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1.
Preparations of DNA from 12 Chlamydia psittaci isolates and one Chlamydia trachomatis strain were compared by restriction endonuclease analysis. Polyacrylamide gel electrophoresis, followed by silver staining, resulted in optimal resolution of fragments generated by digestion. By this technique, four distinct electropherotypes were demonstrated when ovine abortion, ovine arthritis, and avian and Cal10 strains of C. psittaci were examined. Minor profile differences allowed the discrimination of avian isolates derived from psittacine and columbiforme species, and the Cal10 DNA electropherotype was shown to have features in common with these profiles. However, there were no detectable differences in the DNA patterns of eight ovine abortion isolates.  相似文献   

2.
The gene encoding the major outer membrane protein (MOMP) of the psittacine Chlamydia psittaci strain 6BC was cloned and sequenced. N-terminal protein sequencing of the mature MOMP indicated that it is posttranslationally processed at a site identical to the site previously identified in the MOMP of Chlamydia trachomatis L2. The nucleotide sequence of the C. psittaci 6BC MOMP gene was found to be 67 to 68% identical to those of human C. trachomatis strains, 73% identical to that of Chlamydia pneumoniae IOL-207, 79% identical to that of the C. psittaci guinea pig inclusion conjunctivitis strain, GPIC, and 83% identical to that of the C. psittaci ovine abortion strain S26/3. In contrast, the 6BC sequence was found to be greater than 99% identical to the sequences reported for two strains of C. psittaci, A22/M and Cal-10 meningopneumonitis, believed to be of nonpsittacine avian origin. Monoclonal antibody analysis confirmed the nonpsittacine avian origin of A22/M but identified the Cal-10 strain from which the MOMP gene was previously sequenced as a psittacine strain. These results confirm that psittacine and nonpsittacine avian strains of C. psittaci are closely related and distinct from the mammalian guinea pig inclusion conjunctivitis and ovine abortion strains of C. psittaci.  相似文献   

3.
Monoclonal antibodies (MAbs) were produced to four Chlamydia psittaci isolates: NJ1 and TT3 from turkeys, VS1 from a parakeet, and B577 from an ovine abortion. MAbs were tested for reactivity with each isolate by the indirect immunofluorescent antibody technique and for neutralization by an inclusion reduction neutralization technique in tissue culture. Two genus-specific and 14 serovar-specific MAbs were produced. Genus-specific MAbs reacted with all avian and mammalian isolates; however, each failed to neutralize its homologous chlamydial isolate. Turkey isolates NJ1 and TT3 were antigenically similar; serovar-specific MAbs produced to each reacted equally with both isolates yet showed little or no reaction with other serovars. Serovar-specific MAbs to the parakeet and abortion isolates were distinct; no cross-reactions were seen with other serovars. None of the serovar-specific MAbs reacted with an ovine arthritis isolate. Of the 14 serovar-specific MAbs, 13 partially neutralized homologous strains with or without the addition of complement. Because of the high specificity, the serovar-specific MAbs used with the immunofluorescence technique provided a rapid and precise method to identify three serovars of C. psittaci.  相似文献   

4.
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.  相似文献   

5.
The major outer membrane protein gene (omp1) was sequenced for each of six Chlamydia psittaci (guinea pig inclusion conjunctivitis [GPIC]) strains isolated from guinea pigs. Five of the isolates were obtained in the United States during the 1960s and 1970s, including the prototype strain isolated by Murray in 1962. The other isolate was obtained from a guinea pig in England. The nucleotide sequence of the omp1 gene for each strain was identical. The lack of omp1 allelic polymorphism among GPIC isolates suggests that, unlike C. trachomatis, the GPIC agent lacks antigenic variation in the major outer membrane protein.  相似文献   

6.
DNA from a total of 60 Chlamydia trachomatis isolates was examined by restriction endonuclease analysis. Strains from all established biovars and serovars were tested. There was great diversity between the mouse biovar and the lymphogranuloma venereum (LGV) and trachoma biovars. The LGV and trachoma biovar isolates generated similar fragment patterns; however, distinct fragments appeared to be unique to both biovars, thus allowing differentiation of these two major groups. In most cases, strains of the same serovar could be differentiated from one another when a battery of restriction enzymes was used. In addition, in some cases, certain restriction fragments appeared to be characteristic of strains from a particular geographical location. The DNA patterns generated by all C. trachomatis isolates differed greatly from the DNA patterns generated from the Chlamydia psittaci isolates tested, including TWAR, a human C. psittaci strain.  相似文献   

7.
Several molecular techniques were used for comparison of the novel Chlamydia agent, TWAR, with Chlamydia trachomatis and Chlamydia psittaci. Unlike all serotypes of C. trachomatis and most strains of C. psittaci, the eight TWAR isolates examined did not contain extrachromosomal DNA. TWAR was readily distinguished from C. trachomatis or C. psittaci by restriction endonuclease analysis, whereas identical or nearly identical restriction patterns were observed among the TWAR isolates. Southern blot analysis with a gene encoding a portion of the C. trachomatis serovar L2 major outer membrane protein as the probe showed that TWAR, like C. psittaci, contained sequences homologous to this gene. However, while the hybridization patterns were identical for all TWAR isolates, they differed from those of any of the other Chlamydia species tested. A PstI gene bank containing TWAR DNA was constructed in pUC19. Random fragments were purified and used for probing Chlamydia chromosomal digests. All of the five probes tested were TWAR specific, with the TWAR isolates showing identical patterns of homology. Qualitative studies of the DNA homology revealed that TWAR did not have significant homology to any of the Chlamydia strains assayed. Collectively, these results demonstrate that the TWAR isolates represent a single strain or closely allied genotypes and are clearly distinct from any of the other chlamydiae tested.  相似文献   

8.
Fifty-one monoclonal antibodies were prepared by two different techniques against Chlamydia psittaci strain A22 isolated from an ovine abortion. These antibodies were tested for reactivity by the indirect immunofluorescent antibody technique with eleven reference Chlamydia strains (nine C. psittaci, one Chlamydia trachomatis and one Chlamydia pneumoniae). Four classes of specificity were recognized for monoclonal antibodies: genus, species, subspecies and type specificity. The type-specific monoclonal antibodies were non-reactive with ovine arthritis isolates. Twenty monoclonal antibodies were specific for two mammalian strains: ovine abortion A22 and K mouse. Some monoclonal antibodies were reactive with C. pneumoniae strains and non-reactive with C. trachomatis strains. All these monoclonal antibodies were very useful for improving the diagnosis of chlamydial infection, the antigenic analysis and the serotyping of C. psittaci.  相似文献   

9.
The genomes of Chlamydia spp. encode a family of putative outer membrane proteins, referred to as polymorphic outer membrane proteins (POMPs), which may play a role in the avoidance of host immune defenses. We analyzed avian strain 6BC of Chlamydia psittaci by polyacrylamide gel electrophoresis for the expression of POMPs. At least six putative POMPs were identified on the basis of their size (90 to 110 kDa) and labeling with an outer membrane-specific probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Three of the putative POMPs reacted with antiserum raised against a recombinant ovine C. psittaci strain POMP, and two possessed surface-exposed, trypsin-sensitive sites. The POMPs were dependent on disulfide bonds for their maintenance in sodium lauryl sarcosine- and sodium dodecyl sulfate-insoluble complexes but did not appear to be interpeptide disulfide bond cross-linked. The putative POMPs were found to be synthesized during the late phase of the chlamydial developmental cycle, cotemporally with the cysteine-rich doublet periplasmic proteins.  相似文献   

10.
While several diseases associated with Chlamydia psittaci infection have been reported in Phascolarctos cinereus (koala), it is still unclear whether one or more chlamydial strains are responsible. In this study, we provide evidence, obtained by restriction enzyme and gene probe analysis, that two quite distinct strains of C. psittaci infect koalas; one strain was isolated from the conjunctivae, and the other was isolated from the urogenital tract and the rectum. A gene probe, pFEN207, containing the coding sequence for an enzyme involved in the biosynthesis of the chlamydial genus-specific lipopolysaccharide antigen, and a separate probe, pCPML-4N, prepared from a DNA fragment of a koala-infecting strain of C. psittaci, were used to determine the patterns of hybridization in the koala-infecting strains; these patterns were found to be quite distinct from those observed with C. psittaci isolates from other animals. We also demonstrated by hybridization analysis with an avian strain plasmid that all three koala urogenital isolates contain a plasmid and that there is no evidence for the presence of a homologous plasmid in any of the ocular isolates.  相似文献   

11.
A group of 24 Chlamydia psittaci strains isolated from ruminants, belonging to serotype 1 and previously classified as invasive in a mouse model of virulence, was compared to a group of 10 non-invasive strains belonging to serotype 2 by using determination of glucose-6-phosphate and L-malate dehydrogenase zymotypes resulting of the infection of cells by these strains. The serotype 1 or invasive isolates represent a homogeneous group by sharing a unique zymotype which was not observed in the non-invasive strains. On the contrary, the serotype 2 or non-invasive isolates constitute a heterogeneous group in generating 2 different zymotypes. Zymotyping clearly distinguishes the ruminant strains from an avian C. psittaci and two C. trachomatis isolates studied for comparison. Our results suggest the usefulness of the studied molecular approach for chlamydiae typing. Furthermore, it can be used as marker of virulence within the C. psittaci strains isolated from ruminants.  相似文献   

12.
Chlamydia pneumoniae is a human respiratory pathogen. Unlike the other two Chlamydia species, no species-specific antigen has been defined for C. pneumoniae. An immunoreactive clone containing a 0.8-kb fragment was isolated from a C. pneumoniae (AR-39) genomic library by using anti-C. pneumoniae rabbit immune serum. By Southern hybridization analysis of chromosomal digests of the different Chlamydia spp., the 0.8-kb fragment was shown to react specifically with C. pneumoniae. Subcloning of this fragment into the pGEX-1 lambda T expression vector resulted in the expression of a 62-kDa fusion protein. This fusion protein as well as the cleaved C. pneumoniae peptide were recognized by anti-C. pneumoniae rabbit immune serum, while the glutathione S-transferase moiety was not recognized. The fusion protein was used to produce monospecific rabbit antiserum. This antiserum was shown to react with a 76-kDa protein in all C. pneumoniae isolates tested, specifically recognize C. pneumoniae inclusions in tissue culture, and neutralize infectivity of C. pneumoniae in cell culture. No reactivity was observed with Chlamydia trachomatis or Chlamydia psittaci. To isolate the entire coding sequence of the 76-kDa protein, two partially overlapping fragments of C. pneumoniae DNA, a 3.2-kb HindIII fragment and a 1.2-kb PvuII fragment, were isolated, cloned, and sequenced. No significant sequence similarity was found with any previously reported nucleotide or amino acid sequence of the other Chlamydia species. This C. pneumoniae protein containing a species-specific epitope could play a role in pathogenesis and may be useful as a diagnostic tool.  相似文献   

13.
Three touchdown enzyme time release (TETR)-PCR assays were used to amplify different DNA sequences in the variable regions of the 16S and 16S-23S spacer rRNA genes specific for Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci as improved tests for sensitive diagnosis and rapid species differentiation. The TETR-PCR protocol used 60 cycles of amplification, which provided improved analytical sensitivity (0.004 to 0.063 inclusion-forming unit of Chlamydia species per PCR). The sensitivity of TETR-PCR with primer set CTR 70-CTR 71 was 96.7%, and the specificity was 99.6%, compared to those of the AMPLICOR PCR for the detection of C. trachomatis in vaginal swab samples. TETR-PCR for C. pneumoniae with primer set CPN 90-CPN 91 was 90% sensitive and 93.3% specific compared with a nested PCR with primer set CP1/2-CPC/D for clinical respiratory samples. TETR-PCR for C. psittaci with primer set CPS 100-CPS 101 showed substantial agreement with cell culturing (kappa, 0.78) for animal tissue samples. Primer sets were then combined into a single multiplex TETR-PCR test. The respective 315-, 195-, and 111-bp DNA target products were precisely amplified when DNA from each of the respective Chlamydia species or combinations of them was used. Multiplex chlamydia TETR-PCR correctly identified one strain of each of the 15 serovars of C. trachomatis, 22 isolates of C. pneumoniae, and 20 isolates of C. psittaci. The primer sets were specific for each species. No target products were amplified when DNA from C. pecorum or a variety of other microorganisms was tested for specificity. TETR-PCR with primers selected for specific sequences in the 16S and 16S-23S spacer rRNA genes is a valuable test that could be used either with individual primers or in a multiplex assay for the identification and differentiation of Chlamydia species from culture isolates or for the detection of chlamydiae in clinical samples.  相似文献   

14.
The 60-kDa cysteine-rich outer membrane protein genes of Chlamydia psittaci, Chlamydia pneumoniae, and Chlamydia trachomatis have very different 5' ends, but two areas flanking this variable region show absolute sequence conservation. This observation permitted differentiation of the three species of Chlamydia by the polymerase chain reaction (PCR), forming the basis of a diagnostic test for chlamydial infections. The PCR product containing the variable region of the respective 60-kDa CrP genes was also subjected to restriction endonuclease digestion, enabling differentiation of individual type strains of C. psittaci. Differentiation was possible between lymphogranuloma venereum and trachoma isolates of C. trachomatis. The PCR-based diagnostic test was successful with all strains of chlamydiae studied. The PCR primers showed high specificity and did not produce any product with common bacterial pathogens that may share the same sites of infection.  相似文献   

15.
Serotyping of Chlamydia I. Isolates of Ovine Origin   总被引:17,自引:9,他引:8       下载免费PDF全文
Eight chlamydial isolates of ovine origin were tested in a plaque reduction system using homologous and heterologous rooster antisera. The eight isolates could be separated into two separate immunotypes. Type 1 included isolates associated with ovine abortion and one agent recovered from the feces of an apparently normal sheep. Type 2 isolates were associated with polyarthritis and conjunctivitis. These two serotypes were not cross-reactive with several chlamydiae of avian origin. Further application of the plaque reduction test may provide a useful means of typing chlamydiae.  相似文献   

16.
Application of a nested, multiplex PCR to psittacosis outbreaks.   总被引:7,自引:5,他引:7       下载免费PDF全文
We developed a nested, multiplex PCR for simultaneous detection of three species of chlamydiae in human and avian specimens. The PCR was designed to increase sensitivity and to circumvent inhibitors of PCR present in clinical specimens. The target sequence was the 16S rRNA gene. The first-step PCR was genus specific, and the second-step PCR was multiplexed (i.e., had multiple primer sets in the same tube) and could discriminate among Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia trachomatis on the basis of the molecular weight of the amplicon. The limit of detection of each of the two PCR steps was 5 inclusion-forming units. We used PCR and serologic evidence during outbreaks of psittacosis to infer that C. psittaci had been transmitted from birds purchased in pet stores to humans. We also used this method to test both live and dead birds from pet stores for infection with C. psittaci. Compared with culture, the application of PCR to avian specimens increased the rate of C. psittaci detection.  相似文献   

17.
18.
Sequence analyses 5' ends of the 60-kDa cysteine-rich outer membrane protein genes (Omp2) of Chlamydia psittaci and Chlamydia pecorum strains indicate that these species have approximately 70% nucleotide identity. On the basis of this sequence information, PCR primers were designed to allow the specific amplification of DNA extracted from C. psittaci S26/3 (abortion strain), P94/1 (pigeon strain), and C. pecorum W73 (fecal strain) in one reaction tube. By using nested reactions (with primers PCR-D1 and PCR-D2 followed by the specific primers and PCR-D2), 0.6, 0.2, and 8 inclusion-forming units of S26/3, P94/1 (both diluted in tissue culture-negative placental material), and W73 (diluted in culture-negative fecal material) per ml, respectively, were detected. The differentiation of C. psittaci and C. pecorum strains of ovine and bovine origins was carried out, and the results were in agreement with those obtained from AluI restriction enzyme analysis of DNA amplified from corresponding strains by PCR. This approach allows the simultaneous detection and typing of C. psittaci and C. pecorum strains and the identification of samples containing both species.  相似文献   

19.
A method is described which permits the assay of specific secretory immunoglobulin A (sIgA) antibodies produced by guinea pigs in response to ocular infection with the guinea pig inclusion conjunctivitis strain of Chlamydia psittaci (GPIC agent). The enzyme-linked immunosorbent assay (ELISA) developed was shown to be more sensitive and less subjective than the micro-immunofluorescence assay as a means of assaying specific antibody.  相似文献   

20.
P D Gershon  D N Black 《Virology》1988,164(2):341-349
HindIII, PstI, AvaI, and SalI sites were mapped on the genomes of six isolates of capripoxvirus from sheep, goats, and cattle. Genome pairs were aligned by the alignment of cross-hybridizing HindIII fragments and the introduction of padding fragments (pads) at specific locations. The majority of these pads represent the approximate positions of relative deletions or insertions. Three possible phylogenetic networks were generated for seven capripoxvirus isolates by Wagner parsimony analysis of the nonconserved HindIII sites on their genomes, and confidence limits were calculated for the network nodes. Nucleotide sequence divergence values, calculated from the numbers of nonconserved HindIII, PstI, AvaI, and SalI sites on the genomes of typical sheep, goat, and cattle isolates, indicated that the typical sheep and cattle isolates are more closely related to one another than to the typical goat isolate. Nonconserved HindIII, PstI, AvaI, and SalI sites were shown to be distributed throughout the genomes. Evidence that isolates YG-1 and OS-1 are descended from an isolate whose genome arose by recombination is discussed. Terminally repeated regions were identified on each of the capripoxvirus genomes mapped here. By mapping BamHI, ClaI, EcoRI, HindII, HindIII, and SalI sites present within the terminal 10 kb of the genome of isolate InS-1, the terminal repeats of this genome were shown to be between 2.25 and 3.40 kb in length and inverted with respect to one another.  相似文献   

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