INFECTION AND IMMUNITY, May 1993, p. 2037-20400019-9567/93/052037-04$02.00/0Copyright © 1993, American Society for Microbiology

A Mouse Model of Chlamydia pneumoniae StrainTWAR Pneumonitis

ZI-PING YANG,1 CHO-CHOU KUO,l* AND J. THOMAS GRAYSTON1'2

Departments ofPathobiology' and Epidemiology, 2 University of Washington,Seattle, Washington 98195

Received 16 November 1992/Accepted 26 February 1993

Chlamydia pneumoniae is a common cause of acute respiratory infections in humans. We evaluated mice as

experimental animals for C. pneumoniae. Intranasal inoculation of Swiss Webster mice with C. pneumoniaeinduced a prolonged course of lung infection, as demonstrated by reisolation of organisms from lungs (at 42days) and persistence of lung pathology (>60 days). The lung pathology was characterized by patchy interstitialpneumonitis with predominately polymorphonuclear leukocyte infiltration in the eariy and mononuclear cellinfiltration in the later stages of infection. Inoculated mice developed serum immunoglobulin G antibodyresponses and partial resistance against rechallenge inoculation. The other mouse strains tested, Icr,BALB/cAnN, C57BL/6N, C3IIVHeN, and B6C3F1, were shown to be susceptible to C. pneumoniae. The mousemodel should be useful for investigating the immunopathogenesis of C. pneumoniae infections.

Chlamydia pneumoniae strain TWAR has been recog-nized as the third species of Chlamydia (7). So far, only oneserovar or strain (TWAR) has been found. The TWARorganism has been previously established as an importantcause of respiratory infections, being associated with 5 to10% of cases of pneumonia, bronchitis, and sinusitis (5, 6).Infection with TWAR is very common. Population antibodyprevalence studies have shown that more than 50% of adultsworldwide have antibody (5, 6). While antibody is infrequentin children under the age of 5, incidence studies have shownantibody conversion of 6 to 9%/year in children from theages of 5 to 14, with lower rates in older persons (1). Theprevalence of antibody continues to increase throughoutadulthood, and it is highest in the elderly. TWAR infections,which may be more asymptomatic in the young, often resultin pneumonia in the elderly. The organism is susceptible tomacrolides and tetracyclines. Treatment of symptomaticillness is often successful, but eradication of the organism isdifficult. Recently, C. pneumoniae has been associated withatherosclerosis and coronary artery disease (14, 17).No information is available on the pathology of C. pneu-

moniae infections, and no successful animal model has beenreported.Mice are a natural host for two pneumonia-causing chlamy-

dial agents, the meningopneumonitis strain of Chlamydiapsittaci (2, 4, 9) and the mouse pneumonitis strain of Chlamy-dia trachomatis (15). Intranasal inoculation of mice with thehuman biovars of C. trachomatis, i.e., trachoma and lympho-granuloma venereum, has been previously shown to inducepneumonitis (3, 8, 12). In classifying TWAR as belonging to a

separate species, C pneumoniae, we showed that mice weresusceptible to intranasal inoculation with C. pneumoniae (11).Here we report on a mouse model of pneumonitis that

should be useful for studying the pathogenesis of C. pneu-moniae pneumonia.

MATERIALS AND METHODS

C. pneumoniae strain and inoculum preparation. TWARstrain AR-39, a pharyngeal isolate, was used in this study (7).

* Corresponding author.

AR-39 was originally isolated and propagated in HeLa 229cell culture (11). Infected cells were harvested with sterileglass beads and ultrasonically disrupted. The cell culture-grown organisms were partially purified by 1 cycle each oflow- and high-speed centrifugation, resuspended in sucrose-

phosphate-glutamic acid (SPG) buffer, and frozen in 0.5-mlaliquots at -70°C. The inoculum preparations contained 5.3x 10' to 6.6 x 108 inclusion-forming units (IFU) of organ-

isms per ml.Experimental animals. Several strains of 4- to 5-week-old

male mice were used, including outbred mice (Swiss Web-ster and Icr), inbred mice (BALB/cAnN, C57BL/6N, andC3H/HeN), and hybrid mice (B6C3F1) (Simonsen Labora-tory Company, Gilroy, Calif.).

Inoculation of mice. Mice were inoculated with the organ-

ism suspension or SPG buffer by the intranasal route. Micewere lightly anesthetized by ether inhalation to inducehyperventilation. Five drops (0.05 ml) of inoculum was

delivered onto the nostrils by a 23-gauge needle attached toa syringe. Delivery of inoculum was timed to the inhalationphase of respiration. Each mouse was given 2.7 x 107 to 3.3X 107 IFU of organisms unless otherwise specified.

Infectivity assay. Mice were killed by axillary bleedingunder ether anesthesia. Blood was saved to obtain serum forserological assay. Lungs were removed in toto, includingtrachea and bronchi. Lungs were weighed, minced withscissors, and homogenized with a mortar and pestle to makea 10% (wt/vol) suspension in cold SPG buffer. Tissue sus-pensions were centrifuged at 500 x g for 10 min at 4°C toremove coarse tissue debris and were frozen at -70°C untiltested. The infectious titer was assayed by titration of tissuehomogenates in HL cells (13) grown on a 12-mm-diameterround coverslip in a flat-bottom, 1-dram (ca. 4-ml) shell vial.Inoculated cells were incubated at 36°C for 4 days. Infectedcells were fixed in acetone (19) and stained with a Chlamydiagenus-specific monoclonal antibody (CF-2) conjugated tofluorescein isothiocyanate (11). Inclusion bodies werecounted under a fluorescence microscope. The infectivitytiter was expressed as log1o IFU per gram of lung.

Serology. Serum antibody was detected by the microim-munofluorescence test with formalin-fixed whole elementarybodies of AR-39 as antigen (18). Both immunoglobulin G

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TABLE 1. Comparison of susceptibilities of six strains of mice tointranasal inoculation with C pneumoniaea

Result after isolation of organismsfrom lungs'

Mouse GeneticMstoraine back- No. of No. of Mean titer +

grud deaths! positives! SD" (logno. tested no. testedc IFU/g of

lung)

Swiss Webster Outbred 0/5 5/5 5.18 ± 0.46BALB/cAnN Inbred 1/8 4/5 5.11 ± 0.30C3H/HeN Inbred 0/6 6/6 4.94 ± 0.50C57BL/6N Inbred 4/8 3/4 4.85 ± 1.17Icr Outbred 0/8 5/6 4.35 ± 0.75B6C3F1 Hybrid 2/8 4/5 3.92 + 1.12

a Intranasal inoculation was with 2.7 x 10i IFU of organisms per mouse.b At day 11 postinoculation.Denominators exclude those mice that died before day 11 or that were

used for histopathological study.I Averages for positive mice.

(IgG) and IgM serum antibody fractions were measured withfluorescein isothiocyanate-conjugated goat anti-mouse IgGand IgM (heavy-chain specific; Sigma Company, St. Louis,Mo.). Antibodies were first screened with a single dilution ofsera at 1:8. Positive sera were titrated by serial twofolddilutions.

Histopathology. Lung tissues from infected and controlmice were removed from days 2 to 60 and immediately fixedin 10% buffered formalin. Specimens were embedded inparaffin, sectioned, and stained with hematoxylin and eosin.

Determination of intranasal ID50. Serial 10-fold dilutions ofinoculum were prepared in SPG buffer. Each dilution wasinoculated intranasally into four mice. Mice were killed onday 4 postinoculation to obtain lungs for the infectivityassay. The 50% intranasal infective dose (intranasal ID50)was calculated by the method of Reed and Muench (16).

RESULTS

Susceptibilities of different strains of mice to C. pneumoniae.To select the most suitable strain of mouse for our experi-ments, we compared the susceptibilities of the six strains ofmice. On the basis of preliminary studies, each mouse wasinoculated with 2.7 x 107 IFU of organisms and the infec-tivity assay was done on day 11. As shown in Table 1, all sixstrains of mice were susceptible to C. pneumoniae. Nosignificant differences were found among the six strains ofmice in mean titers of organism recovery from lungs (P >0.05 by t test). Deaths were observed with Balb/cAnN,C57BL/6N, and B6C3F1 mice and occurred on days 2 to 7.The highest mortality (four of eight mice) was noted withC57BL/6N mice. Swiss Webster mice showed no deaths,homogeneous susceptibilities, and less individual variationand yielded a high infectivity titer.Primary infection. Swiss Webster mice were used to study

the course of primary infection. At days 2, 4, 7, 11, 15, 21,28, 35, 42, 49, and 60 after inoculation, six to seven micewere euthanized for reisolation of the organism (four to fivemice) and for histopathology (two mice). Blood was saved toobtain serum for serology. Control mice inoculated withSPG buffer were euthanized in parallel at each time point forisolation and histopathological and serological examination.

(i) Clinical observation. The inoculated mice showed weak-ness and decreased activity for the first 2 days. Theyconsumed less food and water than controls. Ruffled fur was

TABLE 2. Duration of lung infections and serum antibodyresponses in Swiss Webster mice inoculated with

C. pneumoniae AR-39

Result of isolation of organismsfrom lungs Serum IgG

Days after ttr(Minoculationa No. of positives! Mean titer + t SD)d

no. testedb SD' (log IFU/ ±S)g of lung)

2 4/4 6.61 ± 0.26 oe

4 4/4 6.39 ± 0.19 07 4/4 6.24 ± 0.52 0

11 5/5 5.18 ± 0.46 28 ± 1.415 4/4 5.25 ± 0.34 48 ± 1.521 4/4 4.80 ± 0.37 108 ± 1.428 3/4 4.98 ± 0.46 147 ± 1.435 3/4 4.28 ± 1.31 128 ± 1.642 2/4 4.09 64 ± 1.849 0/4 0 64 ± 060 0/4 0 16 ± 0

a Intranasal inoculations were with 2.7 x 107 IFU of organisms per mouse.b One control mouse was negative in each period.c Averages for positive mice.d GMT, geometric mean titer; IgM antibody was not detected.e 0 = <1:8.

noted. Mice recovered within 1 week. No deaths wereobserved.

(ii) Isolation. Reisolation of organisms from lung tissueswas positive until day 42 (Table 2). The infectivity titer washighest on day 2 and then decreased gradually. The isola-tions were positive with all of the mice tested until day 21.After day 21, some inoculated mice were negative at eachtest date. Control mice were consistently negative.

(iii) Serology. All inoculated animals developed IgG anti-body. IgG antibody in the microimmunofluorescence testwas first found on day 11 (Table 2). The geometric mean titerwas 1:28. The antibody titers increased rapidly, reached apeak of 1:147 at day 28, and then declined gradually until theend of the experiment at day 60 (1:16). No IgM antibody wasdetected at the lowest dilution tested (1:8). No antibodieswere detected in control animals.

(iv) Histopathology. Gross observation showed a patchydistribution of areas of consolidation in both lungs in the first2 weeks. The gross appearance of the lungs graduallyreturned to normal after 2 weeks. The histopathology of lunginfections with TWAR was characterized by irregularlydistributed interstitial pneumonitis that was most severe ondays 2 and 4. These lesions showed extensive infiltration ofpolymorphonuclear leukocytes, with exudate in alveolarspaces and bronchial lumens (Fig. la). Areas of lung consol-idation were noted. Infiltration was still severe on days 7 and11, with mixed mononuclear and polymorphonuclear leuko-cytes (Fig. lb). After day 15, the infiltrates were mainlymononuclear cells. Mild infiltration persisted through day49. Minimum infiltrates with foci of mononuclear cells werestill noted on day 60. One striking pathological feature wasthe observation of perivascular and peribronchial lymphoidcell accumulations which began on day 11 and persistedthrough day 60, the last day of observation (Fig. lc). Nopathological changes were observed for control mice exam-ined at each time interval (Fig. ld).

Secondary infection. Rechallenge inoculations were con-ducted to see whether mice recovered from primary infec-tions had developed resistance against infection withTWAR. In order to determine the optimal dosage for rechal-

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C. PNEUMONL4E PNEUMONITIS IN MICE 2039

FIG. 1. Mouse lung sections stained with hematoxylin and eosin. (a) Section 2 days after inoculation with C. pneumoniae AR-39. Notemassive infiltrates of polymorphonuclear cells in the alveolar spaces and the terminal bronchi. (b) Section 11 days after infection. Infiltrateswere mostly mononuclear cells with formation of mononuclear cell clusters (arrows). (c) Section 60 days postinoculation showing foci ofperivascular and peribronchial lymphoid cell accumulation (arrow). (d) Representative micrograph of normal mouse lung showing no evidenceof inflammatory reaction. Magnification for all panels, x 140.

lenge, the intranasal ID50 was determined with Swiss Web-ster mice. It was equivalent to 7.0 x 103 IFU. Rechallengeinoculations with 20 ID50 per mouse were given on day 70 or100 after the primary inoculation. Attempted isolations fromlungs at days 70 and 100 were negative with four miceexamined for each time.

The results showed that mice recovered from primaryinfections were partially resistant to reinfections (Table 3).More than half (8 of 13) of previously infected mice resistedreinfection, while only one control mouse was not infectedby the 20 ID50 inoculum when challenged on day 70 afterprimary infection (P < 0.02 by Fisher exact two-tailed test).

TABLE 3. Resistance against reinfection of lungs with C. pneumoniae AR-39 in Swiss Webster mice

Day after Prior Day 70" Day 100"challenge infectiona No. of positives/ Mean No. of positives/ Mean

no. tested titee no. tested titer'

4 No 5/5 5.61 4/5 5.68Yes 3/7 5.15 4/5 4.76

7 No 5/6 4.80 5/5 4.74Yes 2/6 2.98 2/5 4.37

aAt primary infection, one group of mice was inoculated with C pneumoniae (yes) and the other group was inoculated with SPG buffer (no). On days 70 and100 after primary inoculation, all mice were inoculated with C. pneumoniae.

b Day of challenge after primary inoculation.c Mean titer, log IFU/g of lung; averages for positive mice.

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Partial resistance was still noted on day 100 after the primaryinfection. Although the difference was not statistically sig-nificant, the sample size was small.

DISCUSSION

In this study, we demonstrated that six strains of com-monly used laboratory mice were susceptible to intranasalinoculations with C. pneumoniae. Swiss Webster mice werechosen as suitable for studies of C. pneumoniae lung infec-tions, since they yielded high lung infectivity titers, lowindividual variations, and no deaths and since they arerelatively inexpensive. Infections in Swiss Webster micewere shown to be prolonged. Organisms were recoveredfrom lungs until day 42 after inoculation. Inflammatoryreactions were shown to persist until at least day 60. Thisdiffers from our results with pneumonitis induced in SwissWebster mice by the human biovars of C. trachomatis (3,12). With C. trachomatis, isolation became negative on day14 and lung pathology returned to normal by 10 to 14 days.The difference may reflect the pathogenic nature of theorganism, with C. pneumoniae being a respiratory pathogenand C. trachomatis being an oculogenital pathogen.

C. pneumoniae lung infections induced a good serum IgGresponse in mice, and the appearance of IgG antibodycorrelated with the decrease in the numbers of organismsrecoverable from the lungs. Whether the presence of anti-body provides any protective immunity remains to be stud-ied. The pattern of IgG appearance in mice was similar tothat for the infection with the human biovars of C. tracho-matis (3, 8, 12), in which IgG was first detected at 10 to 14days. In this study, we failed to show serum IgM response.IgM response to C. trachomatis lung infection was demon-strated by Harrison et al., but only at a titer of 1:4 (8). Thelowest dilution we tested was 1:8.Although in Swiss Webster mice TWAR organisms could

be recovered from infected lungs for a relatively prolongedtime, the infection was not fatal and the lung histopathologyreturned to normal with minor residual changes. Nor was itpossible by serial passage of infected lung homogenates todevelop a more pathogenic inoculum. Infectivity titers de-clined with passage levels, and there were no reinfectionsafter the fourth passage (data not shown).The histopathology of C. pneumoniae lung infections in

mice was characteristic of interstitial pneumonitis. Theinflammatory infiltrates were predominantly polymorphonu-clear cells in the early stage and mononuclear cells in thelater stage of infection. This situation is similar to that withC. trachomatis pneumonitis in mice. However, one uniquefinding for C. pneumoniae infections is perivascular andperibronchial lymphoid cell accumulations. These were ob-served as early as day 11, and they persisted throughout thecourse of observation (day 60). With C. trachomatis infec-tions, these lymphoid cell accumulations were associatedonly with reinfection (10). Because human pathology of C.pneumoniae infections has not been described, it is notknown whether the same lung pathological changes occur inhuman C. pneumoniae infections.The inbred and hybrid mice tested were susceptible to C.

pneumoniae infections, but we did not follow the course ofdisease. Use of inbred and hybrid mice may provide anadditional tool for studying the immune mechanisms of C.pneumoniae infections.We have described a mouse model of C. pneumoniae

pneumonitis. This model offers a nonfatal, prolonged courseof pneumonitis with similarities to the human disease, which

should be useful for studying pathology, immunity, andtreatment of C. pneumoniae infections. Mice that had recov-ered from C. pneumoniae infections developed partial resis-tance to reinfection. This finding suggests that the mousemodel will be useful for studies of prevention of infection.

ACKNOWLEDGMENT

This work was supported by Public Health Service research grantAI-21885 from the National Institute of Allergy and InfectiousDiseases.

REFERENCES

1. Aldous, M. B., J. T. Grayston, S.-P. Wang, and H. M. Foy. 1992.Seroepidemiology of Chlamydia pneumoniae TWAR infectionin Seattle families, 1966-1979. J. Infect. Dis. 166:646-649.

2. Ata, F. A., E. H. Stephenson, and J. Storz. 1971. Inapparentrespiratory infection of Swiss mice with sulfadiazine-resistant,iodine-negative chlamydiae. Infect. Immun. 4:506-509.

3. Chen, W. J., and C.-C. Kuo. 1980. A mouse model of pneumo-nitis induced by Chlamydia trachomatis: morphological, micro-biological, and immunological studies. Am. J. Pathol. 100:365-382.

4. Francis, T., Jr., and T. P. Mugill. 1938. An unidentified virusproducing acute meningitis and pneumonitis in experimentalanimals. J. Exp. Med. 68:147-160.

5. Grayston, J. T. 1992. Infections caused by Chlamydia pneumo-niae, strain TWAR. Clin. Infect. Dis. 15:757-763.

6. Grayston, J. T., L. A. Campbell, C.-C. Kuo, C. H. Mordhorst,P. Saikku, D. H. Thom, and S.-P. Wang. 1990. A new respira-tory tract pathogen: Chlamydia pneumoniae strain TWAR. J.Infect. Dis. 161:618-625.

7. Grayston, J. T., C.-C. Kuo, L. A. Campbell, and S.-P. Wang.1989. Chlamydia pneumoniae sp. nov. for Chlamydia sp. strainTWAR. Int. J. Syst. Bacteriol. 39:88-90.

8. Harrison, H. R., S. M. Lee, and D. 0. Locas. 1982. Chlamydiatrachomatis pneumonitis in the C57BL/KsJ mouse: pathologicaland immunologic features. J. Lab. Clin. Med. 100:953-962.

9. Horsfall, F. L., Jr., and R. G. Hahn. 1940. A latent virus innormal mice capable of producing pneumonia in its natural host.J. Exp. Med. 71:391-408.

10. Kuo, C.-C. 1988. Host response, p. 193-208. In A. L. Barron(ed.), Microbiology of chlamydia. CRC Press, Inc., Boca Raton,Fla.

11. Kuo, C.-C., H. H. Chen, S.-P. Wang, and J. T. Grayston. 1986.Identification of a new group of Chlamydia psittaci strainscalled TWAR. J. Clin. Microbiol. 24:1034-1037.

12. Kuo, C.-C., and W. J. Chen. 1980. A mouse model of Chla-mydia trachomatis pneumonitis. J. Infect. Dis. 141:198-202.

13. Kuo, C.-C., and J. T. Grayston. 1990. A sensitive cell line, HLcells, for isolation and propagation of Chlamydia pneumoniaestrain TWAR. J. Infect. Dis. 162:755-758.

14. Kuo, C.-C., A. Shor, L. A. Campbell, H. Fukushi, and J. T.Grayston. Demonstration of Chlamydia pneumoniae in athero-sclerotic lesions of coronary arteries. J. Infect. Dis., in press.

15. Nigg, C., and M. D. Eaton. 1944. Isolation from normal mice ofa pneumotropic virus which forms elementary bodies. J. Exp.Med. 79:497-509.

16. Reed, L. J., and H. Muench. 1938. A simple method of estimat-ing fifty percent endpoints. Am. J. Hyg. 27:493-497.

17. Shor, A., C.-C. Kuo, and D. L. Patton. 1992. Detection ofChlamydia pneumoniae in coronary arterial fatty streaks andatheromatous plaques. S. Afr. Med. J. 82:158-161.

18. Wang, S.-P., and J. T. Grayston. 1986. Microimmunofluores-cence serological studies with the TWAR organism, p. 329-332.In J. D. Oriel, G. Ridgway, J. Schachter, D. Taylor-Robinson,and M. Ward (ed.), Chlamydial infections. Cambridge Univer-sity Press, Cambridge.

19. Wang, S.-P., and J. T. Grayston. 1991. Chlamydia pneumoniaeelementary body antigenic reactivity with fluorescent antibodyis destroyed by methanol. J. Clin. Microbiol. 29:1539-1541.

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