synthesized. Nested PCR reactions were performed, in a single tube, by varying the annealing temperature of the amplification cycles. The initial thermal cycles were selected to allow annealing and extension of only the outer primer pair, whilst in later cycles a temperature that allowed inner primer annealing was employed. The inner primers were labelled, one with biotin and the other with fluorescein and consequently the dual labelled amplicon could be immobilized onto antibiotin-coated microtitre plates and detected colorimetrically via an antifluorescein-enzyme conjugate. The assay was found to be sensitive and specific. No cross reactions were observed with C. trachomatis, C. psittaci or other common respiratory pathogens.     

Conserved DNA sequences in chlamydial plasmids

Two 7.4-kb plasmids from Chlamydia psittaci have been cloned and characterized. These plasmids are quite distinct from the 6.2-kb C. psittaci and the C. trachomatis plasmids when compared by restriction endonuclease analysis. The plasmids show considerable cross-hybridization, with only a small region highly conserved and identified as a 4 X 22-bp tandemly repeated region. This sequence is identical in the two size categories of C. psittaci plasmids and differs from C. trachomatis plasmids by only 2 bp in the 22-bp motif. AT-rich clusters 5' to the repeat region which are present in C. trachomatis and Escherichia coli plasmids were absent from both classes of C. psittaci plasmids. Extensive regions are less highly conserved but show a sufficient degree of cross-hybridization to suggest that the plasmids are homologous.