INFECTION AND IMMUNITY,0019-9567/99/$04.0010

Nov. 1999, p. 6145–6151 Vol. 67, No. 11

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Chlamydia trachomatis (Mouse Pneumonitis Strain) InducesCardiovascular Pathology following Respiratory Tract Infection

YIJUN FAN, SHUHE WANG, AND XI YANG*

Laboratory for Infection and Immunity, Department of Medical Microbiology, Faculty of Medicine, University ofManitoba, Winnipeg, Manitoba, Canada R3E OW3

Received 26 June 1999/Returned for modification 28 July 1999/Accepted 12 August 1999

Chlamydia, especially Chlamydia pneumoniae, infection is closely associated with human cardiovasculardiseases. Thus far, however, few experimental studies have been carried out to investigate whether natural C.trachomatis infection can induce cardiovascular pathological changes. In this article, we report that pulmonaryinfection with C. trachomatis mouse pneumonitis strain (MoPn) can induce myocardial and perivascularinflammation and fibrosis in C57BL/6 mice. The pulmonary MoPn infection appeared to be disseminatedsystemically, because chlamydial antigens were readily detectable in multiple organs including the cardiovas-cular tissues. In addition, gamma interferon gene knockout mice with a C57BL/6 genetic background showedsignificant endocarditis and pancarditis characterized by vegetation in aortic valves, interstitial and pericar-dial inflammatory cellular infiltration, and growth of the organisms in the heart following respiratory tractMoPn infection. The results indicate that C. trachomatis can induce cardiovascular diseases following respi-ratory tract infection and suggest that murine MoPn respiratory tract infection may be a useful experimentalmodel for investigating cardiovascular diseases caused by chlamydial infection.

Heart diseases are the most common cause of death indeveloped countries. Chlamydiae, especially Chlamydia pneu-moniae, are highly associated with cardiovascular diseases in-cluding atherosclerosis (8, 11, 17, 21, 29–32). C. trachomatis,although rare, is a cause of bacterial endocardial and myocar-dial diseases (6, 7, 12, 24, 27, 28, 33). Thus far, however, fewexperimental studies have been carried out to directly investi-gate the relationship between C. trachomatis and cardiovascu-lar pathology and to examine the protective factors involvinghost defense against cardiac chlamydial infection. The majorreasons underlying the lack of experimental studies are prob-ably the lack of proper animal models for C. trachomatis-induced cardiovascular diseases and the relatively rare obser-vation of C. trachomatis-related cardiovascular diseasesdocumented in epidemiological and clinical studies.

Very recently, however, Bachmaier et al. reported that theinjection of chlamydial peptides homologous to murine heartmuscle-specific a-myosin heavy chain from various chlamydialspecies induced autoimmune heart disease in mice (2). Theauthors argued that chlamydia-mediated cardiovascular dis-eases were induced by antigenic mimicry of heart muscle-spe-cific proteins by chlamydial antigens. Interestingly, the datafrom that report demonstrated that the peptides from both C.trachomatis and C. pneumoniae exhibited homology to the im-munogenic mouse heart muscle-specific a-myosin motif andthat the peptide from C. trachomatis induced prevalent andsevere cardiac inflammation in mice. The study suggests that C.trachomatis infection could be as efficient in inducing cardio-vascular pathological changes as C. pneumoniae, at least incertain conditions. A key factor in determining the role ofchlamydial infection in cardiovascular diseases might bewhether the organism can reach the cardiovascular systemduring natural infection. We therefore hypothesized that the

relative rarity of documentation of C. trachomatis-associatedcardiovascular diseases might be at least partially because hu-man C. trachomatis strains normally cause ocular and genitaltract infections and thus may not easily disseminate to remoteorgans such as the heart, as happens with C. pneumoniae,which normally causes pulmonary infection. Indeed, previousstudies have clearly shown that pulmonary C. pneumoniae in-fections normally disseminate to multiple organs (1, 9, 10, 22,40).

A mouse model of C. trachomatis pneumonia has been es-tablished in our and others’ laboratories by using the mousepneumonitis strain of C. trachomatis (MoPn) (35, 37, 38). Un-like other C. trachomatis strains, which normally cause ocularand genital tract infections, MoPn naturally causes murinerespiratory tract infection even with a small dose of intranasalinoculator (,100 inclusion-forming units [IFU]). Therefore, interms of the location of natural infection (the lungs), MoPn ismore like C. pneumoniae than it is like other C. trachomatisstrains. However, it is not clear whether the respiratory tractMoPn infection can cause systemic dissemination, in particularto the heart, in immunologically competent mice, especiallyfollowing a short period of infection.

Gamma interferon (IFN-g) is a critical cytokine in hostdefense against pulmonary and genital chlamydial infection inboth human and animal studies (14, 18, 23, 26, 34, 36, 39).Epidemiological studies showed that patients with severe se-quelae of ocular C. trachomatis infection exhibit significantlylower levels of IFN-g production by peripheral blood mono-nuclear cells (3, 15). In vivo administration of exogenousIFN-g promoted the clearance of C. trachomatis infection,while neutralization of endogenous IFN-g with anti-IFN-gmonoclonal antibody (MAb) exacerbated C. trachomatis infec-tion in mice (41, 42). More recently, it was reported thatknockout mice deficient in IFN-g or IFN-g receptor (IFN-gKO mice) exhibit multiorgan infection following genital infec-tion with C. trachomatis, although the heart was not examinedin theses reported studies (4, 16).

The objective of the present study was to explore whetherrespiratory tract MoPn can induce systemic (including cardio-

* Corresponding author. Mailing address: Laboratory for Infectionand Immunity, Department of Medical Microbiology, Faculty of Med-icine, University of Manitoba, Room 523, 730 William Ave., Winnipeg,Manitoba, Canada R3E OW3. Phone: (204) 789-3481. Fax: (204) 789-3926. E-mail: yangxi@cc.umanitoba.ca.

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vascular system) chlamydial infection and, more importantly,whether the infection can cause pathological changes in thecardiovascular system. The results showed that immunocom-petent wild-type C57BL/6 mice intranasally infected withMoPn exhibited multiorgan dissemination of chlamydial anti-gen and a mild but significant inflammatory reaction. In par-ticular, chlamydial antigens were detected in the myocardium(myocytes) and the endothelium of cardiac blood vessels andsignificant fibrotic reactions were found in myocardial andperivascular areas. Furthermore, experiments with IFN-g KOmice showed significant chlamydial growth, massive inflamma-tory infiltrates in the heart, and large vegetations in the aorticvalves following respiratory tract MoPn infection. The datasuggest that respiratory tract C. trachomatis infection can causecardiovascular pathology and that IFN-g plays a crucial role inhost defense against cardiovascular chlamydial diseases. Thedata also suggest that murine respiratory tract MoPn infectionmay be a useful model for the study of cardiovascular diseasesinduced by chlamydial infection.

MATERIALS AND METHODS

Organism. C. trachomatis MoPn was grown in HeLa 229 cells and purified bydiscontinuous density gradient centrifugation with Renografin (Squibb, Prince-ton, N.J.) as previously described (37, 38). The infectivity of the stock chlamydialelementary bodies was determined by infection of HeLa 229 cells and enumer-ation of inclusions that were stained by an anti-chlamydial lipopolysaccharide(LPS) MAb as previously described (37, 38).

Infection of mice. Female C57BL/6 mice were purchased from Charles RiverCanada (St. Constant, Quebec, Canada). Female homozygote IFN-g KO mice (8to 12 weeks old) with C57BL/6 background (C57BL/6-Ifg,tm1Ts.) were pur-chased from Jackson Laboratory (Bar Harbor, Maine). All mice were maintainedand used in strict accordance with the guidelines issued by the Canadian Councilon Animal Care. Mice were kept in a specific-pathogen-free facility for animalsat the University of Manitoba with filtered air flow and autoclaved cage, food,and water. The mice were intranasally inoculated with C. trachomatis MoPn in 40ml of sucrose-phosphate-glutamic acid (SPG) as previously described (37). Theywere sacrificed on various days following infection to examine chlamydial growthand pathological changes in various organs. To analyze the chlamydial infectivityin various organs, homogenates of these organs were subjected to quantitativeculture of chlamydial inclusions by using HeLa 229 cells as previously described(37, 38).

Histopathological and immunohistochemical analysis. Different organs ofmice were removed and fixed in 10% buffered formalin and embedded in par-affin. Sections (5 mm) were cut and stained with hematoxylin and eosin (H & E).The structural changes and cellular infiltration in sections were determined bylight microscopy. Perivascular fibrotic change and fiber deposition in vegetationswere determined by Masson trichrome staining (19). Briefly, tissue sections (5mm) were deparaffinized and successively stained with Weigert iron hematoxylin,mixed solution of Ponceau, acid facsin and orange G, and fast green. Identifiedchlamydial inclusions (antigens) in different organs were subjected to immuno-histochemical staining with an anti-chlamydial LPS MAb. Briefly, tissue sections(5 mm) were deparaffinized and washed with phosphate-buffered saline (PBS) (5min each). After blocking with 0.3% H2O2–100% methanol for 30 min at roomtemperature, the sections were washed with PBS and blocked with 2% goat serafor 30 min. Mouse antichlamydial LPS MAb (primary Ab) was added to thetissue sections and incubated overnight at 4°C. After extensive washing with PBS,goat anti-mouse immunoglobulin G (IgG) (secondary Ab) conjugated withhorseradish peroxidase was added for 40 min at room temperature. Isotype-matched (IgG) naive Ab was used as control in the staining. Finally, the sectionswere washed, and substrate (4-chloro-1-naphthol) (Sigma, St. Louis, Mo.) wasadded. Stained inclusions were visualized by light microscopy. For staining ofinfiltrating CD4 and CD8 T cells in the tissue sections, an Envision system kit(DAKO Corp., Carpinteria, Calif.) was used. The primary Ab used in stainingwas either anti-CD4 MAb YTS-191.1 or anti-CD8 MAb YTS 169 (hybridomaskindly provided by H. Waldmann, Cambridge University). The secondary Ab wasrabbit anti-rat antibody conjugated with horseradish peroxidase. Staining wasperformed as specified by the manufacturer and was completed by incubationwith 3-amino-9-ethylcarbazole (AEC) substrate chromogen, which results in aprecipitate at the antigen sites. Normal rat IgG was used as the negative-controlprimary Ab and showed no staining in the tested tissues.

RESULTS

Intranasal MoPn infection induces systemic chlamydial in-fection and inflammation. Our previous studies showed that

intranasal infection of C57BL/6 mice with MoPn can readilycause pneumonia, demonstrated by pulmonary inflammatorycellular infiltration and recovery of chlamydial organisms fromthe lungs (37, 38). We recently examined whether viable or-ganisms can be recovered in nonlung organs of C57BL/6 micefollowing intranasal infection with 100 to 10,000 IFU of MoPn.The results showed that except for the primarily infected or-gans (the lungs), no viable (infectious) organisms were recov-ered from the organs including the liver, kidneys, and heart at3, 7, 10, and 20 days following intranasal MoPn infection (33a).However, the lack of viable (infectious) organisms in nonlungorgans cannot exclude the possibility that small amounts ofMoPn are temporarily disseminated to these organs and/orthat the disseminated organisms in these organs are inactivatedby the host defense mechanism. To test this possibility, we usedan immunohistochemical method to detect whether chlamydialantigens exist in the nonlung organs of the mice intranasallyinfected with MoPn (2,000 IFU). As shown in Fig. 1, in addi-tion to their abundance in the primarily infected organ (thelung), chlamydial antigens were detected in all the organsexamined, including the liver, kidneys and heart, of 100% ofthe infected mice (18 of 18) from four independent experi-ments in which mouse tissues were examined on day 15 or 20following intranasal infection with MoPn. Staining with a MAbspecific for chlamydial LPS demonstrated irregular shapes ofchlamydial antigen accumulation with few areas of typical in-clusion-like morphology. Tissues from naive mice withoutMoPn infection were completely negative for anti-chlamydialLPS MAb staining (data not shown). Similarly, staining withisotype-matched control Ab (normal rat IgG) of the tissuesfrom MoPn-infected mice also showed negative results (datanot shown). The existence of chlamydial antigens (inclusions)in multiple organs of the mice intranasally infected with MoPnindicates that chlamydial antigens (organisms) were dissemi-nated during respiratory tract infection. The great irregularityin the shapes of chlamydial antigen accumulation (inclusions)in these nonlung organs suggests the inactivation (inhibition)of infectious organisms by the host defense mechanism, whichmay partially explain the observed negativity of chlamydialinfectivity in these organs. It is also possible that the survival oforganisms in these organs was so low that the number oforganisms was below the sensitivity of the assay used for testingchlamydial infectivity in the tissue homogenates (the assay canonly detect chlamydial infectivity higher than 800 IFU/organbecause of the small volume of tissue homogenates which canbe placed in each culture well). In association with the exis-tence of chlamydial antigens, significant infiltration of inflam-matory cells was also determined in most of these organs,although the extent of inflammation was significantly lowerthan that in the lungs (Fig. 1E to G). In particular, 100% of theinfected mice (18 of 18) showed mild to moderate cardiacinflammation. Notably, although chlamydial antigens (inclu-sions) were clearly identified in the kidneys, the inflammationin these organs was not very significant (Fig. 1B and F). Inaggregate, the results indicate that intranasal inoculation withC. trachomatis MoPn causes not only pneumonia but also sys-temic dissemination of the infection. In particular, the exis-tence of chlamydial antigens (inclusions) and infiltration ofinflammatory cells in cardiac tissues suggest that MoPn pneu-monia may cause cardiac pathology.

MoPn infection induces perivascular inflammation, fibro-sis, and myocardial hypertrophy. To further identify whetherpulmonary MoPn infection can cause cardiac and vascularpathological changes, we examined the existence of chlamydialantigens and inflammatory reactions in the area within andsurrounding cardiac blood vessels. The results showed that all

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of the infected C57BL/6 mice exhibited chlamydial antigens inthe endothelium and/or the smooth muscle layers of cardiacblood vessels (Fig. 2A). The chlamydial antigen accumulationalso displayed irregular morphology. Perivascular inflamma-tion with lymphocytes and monocytes/macrophages was de-tected in 80% of the mice (14 of 18) (Fig. 2B). Staining ofinflammatory cells with anti-CD4 and anti-CD8 MAbs showedsignificant infiltration of CD4 cells in the perivascular inflam-mation, with few CD8 cells (data not shown). More interest-ingly, about 60% of C57BL/6 mice with MoPn pneumonia (11of 18) exhibited perivascular fibrosis in their cardiac bloodvessels. The pathological changes in cardiac blood vesselswere remarkably similar to that reported by Bachmaier etal., who showed perivascular fibrosis and inflammation in-duced by C. trachomatis peptides (2). Moreover, 40% of

mice (7 of 18) also showed interstitial fiber proliferation inthe myocardium (Fig. 3B) and about 20% of mice (4 of 18)showed myocardial hypertrophy and enlargement of nucleiof myocytes (Fig. 3D). The results indicate that respiratorytract C. trachomatis infection can indeed induce cardiovas-cular pathology, including inflammation and fibrotic changes incardiac blood vessels and changes in myocardium observed inmyocardiopathy.

IFN-g KO mice show severe myocarditis, endocarditis, andpericarditis. IFN-g is inhibitory for chlamydial growth both invitro and in vivo. We recently found that IFN-g KO mice,unlike wild-type controls, showed significant growth of chla-mydial organisms in multiple organs, including the lungs, liver,and kidneys, following intranasal infection with MoPn, dem-onstrated by constant recovery of viable organisms from these

FIG. 1. Systemic dissemination of chlamydial organisms (antigens) and inflammatory cellular infiltration to multiple organs following intranasal infection with C.trachomatis MoPn. Female C57BL/6 mice (three to five mice/group) were intranasally infected with MoPn (2,000 IFU) and sacrificed on days 15 to 20 followinginfection. (A to D) Chlamydial inclusions (antigens) were detected by immunohistochemical staining with an anti-chlamydial LPS MAb in the liver (A), kidneys (B),heart (C), and lungs (D). (E to H) Histological structure and inflammatory infiltration (arrows) were examined by H & E staining in the liver (E), kidneys (F), heart(G), and lungs (H). The experiments were repeated four times, and similar results were obtained. Representative histological changes are shown. Magnification, 3400(A to D) and 3200 (E to H).

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organs (33a). In the present study, we further examined chla-mydial infectivity in the hearts of intranasally infected IFN-gKO mice. As shown in Fig. 4, unlike wild-type controls, IFN-gKO mice repeatedly showed chlamydial infectivity in the heart.Immunohistochemical staining with anti-chlamydia LPS MAbalso showed chlamydial inclusions in the cardiac tissues of

IFN-g KO mice (Fig. 5B). The morphology of the inclusions(chlamydial antigen accumulations) in the myocardium ofIFN-g KO mice was also variable but more closely resembledthe shape of chlamydial inclusions seen in in vitro HeLa cellcultures. Histological analysis showed much more profoundpathological changes in the heart of IFN-g KO mice comparedwith wild-type mice described above (Fig. 1G). All IFN-g KOmice (17 of 17) displayed massive inflammatory infiltration inthe interstitial areas of the heart (Fig. 5A). Perivascular cellu-lar infiltration, especially surrounding small blood vessels, wasonly occasionally found in IFN-g KO mice. Interestingly, al-though all IFN-g KO mice showed severe cardiac inflamma-tion, most of them (16 of 17) did not show fibrosis in cardiacblood vessels (data not shown). Moreover, 45% of the IFN-gKO mice (4 of 9) whose aortic valves were examined showedchanges of endocarditis characterized by aortic valve vegeta-tions (Fig. 5C). In contrast, none of the infected wild-type mice(0 of 7) whose aortic valves were examined showed vegetation.The vegetations in IFN-g KO mice were large and were seenonly on aortic valves. Vegetations contained mononuclear cellsand polymorphonuclear cells admixed with fiber (Fig. 5D).Chlamydial organisms (antigens) were also detected in thevegetations after histoimmunological staining with anti-chlamydial LPS MAb (Fig. 5E). Inflammatory infiltrationwas also found in pericardial areas of 50% of the IFN-g KOmice (9 of 17), with a trend to more severe inflammation inthe position close to the root of aorta (Fig. 5F and G) andcomposed of a significant number of CD4 cells (Fig. 5H).Five naive (uninfected) wild-type and five naive IFN-g KOmice were also examined for cardiac inflammation, vegeta-tion, and vascular fibrosis, and none of them showed positivefindings. Taken together, the results showed that IFN-g KOmice suffered more serious cardiovascular chlamydial infec-tion than did wild-type mice, suggesting that IFN-g plays acritical role in preventing massive dissemination of chlamyd-ial infection, thus preventing chlamydial endocarditis andpancarditis.

DISCUSSION

In the present study, we used a unique C. trachomatisstrain (MoPn), which normally causes respiratory tract in-fection in mice, to explore whether pulmonary C. trachoma-tis infection can induce cardiovascular pathological changes.The data clearly show that respiratory tract infection with C.trachomatis is able to cause systemic and myocardial chla-mydial infection and, probably more importantly, inducefibrotic changes in cardiac blood vessels. Moreover, thepresent study showed that about 40% of mice exhibitedfibrotic changes in the myocardium and that 20% of miceexhibited myocardiopathy, suggesting that C. trachomatismay be an important causative agent of clinical cardiomy-opathy. Notably, there are reported clinical cases of chroniccardiomyopathy caused by chlamydial infection (25). Thepresent study, on one hand, demonstrates that C. trachoma-tis in certain conditions can induce cardiovascular diseasesincluding perivascular fibrosis and, on the other hand, sug-gests that the intranasal-infection model of MoPn in micemay be useful for investigating cardiovascular diseasescaused by chlamydial infection. This study, using a naturalmurine C. trachomatis infection model, confirmed and ex-tended the very recent finding made by Bachmaier et al. ofvascular inflammatory and fibrotic changes caused by theantigens of C. trachomatis (2). Since C. trachomatis infectionis common in humans and since the heart is one of theorgans infected by the agent, we speculate that the real

FIG. 2. Pathological change of cardiac blood vessels following intranasalinfection with C. trachomatis MoPn. The mice in Fig. 1 were examined forpathological changes in their cardiac blood vessels. (A) Chlamydial inclusions(antigens) in the walls of blood vessels were detected by immunohistochemicalstaining with an anti-chlamydial LPS MAb. (B and C) Perivascular cellularinfiltrations are shown by H & E staining (B), and the fibrotic changes inperivascular areas were determined by Masson trichrome staining (C). Experi-ments were repeated four times, and representative pathological changes areshown. Magnification, 3400.

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prevalence of myocarditis and cardiomyopathy may be sig-nificantly higher than that previously documented. It shouldbe noted, however, that the cardiovascular pathologicalchanges caused by MoPn infection observed in this study arenot the same as the characteristic vascular pathology asso-ciated with C. pneumoniae, i.e., arthrosclerosis. Furtherstudy is required to examine the role of cardiovascular in-fection with MoPn following intranasal inoculation of or-ganism in the formation of arthrosclerosis.

Another very interesting finding in this study is that IFN-gKO mice intranasally infected with MoPn exhibited endocar-

ditis and pericarditis. Endocarditis caused by C. trachomatisinfection has been reported in previous clinical studies (6, 7,24, 33). Several individual cases of chlamydial endocarditiscaused by C. pneumoniae have also been documented in clin-ical practice over the past decade (5, 13, 20). Thus far, how-ever, no experimental study has been carried out to investigatethe mechanism for this endocarditic disorder. In particular, itis not clear why only a few individuals among a large popula-tion with C. trachomatis infection suffer chlamydial endocardi-tis. In the present study, we found that IFN-g KO, but notwild-type, mice showed severe aortic vegetation and pancardi-tis following respiratory tract MoPn infection. The results sug-gest that chlamydial endocarditis, particularly vegetation for-mation, occurs only when massive infection exists and alsosuggest that IFN-g is very efficient in preventing the formationof endocarditis and vegetation. The observation that IFN-gKO mice suffer remarkably more severe cardiovascular infec-tion but significantly less vascular fibrosis may be due to theongoing acute inflammation in these mice; thus, healing orfibrotic reaction does not occur, although the possibility thatIFN-g is involved in the process of fibrotic reaction cannot beexcluded.

In conclusion, the study suggests that disseminated C. tra-chomatis infection can induce cardiovascular diseases includ-ing perivascular fibrosis. Severe endocarditis and pericarditisoccur when the host fails to control the infection, leading tomassive growth of the organism and severe systemic dissemi-nation of the infection. The mouse lung infection model mayprovide a useful system for studying the mechanism and ther-

FIG. 3. Fibrotic and degenerative changes in the myocardium following pulmonary C. trachomatis MoPn infection. Mice were examined for fibrosis by usingMasson’s trichrome staining. (A) Trichrome stain of normal myocardium and blood vessels. (B) Trichrome stain of heart tissue collected from mice intranasally infectedwith MoPn (Fig. 1), showing fibrotic changes in the myocardium and perivascular areas. (C) H & E staining of normal myocardium. (D) H & E staining of themyocardium of MoPn-infected mice, showing myocardial hypertrophy and enlargement of nuclei. Experiments were repeated four times, and representativepathological changes are shown. Magnification, 3400.

FIG. 4. Significant growth of C. trachomatis MoPn in the heart followingintranasal infection. Wild-type and IFN-g KO mice were intranasally infectedwith MoPn (2,000 IFU) and sacrificed on day 15 postinfection. The homogenatesof the cardiac tissues were analyzed for in vivo chlamydial growth as described inMaterials and Methods. Each point represents the mean and standard deviationof log10 IFU for five mice. One of two independent experiments with similarresults is shown.

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apeutic approaches for chlamydia-induced cardiovascular dis-eases.

ACKNOWLEDGMENTS

We thank R. C. Brunham for reading the manuscript and for valu-able discussions.

This work was supported by grants from the Medical ResearchCouncil of Canada (MRC), Manitoba Medical Service Foundation andManitoba Health Research Council. X.Y. holds a salary award (Schol-ar) from the MRC.

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FIG. 5. Severe cardiac pathology in IFN-g KO mice following intranasal infection with C. trachomatis MoPn. IFN-g KO mice were intranasally infected with MoPn(2,000 IFU) and sacrificed on days 15 to 20 postinfection. Photomicrographs of H & E-stained sections show massive interstitial inflammation (A), vegetation on theaortic valve (C), pericardiac inflammation (F), and massive cellular infiltration at the area close to the root of the aorta (G). Immunohistochemical staining withanti-chlamydial LPS MAb shows chlamydial inclusions (antigens) in myocytes (B) and within the vegetation (E). Trichrome staining shows fiber (green) in the aorticvegetation (D). CD4 cell staining shows periaortic infiltration of CD4 cells. (H) The experiments were repeated four times, and similar results were obtained.Representative histological changes are shown. Magnifications, 3400 (A and B) and 3200 (C to H).

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