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A comparative ultrastructural and molecular biological study on Chlamydia psittaci infection in alpha-1 antitrypsin deficiency and non-alpha-1 antitrypsin deficiency emphysema versus lung tissue of patients with hamartochondroma
© Theegarten et al; licensee BioMed Central Ltd. 2004
Received: 02 July 2004
Accepted: 21 September 2004
Published: 21 September 2004
Chlamydiales are familiar causes of acute and chronic infections in humans and animals. Human pulmonary emphysema is a component of chronic obstructive pulmonary disease (COPD) and a condition in which chronic inflammation manifested as bronchiolitis and intra-alveolar accumulation of macrophages is common. It is generally presumed to be of infectious origin. Previous investigations based on serology and immunohistochemistry indicated Chlamydophila pneumoniae infection in cases of COPD. Furthermore, immunofluorescence with genus-specific antibodies and electron microscopy suggested involvement of chlamydial infection in most cases of pulmonary emphysema, but these findings could not be verified by PCR. Therefore, we examined the possibility of other chlamydial species being present in these patients.
Tissue samples from patients having undergone lung volume reduction surgery for advanced alpha-1 antitrypsin deficiency (AATD, n = 6) or non-alpha-1 antitrypsin deficiency emphysema (n = 34) or wedge resection for hamartochondroma (n = 14) were examined by transmission electron microscopy and PCR.
In all cases of AATD and 79.4% of non-AATD, persistent chlamydial infection was detected by ultrastructural examination. Intra-alveolar accumulation of macrophages and acute as well as chronic bronchiolitis were seen in all positive cases. The presence of Chlamydia psittaci was demonstrated by PCR in lung tissue of 66.7% AATD vs. 29.0% non-AATD emphysema patients. Partial DNA sequencing of four positive samples confirmed the identity of the agent as Chlamydophila psittaci. In contrast, Chlamydophila pneumoniae was detected only in one AATD patient. Lung tissue of the control group of non-smokers with hamartochondroma was completely negative for chlamydial bodies by TEM or chlamydial DNA by PCR.
These data indicate a role of Chlamydophila psittaci in pulmonary emphysema by linking this chronic inflammatory process to a chronic infectious condition. This raises interesting questions on pathogenesis and source of infection.
Several species of the family Chlamydiaceae are well-known etiological agents of acute and chronic infections in humans and animals [1, 2]. The first description of chlamydial respiratory disease in humans referred to psittacosis, also known as ornithosis, and dates back to 1879 . Chlamydia (C.) psittaci, the agent responsible for this disease, has had several different names and, according to a recent proposal, should now be called Chlamydophila (Cp.) psittaci . A century later, in 1986, Grayston et al. discovered another chlamydial respiratory agent, strain TWAR, which was later assigned to the species C. pneumoniae [5, 6] currently reclassified as Chlamydophila pneumoniae . Meanwhile, a variety of respiratory conditions in humans has been shown to be associated with this agent. Evidence of Cp. pneumoniae infection based on serology was reported in severe cases of chronic obstructive pulmonary disease (COPD), in which emphysema is dominant [7, 8], as well as in exacerbations of COPD  and in persistent infections of the respiratory tract [10, 11]. The detection rate of Cp. pneumoniae by immunohistochemical staining was elevated in lung tissue from subjects with COPD, but controls were not completely negative . Initially Cp. pneumoniae was thought to be virulent for humans only, but recent descriptions of isolates from horse, koala, frog and reptiles suggest a wider host spectrum and even the possibility of zoonotic transmission [13–15].
Our previous investigations by means of immunofluorescence using a genus-specific antiserum against chlamydial LPS and scanning as well as transmission electron microscopy showed infection of the alveolar parenchyma and the bronchioles by Chlamydia spp. in patients having undergone lung volume reduction surgery for advanced pulmonary emphysema [16, 17]. Accumulation of alveolar macrophages as well as different forms of bronchiolitis and focal pneumonia accompanying emphysematic changes were found regularly . In preliminary examinations using an established nested PCR with DNA hybridization , DNA specific of Cp. pneumoniae was detected in two out of ten cases . But this detection rate was far lower than that in electron microscopy or immunofluorescence using genus-specific antibodies, which showed Chlamydia spp. in over 80% . Because of this fact PCR was extended to other Chlamydiaceae. Here we report the results of a more detailed study involving a larger number of cases and samples including controls.
Lung tissue of adequate quality from patients with advanced emphysema undergoing lung volume reduction surgery was used for the present study [18, 21]. Samples examined by transmission electron microscopy (TEM) included five specimens from alpha-1 antitrypsin deficiency (AATD) and 34 from non-AATD patients. PCR examinations were conducted on six AATD and 31 non-AATD specimens. History showed a status of cigarette smoking with over ten packyears in most of these patients (91.7%). There were two non-smokers among nine patients with AATD. Furthermore patients with hamartochondroma undergoing wedge resection were reviewed for clinical data (A.M.) and histology. Normal lung tissue of 14 non-smokers taken with resection of hamartochondroma was selected as a control group. Statistical analysis was done using SPSS, version 11.5 (SPSS Inc., Chicago, USA) on a PC running Windows XP Professional (Microsoft, Redmond, USA) as operating system. A test value below 0.05 was considered to be statistically significant.
Light Microscopy and Transmission Electron Microscopy (TEM)
Formalin-fixed lung tissue was embedded in paraffin wax (Tissuewax™; Medite GmbH, Burgdorf, Germany), slides of 3–7 μm thickness were cut using a rotatory microtome (Microm GmbH, Walldorf, Germany) and stained by hematoxylin and eosin. For TEM, tissue was fixed in 2.5% buffered glutaraldehyde or 3.5% formaldehyde and embedded in epon after postfixation with osmium tetroxide and block contrastation with uranyl acetate. In the cases of the hamartochrondroma control group, cores with diameters of 0.60 cm and 0.24 cm were obtained from paraffin blocks using a prototypical self-made manual tissue puncher (a device for tissue microarray construction developed by M.W.). These cores were used to select an area well defined by light microscopy for PCR (0.60 cm cores) and TEM (0.24 cm cores). For TEM, tissue was dewaxed with xylene and processed as described above. Semithin sections (prepared on a Reichert Om U3 ultramicrotome; Reichert, Vienna, Austria) were stained with basic fuchsin and methylene blue to define blocks of adequate quality. Ultrathin sections from two to five blocks were stained with lead citrate and examined using a Zeiss EM 900 transmission electron microscope (Zeiss, Oberkochen, Germany).
Polymerase Chain Reaction (PCR)
Tissue from two different resources was used. Firstly, frozen tissue was collected immediately after resection and stored at -80°C (n = 31). Secondly, paraffin-embedded tissue (PET) containing histologically discernible inflammation sites was used, and sections or 0.60 cm cores were dewaxed using xylene (n = 12 and 14 controls). In five cases, material was available as both frozen and PET. DNA was isolated from lung tissue using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to the instructions of the manufacturer. Five μl of the DNA extract were used as template in PCR.
Samples were tested forC. psittaci and Cp. pneumoniae by a modified version of the nested PCR procedure described by Kaltenböck et al. , which targets the ompA gene. The first step was genus-specific amplification using primers 191CHOMP (5'-GCI YTI TGG GAR TGY GGI TGY GCI AC-3') and CHOMP371 (5'-TTA GAA ICK GAA TTG IGC RTT IAY GTG IGC IGC-3'). For the second amplification, we used 1 μl of the genus-specific product and primer combination 218PSITT (5'-GTA ATT TCI AGC CCA GCA CAA TTY GTG-3') / CHOMP336s (5'-CCR CAA GMT TTT CTR GAY TTC AWY TTG TTR AT-3') for C. psittaci, or 201CHOMP (5'-GGI GCW GMI TTC CAA TAY GCI CAR TC-3') / PNEUM268 (5'-GTA CTC CAA TGT ATG GCA CTA AAG A-3'), for Cp. pneumoniae, respectively. The sizes of specific amplicons are: 576–597 bp (genus-specific product), and 389–404 bp for C. psittaci or 244 bp for Cp. pneumoniae after nested PCR. A detailed protocol of the procedure is contained in .
In order to discriminate the different members of the C. psittaci-group , five μl of the final DNA extract served as template for PCR amplification of the 16S rRNA signature region. Amplicon bands were cut out of agarose gels, extracted using the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), and subjected to cycle sequencing using the BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Darmstadt, Germany). The oligonucleotides 16SIGF (5'-CCG CGT GGA TGA GGC AT-3') and 16SIGR (5'-TCA GTC CCA GTG TTG GC-3') were used as amplification and sequencing primers . Nucleotide sequences were determined on an ABI Prism 310 Genetic Analyzer (Applied Biosystems).
Transmission electron microscopy
Detection of Chlamydia spp. in emphysema by TEM and PCR
Fisher Yates test vs. controls
Non AATD emphysema
Polymerase Chain Reaction (PCR)
Strains of Cp. psittaci are known to cause infections in over 130 avian species and 32 other domestic and wild animals. Classical psittacosis represents a systemic disease in psittacine birds of acute, protracted, chronic or subclinical manifestation. Avian strains of the agent are known to be pathogenic to humans, the symptoms being mainly non-specific and influenza-like, but severe pneumonia, endocarditis and encephalitis are not uncommon [24, 25]. The possibility of persistent infection in man was first described in the 1950s .
In the present study and in previous investigations, transmission electron microscopy revealed elementary bodies as well as typical and aberrant reticular bodies, thus indicating active infection alongside persistent infection [10, 16, 17]. Chlamydiae could not be detected in each case, but the rate of positive findings in TEM and PCR (Table 1) was comparable to that of Cp. pneumoniae in atherosclerosis [27, 28]. Pear-shaped elementary bodies as typically found in Cp. pneumoniae infection  were not observed. While the findings of TEM are more indicative of Cp. psittaci infection , it must be noted that this method provides no clear-cut differentiation among Chlamydiaceae species, for even strains of the same species exhibit different morphology at the various developmental stages. Rather unexpectedly, Cp. pneumoniae was detected only in one sample by PCR, not indicating an important role of this agent in the cases examined here. The fact that, apart from psittacosis, Cp. psittaci has not been associated with human respiratory disorders in recent decades may be a question of sensitivity and specificity of detection. Particularly PCR with its capability to specifically identify all individual species of Chlamydiaceae at a detection limit of less than one inclusion-forming unit has opened up new possibilities in this respect.
Higher detection rates of Cp. psittaci in tissue with histological evidence of inflammation in comparison to unselected frozen tissue (6/12 = 50% vs. 7/32 = 21.9%, Fisher Yates test n.s.) indicate an association with regional activity of infection and inflammation. Besides psittacosis, Cp. psittaci has been recently associated with chronic inflammation in patients with ocular adnexal lymphomas . In COPD, activated macrophages and neutrophils produce matrix metalloproteinases which are relevant in the development of emphysema [32, 33]. The release of matrix metalloproteinases was shown to be stimulated by cytokines produced in the course of chlamydial infection  and by chlamydial heat shock protein 60 as well . These findings represent a link to the established pathogenetic concepts in pulmonary emphysema. Cp. psittaci was found at comparable and statistically significant rates in AATD and non AATD emphysema.
The fact that the chlamydial agent present in the emphysema tissue was identified by DNA sequencing as an avian serovar of Cp. psittaci provides an important indication on the source of infection. It is conceivable that the patients were infected through contact with birds, although this could not be verified for lack of relevant data on history. The detection rate of chlamydiae in cases of AATD emphysema vs. non-AATD emphysema was clearly higher, thus indicating a relevant role of Cp. psittaci infection in this disorder, or at least a higher susceptibility of AATD patients for an infection of their lungs with Cp. psittaci. Further investigations concerning smokers and non-smokers, pathogenetic relevance and zoonotic implications are required. In any circumstances, Cp. psittaci has to be considered an underestimated pathogen with considerable importance in public health, although the various facets of its specific impact have yet to be evaluated.
- Peeling RW, Brunham RC: Chlamydiae as pathogens. New species and new issues. Emerg Infect Dis. 1996, 2: 307-319.View ArticlePubMedPubMed CentralGoogle Scholar
- Schachter J: Infection and disease epidemiology. In Chlamydia. Intracellular biology, pathogenesis, and immunity. Edited by: Stephens RS. 1999, Washington: American Society for Microbiology Press, 139-169.Google Scholar
- Ritter J: Beitrag zur Frage des Pneumotyphus. Eine Hausepidemie in Uster (Schweiz) betreffend. Dtsch Arch Klin Med. 1879, 25: 53-96.Google Scholar
- Everett KDE, Bush RM, Andersen AA: Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. Int J Syst Bacteriol. 1999, 49: 415-440.View ArticlePubMedGoogle Scholar
- Grayston JT, Kuo CC, Wang SP, Altman J: A new Chlamydia psittaci strain, TWAR, isolated in acute respiratory tract infection. New Engl J Med. 1986, 315: 161-168.View ArticlePubMedGoogle Scholar
- Grayston JT, Wang S-P: History of Chlamydia pneumoniae (TWAR). In Chlamydia pneumoniae. The Lung and the Heart. Edited by: Allegra L, Blasi F. 1999, Milan: Springer, 1-8.View ArticleGoogle Scholar
- Hertzen Lv, Alakärppä H, Koskinen R, Liippo K, Surcel H-M, Leinonen M, Saikku P: Chlamydia pneumoniae infection in patients with chronic obstructive pulmonary disease. Epidemiol Infect. 1997, 118: 155-164. 10.1017/S095026889600725X.View ArticleGoogle Scholar
- Hahn DL: Chlamydia pneumoniae, asthma, and COPD: what is the evidence?. Ann Allergy Asthma Immunol. 1999, 83: 339-344.View ArticleGoogle Scholar
- Mogulkoc N, Karakurt S, Isalska B, Bayindir Ü, Celikel T, Korten V, Colpan N: Acute purulent exacerbation of chronic obstructive pulmonary disease and Chlamydia pneumoniae infection. Am J Respir Crit Care Med. 1999, 160: 349-353.View ArticlePubMedGoogle Scholar
- Beatty WL, Morrison RP, Byrne G: Persistent Chlamydiae. From cell culture to a paradigm for chlamydial pathogenesis. Microbiol Rev. 1994, 58: 686-699.PubMedPubMed CentralGoogle Scholar
- Hammerschlag MR, Chirgwin K, Roblin PM, Gelling M, Dumornay W, Mandel L, Smith P, Schachter J: Persistent infection with Chlamydia pneumonia following acute respiratory illness. Clin Infect Dis. 1992, 14: 178-182.View ArticlePubMedGoogle Scholar
- Wu L, Skinner SJM, Lambie N, Vuletic JC, Blasi F, Black PN: Immunohistochemical staining forChlamydia pneumoniae is increased in lung tissue from subjects with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2000, 162: 1148-1151.View ArticlePubMedGoogle Scholar
- Berger L, Volp K, Mathews S, Speare R, Timms P: Chlamydia pneumoniae in a free-ranging giant barred frog (Mixophyes iteratus) from Australia. J Clin Microbiol. 1999, 37: 2378-2380.PubMedPubMed CentralGoogle Scholar
- Hotzel H, Grossmann E, Mutschmann F, Sachse K: Genetic characterization of a Chlamydophila pneumoniae isolate from an african frog and comparison to currently accepted biovars. Syst Appl Microbiol. 2001, 24: 63-66.View ArticlePubMedGoogle Scholar
- Bodetti TJ, Jacobson E, Wan C, Hafner L, Pospischil A, Rose K, Timms P: Molecular evidence to support the expansion of the hostrange of Chlamydophila pneumoniae to include reptiles as well as humans, horses, koalas and amphibians. Syst Appl Microbiol. 2002, 25: 146-152.View ArticlePubMedGoogle Scholar
- Theegarten D, Stamatis G, Morgenroth K: The role of persisting infections in the pathogenesis of pulmonary emphysema. Electron microscopy reveals a probable bacterial colonization of the alveolar space and the bronchioles. Pathol Res Pract. 1999, 195: 89-92.View ArticlePubMedGoogle Scholar
- Theegarten D, Mogilevski G, Anhenn O, Stamatis G, Jaeschock R, Morgenroth K: The role of Chlamydia in the pathogenesis of pulmonary emphysema. Electron microscopy and immunofluorescence reveal corresponding findings as in atherosclerosis. Virchows Arch. 2000, 437: 190-193. 10.1007/s004280000242.View ArticlePubMedGoogle Scholar
- Theegarten D, Teschler H, Stamatis G, Konietzko N, Morgenroth K: Pathologisch-anatomische Befunde bei der chirurgischen Lungenvolumenreduktion des fortgeschrittenen Emphysems. Pneumologie. 1998, 52: 702-706.PubMedGoogle Scholar
- Maass M, Bartels CB, Engel PM, Mamat U, Sievers H-H: Endovascular presence of viable Chlamydia pneumoniae is a common phenomenon in coronary artery disease. J Am Coll Cardiol. 1998, 31: 827-831. 10.1016/S0735-1097(98)00016-3.View ArticlePubMedGoogle Scholar
- Theegarten D, Mogilevski G, Anhenn O, Stamatis G, Maass M, Morgenroth K: Evidence for chronic Chlamydia pneumoniae infection in pulmonary emphysema. Eur Respir J. 2000, 16 (Suppl 31): 333s-Google Scholar
- Cassina PC, Teschler H, Konietzko N, Theegarten D, Stamatis G: Two-year results after lung volume reduction surgery in alpha-1-antitrypsin deficiency versus smoker's emphysema. Eur Respir J. 1998, 12: 1028-1032. 10.1183/09031936.98.12051028.View ArticlePubMedGoogle Scholar
- Kaltenböck B, Schmeer N, Schneider R: Evidence for numerous omp 1 alleles of porcine Chlamydia trachomatis and novel chlamydial species obtained by PCR. J Clin Microbiol. 1997, 35: 1835-1841.PubMedPubMed CentralGoogle Scholar
- Sachse K, Hotzel H: Detection and differentiation of chlamydiae by nested PCR. In PCR detection of microbial pathogens: Methods and protocols. Edited by: Sachse K, Frey J. 2002, Meth Mol Biol, 216: 231-246.View ArticleGoogle Scholar
- Crosse BA: Psittacosis: a clinical review. J Infect. 1990, 21: 251-259.View ArticlePubMedGoogle Scholar
- Salisch H, Malottki Kv, Ryll M, Hinz K-H: Chlamydial infections of poultry and human health. Worlds Poult Sci J. 1996, 52: 279-308.View ArticleGoogle Scholar
- Meyer KF, Eddie B: Human carrier of the psittacosis virus. J Infect Dis. 1951, 88: 109-125.View ArticlePubMedGoogle Scholar
- Campbell LA, Kuo C-C, Grayston JT: Chlamydia pneumoniae and cardiovascular disease. Emerg Infect Dis. 1998, 4: 571-579.View ArticlePubMedPubMed CentralGoogle Scholar
- Shor A: A pathologist's view of organisms and human atherosclerosis. J Infect Dis. 2001, 183: 1428-1429. 10.1086/319873.View ArticlePubMedGoogle Scholar
- Chi EY, Kuo CC, Grayston JT: Unique ultrastructure in the elementary body of Chlamydia sp. strain TWAR. J Bacteriol. 1987, 169: 3757-3763.PubMedPubMed CentralGoogle Scholar
- Vanrompay D, Charlier G, Ducatelle R, Haesebrouck F: Ultrastructural changes in avian Chlamydia psittaci serovar A-, B-, and D-infected Buffalo Green Monkey cells. Infect Immun. 1996, 64: 1265-1271.PubMedPubMed CentralGoogle Scholar
- Ferreri AJ, Guidoboni M, Ponzoni M, De Conciliis C, Dell'Oro S, Fleischhauer K, Caggiari L, Lettini AA, Dal Cin E, Ieri R, Freschi M, Villa E, Boiocchi M, Dolcetti R: Evidence for an association between Chlamydia psittaci and ocular adnexal lymphomas. J Natl Cancer Inst. 2004, 96: 586-594. 10.1093/jnci/djh102.View ArticlePubMedGoogle Scholar
- Barnes P: Chronic obstructive pulmonary disease. New Engl J Med. 2000, 343: 269-280. 10.1056/NEJM200007273430407.View ArticlePubMedGoogle Scholar
- Shapiro SD: The macrophage in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 1999, 160: S29-32.View ArticlePubMedGoogle Scholar
- Rasmussen SJ, Eckmann L, Quayle AJ, Shen L, Zhang Y-X, Anderson DJ, Fierer J, Stephens RS, Kagnoff MF: Secretion of proinflammatory cytokines by epithelial cells in response to Chlamydia infection suggests a central role for epithelial cells in Chlamydial pathogenesis. J Clin Invest. 1997, 99: 77-87.View ArticlePubMedPubMed CentralGoogle Scholar
- Kol A, Sukhova GK, Lichtman AH, Libby P: Chlamydial heat shock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-α and matrix metalloproteinase expression. Circulation. 1998, 98: 300-307.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/4/38/prepub
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