J Med Microbiol 56 (2007), 407-417; DOI: 10.1099/jmm.0.46850-0
© 2007 Society for General Microbiology
ISSN 1473-5644
Molecular characterization of Chlamydophila pneumoniae isolates from Western barred bandicoots
Andrei Kutlin1,
Patricia M. Roblin1,
Swati Kumar1,
Stephan Kohlhoff1,
Tracey Bodetti2,
Peter Timms2 and
Margaret R. Hammerschlag1
1 Department of Pediatrics, SUNY Downstate Medical Center, Brooklyn, New York, USA
2 Institute of Health and Biomedical Innovation, School of Life Sciences, Queensland University of Technology, Brisbane, Australia
Correspondence
Andrei Kutlin
andrei.kutlin{at}rcn.com
Received 21 July 2006
Accepted 25 October 2006
Chlamydophila pneumoniae is an obligate intracellular respiratory pathogen that has been associated with pneumonia and chronic bronchitis, atherosclerosis, asthma and other chronic diseases in humans. However, C. pneumoniae is not restricted to humans, as originally thought, and can cause infections in several animal hosts. C. pneumoniae was isolated in cell culture from nine Western barred bandicoots (Perameles bougainville) from Australia. The sequences of five genomic regions were determined, including full-length sequences of the16S rRNA and ompA genes and the ygeD–urk intergenic spacer, and partial sequences of the 23S rRNA and rpoB genes. Sequence analysis of the entire 16S rRNA and ompA genes from bandicoot isolates demonstrated that they were 98.2–98.3 % similar to human isolates, 94.6–99.3 % similar to the equine biovar and almost identical, with 99.5–99.9 % similarity, to the koala biovar. Comparative genotyping of the variable domain 4 region of the ompA gene demonstrated that bandicoot isolates seemed to be identical to the animal genotype that has been recently identified in human carotid plaque specimens. Minor sequence polymorphism observed in ompA, 16S rRNA and rpoB genes of animal isolates, indicating genomic diversity within C. pneumoniae, may have important implications for diagnostic PCR assays leading to false negative results. Forty percent of selected published species-specific PCR assays were found to have sequence variability in primer and/or probe that might affect their performance in detecting bandicoot isolates of C. pneumoniae, or possibly other animal and human strains where minor sequence polymorphisms may be present. The data from this study support the previous observations that C. pneumoniae is not restricted to humans and may be widespread in an animal reservoir with a potential risk of transmission to humans.
Abbreviations: SNP, single nucleotide polymorphism; WBB, Western barred bandicoot.
The GenBank/EMBL/DDBJ accession numbers for the ompA and 16S rRNA genes, partial sequences of 23S rRNA and rpoB genes, and ygeD–urk intergenic spacer from bandicoot C. pneumoniae isolates are DQ358972, DQ444323, DQ465990, DQ460031 and DQ463439, respectively.
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INTRODUCTION
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Chlamydophila pneumoniae is an obligate intracellular bacterium responsible for respiratory infections (pneumonia and bronchitis) in adults and children, affecting up to 70 % of the worldwide population at least once during his or her lifetime (Peeling & Brunham, 1996). Persistent C. pneumoniae infections have been implicated in the development of atherosclerosis, asthma and other chronic diseases in humans (Balin et al., 1998; Blasi et al., 2002; Hahn et al., 1991; Wong et al., 1999).
C. pneumoniae was initially thought to be an exclusively human pathogen. However, several studies demonstrated that C. pneumoniae could also cause ocular, respiratory and urogenital infections in a wide variety of animal species, including koalas, horses, frogs and reptiles (Berger et al., 1999; Bodetti et al., 2002; Hotzel et al., 2001; Jacobson et al., 2004; Storey et al., 1993). Current taxonomic classification divides C. pneumoniae into three distinct biovars: human biovar TWAR, koala biovar and equine biovar (Everett et al., 1999a). The distinction was based primarily on comparative sequence analysis of the 16S rRNA, 23S rRNA and ompA genes, and biological characteristics. Human C. pneumoniae isolates were found to be almost identical to each other, with only 0.1 % difference in 16S rRNA gene and 0.4 % difference in the ompA gene. While some of the animal isolates are almost identical to human strains, the others seem to be genetically more diverse, with up to 6 % ompA gene dissimilarity (Bodetti et al., 2002).
The existence of animal strains of C. pneumoniae raises the issue of possible transmission risk to humans. A recent study by Cochrane et al. (2005), where animal genotypes of C. pneumoniae were identified in human specimens, and human genotypes were detected in koalas, supports the possibility of such transmission. However, no cases of zoonotic C. pneumoniae infections in humans have been described so far.
In this study, C. pneumoniae was isolated from Western barred bandicoots (WBBs) (Perameles bougainville) in cell culture and characterized by sequence comparison of five genomic regions relevant for molecular diagnostics, including 16S rRNA, 23S rRNA, ompA and rpoB genes and the ygeD–urk intergenic spacer. The entire ompA and 16S rRNA genes, which are frequently used in species-specific PCR-based assays (Dowell et al., 2001; Loens et al., 2006), were sequenced and compared to the published sequences of human and animal C. pneumoniae isolates. Genotyping of the bandicoot isolates was performed in the variable domain 4 of the ompA gene, rpoB gene and ygeD–urk intergenic spacer loci.
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METHODS
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Animals.
All WBBs sampled in this study were from wild populations from Bernier Island and Dryandra, Western Australia. Thirty-seven conjunctival, nasal, throat and cloacal swab specimens from twenty-one animals with clinical signs of ocular and/or respiratory disease were examined.
Isolation and propagation of Chlamydiales.
Isolation and propagation of Chlamydiales was performed by cell culture, as previously described (Roblin et al., 1992). Briefly, swab specimens were inoculated onto HEp-2 cell monolayers (ATCC CCL-23) by centrifugation at 1700 g for 1 h. Infected monolayers were then overlaid with Iscove's DMEM medium (Sigma-Aldrich) supplemented with 10 % fetal calf serum (Atlanta Biologicals), 20 mM L-glutamine and 1 µg cycloheximide ml–1, and incubated at 35 °C for 72 h. Up to six passages were performed for each isolate. Following incubation, inoculated HEp-2 cells were fixed in ethanol and stained for specific chlamydial inclusions with the Pathfinder Chlamydia culture confirmation system (Bio-Rad) for 30 min at 37 °C.
DNA extraction.
DNA from the cultured Chlamydiales was extracted using a DNeasy Tissue kit (Qiagen) according to the manufacturer's protocol. Human C. pneumoniae CWL029 (ATCC VR-1310) and TW-183 (ATCC VR-2282) grown in HEp-2 cells were used as a positive control in PCR assays.
Chlamydiaceae-specific PCR TaqMan assay.
The TQF and TQR primers and probe targeting the Chlamydiaceae-specific region of the 23S rRNA gene were as described by Everett et al. (1999b) (Table 1
). PCR was performed using a LightCycler 2.0 (Roche) system with DNA Master HybProbe kit (Roche), containing FastStart Taq DNA polymerase, under the following conditions: an initial denaturation step at 95 °C for 10 min, then 45 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 10 s and extension at 72 °C for 10 s. Product amplification was analysed with the manufacturers' supplied software.
C. pneumoniae-specific PCR TaqMan assay.
The primers QMOMP1, QMOMP2 and QMOMPS probe targeting an 85 bp C. pneumoniae-specific region of the ompA gene are shown in Table 1
. PCR was performed using the Roche system mentioned above, with predenaturation at 95 °C for 10 min, then 45 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 10 s and extension at 72 °C for 10 s.
16S and 23S rRNA genes signature sequencing.
Amplification/sequencing primers used for 16S rRNA gene and 23S rRNA gene products were 16SIGF, 16SIGR, U23F and 23SIGR (Table 1). PCR was performed using Qiagen ProofStart DNA polymerase kit (Qiagen), with initial denaturation at 95 °C for 5 min, then 45 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 60 s, with a final extension for 5 min at 72 °C. Following amplification, the products were separated by electrophoresis in 2 % agarose E-gel (Invitrogen) and visualized using an ultraviolet transilluminator. PCR products were purified with a QIAquick PCR purification kit (Qiagen) and sequenced in both directions (GeneWiz).
ompA gene sequencing.
A DNA fragment containing the entire ompA gene was amplified with CpompA1F and CpompA3R primers (Table 1). PCR was performed using a ProofStart DNA polymerase kit, with an initial denaturation step at 95 °C for 5 min, then 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 60 s, with a final extension for 5 min at 72 °C. Following electrophoresis in 2 % agarose E-gel, PCR products were visualized by using an ultraviolet transilluminator. PCR products containing 1170 bp ompA gene were sequenced in both directions, with overlap, using the sequencing primers shown in Table 1. These primers were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) (Rozen & Skaletsky, 2000).
16S rRNA gene sequencing.
The DNA region containing the entire 16S rRNA gene was amplified with Cp16SF and Cp16SRb primers (Table 1). PCR was performed using a ProofStart DNA polymerase kit, with initial denaturation step at 95 °C for 5 min, then 45 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 60 s, with a final extension for 5 min at 72 °C. Following electrophoresis in 2 % agarose E-gel, PCR products were visualized and purified with QIAquick PCR purification kit. PCR products containing the 1552 bp 16S rRNA gene were sequenced in both directions, with overlap, using the following primers: Cp16SF1, Cp16SR1, Cp16SF2, Cp16SR2, Cp16SF3, Cp16SR3, Cp16SF4 and Cp16SR4 (Table 1). The above primers were designed using the Primer3 program.
rpoB gene sequencing.
A 733 bp section of the DNA-directed RNA polymerase ß gene (rpoB) containing segment, matching a C. pneumoniae-specific PstI fragment (Campbell et al., 1992), was amplified using CprpoBFc and CprpoBRc primers (Table 1), designed using Primer3. PCR was performed using a ProofStart DNA polymerase kit, with the initial step at 95 °C for 5 min, then 45 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 60 s, with a final extension for 5 min at 72 °C. PCR products were purified and sequenced in both directions using the same primer pair.
ygeD–urk intergenic spacer sequencing.
A 621 bp fragment spanning the ygeD–urk intergenic spacer was amplified and sequenced using OUT1 and OUT2 primers (Table 1). PCR was performed using a ProofStart DNA polymerase kit under the above conditions. Prior to sequencing products were purified from 2 % agarose E-gels with a QIAquick PCR purification kit.
Sequence analysis.
The sequences were analysed using BLAST 2 (http://www.ncbi.nlm.nih.gov/blast/bl2seq/) (Tatusova & Madden, 1999) and compared to the Chlamydiales sequences of 16S rRNA, 23S rRNA, rpoB and ompA genes available in GenBank, including three biovars of C. pneumoniae. C. pneumoniae isolates whose sequences were used for comparative analysis are shown in Table 2. ClustalW multiple sequence alignment was performed using the MegAlign 5.0 program (DNAStar). The sequences of the entire ompA and 16S rRNA genes, partial sequences of 23S rRNA and rpoB genes and the ygeD–urk intergenic spacer from bandicoot C. pneumoniae were submitted to GenBank with the accession numbers DQ358972, DQ444323, DQ465990, DQ460031 and DQ463439, respectively.
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RESULTS AND DISCUSSION
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This is believed to be the first report of C. pneumoniae isolated and characterized from a third affected mammalian species. This species, WBBs, are small endangered marsupials whose natural habitat is currently limited to the Bernier and Dorre Islands off the West Coast of Australia. However, uncultured unspeciated Chlamydiales and Chlamydophila pecorum have been previously detected in these animals by molecular-based methods (Bodetti et al., 2003; Warren et al., 2005).
Isolation and speciation
Ten swab specimens (six ocular, three throat and one nasal) from nine WBBs were positive by isolation in cell culture followed by staining with a family-specific mAb against chlamydial lipopolysaccharide. These isolates were propagated by cell culture to 103–104 inclusion forming units ml–1 and were confirmed as Chlamydiaceae by 23S rRNA gene-based PCR. Sequence analysis of the 16S and 23S rRNA signature sequences described by Everett et al. (1999a) revealed that all 10 bandicoot isolates belonged to the C. pneumoniae species and were more than 99.1 % similar to C. pneumoniae isolates of human and animal origin, with only 1–5 bp variations.
ompA gene-based PCR and sequencing
All ten bandicoot isolates tested positive by C. pneumoniae-specific ompA-based, PCR using the originally described protocol (Apfalter et al., 2003), which confirmed them as C. pneumoniae. However, the fluorescence signal was approximately 1.7 times lower than in the human C. pneumoniae control as shown in Fig. 1. An increase in annealing temperature resulted in a negative fluorescence signal for all bandicoot isolates but did not affect the signal for the human C. pneumoniae control (data not shown). Possible sequence variation between bandicoot and human isolates in the area where published primers and probe are meant to bind the target sequence was suspected.
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Fig. 1. Fluorescence signal of species-specific ompA-based real-time PCR of bandicoot C. pneumoniae isolates (WBB).
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To address this performance problem of the ompA-based PCR, the entire ompA gene was amplified and sequenced for all isolates. ompA genes of all bandicoot C. pneumoniae isolates were identical to each other. As seen in Fig. 2, nucleotide alignment of the 85 bp target region used in the ompA-based TaqMan assay did reveal one single nucleotide polymorphism (SNP) in the forward primer and two SNPs in the probe sequence as compared to human isolates of C. pneumoniae. Overall, the entire ompA gene sequence from bandicoot C. pneumoniae was found to be 98.2 and 94.6 % similar to human and equine biovars, respectively (Table 3), and almost identical, 99.9 % similarity, to koala biovar (1 bp difference). This SNP at position 982 resulted in an amino acid substitution from alanine in the koala biovar to proline in the bandicoot C. pneumoniae.
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Fig. 2. Nucleotide sequence alignment of the 85 bp ompA target region used in the C. pneumoniae-specific TaqMan PCR assay. Dots indicate a nucleotide is the same as in sequences of human isolates (TW-183, CWL029, AR-39 and J138).
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Table 3. Sequence similarities (%) in five genomic regions of WBB isolates compared to existing C. pneumoniae biovars/isolates.
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16S rRNA gene sequencing
16S rRNA gene, which is also frequently used as a target for identification and speciation of Chlamydiales and other bacteria, was also amplified and sequenced for all bandicoot isolates. All bandicoot isolates were identical to each other and very similar to human and animal C. pneumoniae isolates as shown in Table 3. Minor nucleotide differences in both 16S rRNA and ompA genes may indicate genomic diversity within animal C. pneumoniae strains, as suggested by Hotzel et al. (2001).
The identification of C. pneumoniae infection in WBBs further proves that this species is not restricted to humans, and may exist in an animal reservoir with a possible transmission risk to humans. Coles et al. (2001) demonstrated that animal strains of koala biovar are able to infect and multiply in human respiratory cells and monocytes. Recently, Cochrane et al. (2005) identified an animal genotype of C. pneumoniae in carotid arteries and peripheral blood mononuclear cell (PBMC) specimens from humans, and human genotype in PBMC specimens from koalas by using nested ompA- and ygeD–urk-based PCR and sequencing assays. The authors suggested that C. pneumoniae might be capable of being transmitted between human and animals.
Genotyping
Data on the genotyping of human and animal strains of C. pneumoniae are limited. For genotyping purposes three genomic targets were selected: variable domain 4 region (VD4) of the ompA gene, the ygeD–urk intergenic spacer and a region of the rpoB gene matching a C. pneumoniae-specific PstI fragment.
VD4 genotyping
VD4 was proposed as a possible genotyping target due to its small size and high degree of variability between human and animal isolates (Cochrane et al., 2005; Wardrop et al., 1999). Bodetti et al. (2002) suggested a genotyping system based on as little as 1 bp sequence polymorphism in the VD4 segment of the ompA gene. We analysed C. pneumoniae VD4 sequences publicly available in the GenBank database and identified 22 sequences, which we separated into 3 human and 7 animal genotype groups (based on their sequence identity within a group) as presented in Table 4. Sequence alignment of the 174 bp region of the VD4 segment demonstrated that bandicoot isolates differed from human isolates by 6–7 SNPs and were identical to previously reported koala and frog isolates (genotype group A5) and, according to the Cochrane et al. (2005) designations, would be assigned to genotype D. Interestingly, the animal C. pneumoniae isolates from genotype group A1 from reptiles and amphibians (Bodetti et al., 2002) were absolutely identical to human isolates from group H1 as shown in Fig. 3.
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Fig. 3. Nucleotide sequence alignment of a 174 bp region within the VD4 segment of bandicoot C. pneumoniae ompA gene with human and animal genotype. Dots indicate a nucleotide is the same as in sequences of human H1 group. The GenBank accession no. for WBB isolates is DQ358972.
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ygeD–urk genotyping
The intergenic spacer region between ygeD and urk genes differs by the orientation of a 23 bp segment in one of the human C. pneumoniae strains (Read et al., 2000) and was used for genotyping of C. pneumoniae (Cochrane et al., 2005). The results of the 320 bp ygeD–urk spacer genotyping are shown in Fig. 4. The bandicoot isolates were identical to CpnIII genotype recently found in humans (Cochrane et al., 2005) and had the 23 bp invertible region in the same orientation as human TW-183, AR39 and J138 strains. There was a 3 bp difference as compared to human isolates.
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Fig. 4. Nucleotide sequence alignment of a 320 bp ygeD–urk intergenic spacer of bandicoot and human isolates. Dots indicate a nucleotide is the same as in sequences of human AR39, TW-183 and J138 isolates, and dashes represent gaps in the sequences. CpnIII is a C. pneumoniae genotype identified in human carotid specimens, GenBank accession no. AY427827 (Cochrane et al., 2005).
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rpoB genotyping
rpoB gene was of relevant interest because of its current use as a target in C. pneumoniae-specific PCR assays (Dowell et al., 2001; Loens et al., 2006). Sequence analysis of the rpoB gene region that matched a C. pneumoniae-specific PstI fragment (Campbell et al., 1992) showed a 1 bp polymorphism as compared to human strains of C. pneumoniae (Fig. 5). This nucleotide change at position 2946 was a silent mutation with no amino acid change (alanine).
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Fig. 5. Nucleotide sequence alignment of a 474 bp region of rpoB gene (matching a C. pneumoniae-specific PstI fragment) of human and WBB isolates. Dots indicate a nucleotide is the same as in human isolates, represented by TW-183, CWL029, AR39 and J138, and dashes represent gaps in the sequence. Primer sequences HL-1 and HR-1 used in the C. pneumoniae-specific PCR assay (Campbell et al., 1992) are shown in bold/underlined.
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C. pneumoniae species-specific PCR assays and sequence polymorphism
The results of this study may have important implications for molecular diagnosis of C. pneumoniae infection in both animals and humans. Since most of the C. pneumoniae-specific PCR assays were developed and evaluated using human isolates, they may not be appropriate for detection of animal strains. Even minor sequence variability in the target regions can significantly compromise the sensitivity of the PCR assay and result in false negative results due to either target amplification failure (primer sequence polymorphism) or target detection failure (probe sequence polymorphism) (Kwok et al., 1990; Stevenson et al., 2005). As we demonstrated in this study a real-time ompA-based PCR assay developed for human strains either generated a lower fluorescent signal or failed to detect bandicoot C. pneumoniae isolates because there was one SNP in the forward primer and there were two SNPs in the probe. Similar assay-performance problems due to target-sequence variability have been recently reported for real-time PCRs for the detection of herpes simplex virus and Listeria monocytogenes (Rodríguez-Lázaro et al., 2004; Stevenson et al., 2005). We analysed several published and currently used C. pneumoniae species-specific PCR assays for the presence of the sequence polymorphism in the target regions as shown in Table 5. We found that 6 out of 15 species-specific PCR assays had sequence variability in primer and/or probe that may affect their performance in detecting bandicoot strain of C. pneumoniae, and possibly other animal and human strains where minor sequence polymorphism may be present.
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Table 5. Sequence variability in the target regions of the selected species-specific PCR assays as compared to bandicoot C. pneumoniae isolates
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The expanding number of animal hosts and isolates of C. pneumoniae, and genetic similarity of animal and human strains raises some interesting questions about the evolution and epidemiology of this pathogen. It is quite possible that C. pneumoniae was primarily an animal pathogen, which was only recently, in evolutionary terms, acquired by humans. Considering almost universal exposure of humans to C. pneumoniae and generally inefficient mode of transmission from person to person (Blasi et al., 1998), one could hypothesize that an environmental source of C. pneumoniae should be in close proximity to humans. While possible zoonotic transmission of C. pneumoniae from exotic animals occupying a unique environmental niche (koalas and bandicoots) to humans seems unlikely, acquisition by contact with animals living in urban or rural areas, including domestic and production animals, either directly or via a transmission vector such as free-living protozoa, could be possible (Essig et al., 1997). However, in addition to the previously described equine biovar (Storey et al., 1993) there have been only two reports of possible C. pneumoniae infection in domesticated animals. Sako et al. (2002) described detection of C. pneumoniae antigens in vascular specimens from dogs and Canderle et al. (2005) reported high prevalence of C. pneumoniae species-specific antibodies in boars.
More targeted research efforts are necessary to determine the prevalence and host range of C. pneumoniae in environmental reservoirs of different geographical regions since most of the published reports on animal strains were simply serendipitous findings. There is also a need for the development of relevant and consistent genotyping system to be used for epidemiological surveillance, pathogenesis, evolution studies and characterization of novel C. pneumoniae isolates.
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ACKNOWLEDGEMENTS
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The Australian veterinarians who did the field work and collected the specimens are gratefully acknowledged.
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INTRODUCTION
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