The pathogenesis of ovine pneumonia

J.

COMPo PATH.

1962.

VOL.

7'2..

49

THE PATHOGENESIS OF OVINE PNEUMONIA 1.

ISOLATION OF A VIRUS OF THE

PLY

GROUP

By D.

L.

DUNGWORTH*t

and

D.

R.

CORDY

Department of Pathology, School of Veterinary .11edicine, University of California, Dal,is. INTRODUCTION

Most aetiological investigations of the common forms of sheep pneumonia have been concerned with the role of Pasteurella spp. as causative agents. Because of conflicting and inconsistent results, there has not been general agreement with respect to the primary aetiological importance of these bacteria. Partly as a result of this fact, and partly by analogy with the causes of pneumonia in other species, it has been suggested frequently that a virus might be the initial incitant of pneumonia in sheep. Isolations of viruses or virus-like agents from sheep pneumonia were reported by Morcos, Zaki and Zaki (1946), Ramirez, Gray and Thorp (1951), Pokorny (1955), and Downey (1957). In none of these cases was the agent well defined, and where experimental transmissions in sheep were performed there was little evidence that the disease produced was typical of enzootic pneumonia. The only well-characterised virus thus far recovered from cases of ovine pneumonia is a member of the psittacosis-lymphogranuloma venereum (PLV) group which was first isolated by McKercher (1952). He produced small lung consolidations by combined intranasal and intratracheal inoculation of one sheep. Boidin, Cordy and Adler (1958), and Hamdyand Pounden (1959) carried out more extensive trials with other strains of PLV group viruses obtained from pneumonic sheep lungs. Boidin et at. consistently produced irregular, patchy consolidations in the lungs 4 days after intratracheal administration of a viral suspension. The lesions had almost disappeared 20 days after inoculation. The addition of P. haemolytica, pleuropneumonia-like organisms (PPLO), or both, to the viral inoculum did not increase significantly the severity of the lung involvement. Hamdy and Pounden, in contrast, reported that they were only able to produce pneumonia consistently by combinations of virus, P. multocida, PPLO, and stress, or P. multocida, PPLO, and stress. Virus alone gave slight pneumonia in 1 of 6 lambs; all lambs were killed 3 weeks after inoculation.

The primary purpose of the work presented in this paper was to study in detail the pathogenesis of the lesion produced by intratracheal administrations of a PLY group virus originally isolated from a pneumonic sheep lung. Further experiments involved an attempt to evaluate more precisely the role of the virus in the production of naturally-occurring pneumonias in sheep.

* Taken in part from a thesis submitted to the Graduate School, University of California, Davis, in partial fulfillment of the requirements for the Ph.D. degree. t Present

address, Department of Veterinarv

~1cdicine,

University of Bristol, England.

50

OVINE PNEUMONIA: PLV VIRUS

MATERIALS AND METHODS

Virus. The sheep pneumomtls virus was a strain (SPB) of the PLY group of viruses originally isolated from a case of acute pneumonia and characterised by Boidin (1957). The virus was propagated by inoculating 0'4 ml. of 10- 2 to 10-" vol./vol. dilutions of infected yolk-sac membrane suspensions into the yolk-sacs of 5- to 7-day embryonating hens' eggs. Tryptose broth (Difco), pH 7'4 was used as diluent. Yolk-sac membranes of embryos dying 3 to 13 days after inoculation were harvested and tested for bacterial contamination by culturing aerobically and anaerobically. Portions of the membranes were smeared, and the smears were examined for the presence of characteristic elementary bodies (see below). The experimental inocula were obtained from the ninth to fourteenth yolk-sac passages. Viral titration. Ten-fold dilutions of samples from the viral suspensions used in the experimental studies were made in tryptose broth, and 0'2 ml. of each dilution was inoculated into the yolk-sac of 5- to 6-day embryonating eggs; six eggs were used for each dilution. Deaths from the third to the thirteenth days after inoculation were considered to be significant. The 50 per cent. embryo lethal dose (ELD 50) was calculated according to the method of Reed and Muench (1938). Isolation of virus from experimental tissues. Portions of tissues were ground in a TenBroeck grinder or in a mortar with sterile sand, and diluted to a 10 per cent. suspension with tryptose broth. In the early isolation attempts tyrothrycin, sulphadiazine and streptomycin (TSS) were added to the broth as recommended by Meyer and Eddie (1956), but later, to increase the efficiency of elimination of bacterial contamination, the streptomycin concentration was increased to 5 mg. per ml. The tissue suspensions were stored at IO°C. for 2 to 3 hours, or occasionally overnight. The supernatant fluid was aspirated and centrifuged at 2,400 r.p.m. for 20 minutes when sedimentation appeared to be inadequate, or when bacterial contamination was suspected to be heavy. Three methods were employed for screening the resulting fluid for the presence of virus. The procedure used initially was the intranasal inoculation of 0'05 ml. into young white Swiss-Webster mice. The mice were from a self-contained colony and were shown to be free from latent pneumonitis viruses (Horsfall and Hahn, 1940; Nigg, 1942) by repeated intranasal passage of lung suspensions. They were killed '7 to IO days after inoculation and the lungs were examined for the discrete, grey-pink consolidations which denoted the presence of virus. The presence of virus in pneumonic lesions was confirmed by examination of stained smears, by inoculation of material into the yolk-sacs of embryonating eggs, or by both methods. The second method was the intracranial injection of 0'03 m!. tissue suspension into mice. When virus was present the majority of the mice died, after exhibiting signs of meningo-encephalitis, between 4 and 13 days after inoculation. Presence of the virus was substantiated by the methods described above. The third and most precise method of viral isolation was the inoculation of 0'4 mL tissue suspension into the yolk-sacs of 7-day embryonating eggs; six to IO eggs were used for each specimen. Only bacteria-free inocula could be used and for this reason primary screening was largely via intranasal and intracranial inoculation of mice. In all procedures 2 or

D. L. DUNG WORTH AND D. R. CORDY

51

3 blind passages were made before the inoculum was considered to be virus free. Examination of smears. Light impression smears were made after washing the portion of yolk-sac membrane in sterile isotonic saline and removing the excess fluid with absorbent paper. They were fixed by mild heat and stained by the method of Macchiavello (1937, quoted by Zinsser, 1940 ). Examination of tissues for the presence of bacteria and PPLO. Portions of pneumonic lesions and mediastinal lymph nodes were examined routinely. Cultures were made on ox blood agar and in screw-capped tubes of trypticase soy broth (Difco). The blood agar plates were incubated in an atmosphere containing 4 to 5 per cent. carbon dioxide. Material from the trypticase soy broth was subcultured on blood agar after a maximum of 72 hours. Pasteurella organisms were identified by the criteria listed by Biberstein, Gills and Knight (1960). Methods for cultivation and identification of PPLO were those described by Adler, Fabricant, Yamamoto and Berg (1958). Experimental animals. The sheep used in the first and third experiments were cross-bred Rambouillet-Suffolk lambs 5 to 6 months of age; the average weight was 70 pounds. They were purchased at different times in 2 lots from nearby ranches in the Sacramento Valley. The sheep used in the second experiment were obtained from a flock maintained at the experimental farm of the School of Veterinary Medicine at Davis. They were of similar age and breed type to those purchased. Selection was based on group uniformity and absence of clinical signs of disease. The lambs were observed and temperatures were recorded for 10 to 20 days prior to experimentation. Blood was taken for serological examination during this period. Post-mortem and histological procedures. The sheep were killed by rapid intravenous injection of8 to 12 m!. ofa concentrated pentabarbitol sodium solution (Beuthenasia, H. C. Burns Co. Inc.). The distribution of lung les.ions was recorded on a diagram of the lung outline. Tissue blocks representing various stages and portions of lesions and different lobes of the lungs were fixed in Zenker-formol solution. Duplicate blocks from a few areas were fixed in 10 per cent. neutral formalin . Five to 10 blocks per lung were taken; each was identified as to its location. Representative blocks, taken from other visible lesions and from the remainder of the tissues and organs, were placed in formalin. Specimens were stored at -23 °C. for subsequent bacteriological and virological studies. Tissue blocks were embedded in paraffin and sections were cut at a thickness of6/L. The stains used were: haematoxylinand eosin, haematoxylin-eosin-azure, Pollak's trichrome, van Gieson, periodic acid-Schiff (P.A.S.), Gram-Weigert, Barbeito-Lopez, Giemsa, Pinkerton-Macchiavello, Noble, and Foot's modification of Bielschowsky's reticulin stain. A few frozen sections were stained with oil red O. Complement fixation test. The complement-fixing antigen was made from McNutts' strain of sporadic bovine encephalomyelitis (SBE) virus according to the method of Topping and Shepard (1946). The SBE virus possesses the group specific, heat stable antigenic component common to all the PLV group viruses and was used because it constituted a convenient source of antigen.

OVINE PNEUMONIA: PLV VIRUS RESULTS

Intratracheal Inoculation rif Lambs with SPB Virus The primary object of this experiment was to study the serial development and regression of lung lesions caused by intratracheal inoculation of SPB virus. Thirty-nine 5-month-old lambs were used, of which 6 were controls. Details of the inocula are given in Tables I and 2. All inoculations were performed with the lambs held in an upright position with their vertebral columns perpendicular to the ground. The control and infected groups were penned separately. Table I gives the pattern of random slaughter for the control lambs and the infected lambs killed during the 20 days following inoculation. Since the experimental lesions had practically disappeared by the twentieth day after inoculation, the remaining 5 lambs were utilised to test the effect of a second exposure to the virus. Therefore, 27 days after the first exposure, 3 lambs were inoculated a second time with virus and 2 were inoculated with normal yolk-sac suspension (Table 2). On the basis of yolk-sac titration, 0·2 ml. of the first viral inoculum contained 105 '68 ELD 50, and the second contained 10 5 '81 ELD 50 per 0·2 ml. The inocula contained neither bacteria nor PPLO. Clinical signs. The most constant and characteristic clinical sign following exposure to the virus was a rapid rise in temperature which showed little variation from one animal to another; Fig. I illustrates 2 typical temperature curves. The marked diurnal variation was due to a 30 to 40°F. fluctuation in environmental temperature. The febrile response in the three lambs which received the second viral inoculation was only slightly reduced compared to the response after the first exposure (Fig. 2). Concurrent with the febrile response the lambs became moderately depressed, many standing with heads and ears drooping slightly. Thirty-six hours after inoculation a jerky abdominal lift was noted during expiration, but the respiratory rate was not significantly increased. This abdominal component of respiration increased in extent up to the fourth or fifth day, at which time it was accompanied by a slight groan in some of the lambs. All signs disappeared rapidly after the fourth or fifth day post-inoculation. At no time were the lambs severely ill, and there was only an occasional dry cough. Post-mortem observations. Significant lesions were confined to the respiratory system and to the bronchial and mediastinal lymph nodes. It is thought that the majority of the lesions were in the ventral portions of the diaphragmatic lobes because of the upright position in which the lambs were restrained during inoculation. All the lambs, including the control group, had small accumulations of mucus or froth in the lower portion of the trachea and into the bronchi for a variable distance. The quantity was greater in the lambs killed within the first 9 days after viral inoculation.

D. L. DUNGWORTH AND D. R. CORDY

53

The earliest macroscopical change in the lung parenchyma, seen 24 hours after inoculation, was the presence of irregular dark red streaks and bands in the ventral portion of the diaphragmatic lobes. A transverse section showed them to be up to 6 mm. in height, several centimetres wide, and often located close to the secondary and tertiary bronchi (Fig. 3). The plane of the b ands tended to run parallel to the base of the diaphragmatic lobe, with the r esult that only a small portion was visible on the pleural surface . The involved areas were homogeneous, dark red, slightly moist and slightly sunken. Border-line increase in firmness was detectable on palpation. By the second day there were small, irregular, mottled grey-red nodules which raised the pleura or bulged on cut section (Fig. 4). These lesions increased rapidly in extent to the third day and then more slowly through the fourth (Fig. 5 ) to the fifth day, at which time the degree of lung involvement was greatest. This comprised 30 to 35 per cent. consolidation of the total lung parenchyma, most of it being in the diaphragmatic lobes. The mature fifth-day lesion differed from those of the second, third a nd fourth d ays primarily in extent. The lungs failed to collapse and were heavier than normal. Viewed from the surface, the irregular but sharply defined consolidations were a dull blue-pink, raised I to 2 mm. and a few millimetres to several centimetres in diameter. There was minimal alveolar emphysema in the adjacent lung tissue. On palpation, the lesions were solid, bulging and irregularly lumpy. A cross-section of the consolidations revealed fleshy, mottled grey-pink tissue which consisted of approximately 2 mm. diameter pale grey foci with deeply crenated borders, separated by narrow, dull red zones. The cut surface glistened m oistly; the bronchi and bronchioles exuded a small quantity of white froth or slightly opaque mucus when compressed. The lesions regressed after the fifth day; they passed through stages similar to those seen in their development, but in the reverse order. Whereas the members of each group of lambs killed up to the fifth d ay showed only minor variations one from anoth er, those killed from the seventh to the twentieth day exhibited considerable differences. The bulky fifth-day lesion decreased in size, becoming level or slightly depressed from the ninth d ay onwards. The obvious mottling disappeared gradually, to be replaced by a homogeneous grey, then dark red tissue. The lesions became streaky once more, assumed the appearance of alveolar collapse and could not be distinguished from normal tissu e by palpation (Fig. 6). The time taken for the lesion to be resolved, except for the collapsed streaks I to 2 mm. wide, varied from 13 to more than 20 days after inoculation. Pasteurella organisms, PPLO, or both, were regularly associated with th e lesions persisting after the thirteenth day (Table I). In those cases, the bronchi and bronchioles contained a small quantity of mucopus. Resolution seemed to be almost complete by the thirtieth day after infection. There was no evidence of pleuritis in any of the necropsies.

---.

"

"

460 435

"

"

" " "

45 0 442

46 4 37 2 434

" "

"

474

"

" "

475 44 8

440 44 6 473

"

" "

" "

SPBl virus

Inoculum (8ml . intratracheally )

449 471 472

Infected 441 453 45 8

Lamb number

i

I

I

9 9

7 7

5 5 5

4 4 4

3

Dead on m orning of secon d day 3 3

2 2

1 1 1

Killed days postinoculation

1:8 1:8

< <

< < <

1:8 1:8 1:8 1:8 1:8 1:8 1:8

< < < <

1:8

AC

1:3 2

AC

1:1 6 1:8 1:8

1:8 1:8

AC

AC 1:8 1:8

AC

AC3 AC

NT

1:8 1:8 1:8

< < <

of slaughter

AC

AC

1:8

1:8 1:8 1:8

1:8 1:8 1:8

Day

< < < < <

< < < <

< < <

Pre-inoculation

Complement fixa tion titre

-

-

+ + + + + + + + + -

-1-

+

NT NT

NT' NT NT

Virus

I

I

i

P. multocida

-

P. haemolytica

-

-

-

+

+ -

-

-

+

-

-

NT

-

-

NT

'"d

-

~

::i

~

z

o

s:::

C

t'1

;;

Z

'"d

o ::i z t'1

-

+ +

-

-

+ +

PPL O

~

U1

E. coli, haemolytic streptococcus Unidentified Gram negative rod P. haemolytica

-

-

P. multocida

-

Bacteria

Isolation of micro-organisms f rom lung lesions

INTRATRACHEAL I NOCULATION OF LAMBS \YITH SPB VIRUS

TABLE

I

"

"

"

"

"

IYS"

" " "

"

"

" "

"

SPB'virus

Inoculum (8 mi. intratracheally )

6 10 21 21 30

2

20 20 20

15 15

13 13

II

II

Killed days postinoculation

2

(Continued)

1:8 AC 1:3 1:8 1:16

< < < 1:8 1:8 1:8 AC 1:16 < 1:8

1:8 1:16

<

< < <

1:8 1:8

< <

Pre-inoculation

1:3 1:16 1:8

1:8 1:8

I

I

I

...

.. . .. . .. . .. .

.. .

-

-

-

-

-

-

-

-

NT

Virus

-

.. . ... .. . ... ... . ..

-

P. haemolytica P. multocida

P. haemolytica

-

P. haemolytica

-

P. haemolytica P. multocida

Bacteria

I

Isolation of micro-organisms from lung lesions

... ... ... ... . .. ...

+ + + -

-

+ + + -

PPLO

3 AC anti-complementary. " IYS- IO per cent. normal yolk-sac suspension.

NT < 1:8 < 1:8 < 1:8 < 1:8 1:8

<

<

1:64 1:128

1:64 1:128

Day of slaughter

Complement fixation titre

I

'SPB -- IO per cent. yolk-sac suspension of th" 9th egg passage. NT - not tested.

Control 479 45 1 45 2 466 43 0 373

43 1 445 462

36 4 454

4 65 47 6

46 9 43 8

Lamb number

TABLE

Ij

c..,~

U·

~

::0

C'l

o

?='

~

Ij

~

::z:

~

~

~

Ij

r

OVINE PNEUMON IA: PLV V IRUS TABLE

2

L AMBS RECEIVING 2ND INTRATRACHEAL I NOCU LATIO N 27 DAYS AFT ER 1ST EXPOSURE

I

I

L amb i number

- -,I- 480

i

436 468

I I

lSt inoculum (intratracheal)

8 m!. 9th passage SPB (10_ 1 di ln.) -

,

- -

439 !

,,-

I

-

433

I

--

--

-

" " "

"

- -

- - - -

'" IYS -

2nd inoculum (intratracheal )

8 m!. IYS'"

_... -

"

- -

Co mplement fixation Killed titre ! days after 2nd inocuDays of Pre-inoculation , lation ( 1st ) slaughter

I

I

3

4

"

- -

4

--

4

- -

"

<

1:8

- -- - 1:8

1:16

AC

3

8 m!. 11th passage SPB (10_1 diln. ) --.-

i

<

1:8

AC

<

1:8

<

1:8 1:8 1:64

10 per cent. normal yolk-sac suspension

The bronchial and mediastinal lymph nodes were approxim ately one-and-one-half times normal size by the first day after viral inoculation, and from the second day onwards they were enlarged from one-and-one-half to three times. All were oedematous, a few were reddened. Nodes associated with lungs which contained almost completely resolved lesions were still slightly enlarged. Lamb 472 died during the morning of the second day as the result of a septicemia supervening during the height of the fever. Escherichia coli and haemolytic streptococci were cultured from the organs. Histopathology. Foci of catarrh al bronchiolitis comprised the basis of the first-day lesions. The surrounding alveoli were partially or completely collapsed, their lumina contained a small a mount of oedema fluid and variable numbers of neutrophils and macrophages, and their septa were thickened and hyp eraemic. Confluence of the m ajority of the foci produced the streaky lesions detected macroscopically. Since the control lung of the second day showed similar but less extensive and less acute changes, the effects described above were presumed to be due, at least in part, to the non-viral component of the inoculum. Frozen sections stained with oil r ed 0 revealed small numbers of fat-containing m acrophages in the infected lungs on the first day, but not on the second. There were no significant lesions in the lungs of controls killed after the second day. The lesions present on the second day were unequivocally produced by the virus. Confluence of fo ci with bronchiolar distribution produced large areas of involvem ent (Fig. 7) in which acute inflamm atory phenomena predominated. The bronchi and larger bronchioles were little affected. They contained small accumulations of oedema fluid , mucus and neu trophils, and there was increased prominence of mucus-secreting epithelial cells. The terminal and respiratory bronchioles, however, were packed with

57

D. L. DUNGWORTH AND D. R. CORDY

Fig.

I.

108 107 106

u.:

0

IJ..J

0::: ~

r-


0:::

IJ..J

a...

::E

IJ..J

r-

103

r

102 101

INOCULATION

100 -2

-I

0

2

3

4

5

DAYS

6

7

8

9

10

II

12

Temperature response to intratracheal inoculation of SPB virus. - - - - - L a m b No. 476; ··························Lamb No. 465.

masses of neutrophils, a small amount of oedema fluid and a few macrophages. A few neutrophils were infiltrating the hypertrophic and hyperplastic epithelium (an occasional mitotic figure was observed in the epithelial cells from the third day onwards). Cilia, where present, were matted together and the free surface of many epithelial cells was prominently convex. There were perivascular and peribronchiolar accumulations, approximately 5011- wide, which consisted of lymphocytes, plasma cells and reticulo-endothelial cells. The first two types of cells predominated; neutrophils were present in small numbers. The cellular accumulations were particularly prominent around the arterioles whether these were by themselves (pulmonary artery branches), or in the peribronchiolar region. The vessel walls were thickened by acidophilic material, the endothelium was swollen, there was pavementing ofleucocytes along the damaged endothelium in some instances and neutrophils, plus a few lymph-

OVINE PNEUMONIA: PLV VIRUS

Fig.

2.

108 107 I I

I

106 u.:

0

w 105

a:: ~

f
t5 104

CL

:,; w

f-

I

103 KILLED 102 2nd. INOCULATION

101 100

-2 -I

0

2

3

4

5

6

7

8

DAYS

9

10 25 26 27 28 29 30 31

32

Temperature response to 2 successive intratracheal inoculations of SPB virus at an interval of 27 days. - - - - - L a m b No. 4 3 3 ; · · ············· .. ·····.. Lamb No. 439.

ocytes, were infiltrating the walls (Fig. 8). The perivascular and peribronchiolar cell accumulations originated from the vascular lumina, and also by proliferation of existing mononuclear cells in these locations. Proliferation in situ became increasingly significant in later stages, as evidenced by increasing numbers of mitotic figures. The alveoli for a varying distance surrounding the respiratory bronchioles were packed with an exudate similar to that in the bronchioles themselves. As the distance from the bronchioles increased, the hyperaemia, oedema and haemorrhage became more prominent, and the numbers of neutrophils less so. The exudate in the peripheral portions of the foci was composed almost entirely of high protein content oedema fluid and small numbers of erythrocytes. The septal capillaries were hyperaemic and contained excessive numbers of neutrophils. Small numbers of macrophages were contained in the alveolar exudate and there was beginning proliferation of the alveolar cells, but these components were overshadowed by the acute exudative ones. Prominent oedematous thickening of the interlobular septa and subpleural connective tissue occurred

D. L. DUNGWORTH AND D. R. CORDY

59

in the involved areas. The lymphatics were distended by oedema fluid containing a few fibrin strands and scattered mixed inflammatory cells. Similar cells infiltrated the oedematous connective tissue. The lesions on the second day were the only ones in which there was noticeable involvement of the interlobular and subpleural zones. The subsequent development of the lesions from the second through to the fifth day consisted of a progressive increase in the numbers of large mononuclear cells and a diminution in the zones of hyperaemia, oedema and neutrophilic exudation. The foci of cellular consolidation surrounding the respiratory bronchioles were seen macroscopically as the I to 2 mm. diameter grey foci, and the peripheral zones of hyperaemia and oedema constituted the intervening narrow, dark red bands. Proliferation of the alveolar cells was visible on the second day and increased rapidly through to the fifth. It occurred primarily in the alveoli adjacent to the respiratory bronchioles, from which it spread peripherally. In the early stages the alveolar cells v.,-ere attached to the septa. They were hyperchromatic, plump, cuboidal or rounded, and occurred either singly, in groups, or as a partial lining to the alveoli (Fig. 9). This marked the beginning of epithelialisation. Binucleate forms or small syncytial cell masses were commonly found; sometimes they appeared to have been detached into the alveolar lumina. Several high-power microscopical fields exhibited one mitotic figure in the proliferating cells and a few showed two. Infrequently, hyperchromatic cells still attached to the septa appeared to contain phagocytised neutrophils, although it is possible that the cells were lying against the septa rather than attached to it. Concurrent with proliferation of cells lining the alveoli, increased numbers of macrophages appeared free within their lumina. Whereas the former had more densely staining, centrally loca ted nuclei, slightly basophilic cytoplasm, and indistinct or ina pparent cell boundaries, the latter tended to possess eccentrically placed nuclei, abundant acidophilic cytoplasm and distinct cell boundaries. The free macrophages frequently contained neutrophils or other debris. In a few instances, what could be interpreted as transitional forms between the attached alveolar cells and the free macrophages were detected. The progression of the process described above led to the peak involvement of the fifth day. At this time there were large, confluent densely cellular areas with little indication of alveolar pattern when stained with haematoxylin and eosin. A reticulin stain (Foot's) revealed that the alveolar structure was well preserved in the solid areas of the lesion (Fig. IO). It also confirmed that the proliferating cells were on the lumen side of the reticulin framework of the septa. Lymphoid tissue around small vessels was increased in width and compactness at this stage and in the larger accumulations (I SOfL diameter) a follicular structure was apparent. In the latter a few mitotic figures were apparent. The majority of the vessels with lymphoid aggregations were associated with bronchioles. The

60

OVINE PNEUMONIA: PLV VIRUS

alveolar areas were composed of compact cell masses with only narrow, irregular clefts present to indicate alveolar and alveolar duct lumina. The majority of the cells resembled the proliferated alveolar cells which were described previously. At least one, and as many as four mitotic figures were seen in most high-power microscopical fields. Neutrophils and lymphoid cells were distributed in small numbers throughout the lesion. In the few areas where the alveolar pattern was visible, cuboidal cells formed a continuous alveolar lining (epithelialisation), and variable numbers of macrophages and neutrophils were present in the lumina (Fig. I I). The lesions gradually regressed from the seventh day onward, as illustrated in Figures 12 and 13. The pattern of resolution in the lungs of the animals killed from the seventh to the thirtieth day was similar, but the rate at which it had occurred varied considerably. Resolution was effected by gradual disappearance of the excess cellular components present in the mature lesion, by reappearance of an alveolar pattern and by re-aeration of the tissue. The first indication of resolution (seventh day) was a decreased cellular density and reappearance of a recognisable alveolar architecture. It was first noticed at the periphery of the lesion and in portions of consolidated lobules farthest removed from the respiratory bronchioles. Concurrent with the decrease in cellular density there was an increase in vascularity. Following closely the changes described above, irregular alveolus-like spaces of varying sizes were seen. These spaces were separated by structures varying from normal alveolar septa to wide cellular zones (Fig. 14). In some instances, it was possible to see that the alveolar cuboidal lining had become flattened and merged into the thickened septa, whereas in other instances the cuboidal cells were becoming detached irregularly from the septa. Extension of the process described above led first to a mottled distribution of areas resembling a diffuse interstitial pneumonia, and thcn to complete resolution, except for the narrow confluent lesions which were the last macroscopically visible traces. The areas resembling interstitial pneumonia were shown, by reticulin staining, to consist of groups of microatelectatic alveoli containing mononuclear cells. Resolution was not followed in detail beyond the twentieth day. A reticulin stain of the twenty-day lesion demonstrated that the alveolar structure was still well preserved, but the reticulin network was broader and formed a more obvious network than normal. This was partly due to alveolar collapse, but there were some areas, particularly at the periphery of the lesions, where the reticulin network appeared to be incorporating cells attached to the septa into the intimate structure of the septa. The perivascular and peribronchiolar accumulations of lymphoid tissue continued slowly to increase in size as long as active cellular consolidations were present. They were prominent, however, only in lesions persisting to the twentieth day, at which time there were eccentrically placed peri-

D. L. DUNGWORTH AND D. R . CORDY

61

bronchiolar lymphoid follicles measuring up to 320fL in diameter. The presence of Pasteurella spp. and PPLO was associated with the lesions which remained active for a longer duration. Histologically, there was a slight increase in the vascular and neutrophilic response. A Barbeito-Lopez stain revealed small numbers of bacteria dispersed infrequently in the lumina of the terminal bronchioles . The early reaction in the mediastinal and bronchial lymph nodes was an acute serous lymphadenitis which was at a peak on the second day. Subsequently there was a decline in the acute inflammation with a progressive hyperplasia of reticuloendothelial and lymphoid elements. From the fifth day onwards the appearance of lymphoid hyperplasia was fairly constant. It lasted while active lesions were present in the lung and did not begin to regress until resolution was well advanced. Demonstration of viral elementary bodies in tissue sections. Giemsa, Noble's and Pinkerton's stains were used in an attempt to demonstrate the presence of elementary bodies in lung sections. Only Noble's stain was satisfactory, but even with this stain elementary bodies were not demonstrated unequivocally after the second day. In second-day lesions elementary bodies were distributed irregularly in small numbers, usually in alveoli containing many neutrophils. Occasionally elementary body plaques were seen within the cytoplasm of large macrophages; at other times it was impossible to associate them with any defi nite cell body. A few neutrophils contained elementary bodies. Attempted demonstration of elementary bodies in tissue sections would normally b e of little practical value in consequence of the small size of the particles, the ease with which they could be confused with other granular material in an inflammatory lesion, and the difficulty of staining them specifically. Lesion produced by reinoculation '!f virus. Four days after the second inoculation the lungs contained the characterisitic irregularly nodular, solid mottled lesions. The extent of involvement in lamb 433 was the same as in the group killed 4 days after the first inoculation, but in the other two it was approximately 40 percent. reduced. Histologically, the components of the lesion were the same as those present four days after one exposure, but the relative proportions were different. The most obvious difference was the greater width (approximately two-fold) of the perivascular and peribronchiolar accumulations of mononuclear cells. All variations from small macrophage (monocyte) type cells to plasma cells and lymphocytes were noted; plasma cells were particularly prominent. Other less obvious differences were the greater degree of alveolar oedema and septal hyperaemia, and reduced proliferation of alveolar cells. Chronic lung lesions. A fe w small lesions were present in some of the lungs of both control and infected groups of lambs. The majority of them were narrow bands of alveolar collapse which were mainly in the apical lobes and to a lesser extent in the cardiac lobes. A few of them consisted of grey-pink consolidations whoes surface was

OVINE PNEUMONIA: PLV VIRUS

depressed below the normal level of the pleura. These lesions were distinguished from the experimentally induced ones by their location, and, at least up to the seventh day, by their morphology. Histologically, they consisted of areas of collapsed alveoli containing variable numbers of alveolar cells and free macrophages. Accumulations of neutrophils and mucus were commonly found in the terminal bronchioles. Large peribronchiolar lymphoid foci occurred with variable frequency, but they were not large enough to constrict the lumina of the bronchioles. The more consolidated foci had greater numbers of alveolar cells and macrophages. Examination of smears of experimental lung lesions for elementary bodies. Macchiavello-stained smears revealed the presence of elementary bodies in the cytoplasm of macrophages and neutrophils up to the fourth day. Beyond that time they could not be detected with certainty. At the fourth day careful search was necessary before elementary bodies were found. Viral isolations. The majority of the isolations were performed by intranasal inoculation of lung suspensions into mice. Virus was not isolated from lesions later then the seventh day following inoculation. The use of a more sensitive method for detecting the virus might have resulted in its recovery from slightly older lesions. Bacteriological investigations. Table 1 gives the results in detail. From the 27 lesions cultured there were II which yielded no organisms, 10 which gave pasteurellae and 12 from which PPLO were isolated. Of the pasteurellae, 6 were P. haemolytica and 4 were P. multocida strains. Pasteurella spp. and PPLO were both recovered from 8 of the 27 specimens. The majority of the isolations were made from the older lesions and the presence of the microorganisms was associated with a reduced rate of resolution. Serological investigations. Twenty-nine of the pre-inoculation sera were negative to the complement fixation test in a dilution of 1:8, 7 were anti-complementary and 3 had a titre of I :16 (Tables I and 2). Subsequently, in 9 infected lambs there was a rise in titre to between I: 1 6 and I: I 28. The fact that the rise was consistent only on the eleventh and thirteenth days would seem to indicated sufficient antigenic stimulus merely for transient detectable antibody production.

Inhalation of Nebulised SPB Virus This experiment was designed to test, on a small scale, the effect of inhaling virus-containing droplets. Thus, the possible complications caused by several millilitres of yolk-sac material flowing down the trachea were obviated. Three ewe lambs, 9 months old, were used; a fourth served as a clinical control. A 10 per cent. suspension of yolk-sac material containing fourteenth passage SPB virus was centrifuged at 1500 r.p.m. for 10 minutes and the fluid beneath the superficial layer of lipid material was used for nebulisation. The suspension contained 106. 33 ELD

D. L. DUNGWORTH AND D. R. CORDY

50 per 0·2 ml.; cultures for bacteria and PPLO were negative. A poultry vaccine sprayer (American Scientific Laboratories Inc., Madison, Wis.) was used to nebulise the fluid into a 30" x 24" plastic bag which enclosed the lamb's head. Thirty-six ml. of viral suspension were sprayed into the bag over a period of 40 minutes. The bulk of the material precipitated on the side of the bag, but a fine mist persisted during the course of inhalation. The 3 lambs were killed 4, 7, and IO days after exposure to the virus (Table 3). TABLE 3 INHALATION OF NEBULISED SPB VIRUS

Lamb number

Nebulised SPBl

797 793 700

lSPB 2NT -

Killed days postinoculation

Inoculum ( inhalation)

I

VIrus

I

"

I

"

I I

I

I

Isolation of microorganisms from lung Bacteria

I PPLO

Isolation 0/ virus from mediastinal lymph nodes

4

I

-

-

NT"

7

I

-

---

-

-

-

10

I

I

I

10 per cent. yolk-sac suspension (centrifuged) of the 14th egg passage. not tested.

Clinical signs. The temperature response was identical to that obtained in the first experiment. Lamb number 700, the worst affected, showed other clinical signs similar to those described previously. The signs in the remaining 2 lambs were much less pronounced. Post-mortem observations. The lungs of the lamb killed on the fourth day after exposure failed to collapse on opening the thoracic cavity. The trachea, bronchi and larger bronchioles contained small, irregularly scattered accumulations of mucus and froth. The entire substance of the lung parenchyma was diffusely studded with numerous, discrete, reddish-grey foci, 0'5 to I mm. diameter, which were seen most clearly from the pleural aspect. There was no apparent preferential distribution of the lesions in any part of the lungs at this stage. By the seventh day, the lesions had increased in size and distinctness and were visible as grey, bulging shot-like nodules I to 2 mm. in diameter (Fig. 15). The larger ones were surrounded by a narrow, slightly emphysematous zone. The foci were distributed throughout all lobes, but appeared to be more numerous in the superficial portions of the parenchyma. There were a few, small, dark red collapsed foci along the cranial edges of the anterior lobes as shown in Fig. 15. The lesions present on the tenth day had diminished considerably. Only faint grey-brown specks were detected, distributed in the same manner as the earlier lesions. The uterus ofthis animal contained a foetus of approximately 30 cm. E

OVINE PNEUMONIA: PLV VIRUS

crown-rump length. There were no lesions in either the foetus or placenta. The mediastinal and bronchial lymph nodes were enlarged and presented the same appearance as nodes from lambs at similar stages after intratracheal inoculation of the virus. In all 3 necropsies there were no lesions of significance other than those already described. Histopathology. The lesions observed on the fourth day consisted of small, irregularly distributed cellular foci varying from 40 to 250JL in diameter. The majority of the foci surrounded respiratory bronchioles; a few occurred at the alveolar duct level. The degree of discreteness of the foci varied. Oedematous subpleural and interlobular connective tissues contained varied mononuclear cells. The cellular components of the foci were similar to those described at the same stage after intratracheal inoculation. By the seventh day the foci were larger (up to 800JL diameter) and more sharply demarcated from the surrounding parenchyma (Fig. 16). There was a greater proportion of lymphocytes and plasma cells than at the same stage in the first experiment. In the apical and cardiac lobes the lesions were more active than in the diaphragmatic lobes. Hyperaemia and oedema were still present, and the numbers of neutrophils in the respiratory bronchioles and adjacent alveoli were greater. The collapsed lesions which were noted macroscopically were composed of several peri bronchiolar foci with the intervening and surrounding alveoli collapsed. The collapsed foci seemed generally to abut against either the subpleural connective tissue or the interlobular septa. The lesions were greatly reduced in size by the tenth day. The perivascular and peribronchiolar mononuclear cell accumulations appeared to be the only residuum of focal involvement in some instances. In other instances small cellular foci, which were similar to those of the seventh day, remained. Bacteriological and virological investigations. These are summarised in Table 3. Neither bacteria nor PPLO were isolated from the lungs. Intracranial inoculation of suspensions into mice followed by 2 passages of mouse-brain material through yolk-sacs of embryonating eggs failed to demonstrate the presence of virus in the mediastinallymph nodes of two lambs and in a composite sample of portions of cotyledons from the pregnant lamb. Effect of Environmental Stress on the Results of Intratracheal Inoculation of SPB Virus, P. haemolytica, and a Combination of Both. The purpose of this experiment was to investigate the effect of environmental stress on sheep inoculated intratracheally with SPB virus, with P. haemolytica, and with a combination of both. Thirtysix s-month-old lambs, whose sera were negative for the presence of complement-fixing antibodies to the PLV group of viruses, were divided into 6 equal groups and treated as shown in Table 4. The

65

D. L. DUNGWORTH AND D. R. CORDY TABLE 4

THE EFFECT OF ENVIRONMENTAL STRESS ON THE RESULTS OF I NTRATRACHEAL INOCULATION OF SPB VIRUS, P. HAEMOLYTICA, AND A COMBI NATION OF BOTH

' urn Group and znocu

L am b

number

13 18 13 26 13 04 13 21 133 2 133 6

c ontrol (stressed)

V Irus (stressed)

P. haemolytica (non-stressed)

P . haemolytica (stressed )

Virus' + P. haemolytica (s tressed)

I

inoculation

- - -- - --- -7 7

.. . .. . ., . .. .

Evidence of pneumonia * 1

Clinical

-

-

6 6 14 15 33 34

13 01 13 25 13 22 13 17 13 20 1334

7 7 20 ., .

... . ..

-

13 24 1302 13 19 13 0 7 1309 13 29

7 8 20

--

13 14 1333 1306 133 1 13 10 133 0

...

.. . . .. 6 G

14 IS

IS

33 34

-

. ..

I

i

-

f-

---

I

--

-

-

. ..

- --

_-

.. .

-

r" L

-,I

f-

. .. . .. . ..

-

-

-

.-

...

"

+ + + -

t--

+

. . .. . "

I

+J_

:33 34 6 6 14

Post-mortem

--

13 13 1337 13 05 1327 13 12 13 16

13 11 13 28 1303 13 15 13 13 1335

V irus -1- P. ltaemoll'liea (non-stressed )

Killed

days aifiter virus

II i

. ..

++ + +

-

-1-

+-

+ + +

+-

+ + +

-

* For details see text. viral inoculum consisted of6 ml. of a 10- 2 yolk-sac suspension ofthirteenth passage SPB virus containing 10 5·85 ELD 50 per 0'2 ml. Neither bacteria nor PPLO were obtained from cultures of the inoculum. The intratracheal inocul ation was performed as described in the first experiment. The P. haemolytica suspension was inoculated 40 hours after the virus and the stressing procedure followed immediately. The strain of P. haemolytica was one which had been isolated from a case of acute ovine pneumonia and classi-

66

OVINE PNEUMONIA: PLV VIRUS

fied as Type 3 (Biberstein et al., Ig60). Five ml. ofa 20-hour brainheart infusion culture were administered intratracheally to each of the appropriate lambs. The culture, by direct plate count, contained 8·2 x 10 9 viable organisms per ml. The groups of lambs to be stressed were placed in separate sections of an open cattle truck and taken for a 220-mile journey lasting approximately 9 hours. The maximum environmental temperature (in the shade) was 95°F. Three days after the journey (5 days after viral inoculation) the lambs were placed in a 36°F coolroom for I2 hours. The lambs were killed in the pattern shown in Table 4. Since there were no significant lesions in the control or pasteurella groups on the seventh day, not all the lambs in these groups were killed. The remaining 3 lambs in each of the pasteurella groups were observed clinically for a total of 30 days, during which time there was no evidence of disease. Clinical signs. The control lambs and those receiving only P. haemofytica showed no clinical signs. The lambs inoculated with virus responded with the characteristic temperature rise. The addition of P. haemo£vtica, with or without stress, had no effect on this response, with the exception of one lamb. This animal (1331), developed a second febrile reaction which was first noted 24 hours after removal from the cool-room. The temperature rose to 106·3°F. by the second day and slowly subsided to normal over the course of the next 5 days. This lamb was found subsequently to have a resolving fibrinous pleuritis. The lambs receiving virus were moderately depressed on the morning of the second day and there was occasional coughing at this time which persisted in lessening degree to the seventh day. The accelerated jerky respiration described in the first experiment was not evident. Post-mortem observations. There were no significant lesions in the stressed and non-stressed lambs which received P. haemofytica and in the control lambs. The pattern of the lesions produced by the virus in the stressed lambs was the same as described in the first experiment. There were no apparent residual lesions at the thirty-third and thirty-fourth days. With the exception oflamb 133 I the combination of virus and P. haemofytica, with or without stress, did not result in an effect different from that produced by the virus alone. The predominant lesion in lamb 1331 was a chronic fibrinous pleuritis of the right side of the thoracic cavity. Microscopically the pleural lesion was seen to be composed of a broad zone of serofibrinous exudate undergoing organisation by granulation tissue from both surfaces. The major change in the underlying lung parenchyma was alveolar collapse. Virological investigations. Suspensions from portions of the right and left diaphragmatic lobes, pleural exudate and mediastinal lymph nodes of lamb 133 I were passaged twice through yolk-sacs of embryonating eggs. Virus was not recovered from any of the specimens. Strains of a virus of the PL V group were isolated from 3 of 5

D. L. DUNGWORTH AND D. R. CORDY

faecal samples taken from lambs at the start ofthe experiment, before there had been experimental exposure to the virus. Details will be given in a later paper. Bacteriological investigations. Neither pasteurellae nor PPLO were obtained from any of the lung lesions. DISCUSSION

The first two experiments indicate that the virus is consistently capable of producing an extensive and characteristic pneumonia of short duration. These results amplify and confirm those of Boidin et al. (1958). The conflicting report of Hamdy and Pounden (1959) may have been due to delaying death of the lambs until 3 weeks after inoculation, by which time the lesions would have been able to resolve. The initial response to the virus was an acute inflammatory reaction which was at its peak at approximately the second day. This was probably associated with the rupture of intracellular vesicles containing elementary bodies at the end of the first viral developmental cycle, as shown for murine pneumonitis by Gogolak (1953)· The lesion from the second to the fifth day was characterised by increasing proliferation of cellular elements originating from the alveolar septa. Cells which appeared to be proliferating while attached to, or part of~ the alveolar wall were designated alveolar cells. Large mononuclear cells which were free within the alveolar lumina, many of them obviously phagocytic, were referred to as macrophages. The macrophages appeared to arise mainly by hyperplasia of reticulo-endothelial cells which are normally present in the interalveolar septa (Baillif, 1960). Although electron microscopy has shown that there is a continuous epithelial lining to the alveolus (Karrer, 1956; Watrach and Vatter, 1959), the nature of the epithelial cells and their relationship to alveolar macrophages has not been resolved. The exact origin of the cuboidal cells producing the alveolar epithelialisation and their relationship to the macrophages is also unresolved. The remaining feature of the lesion was the development of perivascular and peribronchiolar mononuclear cell accumulations. The cells were derived largely from proliferation of lympho-reticular elements in situ, but during the early acute inflammatory phase of the reaction some originated from the lumina of the blood vessels. The lesion resolved rapidly after the fifth day. The rapidity of resolution was associated with limited viral multiplication after the second day. This was evidenced by the rapid fall of the animal's temperature, the inability to demonstrate the virus in smears after the fourth day and the failure to recover the virus from lung lesions after the seventh day. The SPB virus produced a pneumonia which morphologically resembled those due to other members of the PLV group, such as described in man (Lillie, 1933; Wolins, 1948), mice (Gogolak, 1953), cats (Baker, 1944), goats (Ishitani, Sugawa, Mochizuki and Shibata,

68

OVINE PNEUMONIA: PLV VIRUS

1953) and cattle (Matumoto, Omori, Harada, Inaba, Morimoto, Ishitani and Ishii, 1955). The morphological classifica tion of the lesion presents considerable difficulty because the problem becomes partly one of semantics. The distribution oflesions was bronchopneumonic in pattern and lobar involvement arose by confluence. The intralobular interstitium was involved in the process, according to the description of the pathogenesis of interstitial pneumonia by Gross (1960), and on these bases the lesion could b e classified as an interstitial bronchopneumonia, a term which has been used previously to describe pneumonia due to psittacosis agents. The lesion also falls within the group of hyperplastic pneumonias in which there is alveolar epithelialisation (jarrett, 1956). On an aetiological basis, the lesion belongs to the group of viral pneumonias (Reimann, 1954). The production of pneumonia by a second intratracheal inoculation of virus indicated that the degree of immunity produced by the first inoculation was slight. This suggests that natural exposure to the virus by inhalation would not render the animal immune to a second exposure by th e same route. The lungs of slaughtered lambs frequently contain, in the anteroventral regions, small lesions consisting of varying degrees of collapse and consolidation. Fifteen to 90 per cent. of lungs may be thus affected (Pounden, Bell, Edgington and Thomas, 1956; McGowan, Moulton and Shultz, 1957; Downey, 1957; Hamdy et at., 1959). These so-called slaughter-house lesions are generally an incidental finding and are usually referred to as being chronic in the pathological sense, but histopa thologically they may show acute inflammatory components. They will be referred to in this discussion in the more general sense as lesions of subclinical pneumonia. The resolving lesion produced by the SPB virus was indistinguishable from the m ajority of the subclinical on es. The reaction elicited by the SPB virus, however, is not pathognomonic, and undoubtedly there are other agents, or combinations of agents, capable of causing the lesions found at slaughter. It has not been possible to obtain an exact idea ofthe proportion of subclinical lesions in whose production a PLY group virus plays a part. Virus was isolated from 2 of 10 lesions by Boidin et at., (1958) and I of 8 lesions by Hamdy et at., ( I959). In a corollary to the present work there wer e no isolations from 16 lesions. The probability of recovery is low, however, since the virus could not be recovered more than 7 d ays after heavy experimental exposure (first experiment). The inference is that a PL V group virus is one of the agents responsible for the production of subclinical lung lesions. The significance of the virus in the pathogenesis of acute enzootic pneumonia has not been d efinitely established exp erimentally. In outbreaks of the disease there is an interplay of many factors and it is difficult to reproduce this experimentally, a fact illustrated b y the failure to precipitate a clinically severe pneumonia in the third experiment. There can be little doubt that bacteria, particularly

D. L. DUNGWORTH AND D. R. CORDY

69

P. haemolytica, play the most important part in the ultimate production of the severe outbreaks of pneumonia. It is suggested that the relatively benign lesion caused by the PLV group virus predisposes to acute bacterial exacerbation, in accordance with evidence presented by McGowan et al. (I957), and Carter and Rowsell (1958) for pneumonia in lambs and calves respectively. CONCLUSIONS

Intratracheal inoculation of a virus (SPB strain) of the psittacosis-lymphogranuloma venereum (PLV) group originally isolated from a pneumonic sheep lung resulted in an extensive but clinically mild pneumonia. Maximum involvement, which produced consolidation of 30 to 35 per cent. of lung parenchyma, occurred approximately 5 days after inoculation. Thereafter the lesions regressed; the time interval between exposure and almost complete resolution varied from 13 to 20 days. Consolidations arose by confluence offoci with bronchiolar distribution. An early acute inflammatory response was followed by proliferation of cellular components of the interalveolar septa which gave rise to large numbers of macrophages and to alveolar epithelialisation. Perivascular and peribronchiolar accumulations of mononuclear cells were also formed. Minimal immunity was evidenced by 3 lambs which received a second intratracheal inoculation. Miliary lung foci resulted from exposure of lambs to nebulised viral suspension. Pasteurella haemolytica and environmental stress, singly and together, exacerbated the viral lesion in only I of 30 lambs. The viral lesion was indistinguishable from many of the small lesions found incidentally in the antero-ventral pOliions of the lungs of slaughtered lambs. It is suggested that by the production of such lesions the virus predisposes to an acute bacterial pneumonia. ACKNOWLEDGMENTS

We wish to thank Dr. D. G. M cKercher for his advice and assistance with the virological portion of these studies, and Mrs. Lillian M. Frazier and Miss Elizabeth Nelson for their able technical assistance. REFERENCES

Adler, H. E., Fabricant, J., Yamamoto, R., and Berg, .Jeri (1958). Amer ]. vet. Res., 19, 440. Baker, J. A. (1944).]. expo Med., 79, 159. Baillif, R. N. (1960). Ann. N. Y. Acad. Sci., 88, 3. Biberstein, E. L., Gills, Marjorie, and Knight, H. ( 1960). Cornell Vet., 50, 28 3. Boidin, A. G. (1957). Ph.D. Thesis, University of California, Davis. Boidin, A. G., Cordy, D. R., and Adler, H. E. (1958). Cornell Vet., 48, 410. Carter, G. R., and Rowsell, H. C. (1958).]. Amer. vet. med. Ass., 132, 187. Downey, N. E. (1957) . .N. Z. vet.]. , 5, 128.

OV1NE PNEUMONIA: PLV VIRUS

Gogolak, F. M. (1953).]. infect. Dis., 92, 254. Gross, P. (1960). A. M. A. Arch. Path., 69, 706. Hamdy, A. H., and Pounden, W. D. (1959). Amer. ]. vet. Res., 20, 78. Hamdy, A. H., Pounden, W. D., and Ferguson, L. C. (1959). Ibid., 87. Horsfall, F. L., and Hahn, R. G. (1940).]. expo Med., 71, 391. Ishitani, R., Sugawa, Y., Mochizuki, H., and Shibata, D. (1953). Exp. Rep. Govt. expo Sta. animo Hyg. (Tokyo), No. 27,126 Jarrett, W. F. H. (1956). Brit. vet. ]., 112,431. Karrer, H. E. (1956).]. biophys. biochem. Cytol., 2, 241. Lillie, R. D. (1933). U. S. Nat. Inst. Hlth Bull., No. 161,1. Matumoto, M., Omori, T., Harada, K., Inaba, Y., Morimoto, T., Ishitani, R., and Ishii, S. (1955). ]ap.]. expo Med., 25) 23. McGowan, B., Moulton, J . E., and Shultz, G. (1957). ]. Amer. vet. med. Ass., 131, 318. McKercher, D. G. (1952). Science, 115, 543. Meyer, K. F., and Eddie, Bernice U. (1956). In Diagnostic Procedures for Virus and Rickettsial Diseases, 2nd. ed., p. 416. Amer. Publ, Hlth Ass., New York. Morcos, Z., Zaki, O. A., and Zaki, R. (1946). Vet. Med., 41,202. Nigg, Clara (1942). Science, 95, 49. Pokorny, V. (1955) . Sborn. ces. Akad. zem'ed. Ved. Zivoc. Vyr., 28, 271. Pounden, W. D., Bell, D. S., Edgington, B. H., and Thomas, D. L. (1956). ]. Amer. vet. med. Ass., 128, 298. Ramirez, A. M., Gray, M. L., and Thorp, F. (1951). Amer. ]. vet. Res., 12, 201. Reed, L. J., and Muench, H. (1938). Amer. ]. Hyg., 27, 493. Reimann, H. A. (1954). Pneumonia, Charles C. Thomas; Springfield, Ill. Topping, N. H., and Shepard, C. C. (1946). Publ. Hlth Rep. (Wash.), 61, 701. Watrach, A. M., and Vatter, A. E. (1959). Amer. ]. vet. Res., 20, 723. Wolins, W. (1948). Amer. ]. med. Sci., 216, 551. Zinsser, H. (1940). In Virus and Rickettsial Diseases, Harvard School of Public Health Symposium, (June, 1939), p. 896. Harvard University Press; Cambridge, Mass. [Received jor publication, May 27th, 1961]

;;'

o

~"...,

;;;

~

One day after intratracheal inoculation of SPB virus; transverse section through the right diaphragmatic lobe. Lesions homogeneous, dark red , slightly depressed. Fig. 4. Second day; transverse section through kft diaphragmatic lobe. Small, irregular, pale foci of consolidation separated by na rrow, dark red zones.

Fig. 3.

Fourth day ; extension of the lesion from the second day. Twentieth day ; small, collapsed residual lesions.

Fig. 5. Fig. 6.

?

-<

t:1

;C

o

n

'"

?

t:1

:.-:

>-

:t

..,'"

o

~

C'l

Z

c::

t:1

r

OVINE

Pig. 7. Fig. 8.

PNEUMO~IA:

PLV VIRUS

Two days after intratracheal inoculation of SPB virus. Bronchiolar distribution of lesions, predominance of acute inflammatory phenomena. H. & E. X 40. Higher magnification of a portion of Fig. 7 showing arteriolar involvement. H. & E. X 400.

D. L. DUNG WORTH AND D. R. CORDY

Fig. g. Fig.

10.

F ourth day ; prol iferation of alveolar cells produci ng small synci tial masses and partial "epithelialisation " of alveoli. H. & E. X 400. Fifth day; reticulin stain reveals well preserved alveol a r structure and cellular elements lying on lumen side of reti culin framework. Foot's reticulin stain. X 100.

(WINE PNEUMONIA: PLV V IRUS

Fig.

I. Fifth day; maximum in vo lvem en t. Epi thclia liscd a lveoli ,,"il h lIwir lumina fIlled hy macrophages and neutrophils. H. & E. X 400. Fig. 12. Ninth clay; c1isappf'arancc or e "Ct'ss cellul a .' componen ts, commencing reaeration. H. & E. X '4. 1

D. L. DUNGWORTH AND D. R. CORDY

Fig. ,:,. Twentieth day; j'('sollltiol1 almost complej('. dark hands remain. H. & E. X '4.

or

collapsed alveoli

Fig. 1.1. ~inth day~ lo()senin,Q," and dilninlltion of'ahToIar e,udate, app('nranCt' ()raln~()lar

spaces. II. & E.

XW(l.

(>VINE PNE UMON IA: PLV VIR US

Fig. I:i. r-,liliarr. grn·. shot· likl" nod"!<-,, 7 d :l" s aftl"r inh a la rinn of nt'hu1i ~('d SPB , ·irlls. Fig-. IG. Sec tion frolll Ihe IlIng- in Fi g . 1 0). Bronchiolar dislrii>lIlion of cOlllpacl. discrct<· cellular {(lei. H. & E. X +0.