THE PATHOGENESIS OF PS1TACOSIS

SERIAL STuDIES ON RHESUS MoNKETs EXPOSED TO A SMALL-PARTICLEAEROSOL OF THE BoRG STRIN

CmrAmn MAsLco]EL. MCGAvLAN, M.C, USAR,* CLh W. BrEA, D.VM,RIcHAD F. BzEDTn, PHD., AD FRST IETENANT Rom= AL NA.AuRA, V.C, USAR.

From the United States Army Chemical Corps, Fort Detricik, Frederick, Md

In I930, shortly after the virus of psittacosis was isolated, Rivers andBerry1 showed that rhesus monkeys were susceptible to the "Wenz C"strain after intranasal or intratracheal inoculation of homogenates ofinfected mouse liver and spleen. The monkeys developed a pneumoniathat had radiologic and microscopic similarities to the alterations seenin hurman cases of psittacosis. In I934 Rivers and Schwentker2 describedattempts to immunize monkeys by parenteral inoculation of the livingvirus. Some increase in resistance to subsequent intratracheal challengewas found. We have found no further references to studies in monkeys.

Sequential morphologic investigations of the pneumonia produced bythe members of the psittacosis-lymphogranuloma group of viruses arefew. The data from man are terminal.- De Gara and Furth6 describedthe pneumonia produced in mice after intranasal instillation of a menin-gopneumotropic virus isolated from a fatal human case. Weiss7 studiedthe developmental cycle of 3 members of the psittacosis-lymphogranu-loma group in the lungs of mice, after intranasal inoculation, as well asin embryonated hens' eggs. Gogolak,8 using a purified preparation ofmurine pneumonitis virus, documented the pathogenesis of the pneu-monia, and the effect of aureomycin, in mice exposed to a highly con-centrated inoculum in a cloud chamber.

Recent refinements in virologic and aerobiologic techniques allowexposure of animals to infectious aerosols of known characteristics andrelatively accurately calculated doses. Thus a versimilitude betweenthe artificially produced and the naturally occurring diseases may beachieved, and pathogenetic studies may become more informative. Ourexperiments were done in an attempt to document the pathogenesis ofpsittacosis in monkeys exposed to an infectious aerosol of known char-acteristics, with particular emphasis on the incipiency, development, andresolution of the pneumonia.

Accepted for publication, January i8, I962.* Present address: Department of Anatomy, Washington University School of Medicine,

St. Louis io, Mo.

653

MC GAVRAN ET AL.

MATERIAL AND METHODS

A partially purified egg slurry of the Borg strain of psittacosis virus 9 was used inthese experiments. It was frozen in 3 to 4 ml. lots and held at-20 C. until used.Sorenson's buffer, containing Io per cent egg yolk (v/v) at pH 7.6, was the diluent.The presence of virus was determined by intracerebral (IC) or intraperitoneal

(IP) inoculation of serial dilutions of impinger samples,* blood or triturated tissuesinto each of io Swiss-Webster mice (io to I4 gm. body weight). The mice werechecked daily and deaths recorded. Titers were calculated according to the methodsof Reed and Muench10 and expressed as median mouse IC or IP lethal doses(MICLD50's, MIPLD50's). These two expressions of virus concentration are notidentical. The MIPLD50 is approximately Y2 to i log lower than the MICLDm.

Twenty-four rbesus monkeys (Macaca mulatta), with radiographically clear lungsand negative serum complement-fixation titers for psittacosis, were used. Theyweighed from 2 to 5 kg., were caged individually, and fed Purina Monkey Chow andwater ad libitum. Rectal temperatures were recorded daily with a thermister probe(Tri-R Instrument Company, Jamaica, New York) from 2 days before to 26 daysafter exposure. Chest roentgenograms were taken before exposure and sequentiallyafter the I3th day.The infectious aerosol was continuously disseminated into the 543-liter spherical

section of a Reyniers chamber by a modified Vaponefrin nebulizer.11 The relativehumidity of the aerosol ranged from 26 to 30 per cent and the temperature from 21Ito 220 C. The median diameter of the aerosol particles was i to I.5 p.

Samples of the aerosol were obtained at 3 suitable intervals with Shipe impingers,1'and the fluid therein was assayed for virus. The monkeys were anesthetized withCombutal® (Abbott) and exposed individually for 5 minutes. The projected inhaleddose per monkey was between s,ooo and Io,ooo MICLD5o's. This range was chosento attain uniform infectivity. On the basis of previous experiments, we knew thatthe median infective dose of this strain of psittacosis virus for rhesus monkeys wasless than So MICLD50's. The individual monkey's dose was calculated by multiplyingthe concentration of virus per liter of aerosol by 5 times the respiratory volume perminute as determined from Guyton's formula.12One monkey was killed by intravenous injection of Combutalg on each of the

following days: o, 2, 3, 5, 6, 7, 8, 9, 10, I2, 14, i6, 19, 20, 21, 22, 23, 26, 34, and 37.Four monkeys were killed on day 29. Serum from each monkey was frozen for sero-logic tests. Complete necropsies were done. Samples of lung, liver, spleen, andtracheobronchial lymph node were removed aseptically, weighed, triturated in glassgrinders, diluted in egg yolk buffer, and assayed for virus. Smears of the lung, liver,and spleen were fixed in absolute ethyl alcohol, acetone, and buffered formalin. Tissueswere fixed in io per cent formalin buffered to neutrality with sodium phosphate. Thelungs were gently inflated with the same fixative. Paraffin sections, 4 to 6 j thick,were stained with hematoxylin and eosin and a modified Giemsa stain. The smearswere stained by the May-Griinwald modification of the Giemsa method.

RESULTS

The calculated average inhaled dose of virus was between 4,ooo and5,000 MICLDso's. During the course of the disease the monkeys did notlook sick, nor did any die of psittacosis. They did not cough, sneeze orhibit signs of respiratory difficulty. Variation in food intake and spon-

* Measured amounts of the infectious aerosol are withdrawn into a device designatedan impinger and the particles therein trapped in a smal volume of suitable fluid-in thisinstance, Sorenson's buffer.

654 Vol. 4o, No. 6

PATHOGENESIS OF PSITTACOSIS

taneous activity were difficult to evaluate. No decrease in resistance tobeing caught and held was noted.

Text-figure i is a composite temperature chart showing the medianand range of the temperatures of all the living monkeys. If a tempera-ture above 39.5° is a fever for this group of monkeys, then these mon-keys were febrile from days io through I5. Text-figure 2 is the tempera-ture curve of a monkey. It shows an early mild rise from days 3 through7, and a second pronounced rise from days io through I4. This biphasicresponse is suggested in Text-figure i, but it is not as pronounced.

*1t

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, s--1-a a 5 * a7t 9 a aU 1 3 Ua = 2525 7

T.ExT-nGuitE I. Temperature of monkeys with psittacosis; a chart showing the medianand range of the temperatures of all the living monkeys.

The complement-fixation titers were negative through the eighth day.Thereafter they rose from levels of 1/40 and i/i6o to I/640 and I/1280by the end of the third week. During the fourth and fifth weeks theyfell to levels of 1/320 and i/i6o. The results of the assay of the bloodand tissues for virus are shown in Text-figure 3. Some of the negativeresults, particularly in the lung, may have been due to sampling in-adequacies.

DAYS

TFX-FIGURE 2. The temperature curve of a single monkey, showing an early mild risefrom day 3 through day 7, and a second pronounced rise from day io through day I4.

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656 MCGAVRAN ET AL. Vol. 4o, No. 6

Gross Observations

Through day 7 no gross changes were found. On day 8 two small, 0.2to 0.3 cm., well-demarcated, firm, pale pink areas were seen in the rightapical lobe. A similar lesion was found in the left apical lobe on dayio (Fig. i). By the I3th day (Fig. 2) the lesions had enlarged and were

DAYS

ORGAN 0 2 3 -5 6 7 8 |9 10 12 14 16 19 20 21-122 23 26 29 34 37

LUNG _iEiEIXIiiEi wf _ _TS NODE" |-i i -

LIVER _ +__ __++ __+ + _______

SPLEEN - IuIhEm|it*EhEE -

*not assayed + virus -recovered * bands emphasize time**Tracheobroncbial - v i rus not recovered during which virus was

lymph node recovered.TIx-FIuRE 3. The presence of psittacosis virus in various organs during the course of

the disease, as determined by the assay of blood and tissues for the virus.

detectable radiographically. The maximal extent and character of thepneumonia is shown in Figures 3 to 5. These are from the monkeyskilled on days i 6 and 2I. Resolution became apparent by day 26, andby day 29 was well advanced (Fig. 6). By days 34 and 37 the resolutionwas complete. By comparing the earlier roentgenograms with the grossalterations, it was still possible to find faintly gray and slightly firmerpatches of lung beneath minimally thickened pleura. Without such fore-knowledge the changes were easily overlooked.When the data from the entire group were compiled and the volumes

of the lobes considered, the distribution of pneumonic foci was ratheruniform. Three of the 7 lobes of the monkey lung, the right cardiac andthe right and left intermediate lobes, are small. Their combined volumedoes not equal that of the pair of apical lobes, and is approximately equalto a single diaphragmatic lobe. The foci were evenly distributed withineach lobe. Para-hilar involvement was no more common than wasperipheral.

During the third week fibrinous pleuritis overlying the areas of pneu-monia was a common finding. Occasionally, during the third and fourthweeks, a few ml. of clear fluid were found in the pleural cavities. Thetracheobronchial lymph nodes enlarged during the second week and

PATHOGENESIS OF PSITTACOSIS

diminished in size during the fourth and fifth weeks. No meningealexudate was found, and the spleen did not enlarge appreciably.

Microscopic ObservationsWe found no characteristic inflammatory changes until the third day,

when small aggregates of monocytes with a few neutrophils were seenalong the walls of the respiratory bronchioles (Fig. 7). The site of reac-tion remained the same during the next 5 days. The extent graduallyincreased by involvement of the rudimentary alveoli (Fig. 8) arisingfrom the respiratory bronchiole and by contiguous centrifugal spreadinto the adjacent alveoli. The inflammatory reaction did not spreaddistally along the respiratory bronchiole in a preferential manner. Ini-tially, the distal portion of the primary lobule-the alveolar ducts,atriums, and alveoli-was not involved.By day io to day I2, when the lesions were becoming visible grossly,

the microscopic pattern was rather consistent (Fig. 9). The center ofthe lesion consisted of the involved respiratory bronchiole and the ad-jacent alveoli filled with an exudate composed of mononuclear cells, afew neutrophils and, rarely, some extravasated erythrocytes enmeshedin a fibrinous net (Fig. io). More peripherally, the neutrophil exudatedecreased, and hypertrophy and hyperplasia of the alveolar epithelialcells became conspicuous (Fig. ii). At the advancing edge, only theepithellal hyperplasia and a few mononuclear leukocytes were seen(Fig. I2). Many of the mononuclear cells in the exudate bore a likenessto the hyperplastic epithelial cells.At the peak of the reaction, I4 to i6 days after exposure, the alveoli

were filled with an exudate rich in fibrin (Fig. I3) and the inflammationcontinued to spread peripherally. No capillary thrombi or necrosis ofalveolar walls were seen. Centrally, in the respiratory bronchioles, theexudate began to organize, and these cores developed a squamous to lowcuboidal epithelial covering (Fig. I4). This did not happen in the alveoli.The resolution of the pneumonia began earlier than either the gross

or the radiographic findings had indicated. Between days i6 and 20clearing began centrally, in respect to the individual lesion, not in re-spect to the anatomic structure of the lung, and spread peripherally.Thus foci of confluent lobular pneumonia were found in which centralclearing was marked, while the periphery was still maximally inflamed.The neutrophils disappeared, and the mononuclear cells were replacedby recognizable macrophages (Fig. I 5). Plasma cells and a few lympho-cytes appeared in the residua. The perivascular and peribronchial spacesbecame prominent because of an increase in the latter cell types (Fig.I6).

657Jcx, z96

6MC GAVRAN ET AL.

By the end of the fourth week the alveolar exudate was gone. Coresof organized exudate were still present in the respiratory bronchioles,and the bronchovascular tracts were still accentuated (Fig. I7). Thefibrinous pleuritis was mostly fibrous by this time. At the end of thefifth week the organized cores in the bronchioles had disappeared. We donot know how they were resolved. Actual lysis seemed improbable.Perhaps the pumping action of the lung pushed them proximally andthey were removed by diary action. The alveolar walls returned to nor-mal, the perivascular and peribronchial infiltrate disappeared, and onlythe remnants of the fibrous pleuritis remained.The nges in organs other than the lung were not conspicuous.

Medullary hyperplasia of the tracheobronchial lymph nodes occurredduring the second and third weeks. During the first 2 weeks small groupsof hyperplastic Kupffer cells were seen. During the fourth week smallfoci of hepatocellular necrosis and mononuclear reaction were found inthe livers of 3 monkeys. The splenic alterations were slight and difficultto assess. Pertinent negative features were the absence of inflammatoryreaction in the meninges, pericardium, peripheral lymph nodes and bonemarrow.

Intracytoplasmic aggregates of psittacosis virus were hard to find.After prolonged search of Giemsa-stained sections, we found minute,round to oval, 04 to 2 IL, deeply basophilic bodies, occasion y Sur-rounded by a clear halo in alveolar epithelial cells and mononuclear cellsin the inflammatory exudate (Figs. i8 and i9). The virus aggregateswere more readily found in the smears from the lung, particularly inthose made from grossly obvious lesions. We did not find morphologicallyacceptable evidence of the virus in sections or smears of the tracheobron-chial lymph nodes, the liver or the spleen at any time, nor in the lungafter the end of the third week.

DIscussIoNWe cannot be categorical about the absence of an initial inflamma-

tory reaction in the alveoli, as it is obvious that our method of examina-tion did not encompass each one. But if such alveolar involvementoccurred, under the conditions of this experiment, and played a signifi-cant role in the initiation of infection, we should have found a few foci.In fact we found none.Though many references to "alveolar retention" of aerosol particles

exist,13 we do not think they are applicable to studies such as ours forseveral reasons. First, they are based on indirect methods of measure-ment, and secondly they deal with inert rather than microbial particles.Theoretical and experimental data indicate that the flow of air is very

658 Val. 4o, No. 6

PATHOGENESIS OF PSTACOSIS

slow in the respiratory bronchiole and that it stops in the alveolar ducts."Thus only submicronic particles, that behave as gases, may be expectedto diffuse into the alveoli.'5 Larger particles, I to 5 p, may be expectedto impinge proximally along the respiratory bronchiole. This structureis devoid of the protective covering-ciliated columnar epithellum andthe mucous blanket-in its distal portion, and thus virus can come intodirect contact with naked cells.

Psittacosis is not the sole infection in which the respiratory bronchioleis the site at which infection begins. Previous work,"' recently repeatedand confirmed by one of us (MHM), has shown that, in monkeys exposedto an aerosol of i p particles containing Pasteurella tularensis, the initialinflammatory reaction is in the respiratory bronchiole.The contiguous centrifugal spread of infection from the respiratory

bronchiole is explicable in that the walls of the rudimentary alveoli areno more impervious than those of the true alveoli. The pores of Kohnare present in both. Perhaps lymphangitic spread plays a part. Ourstudies do not evaluate this mechanism. The resulting pneumonia isanatomically lobular, and confluent lobular pneumonia results from thecoalescence of adjacent foci or progressive spread from a single focus.On the basis of our observations and the pertinent reports in the litera-

ture,"3'5 we do not think that the oft-repeated description of psittacoticpneumonia as an interstitial pneumonitis is accurate. With the exceptionof the hypertrophy and hyperplasia of the alveolar epithelial cells (andthey are not actually interstitial), the inflammatory reaction is intra-luminal. Only in the latter phases of resolution, when the interstitialspaces about bronchioles and vessels are filled with the effluvium, is therean actual interstitial component. Similar changes are seen, however, dur-ing the resolution of many pneumonias.The recovery of psittacosis virus from the blood, liver and spleen

re-emphasizes the systemic nature of this infection. It is of interest thatvirus persists for more than 2 weeks after the appearance of serum anti-bodies. Presumably the intracellular location protects the virus from thehumoral antibodies though spread and infection of other cells is in-hibited. The onset of resolution correlates reasonably well with theappearance of significant levels of complement-fixing antibodies.

SUMARY

Twenty-four rhesus monkeys were exposed to a small-particle aerosolof the Borg strain of psittacosis virus. The average inhaled dose wasapproximately 5,ooo MICLDwo's, a uniformly infective dose. None of themonkeys died of psittacosis. Serial morphologic studies showed that theinitial site of infection was in the respiratory bronchiole. Subsequent

659Jun, z962

66o MC GAVRAN ET AL. Vol. 40, No. 6

contiguous centrifugal spread resulted in a lobular pneumonia. Themaximal extent of the inflammatory reaction was reached by the 14thto i 6th days and was coincident with the onset of resolution. Resolutionwas complete by the end of the fifth week. Anatomic evidence of infec-tion was scant in organs other than the lung. A few minute mononucleargranulomas were found in the liver. Intracytoplasmic inclusions of thevirus were found in the alveolar epithelium and in the mononuclearleukocytes in the alveoli. These findings are illustrated and their signifi-cance discussed.

REFERENCESi. RiERs, T. M., and BERRY, G. P. Psittacosis. IV. Experimentally induced in-

fections in monkeys. J. Exper. Med., I930, 54, 120-144.2. RIvERs, T. M., and SCHWENTKER, F. F. Vaccination of monkeys and labora-

tory workers against psittacosis. J. Exper. Med., 1934, 60, 2II-238.3. LILLIE, R. D. The Pathology of Psittacosis in Man. National Institute of

Health Bulletin No. i6i. U.S. Govt. Printing Office, Washington, D.C., 1933,pp. i-66.

4. BLNioRD, C. H., and HAUSER, G. H. An epidemic of severe pneumonitis in thebayou region of Louisiana. III. Pathological observations. Report of autopsyon 2 cases with brief comparative note on psittacosis and Q fever. Pub. HealthRep., I944, 59, I363-I373.

5. Yow, E. M.; BRENNAN, J. C.; PRESTON, J., and LEvy, S. The pathology ofpsittacosis; a report of two cases with hepatitis. Am. J. Med., 1959, 27,739-749.

6. DE GARA, P. F., and FUTRTH, J. Pneumonia produced by meningopneumotropicvirus. Report of a fatal case, with observations on the interrelationship ofpsittacosis-like viruses. Arch. Path., I948, 45, 474-493.

7. WEISS, E. The extracellular development of agents of the psittacosis-lympho-granuloma group (Chlamydozoaceae). J. Infect. Dis., 1949, 84, I25-149.

8. GOGOLAK, F. M. The histopathology of murine pneumonitis infection and thegrowth of the virus in the mouse lung. J. Infect. Dis., 1953, 92, 254-272.

9. OLsON, B. J., and LARSON, C. L. An epidemic of severe pneumonitis in bayouregion of Louisian . V. Etiology. Pub. Health Rep., I945, 60, 1488-i503.

io. REED, L. J., and MUENCH, H. A simple method of estimating fifty per centendpoints. Am. J. Hyg., 1938, 27, 493-497.

ii. RoSEBIRY, T. Experimental Air-borne Infection. Williams & Wilkin Co.,Baltimore, 1947, 222 pp.

I2. GUYTON, A. C. Measurement of respiratory volumes of laboratory animals.Am. I. Physiol., I947, I50, 70-77.

I3. MrrCHELL, R. I. Retention of aerosol particles in the respiratory tract; areview. Am. Rev. Resp. Dis., I960, 82, 627-639.

14. LANDHAL, H. D. On the removal of air-borne droplets by the human respira-tory tract. I. The lung. Bull. Math. Biophysics, I950, 12, 43-56.

I5. MoRROw, P. E. Some physical and physiological factors controlling the fateof inhaled substances. I. Deposition. Health Physics, 1960, 2, 366-378.

Jue, I!#62 PATHOGENESIS OF PSITTACOSIS 66i

I6. RooNEY, J. R., and BLUNDELL, G. P. Pathogenesis of aerosol-induced infec-dons in Macaca mulatta. L Morphological evaluation of pulmonary lesionsinduced by Pasteurella tularensis. (Abstract) Program of the American So-ciety of Clinical Pathology, Chicago, I960.

We wish to thank Mr. John Ray and Miss Einor Brown for their able assistance.

[Illustrations folow]

MC GAVRAN ET AL.

LEGENDS FOR FIGURES

Except where indicated, photomicrographs were prepared from sections stainedwith hematoxylin and eosin.FIG. I. An early lesion in the left apical lobe from the monkey killed on day Io.

The dark horseshoe-shaped mark (arrow) at the periphery is formed by twosmall bronchioles and their associated vessels. X 5.

FIG. 2. The right apical lobe at day I2. The differing density of the infiltrate is wellshown, with the central solid area, while the periphery gradually fades intonormal parenchyma. X 3.

FIG. 3. The fresh, uncut lungs on day i6. Both apical lobes are extensively involved,as indicated by the gray consolidated areas. Note the small right costophrenicangle lesion with a distinct fibrinous pleuritis. The moderate enlargement of thetracheobronchial lymph nodes is seen at the attachment of the right apical lobe.X I.S.

662 Vol. 4o, No. 6

PATHOGENESIS OF PSITTACOSIS 663June, 1962

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FIG. 4. The fixed. cut surfaces of the apical and diaphragmatic lobes from the lungsshown in Figure 3. The homogeneous. pale. nongranular character is apparent.as well as the sharp delimitation. In the right lower lobe the inverted V-shapedinfiltrate is the only one seen that followed the course of the larger bronchi.X I.5.

FIG. D. The left diaphragmatic lobe from the monkey killed on day 2I. There isdiminishing consolidation from the costophrenic angle to hilus. The small bronchiwithin the pneumonia are patent. and fibrinous-fibrous pleural thickening ismanifest. X 2.

FIG. 6. The left apical lobe. day 29. The demarcation (arrow) between an involvedand an uninvolved area is still distinct though resolution has progressed to partialaeration of the affected portions. X 3.

664 Vol. 40, No0. 6

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PATHOGENESIS OF PSITTACOSIS

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FIG. 7. An example of the initial (day 7) inflammatory reaction in psittacosis. The mid-portion of a respiratory bronchiole is seen in cross section with the exudate along thewall and in the rudimentary alveoli. The beginning of extension into the adjacentalveoli appears to the right. X 260.

FIG. 8. The exudate, filling a rudimentary alveolus. is dominantly monocvtic. Day 6. X 840.

665June, I962

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666 MC GAVRAN ET AL. VOI. 40, No. 6

FIG 9. The appearance of a pneumonic focus on day I2. Details illustrated in Figures Ioto 12. X 80.

FIG. IO. The mononuclear exudate fills the respiratory bronchiole, the rudimentary alveoliand the adjacent air spaces. The initial deposition of fibrinous precipitates is seen inthe alveoli. X 260.

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PATHOGENESIS OF PSITTACOSIS

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epithelial cells. X 260.

FIG. I2. Detail of the hyperplastic alveolar epithelial cells. some multinucleated, at theedge of a pneumonic focus. Virus inclusions were never identified in these hyperplasticcells. X 840.

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FIG. I3. The appearance of many of the alveoli at day I4. A fibrin-rich exudate fills them.but does not organize. Some resolution has occurred in the alveoli to the right, wheremacrophages are present. X 260.

FIG. 14. There is distinct peripheral organization of a branching bronchiolar plug and itscentral inflammatory core. Day 21. X 240.

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FIG. I 5. Early clearing at day i6 is accompanied by partial resolution of the alveolarexudate. thickening of the perivascular septums with -retreating" cells. and organiza-tion of the fibrinous exudate in the respiratory bronchioles in the upper and lowerleft corners. X So.

FIG. i6. Detail from Figure I D to iUustrate the change in ceH types in the alveoli and theinitial organization at the periphery of the bronchiolar exudate. upper right. X 240.

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FIG. I 7. At day 29 the alveoli have cleared. and only the fibrous cores remain in the respira-tory bronchioles. X 8o.

FIGS. i8 and I9. Intracytoplasmic Virus inclusions stained with Giemsa stain. The halo sur-rounding the plaque in Figure I8 is characteristic. Multiple inclusions are seen in Figure19. X I,200.

670 Vol. 40, No. 6

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