A STUDY OF THE PATHOLOGY OF THE
CH§CK EMBRYO INFECTED WITH A

TRANSMISSIBLE AGENT ISOLATED
FROM SHEEP LUNGS

Thesis {or The Dagwo of M. S.
MiCZ‘fiGAN STATE COLLEGE
"-a -. .. .T In \A 3'5. .Pz '
{axaaxanam I . Examine;

1950

 

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thesis entitled

 

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A Study of the Pathology of the Chick Embryo
Infected with a Transmieei‘ble Agent Isolated
from Sheep Lungs.

presented by

Alexander M . Ramirez

has been accepted towards fulfillment
of the requirements for

M .S g .. degree in_An1ma.LPatholog

s. - {’4 re)- L...a: - some.

 

Date

0-169

 

 

March 11+, 1950

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-- I

A STUDY OF THE PATHOLOGY OF THE CHICK EMBRYO
INFECTED WITH A TRANSMISSIBLE AGENT ISOLATED FROM
SHEEP LUNGS

by

Alexander M.4§amirez

A Thesis
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements for
the degree of

MASTER OF SCIENCE

Department of Animal Pathology
1950

THESIS

 

I.

II.

III.

IV.

V.

VI.

VII.

TABLE OF CONTEKTS

Acknowledgment

Introduction

Review of Literature

Materials and Methods

Results

Discussion

Summary and Conclusions

Bibliography

Figures

Page

14

18

21

22

26

ACKNOWLEDGKENT

I wish to express my sincere and respectful
appreciation to Dr. Frank Thorp, Jr. who gave constant
guidance in this work. The scientific basis he gave

will always be remembered with gratitude.

Thanks also are given to Miss Sylvia Laine for
the valuable aid in technicological training and assis-
tance in preparation of this manuscript. Appreciation
is extended to Miss Esther Smith and tr. M. L. Gray for
taking the photOgraphs, to Dr. Tsuling Chow for teaching
me the technique of working with chick embryos and to
Dr. R. D. Earner and Dr. H. W. Dunne for helping with
the micropathology and editing the manuscript.

In addition, I wish to thank Dr. RusseILA. Runnells
for his suggestions in examination of the slides prepared

for this thesis.

INTRODUCTION

Pneumonia commonly occurs among all species of domestic
animals. Since most pneumonias are caused by bacteria and
viruses, it is readily understood that the inflammatory
exudate of one sick animal can easily contaminate the feed
and water of all the healthy animals in the herd or flock.
This as well as contact with each other accounts for the
presence of several cases of pneumonia among a group of

animals during a short period of time.

An analysis of mortality among lambs showed that
the deaths resulting from pneumonia tended to be the high-
est of all the diseases with which they are affected. At
the Michigan Agriculture Experiment Station, Venkatachalam
gt_§l'(l949) made a survey of lamb mortality occuring the
first 56 days of life. Pneumonia accounted for 8.7% of
the total number of dead lambs. There were several of
these lambs from which no bacteria were cultured, so a
virus etiology was suspected. A considerable number of
pneumotrophic viruses have been isolated from both man
and animals, but relatively little has been done in the
study of the etiology of sheep pnuumonia.

The purpose of this investigation was two fold:
to determine whether or not a transmissible agent other
than bacteria was present and if so, to determine if it

would grow in the developing chick embryo and produce lesions.

REVIEW OF LITERATURE

The first report of pneumonia in sheep caused by
an unknown agent was made by Friedberger and Frohner (1910).
These investigators reported an enzootic catarrhal pneumonia
of lambs in which the main symptoms and lesions were fever,
pneumonia and dullness of the lungs. "Postmortem revealed
lobular catarrhal-desquamative foci of inflammation", some-
times accompanied by caseous necrotic areas. In some

cases pleuresy and purulent fibrinous pericarditis were

observed.

Mitchell (1915) described the path010gical and

 

clinical findings in "Jagziekte" or Chronic Catarrhal
Pneumonia of Sheep. Besides the classical lesions, he
mentioned the presence of small grayish translucent nodules
on the surface of the lungs. The cut sections of the lungs
also showed nodules. In long standing cases larger nodules
were observed. When these nodules were cut a mucoid exudate

and blood oozed to the surface of the section.

The most important microscopic changes were found
in the sections of the nodules, which were spherical or
oval in shape. The center of the nodules consisted of
two types of cells, those with large nuclei resembling
plasma cells and a second type which resembled fibroblasts.
This central zone was surrounded by a narrow zone of fibro-

blasts arranged in concentric layers and gave the appear-

2

ance of encapsulating the nodule. In some sections pro-

liferation of the bronchial epithelium occurred.

From observation and experiments, the author con-
cluded that this type of pneumonia in sheep was caused by

a virus.

Cowdry (1925) conducted controlled experiments to
determine if there was any difference in the suscepti-
bility of South African and American sheep to pneumonia.

He reported the occurrence of a specific cattarrhal pneu-
monia in South African sheep which was called Jagziekte.
Large areas of the lungs were edematous and inflamed. The
difference between American and South African sheep pneu-
monia seemed to be significant. In about 53 per cent of
the South African animals, the interalveolar tissue was
definitely thickened beyond the range of variation noted

in the lungs of American sheep. The thickenings occurred
in localized areas which were several millimeters in diameter.
The thickening was caused by the engorgement of the alveolar
capillaries and by the accumulation of macrophages and
lymphocytes. many of the macrophages passed into the al-
veolar lumina where they assumed the appearance of typical
epithelieid cells. It was found that these infiltrative

and exudative changes were primary to the proliferation

of epithelium which always arose in parts of the lungs

thus modified. The author was of the opinion that the

disease was contagious. He observed that it was trans-

3
missible to healthy sheep on the introduction of an animal
known to have this type of pneumonia. He further observed
that most probably the causative agent was a virus associated
with a gram negative bacillus and a gram positive diplo-

coccus.

Henderson (1929) described a contagious Pleuropneu-

 

monia of goats. A ceccobacillus could be found in infec-
tive blood during the early stages of the disease, but
this microorganism was not established as the etiological

agent.

Dungal (1931) made a study of contagious pneumonia
of sheep in Iceland. The cause of this disease was found
to be a bacterium resembling the Pasteurella group. Its

relationship to the Pasteurella group was discussed.

Mcissner and Koser (1931) (Germany) reported that

 

about 50 per cent of the lambs suffered from pneumonia.
From these cases several organisms were isolated, such as
g, bipolaris ovisepticum (43%), g, ggli (23%) and anaerobes.
From the pneumonia lungs that did not show bacteria, the

cause of death was ascribed to dietetic errors.

Walker (1931) reported that contagious pleuropneu-
monia of goats in Kenya could be experimentally reproduced
by injecting the filtered pleural exudate into the trachea

of normal goats.

Delpy (1932) described the presence of Epizootic

4
Pneumonteritis in sheep of Persia, which differed from
the other diseases of sheep. This disease was insidious
in nature, and usually no symptoms were present until a

few hours before the animal died. The disease was con-

fusing since it occurred in three different forms; a pneu-
monic type, an intestinal type and a type having the
classical lesions of hemorrhagic septicemia. Cultures

from the blood of sick animals showed the presence of a
Pasteurella and an unidentified gram-negative bacillus,
closely related to the coli group. A.bacterin was prepared

from these two organisms which gave protection after injec-

tion into sheep.

Stylianopoulos (1933) reported pleuropneumonia of
goats in Greece, which appeared similar to that described
for sheep from Asia Minor. The disease caused a heavy
mortality which varied from 65 to 98 per cent, being more
severe during the winter. The etiological factor was thought
to be a virus that did not survive long in vitro. The
virus was present in the infected lung and in the pleural
exudate. Experimental infection could be accomplished by

pulmonary passage. The incubation period varied from

three to six days.

Doukaloff (1935 (Russia) reported that infectious

 

pleuropneumonia of goats caused considerable loss. Artificial

infection was performed by inoculating liver or spleen

emulsions intratracheally or subcutaneously with or without

5

filtration, proving that the cause was a filterable virus.

Kiseleff (1935) (Russia) transmitted pleuropneumonia

 

of goats by injecting animals with infected liver, lymph
nodes, spleen, lungs, heart and kidneys. The author con-
cluded that the causative agent was a virus. However,

the data given in support of this statement was insuf-
ficient. Attempts were made to prepare a vaccine using
edematous fluid taken from the site of subcutaneous in-
oculations of experimentally infected goats, but the results

obtained were inconclusive.

Gilbert (1937) reported infectious pleuropneumonia
of goats in Palestine. The incubation period was less
than 48 hours. The causative agent was a virus, shown to
be communicable to sheep which were less susceptible than
goats. The disease was not fatal unless the animals were
exposed to severe cold weather or if they were in poor
condition. The mortality in goats was from 60 to 80 per

cent while sheep mortality ranged from 40 to 50 per cent.

glakemore and Bosworth (1938) described Jagziekte

 

 

of sheep in England, in which the most characteristic
symptom was the tendency of fluid to run from the nostrils
when the head was inclined. This fluid was of a clear
serous character. The gross lesions were found to be con-
fined to the lungs and smaller bronchi. The lungs were in-
creased in size and showed extensive areas of consolidation.

A striking feature of these lesions was that they were

6
water-logged and when squeezed much fluid was expressed.
They were grayish in color. The ventral parts of the
lungs were involved and the cut surface also revealed
small focci scattered throughout the apparently normal
tissue. The microscOpic changes were identical to the

ones described by Cowdry (1925).

Baumann (1938) stated that infectious pleurOpneu-
monia in goats had been recognized in Algiers, Germany,
Italy and Greece and the etiological agent was said to be
a filterable virus although a variety of bacteria, pre-

dominantly Pasteurellae, were isolated in advanced cases.

Montgomerie, Bosworth and Glover (1938) described

 

a enzootic form of pneumonia in sheep that occurred in
North Wales where this disease showed a fairly regular
seasonal incidence. The macroscopic and microscopic
characteristics as well as the bacteriological picture
were described. In most of the cases, a Pasteurella-like
organism was isolated but in few instances bacteria were
not found. It was suggested that an outbreak might be
precipitated by sudden environmental changes such as in-

clement weather.

La_.n_gham 31 g; (l942)reported on a study of the
pathology and bacteriology of the lungs of nine cattle,
twelve sheep and ten pigs which were affected with broncho-

pneumonia. Lesions were confined mostly to the apical and

7
cardiac lobes of the lungs and tie disease appeared to
start as an inflammatory process of the bronchioles.
Pasteurella oviseptica was isolated from the lungs of eight

of the twelve sheep.

ngg (1945) reported contagious Pleuropneumonia of
goats, a specific caprine disease in India, which was re-
sponsible for a mortality varying from 60 to 100 per cent.
The symptoms, course of disease, pathology and epizootiology
were similar to the description given by Langley (1940).
The etiological agent appeared to be a filterable virus

which was-not transmissible to sheep.

Eggggg, gt E£.(1945) described contagious Ovine
Pneumonia, the cause of which was found to be a filterable
virus. On experimental inoculation this virus was patho-
genic for rabbits but not for guinea pigs, white rats,
mice or pigeons. Secondary organisms were isolated which
played an important part in complicating the specific
disease. A formalized vaccine was prepared from liver

and spleen tissue which protected sheep against the infection.

MATERIALS AND METHODS

The agent isolated in this study was obtained
from a female Suffolk lamb which showed the following
lesions at necropsy: bilateral pneumonia with emphysema,
congestion and red hepatization. There was a dull appear-
ance and marked interstitial edema of both lungs. The
interlobular septa were thickened in the consolidated
areas. The bronchi contained abundant dirty grayish
exudate and the mucosa was congested. Similar changes were
found in the trachea. Sections of the consolidated areas
and the congested zone were removed and fixed in Zenker's

solution.

The microscOpic picture of the lungs showed a great
degree of congestion, edema and perivascular cuffing with
lymphocytes and macrophages (Fig. l). A serous fibrinous
exudate was present'on the thickened pleura. Below the
pleura a few giant cells were seen. Cultures made from
the lung of this sheep were negative for bacteria. The
tissue was stored for a month in a deepfreezer (-25° C.)

prior to its use in this study.

Three grams of lung tissue were ground in a mortar
and pestle and mixed with three mls of nutrient broth.
This suspension was treated with penicillin and strepto-
mycin to avoid the possibility of contaminants (Rose,
Pearce and Molloy, 1946). One thousand units of penicillin

and 0.1 mg of streptomycin were used for each gram of

9
tissue. After the first two passages of the lung material

in the chick embryo the use of antibiotics was discontinued.

Smith (1935) and Burnet (1936) showed that some
strains of Influenza virus grew on the chorioallantoic
membrane of chick embryos. Attempts were made to cultivate
a transmissible agent on the chorioallantoic membrane of

the deve10ping chick embryo.

Supply 32 eggs --- Fertile eggs from healthy white
Leghorn hens were used inthis study and were purchased

from the Poultry Department at dichigan State College.

Preliminagy Incubation --- One to three day old
eggs were incubated for 12 days, at a temperature of 990 F.
and a humidity of 86 per cent. The incubator used was of
an electric type manufactured by the Chicago Scientific
Company*. The eggs were rotated twice a day during the
incubation period. It was observed that the chorioallantoic

surface was increased when the humidity was low and decreased

when high.

Candling g; the eggs --- This was done in a dark

 

room with a microscope lamp.

The eggs were candled after three days of incubation
to eliminate the infertile ones and the dead embryos. The
candling was repeated on the twelfth day and the location

 

——

*Chicago, Illinois.

10 '
of the original air sac was marked. An area devoid of
large blood vessels in the chorioallantoic membrane,
where the artificial air space was to be made, was also

marked on the shell.

Tincture of iodine was appdied to the area where
the egg shell was to be drilled. The eggs were opened

with a stationary electric motor driver drill and disc.*

The shell was cut with care to avoid injury to the under-
lying shell membrane and chorioallantoic membrane. The
methods chosen were the ones used by Beveridge and Burnet
(1946), Dunham (1942 and Alexander (1938). An oval open-
ing 3 mm. by 2 mm. was made, by removing part of the shell
with a side to side movement of the rotating disc. The
surface of the egg over the air space was drilled to make

a hole sufficiently deep to perforate the shell.

The inoculation of the chorioallantoic membrane
was made according to methods of Burnet (1946). Twelve
day old embryos were used. The eggs were inoculated within
three quarters of an hour after removal from the incubator
to avoid adhesion of the chorioallantoic membrane to the
shell membrane. A slit was made in the shell membrane
avoiding injury to the chorioallantoic membrane. The separ-
ation of the chorioallantoic membrane was made by the pro-
duction of the artificial air Space. This was done by
gentle suction with a rubber bulb that was applied to the

 

*Chicago Wheel & Mfg. Co., Chicago, Illinois.

 

 

ll
groove over the original air space which caused the
chorioallantoic membrane to drOp from the shell membrane.
The inocula were checked for sterility before each inocula-
tion. Two tenths ml of the inoculum was placed upon the
chorioallantoic membrane with a tuberculin syringe and an
18 gauge needle. A drop of melted paraffin was used to
seal the opening in the shell. Every third egg served as
a control and was inoculated with 0.2 ml of nutrient broth.
After inoculation, the eggs were placed in special racks.
Care was taken not to disturb the original position of
the air space. These racks were then placed in the in-

cubator and the eggs were candled twice a day.

Some of the embryos were examined each day follow-
ing inoculation up to and including the eighth day. To
examine them, the surface of each egg was brushed with
iodine and the paraffin removed by flaming with a Bunsen
bruner. The egg shell was broken with sterile forceps
and a very small opening made so that a platinum loop
could be passed over the chorioallantoic membrane. Bacterial
contamination was checked by agitating the 100p in nutrient
broth and incubating at 37° F. for 72 hours. The eggs
were then placed on a suitably moulded pad of cotton moisten-
ed with Phemerol. The shell was broken away to the level
of the drOpped chorioallantoic membrane. The chorioallantoic
membranes were clipped away at the boundary of the artificial
air space in order to remove the entire inoculated area.

The chorioallantoic membranes which showed lesions on the

12
surface were saved for passage. The membranes were cut
with a sterile scissors and forceps. ‘These membranes were

placed into screw tap vials and kept in the deepfreezer.

To prepare the chorioallantoicmembranes for egg
passage, 3 or 4 grams of membrane were added to 3 or 4

mls of broth and the mixture ground in the Waring Blender.

This procedure was used to carry the transmissible

agent through nineteen egg passages.

Before tissues were removed for histological study,
Holly's solution was poured into the eggs while they were
kept in the original positions on the rack. They were dis-
turbed as little as possible during the following hour of
fixation to avoid wrinkling and distortion of the membranes.
Then the partially fixed tissues were removed from the eggs.

and further fixed in Holly's solution for five or six hours.

After fixation in Holly's solution (mallory, 1938)

the tissues were treated as follows:

Washed in water .24 hrs.
Alcohol 80% 12 hrs.
Alcohol 95% 6-8 hrs.
Alcohol 100% 1-3 hrs.
Cedar Wood Oil 36 hrs.
Paraffin (2 changes) 24 hrs. MtP. 49-500 C.

Imbedded in Paraffin 31.2. 56° c.

13
Since some of the tissues were thin the length of
time the tissues were kept in alcohols varied from Mhllory's

(1938) original method.

Before passing to cedar wood oil, the organs were
removed and sections of 5 to 7 mm cut for infiltration and
embedding. Tissue sections 5-7 micra in thickness were
cut. The following organs were studied: chorioallantoic
membrane, lungs, heart, liver, spleen, kidney and brain.

Several sections of each organ were examined for lesions.

Sections were stained with Harris' Hematoxylin and
Basin (Mallory, 1938) for general histological study. use
Callum's (1919) modification of Goodpasture's method for
staining gram positive and gram negative bacteria in tissue

sections also was used.

14
RESULTS

Macroscopic Lesions --- The eggs were candled and examined

 

daily after inoculation. After three or four days incuba-

tion the infected embryos showed the following symptoms

and lesions.

1.

2.

3.

4.

5.

6.

7.

8.

Sluggish movements to the extent that in some cases
the embryo appeared dead.

The large blood vessels of the chorioallantoic mem-
branes were less prominent than those of the con-
trol eggs.

Upon Opening the shells the infected embryos, in
most cases, were living, but upon exposure to the
air and lower temperature death occurred much sooner
than in the controls.

The infected embryos were small in some cases being
about half that of the controls. (Fig. 2)

In infected embryos the abdominal wall failed to
enclose the visceral organs.

The lesions were most prominent in those cases in
which the embryos had succumbed to the infectious
agent.

The mortality rate could not be determined since
most of the eggs were opened for pathological examina-
tion at various times. However when death occurred
it was approximately on the seventh day following
inoculation.

The chorioallantoic membrane was congested and showed

15
hemorrhages varying in size from petechiae to
ecchymoses. The membrane was thickened, gelatinous
and edematous. Areas of opacity or "packs" were ob-
served. (Fig. 3) These areas were either homo-
genous or granular in appearance. On these patches,
particularly on those lying along the large blood
vessels, the tissue had proliferated to form yellow-
ish white nodules. (Fig. 4) These nodules varied
in size from one to two millimeters in diameter and
showed a dark grayish necrotic spot.

9. Consistent changes were present in the lungs. It
was difficult to remove the lungs from the body
cavity due to the presence of a serofibrinous exudate
between the lungs and ribs. The lungs appeared to
be slightly increased in size, were swollen, and
when out appeared more flaccid than those of the
controls.

10. The lesions observed in the spleen and brain were
not constant. Some of the spleens appeared to be
increased in size. The medulla oblongata appeared
to be swollen, edematous and exhibited petechial

hemorrhages.

Microscopic Lesions --- The chorioallantoic membrane showed

 

a thickening of the ectodermal layer and in some instances
the cells were necrotic. (Fig. 9) In the mesenchymal
layer, formations of epithelial pearls appeared which often

16
resembled the tumor cells of squamous cell carcinoma and
in other instances "Hassall's" corpuscles of the thymus.
(Fig. 6) The epithelial pearls appeared as irregular bodies
distributed at intervals between engorged blood vessels.
These were located in different zones of the mesenchymal
layer and most of them were situated deep in the mesoderm.
The larger pearls showed the greatest degree of degenera-
tion and the nuclei exhibited pyknosis, karyorrhexis and
karyolysis. The nuclei located in the center of the epithelial
nests were round, while those near the surface tended to
be oval or elongated and parallel to the periphery. (Fig. 7)
The chromatin of the nuclei in some instances was located
at the periphery. Balloon degeneration of the epithelial
cells was noticed. (Fig. 8) Concentric proliferation of
connective tissue was observed around these epithelial
pearls. (Fig. 7) In the older lesions the mature fibro-
blasts occupied the periphery of the pearls while the more
immature fibroblasts were located in the zone next to the
old concentrically arranged connective tissue. Other ep-
ithelial nests showed complete necrosis and sometimes
only a hyalinized zone remained. (Fig. 7) There was uSually
a collection of eusinophilic polymorphnnuclear leucocytes
in the mesoderm near these pearls. Vascular dilatation of
the blood vessels, areas of hemorrhage, proliferation of
connective tissue and in some, infiltration of heterophiles
surrounding the mesenchymal blood vessels were observed.

It was significant that all the areas in which the epithelial

l7

pearls were found corresponded to the "peck" lesions located
on the surface of the chorioallantoic membranes. The changes

were in sharp contrast with that shown by the normal chorio-

allantoic membrane (Fig. 5).

In the early passages of the virus, the embryos
showed pneumonia which was characterized by the presence
of serous exudate in the bronchi, followed by a thickening
of the alveolar walls. (Fig. 11) The capillaries showed
congestion, "perivascular cuffing" by leucocytes. (Fig. 13)
Below the pleura and along its entire length hemorrhage was
often seen. Edema was present in the interstitial tissue
and after each passage of the agent this lesion was more
pronounced. (Fig. 12) Another feature noticed in early
stages was the presence of a heaty fibrinous layer of exudate
over the thickened pleura. Lymphocytes were present in
this fibrinous exudate. (Fig. 15) Around the alveolar
openings, hyalinization of the muscle fibers was noticed.
(Fig. 14) Many of the bronchi showed an increase in thick-
ness of the lamina propia. There was probably epithelial
proliferation since some of the cells seemed to be incar-
cerated in the form of "nests" by the connective tissue of
the lamina prepia. (Fig. 16) These changes appear in sig-

nificance when compared to the lungs of normal chick embryos.

(Fig. 10)

All of these features were observed in the nineteen

passages of the agent through the chick embryo.

18
DISCUSSION

The isolation of a transmissible agent from pneu-
monic lungs of sheep, capable of growing on the chorio-
allantoic membrane of the developing chick embryo had not
been reported. Since the agent was pneumotropic, its patho-
genic capacities for the chick embryo were compared to those
of known agents that caused respiratory infections in

goats and fowls.

The most characteristic lesion on the deve10ping
chorioallantoic membrane was the proliferative process which
occurred, at first in the form of epithelial pearls, and
later degeneration characterized by necrosis and hyaliniza-
tion. Epithelial pearls had been observed by Longley (1946)
in the mesenchymal layer, after inoculating the chorioal-
lantoic membrane with pleuro-pneumonia organisms obtained
from goats. During this investigation the tissue was cul-
tured for pleuropneumonia organisms. However, no evidence

of these forms were found in the cultures nor in the tissue

sections.

Tang's: 21 (1936) observed that pleuropneumonia
organisms when inoculated on the chorioallantoic membrane
of chick embryos, failed to produce lesions in the internal
organs. In this investigation lesions were observed in the

lungs after chorioallantoic inoculation of the transmissible

agent.

19

Brandly, Tharp and Prickett (1948) studied the re-
sponse of chick embryos to tissues of chickens affected
with the avian leukosis complex and to tissues of normal
birds. They reported that sections of chorioallantoic
membrane showed keratinisation, vacuolization and "pilling
up" of the ectodermal cells. There was a general thicken-
ing of the mesoderm in which the developing cells resembled ~
the granulocytic series. Tumor formation with necrotic
foci were seen at times. Epithelial pearls were frequently
observed in the mesenchyme. Leucocytic cells when present,

were usually in small nests, perivascular in position.

Woodruff and Goodpasture (1951) studied fowl—pox

 

infection of the chorioallantoic membranes and reported
that lesions were characterized by a mfaked hyperplasia

of the ectodermal layer and by a thickening of the mesen-
chymal layer. Hyperplasia of entodermal cells was also
noticed together with the presence of inclusion bodies.

In the present investigation no proliferation of the ento-
dermal layer was seen nor were inclusion bodies observed.
Histologically, the lesions appeared to represent primarily
the response of the ectodermal epithelium to the virus

with secondary changes in the other layers.

The "curled" position of embryos as reported by Fab-
ricant (1949) and Loomis (1949) following inoculation with

bronchitis virus was not observed in this investigation.

20
The pathological changes in embryos were noted after
the third passage of the transmissible agent isolated from

sheep lungs.

The only marked lesions in the internal organs
were seen in the lungs. The spleen was increased in size
in some cases (aplenomegalia) but no microscopic changes

were observed.

When the chicks were old enough to hatch, they ap-
peared to be too weak to break the shells and most of those
that did hatch, lived only a short time.

The infected embryos were much smaller than the
normal ones. This indicated that apparently there was
interference with the metabolic processes. The chick embryo
passage of a non bacterial transmissible agent and the ac-

companying lesions suggested a pneumotropic type of virus.

2.

3.

4.

5.

6.

21
SUMMARY AND CONCLUSIOE

A non bacterial transmissible agent was isolated from
pneumonic lungs of sheep. This agent was maintained
on the chorioallantoic membrane of developing chick

embryos through 19 serial passages.

By passage of this agent in chick embryos pneumonia

was produced in the embryonic lung.

A constant and characteristic macros00pic change was

the presence of "pocks" on the chorioallantoic membrane.

The most persistent and distinctive microscopic lesions
of the chorioallantoic membrane were the "epithelial
pearls" formed by proliferation of the-ectodermal cells
in the mesenchyme. Necrosis of the ectodermal layer
and necrosis and hyalinization of the epithelial pearls

were also observed.,

An apparent interference of growth processes in the

chick embryos was observed.

The agent isolated in this study suggested a pneumo-

tropic type of virus.

22
BIBLIOGRAPHY

Alexander, R. A.
Studies on the neurotropic virus of horse sickness.
VI PrOpagation in the developing chick embryo.
Onderstepoort Jour. Vet. Sci. & An. Ind. 11:9-19,
1938.

Brandly, C. A., Frank Thorp, Jr. and C. 0. Prickett,
Response of chicken embryos to tissues of chickens
affected with the Avian Leukosis Complex and to
tissues of normal birds. Poultry Sci. 28:486-498,
1949.

Bmmmm,R.
Pathological anatomy of contagious pleurOpneumonia
of goats. Vet. Rec. 50:1510, 1938.

Bawa, Ho So .
Contagious pleurOpneumonia of goats with special
reference to immunization. Indian Jour. Vet. Sci.
& Anim. Hush. 16:1-10, 1945.

Beveridge, W. C. B. and F. M. Burnet,
The cultivation of viruses and Rickettsiae in the
chick embryo. His Majesty's Stationary Office.
London. Special Reports series No. 256, 16, 1946.

Blakemore, F. and T. J. Bosworth
The occurrence of Jagziekte in England. Vet. Rec.
53:35-36, 1941.

Burnet, F. M.
Influenza virus on the developing egg. Brit. Jour.
Exp. Path. 17:282-293, 1936.

Cowdry, E. V. .
Studies on the etiology of Jagziekte. Jour. Exp.
Med. 42:323-346, 1925.

Laboratory technique in Biology and Medicine. ed. 3.
The Williams & Wilkins 00., Baltimore. 117—118, 1948.

Delaplane, J. P. and H. 0. Stuart
Studies of infectious bronchitis. Rhode Island Agric.

Exper. Sta. Bull. 273, 1939.

23

Delaplane, J. P. and H. 0. Stuart
The modification of infectious bronchitis virus of

chickens as the result of propagation in embryonated
chicken eggs. Rhode Island Agric. Exper. Sta. Bull.
284, 1941.

Delpy, L.
Epizootic of pneumoenteritis in sheep in Persia.

Rev. Gen. Med. Vet. 41:398-407, Abstracted from
Vet. Bull. 3:319, 1933.

Doukaloff, I. A.
Infectious pleuropneumonia in goats. Soviet Vet.
No. 9, pp. 55-61, Abstracted from Vet. Bull. 7:70,

1937.

Dungal ’ NO
Contagious pneumonia in sheep. Jour. Comp. Path.

& Therap. 44:126-143, 1931.

Dunham, We Be
Egg inoculator and shell membrane teaser for virus

culture. Sci. 95:609, 1942.

Friedberger and Frohner's
Text book of Veterinary Pathology. Hurst and Blackett,

London. 2:74, 1910.

Gilbert, So Jo
Infectious pleurOpneumonia of goats in Palestine:

Communicability to sheep. Jour. Comp. Path. &
Therap. 50:33-38, 1937.

Henderson, W. H.
Annual Report of the Veterinary Department for the

year 1929. Lagos Govt. Printer. 84. Abstracted
from Vet. Bull. 1:328-329, 1931.

Kiseleff, E. V.
Vaccination against pleuropneumonia of goats. Lagos
Govt. Printer. 84. Abstracted from Vet. Bull.

7:70, 1931.

Langham, R. F., Frank Thorp, Jr., R. T. Ingle, and L. B. Sholl
Some observations on the pathology of pneumonia in
the food-producing animals. Amer. Jour. Vet. Res.

3:139-145, 1942.

Longley, E. 0.
Contagious pleurOpneumonia of goats. Indian Jour.

Vet. Sci. 10:127-197, 1940.

Loomis, L. N.
Pathology of the chick embryo infected with
infectious bronchitis virus. Thesis, School of
Graduate Studies, Hichigan State College, 1949.

MacCallum, William G.
The pathology of the pneumonia in the United States
Army camps during the winter of 1917-1918. Hono-
graph No. 10. Rockefeller Institute of Medical
Research, New York. 47, 1919.

Mallory, Frank Burr
Pathological technique. W. B. Saunders, Philadelphia.

1938.

Miessner, H. and A. Koser
The 7th and 8th Reports of the imperial center
(Germany) for combating diseases of breeding
animals for the period lst April 1929 to 31st
March 1931. Deuts tierarztl Wesch. 39:693-697.
1931. Abstracted from Vet. Bull. 2:530-532, 1931.

Mitchell, D. T.
Investigation into Jagziekte or chronic pneumonia
of sheep. South Africa Dept. Agr. Dir. Vet. Res.
Reports 3-4, 585, 1915.

Montgomerie, R. E., T. J. Bosworth and R. E. Glover
Enzootic pneumonia in sheep. Jour. Comp. Path.
and Therap. 51:87, 1938.

HOIOOI, 20, O. A..de1 and Re Zaki
Contagious ovine pneumonia caused by a filterable

virus. Vet. Med. 41:202-204, 1946.

Rose, H. M., E. Pearce and E. MOlloy
Effects of penicillin and streptomycin on bacterial
contamination of chick embryo inoculated with un-
filtered sputums. Proc. Soc. Exp. Bio. and Ned.
62:124-127, 1946.

Smith, W. -
Cultivation of the virus of influenza. Brit. Jour.
EXP. Path. 16:508. 1955.

Stylianopoulus, M.
PleurOpneumonia of goats in Greece. Rev. Gen. Med.
Vet. 42:401-416. 1933. Abstracted from Vet. Bull.

4:379-380, 1934.

Tang, F0 F0, Ho Wei and Jo Edgar
Further investigations on the causal agent of bovine
pleurOpneumonia. Jour. of Path. & Bact. 42:45-51,

1936.

25

Venkatachalam, G., R. H. Nelson, Frank Thorp, Jr., R. W.
Luecke and M. L. Gray
Causes and certain factors affecting lamb mortality.

Jour. Anim. Sci. 8:392-397, 1949.

Walker, J.
Contagious Pleuropneumonia of goats in Kenya.
Ann. Rept. Dept. Agr., 1931. Abstracted from
Vet. Med. 41:202-204, 1946.

Woodruff, A. M. and E. W. Goodpasture
The susceptibility of the chorioallantoic membrane

of chick embryos to infection with the fowl—pox
virus. Amer. Jour. Path. 7:209, 1931.

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111ng from which the transmissible
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Fig. 20

Two embryos each 19 days old. The con»
trol shown at the left is twice the
size of the infected embryo.

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Fig. 3o

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Ohcriosllsntoio membrane showing edema,
hemorrhage and the presence of opaque
uses. 6 I. Ansco color print.

ahorioallantoio membrane showing a
large yellowish white nodule with
hemorrhage. Note the engorged blood
vessels. 8 lb Anson color print.

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Fig. 5.

A normal chorioallantoic membrane of
an 18 day old embryo. M. 70 I.

 

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Fig. 5

 

Fig. 6. Chorioallantoic nembrane showing the
epithelial pearls, thickness of the
mesodernal layer, congestion of capu
illaries and edema with extensive
leucoytic infiltration. H&E. 11671.

   

 

 

Fig. 7. - Chcrioallantoie membrane showing
epithelial nests with cells in
various stages of degeneration,
connective tissue around the nests
and congestion of capillaries.
REE. 170 x.

 

 

 

 

 

 

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Charioallsntoic membrane showing a

more severe degeneration of the
epithelial nests. Balloon degenerao
tion and hyalinisation of the epithelial
cells. REE. 170 I;

 

 

Fig. 8

 

 

 

Fig. 90

Chorioallantoie membrane with necrosis
of ectodermal layer and epithelial
pearls; also extensive leucocytio in-
filtration. H.323. 70 I.

 

 

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Fig. 10.

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Fig. 11. Infected lung showing alveoli filled
with serous exudate and a few red
blood cells. Remaining lung tissue
shows edema. HdE. 100 I.

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Fig. 12. ' Infected lung showing serous exudate,
involving the entire organ. The
alveolar walls are thickened and
capillaries congested. Kill. 100 I.

 

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Showing hyalinization of muscle fibers
at the site of the alveolar openings.
Congestion of the capillaries is also
evident. 3&3. 480 I.

 

 

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The pleura is covered with a thick
layer of fibrinous exudate. In-
filtration of lymphocytes is seen
together with the fibrin. BEE.
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Fig. 16. A bronchus showing increase in thick-
ness of the lamina propia. Epithelial
nests in this zone seem to be incar-

cerated by connective tissue. 3&3.
170 I.

 

 

 

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