Proteins present in the outer membrane of chlamydiae that are involved in mucosal epithelial cell infection must clearly be identified and characterized if we are to understand and modify the pathogenic mechanisms utilized by these organisms. We have identified and isolated a family of four genes encoding putative outer membrane proteins (POMPs), a group of proteins of approximately 90 kDa present in the outer membrane of the subtype of Chlamydia psittacithat causes ovine enzootic abortion (strain S26/3). These proteins, although minor components, are major immunogens, as shown by the immunoblotting of chlamydial outer membrane complexes with postabortion sheep sera, and are therefore potential diagnostic and/or protective antigen candidates. Immunoblotting of the expressed amino- and carboxy-terminal halves of one of the POMPs with postabortion sheep sera showed that the major humoral immune response appeared to be directed solely against the amino-terminal half. This result, in combination with the positive immunofluorescence staining of S26/3-infected cells using POMP-specific (specific to the amino-terminal half of the proteins) monoclonal antibodies, suggests the probable surface localization of the POMPs and, more specifically, the surface exposure of the amino-terminal half of these proteins. The four pomp genes are highly homologous, sharing 82 to 100% similarity with each other (two of the genes are identical). Genes with strong and weak homologies were also detected in C. psittaci avian and feline pneumonitis strains, respectively. Nopomp homologs were found in strains of C. trachomatis and C. pneumoniae, but this does not preclude their existence. The absence of homology with various subtypes of C. pecorum, which complicate the diagnosis of the ovine abortion subtype, indicates the possible suitability of the these 90-kDa proteins as serodiagnostic antigens.

Analysis of the genomic DNAs of chlamydial isolates from sheep, cattle, and pigs was performed by Southern blot hybridization and by restriction endonuclease (RE) profiling of DNA amplified by PCR. The hybridization probes were derived from whole genomic DNA, the major outer membrane protein (MOMP) gene, the 16s rRNA gene, and an avian Chlamydia psittaci isolate plasmid. The PCR analysis used targets in the MOMP gene, the 16s rRNA gene, and the 60-kDa cysteine-rich protein gene. Together, the results showed that although there was considerable heterogeneity in the DNA sequence in the MOMP gene region, all the isolates had the same underlying total genomic RE profiles and yielded identical RE profiles for the rRNA and 60-kDa-protein gene regions. Most of the isolates were found to hybridize with the plasmid probe. Comparison of the MOMP sequence of one of the isolates (P787) with that of a known Chlamydia pecorum strain together with the results of the RE analyses allowed the conclusion that the isolates should all be classified within this new species.The genus chlamydia has recently been expanded, largely because of the application of measurements of genomic relatedness to discriminate further the various chlamydial species and types. Currently, there are four species recognized, Chlamydia trachomatis, C. psittaci, C. pneumoniae, and the newest grouping of ruminant strains proposed by Fukushi and Hirai and called C. pecorum (10, 12). Recent multistrain comparisons of the gene sequences of the major outer membrane protein (MOMP) yield phylogenetic trees which support these four species groupings (4, 22,40). However, DNA homology measurements (5, 9) indicate that the species C. trachomatis and C. psittaci could be subdivided further if it is accepted that at least 70% sequence homology is necessary for isolates to be considered as belonging to the same species (20).An important aspect of any taxonomic system is that it should be based on criteria which can easily be applied in a standard fashion by all laboratories and which will allow accurate and rapid identification of isolates. The biological and immunological methods which have been used to identify species and subspecies types of Chlamydia are time-consuming, require considerable skill and experience, and are limited by the problems of generating and standardizing serological reagents. Recently, methods based on identifying variations in DNA sequences, usually through the use of restriction endonuclease (RE) analysis, have been applied to the problem of species and strain identification. When a chlamydia] isolate has been cultured, these methods can be applied either at the whole-genome level (9, 27) or to subgenomic fragments by Southern blotting (11, 18,42). However, a substantial advance has come with the use of the PCR (33), which can be used both to detect the presence of chlamydiae in clinical samples by the amplification of specific chlamydial genes and to identify the chlamydial species and type by analysis of sequence variation in the amplified pro...

The putative outer membrane location of the OMP90 (formerly POMP) family from the ovine abortion strain of Chlamydia psittaci was investigated by immunoelectron microscopy. Using a non-embedding technique, antigens were shown to be localised on the outer membrane surface of both elementary and reticulate bodies, the infectious and non-infectious forms of Chlamydiae respectively. Antibodies affinity-purified against the expressed amino- and carboxy-terminal halves of one of the family members. OMP90A, demonstrated that the amino half is surface-exposed while the carboxyl half is most probably localised internally. Surface localisation on elementary bodies indicates the importance of these proteins as protective antigen candidates.

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The major outer membrane protein (MOMP) gene from an ovine abortion strain of Chlamydia psittaci (S26/3) has been cloned and sequenced. The gene shows the features of other chlamydial MOMPs but comparison with the previously reported sequence for the ovine abortion isolate A22/M has revealed substantial sequence divergence which is clustered into the same four intramolecular regions as the sequence variation found between C. trachomatis serovars. Subsequent restriction enzyme analysis of A22/M DNA has shown that it has an avian‐type genomic profile and thus the comparison is between types rather than between Strains.