Isolation Of Chlamydia Psittaci Research Articles

Genomic analysis of Chlamydia psittaci from a multistate zoonotic outbreak in two chicken processing plants.

Four Chlamydia psittaci isolates were recovered from clinical specimens from ill workers during a multistate outbreak at two chicken processing plants. Whole genome sequencing analyses revealed high similarity to C. psittaci genotype D. The isolates differed from each other by only two single nucleotide polymorphisms, indicating a common source.

Chlamydia bacteriophages

Phages are called "good viruses" due to their ability to infect and kill pathogenic bacteria. Chlamydia are small, Gram-negative (G-) microbes that can be dangerous to human and animals. In humans, these bacteria are etiological agents of diseases such as psittacosis or respiratory tract diseases, while in animals, the infection may result in enteritis in cattle and chronic bowel diseases, as well as miscarriages in sheep. The first-known representative of chlamydiaphages was Chp1. It was discovered in Chlamydia psittaci isolates. Since then, four more species of chlamydiaphages have been identified [Chp2, Chp3, φCPG1 φCPAR39 (φCpn1) and Chp4]. All of them were shown to infect Chlamydia species. This paper described all known chlamydiaphages. They were characterised in terms of origin, host range, and their molecular structure. The review concerns the characterisation of bacteriophages that infects pathogenic and dangerous bacteria with unusual, intracellular life cycles that are pathogenic. In the era of antibiotic resistance, it is difficult to cure chlamydophilosis. Those bacteriophages can be an alternative to antibiotics, but before this happens, we need to get to know chlamydiaphages better.

The Chlamydia psittaci Genome: A Comparative Analysis of Intracellular Pathogens

Background Chlamydiaceae are a family of obligate intracellular pathogens causing a wide range of diseases in animals and humans, and facing unique evolutionary constraints not encountered by free-living prokaryotes. To investigate genomic aspects of infection, virulence and host preference we have sequenced Chlamydia psittaci, the pathogenic agent of ornithosis.ResultsA comparison of the genome of the avian Chlamydia psittaci isolate 6BC with the genomes of other chlamydial species, C. trachomatis, C. muridarum, C. pneumoniae, C. abortus, C. felis and C. caviae, revealed a high level of sequence conservation and synteny across taxa, with the major exception of the human pathogen C. trachomatis. Important differences manifest in the polymorphic membrane protein family specific for the Chlamydiae and in the highly variable chlamydial plasticity zone. We identified a number of psittaci-specific polymorphic membrane proteins of the G family that may be related to differences in host-range and/or virulence as compared to closely related Chlamydiaceae. We calculated non-synonymous to synonymous substitution rate ratios for pairs of orthologous genes to identify putative targets of adaptive evolution and predicted type III secreted effector proteins.ConclusionsThis study is the first detailed analysis of the Chlamydia psittaci genome sequence. It provides insights in the genome architecture of C. psittaci and proposes a number of novel candidate genes mostly of yet unknown function that may be important for pathogen-host interactions.

Isolation of Chlamydia psittaci from Domestic Cats with Oculonasal Discharge in Japan.

Eight strains of Chlamydia psittaci were isolated in Japan from the nasal and conjunctival swabs of six household cats using the L929 cell line of mouse fibroblast origin. The isolates were identified as C. psittaci on the basis of the formation of characteristic inclusion bodies in the cell culture detected by Giemsa stain and immunofluorescence. Comparison of nucleotide sequences of the ompA gene amplified from the three isolates with the published sequence of feline FEPN strain of C. psittaci showed almost 100% homology.

Experimental enteric infection of gnotobiotic piglets with a Chlamydia psittaci strain of avian origin.

The pathogenicity of a Chlamydia psittaci isolate of pigeon origin was assessed using a litter of gnotobiotic piglets. At 3 days of age, six piglets were inoculated intragastrically with egg-grown chlamydiae, the remaining six pigs were sham-inoculated. The animals were observed for clinical signs, and they were killed and necropsied sequentially between 4 and 15 days of age. Clinical manifestations consisted of slight softening of the faeces between 6 and 10 days post-inoculation (DPI). Immunohistochemistry revealed chlamydial replication predominantly in the small intestine, initially within villous enterocytes, after 4 DPI mostly in the lamina propria. Histopathology showed villous atrophy and increased numbers of inflammatory cells in the gut up to 6 DPI. Chlamydial stages of normal morphology were identified within enterocytes using transmission electron microscopy. An enzyme-linked immunosorbent assay (ELISA) run on faecal samples revealed shedding of chlamydial antigen from 3 until 11 DPI. Systemic dissemination of Chlamydia occurred to a limited extent according to polymerase chain reaction and immunohistochemistry results of several extraintestinal organs. Corresponding histopathological changes were minimal. Sera of all pigs were negative for anti-chlamydial antibodies using a complement fixation test. In conclusion, inoculation of this isolate in gnotobiotic piglets resulted in a productive enteric infection with mild lesions, weak systemic dissemination, and faecal shedding, indicating the pig as a potential host for avian chlamydiae.

Genomic relatedness of Chlamydia isolates determined by amplified fragment length polymorphism analysis.

The genomic relatedness of 19 Chlamydia pneumoniae isolates (17 from respiratory origin and 2 from atherosclerotic origin), 21 Chlamydia trachomatis isolates (all serovars from the human biovar, an isolate from the mouse biovar, and a porcine isolate), 6 Chlamydia psittaci isolates (5 avian isolates and 1 feline isolate), and 1 Chlamydia pecorum isolate was studied by analyzing genomic amplified fragment length polymorphism (AFLP) fingerprints. The AFLP procedure was adapted from a previously developed method for characterization of clinical C. trachomatis isolates. The fingerprints of all C. pneumoniae isolates were nearly identical, clustering together at a Dice similarity of 92.6% (+/- 1.6% standard deviation). The fingerprints of the C. trachomatis isolates of human, mouse, and swine origin were clearly distinct from each other. The fingerprints of the isolates from the human biovar could be divided into at least 12 different types when the presence or absence of specific bands was taken into account. The C. psittaci fingerprints could be divided into a parakeet, a pigeon, and a feline type. The fingerprint of C. pecorum was clearly distinct from all others. Cluster analysis of selected isolates from all species revealed groups other than those based on sequence data from single genes (in particular, omp1 and rRNA genes) but was in agreement with available DNA-DNA hybridization data. In conclusion, cluster analysis of AFLP fingerprints of representatives of all species provided suggestions for a grouping of chlamydiae based on the analysis of the whole genome. Furthermore, genomic AFLP analysis showed that the genome of C. pneumoniae is highly conserved and that no differences exist between isolates of respiratory and atherosclerotic origins.

Species identification of Chlamydia isolates by analyzing restriction fragment length polymorphism of the 16S-23S rRNA spacer region.

The genetic diversity of the 16S-23S rRNA spacer region of 12 Chlamydia pneumoniae isolates, 7 Chlamydia trachomatis isolates (human biovars: the trachoma serovars B and C, the urogenital serovars D, E, and F, and the lymphogranuloma venereum serovar L2; and a mouse biovar), 6 Chlamydia psittaci isolates (5 avian isolates and 1 feline isolate), and one Chlamydia pecorum isolate was studied. The 16S-23S rRNA spacer region was amplified by PCR and digested with the restriction enzymes MseI, PstI, AluI, BglII, NlaIV, and BclI. All 26 isolates could be amplified by using one genus-specific primer pair, yielding an amplicon with a size of 803 bp. The analytical sensitivity of the PCR assay was < or = 100 inclusion-forming units per reaction. Digestion with MseI or AluI made it possible to differentiate the four species from each other, the C. trachomatis human biovars from the mouse biovar, and the C. psittaci avian isolates from the feline isolate. The MseI profiles were more distinct than the AluI profiles. Phylogenetic analysis of the results for all enzymes combined supported the current classification of four Chlamydia species: C. pneumoniae, C. trachomatis, C. psittaci, and C. pecorum. Phylogenetic analysis also suggested subdivision of isolates of C. trachomatis and C. psittaci into subgroups that coincide with their host range. In conclusion, we have developed an easy and rapid method for species and subspecies identification of Chlamydia based on restriction fragment length polymorphism analysis of the 16S-23S rRNA spacer region.

Two new serovars of Chlamydia psittaci from North American birds.

Five Chlamydia psittaci isolates (1 turkey, 1 psittacine, 1 human, and 2 pigeon isolates) failed to react with serovar-specific monoclonal antibodies to known avian and mammalian C. psittaci serovars and were presumed to represent 1 or more new serovars. The isolates were characterized using restriction endonuclease analysis of the whole genome, polymerase chain reaction-restriction fragment length polymorphism of the major outer membrane protein genome, monoclonal antibody comparisons, and growth in tissue culture. Monoclonal antibodies were produced to the human isolate (MP) and to the psittacine isolate (VS225). The monoclonal antibody results show that the isolates represent 2 new avian serovars (serovars E and F). The restriction fragment length polymorphism analysis of the major outer membrane protein genome demonstrated that the isolates are distinct. The whole genome restriction endonuclease analysis data and the growth patterns in tissue culture indicate that the new serovars are similar to avian serovars recognized previously. A subspecies monoclonal antibody that reacted with serovars A and B also reacted with serovar E, indicating that these serovars are closely related. The results show that these isolates represent 2 new avian serovars, making them the fifth and sixth avian serovars identified in North American birds.

Analysis of partial 16S rRNA nucleotide sequences of Chlamydia pecorum and C. psittaci

Partial 16S nucleotide sequences of Chlamydia psittaci isolates S26/3 (abortion), P94/1 (pigeon) and Chlamydia pecorum isolates W73 (enteric) and E58 (encephalomyelitis) were determined. Analysis of these data indicates very high levels of interspecies sequence conservation, with C. psittaci being more closely related to C. pecorum than to C. pneumoniae or C. trachomatis. Restriction enzyme analysis of nucleotide sequences indicated that BslI can be used to clearly distinguish C. psittaci and C. pecorum isolates. Psittacine and non-psittacine (pigeon) avian isolates of C. psittaci were distinguished using MaeI.

Analysis of partial 16S rRNA nucleotide sequences of Chlamydia pecorum and C. psittaci.

Partial 16S nucleotide sequences of Chlamydia psittaci isolates S26/3 (abortion), P94/1 (pigeon) and Chlamydia pecorum isolates W73 (enteric) and E58 (encephalomyelitis) were determined. Analysis of these data indicates very high levels of interspecies sequence conservation, with C. psittaci being more closely related to C. pecorum than to C. pneumoniae or C. trachomatis. Restriction enzyme analysis of nucleotide sequences indicated that BslI can be used to clearly distinguish C. psittaci and C. pecorum isolates. Psittacine and non-psittacine (pigeon) avian isolates of C. psittaci were distinguished using MaeI.

Characterization of avian Chlamydia psittaci strains using omp1 restriction mapping and serovar-specific monoclonal antibodies

In the present study, 60 avian Chlamydia psittaci isolates were characterized using restriction fragment length polymorphism as well as serovar-specific monoclonal antibodies, enabling a comparison between the two characterization methods. Sixty avian C. psittaci isolates were characterized by Alul restriction mapping of the major outer membrane protein gene omp1 obtained after amplification by the polymerase chain reaction. The 60 avian C. psittaci strains were also characterized using serovar-specific monoclonal antibodies in a microimmunofluorescence test. Digestion of 60 avian C. psittaci omp1 amplicons by Alul generated 5 of the 6 known distinct restriction patterns (A, B, D, E and F). Restriction pattern C was not observed. Serotyping revealed 4 avian C. psittaci serovars (A, B, C and D). None of the 60 isolates was typed as serovar E. AluI restriction patterns A, B, D and E corresponded in 98% of the cases to serovars A, B, C and D, respectively. One isolate, classified as serovar A, generated restriction pattern F instead of A. Genotyping enabled a more precise differentiation of avian C. psittaci serovar A strains. Serovar A strains were divided into two groups according to their Alul restriction pattern (A or F). For epidemiological studies, genotyping can thus be a highly valuable alternative to serotyping, especially when applied directly to the clinical samples.

Analyses of the genomes of chlamydial isolates from ruminants and pigs support the adoption of the new species Chlamydia pecorum.

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.

Usefulness of omp1 restriction mapping for avian Chlamydia psittaci isolate differentiation

Sixty-five avian Chlamydia psittaci isolates collected worldwide, including 27 previously characterized reference strains, were analysed by restriction mapping of the major outer membrane protein gene ( omp1) obtained after DMA amplification by PCR. They were compared to 2 ruminant isolates, a feline pneumonitis and a guinea pig inclusion conjunctivitis (GPIC) isolate. According to their omp1 restriction patterns, avian strains were heterogeneous in that they exhibited 6 and 4 distinct patterns using AluI and MboII restriction enzymes, respectively, thus defining 7 groups. However, 84% of the studied strains belonged to groups 1 to 4, which share a specific fragment triplet of 411, 282 and 102 base pairs in their AluI digestion patterns. Comparisons with serologfcal classifications showed a strict correlation and allowed further intraserovar differentiation. Furthermore, this classification based upon a single gene ( omp1) roughly correlated with the data obtained by RFLP of native DNA and DNA/DNA hybridization studies. There was no host or geographic specificity in the pattern exhibited by these strains. The ruminant feline pneumonitis and GPIC C. psittaci isolates were clearly distinguished from each other and the avian strains. Moreover, this method was clearly able to identify dubiously designated strains as well as mixtures of isolates within a single sample. In conclusion, this PCR approach based upon omp1 restriction mapping enables the differentiation of avian C. psittaci isolates and can be proposed as a taxonomic and epidemiologic tool.

Primary pathogenicity of an European isolate of Chlamydia psittaci from turkey poults

Chlamydia psittaci was isolated as the sole pathogenic agent from a severe outbreak of respiratory disease in a commercial broiler turkey farm in the Netherlands. The mortality rate in the flocks was 35%. Clinical signs included conjunctivitis, swelling of the sinus infraorbitalis and sneezing. Cloacal excretion of chlamydia was demonstrated in twelve out of fifteen birds examined by a direct immunofluorescence test. In all the fifteen birds antibodies against Chlamydia psittaci were detected in the sera by a competitive ELISA. At necropsy sinusitis, rhinitis, airsacculitis, pneumonia, pericarditis and enlargement of the liver and spleen were found. Chlamydiae were demonstrated in the sinus material of all and in conjunctival smears of eight of the fifteen examined birds. Chlamydiae were isolated from all the examined birds after one to three passages on Buffalo Green Monkey (BGM) cell cultures using samples taken from lung, liver and spleen. No other pathogens were isolated. The chlamydia isolate was typed using a panel of serovar-specific monoclonal antibodies in a micro-immunofluorescence test. The isolate belonged to the avian Chlamydia psittaci serovar D. Experimental inoculation with this isolate of 7-day-old specific pathogen free (SPF) turkeys resulted in severe clinical signs, with mortality and extensive pathological lesions, similar to those seen in turkeys from the examined broiler turkey farm. From the data it was concluded that this Chlamydia psittaci isolate can cause severe disease in turkeys.

Comparison of ovine abortion and non-abortion isolates of Chlamydia psittaci using inclusion morphology, polyacrylamide gel electrophoresis, restriction endonuclease analysis and reactivity with monoclonal antibodies

Twelve reference and four Northern Ireland ovine Chlamydia psittaci isolates including ovine abortion, faecal, conjunctivitis and arthritis isolates were compared. Inclusion morphology was shown to provide a useful means of differentiating the abortion and the non-abortion isolates studied. Identical SDS-PAGE polypeptide profiles were produced by the ovine abortion isolates. The polypeptide profiles of the non-abortion isolates were similar to one another and clearly distinct from the abortion isolate profiles. The restriction endonuclease profiles of the abortion isolates were remarkably similar whereas different profiles were produced by most of the non-abortion isolates. Monoclonal antibodies were prepared and characterized. A number of these reacted with all the isolates of chlamydia tested. Three mAbs reacted exclusively with the ovine abortion isolates while four mAbs reacted exclusively with a number of the faecal isolates.

Antibody Prevalence and Isolation of Chlamydia psittaci from Pigeons (Columba livia)

The isolation of Chlamydia psittaci and serological detection of Chlamydia-specific antibodies in racing pigeons and pigeons from public parks is described. Several serological methods (complement fixation test, indirect microimmunofluorescence test, and enzyme-linked immunosorbent assay) were compared with bacteriological techniques (isolation both in embryonated eggs and McCoy cell monolayers). Tests confirmed that 28.6%, 33.5%, and 35.9% of the pigeons, respectively, were seropositive by the tests mentioned above. Chlamydiae were isolated from 13% of the fecal specimens in ovocultures and from 18% of the fecal specimens in cell cultures. No significant differences between the two groups of pigeons were found (at a 95% confidence level, alpha = 0.05) using the hypothesis test of the difference between proportions of two populations. The serological and bacteriological techniques used are compared and discussed.

Serotyping of European isolates of Chlamydia psittaci from poultry and other birds.

A panel of five serovar-specific monoclonal antibodies which distinguish the five known avian serovars of Chlamydia psittaci was used to serotype 45 European avian Chlamydia psittaci isolates. Chlamydial antigen was grown in Buffalo green monkey (BGM) cells or in embryonated chicken eggs and was then inoculated into BGM cells. Serotyping was performed in an indirect immunofluorescence test. The 45 European isolates included 22 isolates from the order Psittaciformes, 9 isolates from the order Columbiformes, 6 isolates from the order Galliformes, 5 isolates from the order Passeriformes, and 3 isolates from the order Anseriformes. All of these were successfully serotyped. No additional serovars were found. One isolate from a duck and two isolates from psittacine birds gave positive immunofluorescences with two monoclonal antibodies considered to be specific for two different serovars. These three isolates were cloned by an agar overlay method. Serotyping of the clones demonstrated that the duck and one psittacine bird each were infected with two different serovars. After cloning, one isolate from a psittacine bird reacted only with serovar A. From these results it was concluded that this serotyping system allows the classification of all isolates tested so far. The results show that similar serovars are prevalent in avian species in Europe and the United States. The results also indicate that birds from a certain order are more susceptible to a distinct serovar. The use of a panel of serovar-specific monoclonal antibodies in the immunofluorescence test provides a reliable method for serotyping avian isolates. Monoclonal antibodies to new avian isolate serovars can easily be added to the panel, which makes the system useful for epidemiological studies.

Antigenic and morphological differentiation of placental and intestinal isolates of Chlamydia psittaci of ovine origin

Ewe placental and lamb intestinal isolates of Chlamydia psittaci recovered from flocks affected with ovine enzootic abortion were examined by inclusion morphology, indirect immunofluorescence (IIF) and immunoblot analysis. Chlamydiae recovered from the faeces of sheep from two flocks free of clinical disease were also examined. In cell culture ovine abortion (OA) and intestinal isolates were distinguishable by inclusion development and morphology. Similarly, in two-way IIF tests with one week mouse antisera isolates fell into two distinct groups: abortion or intestinal. Immunoblotting with convalescent sheep abortion antiserum identified 30 out of at least 40 silver staining polypeptides as antigenic both in OA and intestinal isolates. The serum produced a similar reaction pattern to the resolved proteins of each OA isolate, indicating a higher degree of antigenic conservation among these isolates. Considerable cross reactivity between the OA and intestinal isolates was identified, but the serum also showed apparent molecular weight differences between antigens of the two types in the 87–116 kDa, 38–44 kDa and 26–28 kDa regions. Furthermore, the immunoblotting analysis revealed heterogeneity among the intestinal isolates, particularly in antigens between 87–116 kDa and 38–44 kDa.

Restriction pattern of the major outer-membrane protein gene provides evidence for a homogeneous invasive group among ruminant isolates of Chlamydia psittaci

Thirty-six ruminant isolates of Chlamydia psittaci, previously classified as invasive or non-invasive in a mouse model of virulence, were compared by analysing AluI restriction patterns of the major outer-membrane protein (MOMP) gene after DNA amplification by the polymerase chain reaction. The 24 invasive isolates, although from various origins, all belonged to serotype 1 and represented a strictly homogeneous group sharing a specific MOMP-gene restriction pattern that was not observed in the non-invasive strains. On the other hand, the 12 non-invasive strains, although all belonging to serotype 2, constituted a heterogeneous group with eight distinct MOMP-gene restriction patterns. However, all eight patterns shared a 180 bp fragment or the corresponding restricted fragments of 110 and 70 bp. MOMP-gene restriction patterns also clearly distinguished the ruminant strains from an avian C. psittaci isolate, a C. pneumoniae isolate and two C. trachomatis isolates which were studied for comparison. The homogeneous character of the invasive C. psittaci strains argues strongly for their genetic relatedness. Our results illustrate the usefulness of the MOMP-gene restriction mapping in typing chlamydiae.

Comparison of isolates of Chlamydia psittaci of ovine, avian and feline origin by analysis of polypeptide profiles form purified elementary bodies

The single species Chlamydia psittaci is a diverse grouping which contains several different types of chlamydial strain for which there is no generally accepted typing method. The results obtained when profiles of polypeptides from purified elementary bodies are compared are consistent with type designations obtained using other criteria. However, the method still requires large scale culture and extensive purification of the chlamydial cells.