i.
iX .

Tii2 ::i s ? c i a t ;:c l c g y

op

cgltagicus

pcar.iriA n; a:TVV”!

TI uj rliOPAGATICi; OP Tax, PYIOLCGICAI AGLi.'T ( V IR U S)
n; 22m chicj: m i m Y C

by
Tsu-ling Chov/

A Phesis
Submitted to the School of Graduate Studies of
Hi chican State College of Agriculture and
Applied Science in partial fulfillment
of the recuirementc for the decree
of
DOCTOR CP PHILOSOPHY

Department of Animal ratholony
1950

TABLE CF CCLTEIJTS

-

ACI-lKC./LSDCrEISI'T

xlvTRCDUC TI Gil

1

ETIOLOGY AL2) ..AYLCGELESIS

4

REVIEW OP LITER AT LiRE

8

ILITER IAL .ALL LET.-.'CDS

26

RESULTS

05

AISTCLATACLOGY

;E6

liscussioi;

49

su: EARY

67

JLLUSTEATIGi.S

6S

BIBLICGEAIL'CY

109

She writer who was sent to the United States to
pursue graduate study under the cooperation of Lichican
State College and the Agriculture Association of China
•fishes to express his gratitude and appreciation for this
benefit,

lie is deeply indebted to Dr. Prank Thorp, Jr.,

'major professor, for his impressing guidance and persistent
encouragement.

Dr. Thorp fires tne writer’s imagination

as he conveys his critical ability.

Also the writer wants

to display his gratitude and thankfulness to Dr. A. A.
Hartnells, Head of t.-.c Department of Animal _athology, and
Dr. C. A. Uoppert, professor of biochemistry for their
critical reading of the thesis.

The writer feels that it

was his privilege tc have years of association with hr.
m. 1. Gray who has taken much interest in -this. Investigation.
Finally, the writer sincerely thanks ail the friends in
the department.of Animal Pathology 'for their friendliness
•throughout all the years of his stay at M c h i g a n State College

IITTRC DU CTI Cl?
Contagious ecthyma is a specific viral disease of
the shin of sheep.

Primarily the stratified epithelium

of the shin is attacked by the virus which results in pro­
liferative and vesicular formations.

Secondarily, the

lesion is invaded by pyogenic bacteria causing pustular
formation and tissue necrosis.
The disease-has been reported in almost all parts
of the world, Annain, India, East Indies, Peru, U. S. A.,
Australia, Hungary, Yugoslavia, Holland, Belgium, 'Jest
Africa, South Africa, liorocco, Germany, Italy, Greece,
Erance, England and Scotland.
This disease is commonly jmown as sore mouth or
contagious ecthyma of sheep.

Classically it is termed

"dermatitis vesiculoso-pustulosa necroticans ovium".
Synonyms such as necrobncillosis, impetigo labialie,
malignant aphtha, orf, lip-and-leg ulceration, 3cabby mouth,
acute facial dermatitis, contagious sore lips, pustular
stomatitis and ensootic necrosis of the sheep do not
exactly express the correct idea of the viral vesicular
formation and the specific location of the lesions on
the body.

ITecrobacillosis, for example, implies bacterial

infection with necrotic formation which differs fundamental­
ly from viral sore raouth disease.

Impetigo labialis re­

fers to the localised form of pustular formation on the
labia, however, it in no.way indicates the viral origin.
The designation malignant aphtha may be confused with foot-

and-moutli disease.

Aphtha is characterized "by an eczema­

like lesion, but sore mouth is of the vwriola-vaceina type.
Orf is a good term, tut unfortunately was not used in the
United States,

lip-and-leg ulceration is not appropriate

because sore mouth disease does not present an ulcerative
formation unless secondary infection takes place.

Scabby

mouth is too general a term there being other causes for
scabby mouth.

Bcthyma is primarily a pustular eruption,

while thi3 disease is principally characterized by a
vesicle formation usually followed by secondary invaders.
i

It would be better to apply! the term "contagious vesiculopustular dermatitis" to this disease.
The disease was first noted in the latter part of
the nineteenth century, although its previous existence
night be assumed.

Zeller (1920) first investigated the

possibility of a virus etiology.

This was verified by

Aynaud in 1921 who concluded it was due' to a filtrable
virus.

About the same time in Greece, Blanc et al (1922)

studied a similar condition which affected goats.

They

teiieved that the same viru 3 affected both the sheep and
the goats.

This disease vm s less widespread during the

summer months.

Animals of all ages were susceptible.

Since the disease v/as so widely distributed throughout
the sheep growing areas of the world mid animals of all
ages were affected, the con lition was deemed worthy of
study’’.
The study’- of the his fcopathological changes in sheep

infected "by this virus constitutes the first part of
this investigation, and the attempts to propagate the
virus and to produce the lesions in the chick embryo
composes the second part.

ETIOLOGY A17D PATHCGEHESIS
The primary infective agent of this disease is a
filtrable virus which is present in the lesions only,
particularly the vesicular contents and the scabs.

This

virus has a relatively large size, and is filtrable
through Berfefeld filter V and Chamberland L. Handler
filters give inconsistent results.

The viral agent does

not pas.: through Berhefeld 17 and Chamberland Lg filters.
Therefore, the disease can not always be produced success­
fully by artificial inoculation with these filtrates.
The virus is rather resistant to desiccation.

It retains

infectiveness for about two years in a dry sto.te.

Infected

pastures contain the etiological material for two to three
months or longer, although no definite period has been
determined.

The virus is easily hilled by common chemical

disinfectants, however it is attenuated slowly by ether
of chloroform.
The disease belongs to variola-vaccinia group but
is not identical to them as was proved by Aynaud (1925),
Glover (195.10 and Bennett e_t al (1944).

Artificial in­

fection in sheep and goats of all ages could be produced
by emulsified crust material.

Inoculation could be per­

formed by scarifying or puncturing the shin.
An important accessory factor in the etiology is
the portal of entry of the virus.

It can be the wounds

or abrasions of the shin which occur commonly during
grazing.

Other factors which can lower the resistance

of the tissues such as exposure to dampness, exhausting
journey, insufficient food and lambs suckling the ewes.
Soft skin devoid of viool or hair is the most favor­
able location for the viral esion.

The lesions after

healing leave no scar or any sign of the disease provided
there are no deep erosions formed.

Complications, in­

volving the tongue, omasum, reticulum, intestines, liver
or lungs due tc secondary invaders, have been reported
(Newsom and Cross, 1951).
crosis and ulcers.

The evident lesions were ne­

Transmission to man under natural con­

ditions had been reported (Hatzioles, 1950; Branderburgh,
1952; ITev/som and Cross, 1954), however it rarely occurred.
Eorses, calves, dogs, and monkeys have been mentioned as
susceptible (Hutyra, et al, 1949).

nevertheless, Howarth

(1929) stated conclusively that cattle, pigs, dogs and
rabbits were not susceptible.
The primary macroscopic lesions develop after an
incubation period which w r i e s from one to four days.

The

lesions always occur on the non-wooly area of the skin.
In general, three types can be distinguished according to
the location of the lesions, namely, labial type, padolic
type, and genital type.
one.

The labial type is the most common

The lesions are confined to the lips and the com­

missure of the mouth, but they quite often occurron the
nose.

The symptoms begin with a circumscribed red spot

termed a macule.

It continues to swell and increases in

height and becomes a papule.

Soon the papule becomes

vesicular in nature.

Meanwhile the secondary bacterial

invaders gain entrance into the lesion and change the vesicle
into a pustule.

This secondary infection hastens the pro­

gress of the stages to such a degree that sometimes the
vesicular stage is so short and insignificant that it is
often not observed, particularly under natural conditions.
Moreover, it is not uncommon to find ulcers which tend
to coalesce.

The pustule, either ruptures or remains in­

tact and dries gradually, so that a crust is formed on
top of the pustule.

The whole period, from macule to scab,

lasts from one to two weeks.
three weeks.

The scab loosens in two to

If the whole flock is affected, the disease

persists for about four to six weeks.

The morbidity of

the lambs in the flock may be as high as 95f>.

Death does

not ensue unless secondary infection sets in.

The labial

type is usually benign in form.

If severe secondary in­

fection takes place under conditions of poor management,
the lesions may spread over both lips and not frequently
may spread to the non-woolly parts of the head such as the
eyelids and ears.

Suckling lambs often iniect the teats

of the ewes during nursing.
The padolic type occurs less frequently than the
labial type.

The favorite location of such lesions is

the coronet.

Due to the anatomical location, it always

results in erosions and ulcers with extensive suppura­
tion and lameness.

The genital type is rare.

It is

characterized, in the female, by painful swelling of the

labia sometimes with development of ulcers.

It often in­

volves the under surface of the tail, adjacent skin, udder
and teats.

In the male, the prepuce is primarily affected

v/ith reddening and swelling.

The lesions also develop

into ulcers which may coalesce to form a large suppurat­
ing area, sometimes involving the entire prepuce.

The

progress of the lesion does not depend necessarily upon
the time element because a two-day old lesion of one sheep
did not show less advanced changes than the three-day
lesion of another sheep.

The seriousness of the infection

v/as mainly contingent up on the manner of management
and the resistance of the host.

REVIEW OF LITERATURE
For many years there has existed a misconception
concerning the nature of contagious ecthyma of sheep.

It

comprised a group of shin diseases under the name of "necrohacillosis".

A virus etiology was not yet known.

Host

of the reports and experimental work incriminated the
bacterium "Bacillus necrophorus".

Due to the resulting

confusion, it seemed desirable to divide the literature
resume into two groups.

First, that which presumably

dealt v/ith the disease solely in connection v/ith the ne­
crosis bacillus; and secondly, that which definitely dealt
with the true etiology.
’
/alley of England (1390) was probably the first to
have reported contagious dermatitis or "Orf", an eruptive
disease seen mostly among lambs.

According to this report

other veterinarians had termed the disease "Carbuncle of
the Coronary Band".
"I'outh and Foot",

All names such as "Hair and Hoof",

"Carbuncle of the Coronary Banu", and

"Orf" which were employed by shepherds and veterinary sur­
geons in different localities were placed under one term,
contagious dermatitis.

The author transmitted the disease

from infected to susceptible animals.

V/alley described

the symptoms and course as follows:
"In the early stages the lesion is cir­
cumscribed, and presents itself as a
local inflammation involving the skin
and to some extent the subcutaneous
tissue.
The skin of the affected part, usually of

9
the coronet and the lips in the first
place, is swollen, hot and tender, and,
where the colour can be seen, red; in
a few days a breach of the surface takes
place, serum oozes from it, and a sore
is quickly formed which tends to spread
and to propagate itself to the skin of
any part devoid of v/ool with which it
comes in contact.
The area involved in
the diseased processes at the outset
does not usually exceed an inch in di­
ameter, but as the malady progresses
the whole of the lower part of the
limb may become involved, either as
the result of pez’ipheral extension or
by coalescence of discrete centres of
disease.
From being of the nature of a compara­
tively healthy sore, the lesion assumes
a somewhat malignant aspect.
The cu­
taneous papillae become hypertrophied
and congested, and readily bleed on
leing scraped.
A purulent fluid of a
dirty grey color and possessing a very
unpleasant odour is constantly dis­
charged and this collects on the sur­
face of the sore, and with the villosities produces a repulsive condi­
tion —
a condition, in fact, close­
ly allied to that v/hich is seen in
grease of the leg of the horse, or
canker in the foot of that animal.
In addition to these diffused sores,
others of a much smaller extent may form
on skin of the leg, and even on the
arm, but these isolated sores do not as
a r le present the foul conditions Just
described, as the discharges, becoming
inspissated, form a crust on the surface;
and the same remark applies, in the
main, to the labial sores.
The histopathological changes of the skin were il­
lustrated by a microscopic section v/hich showed normal
dermis, connective tissue core in an enormously hypertrophied
papilla, irregularly thickened epithelium, remains of
extravasated blood and concentrically arranged masses of
epithelium.

10
Williams of England (1894) (cited by Kohler, 191C)
described a disease v/hich occurred particularly in lambs
but occasionally in sheep, manifested by inflammation
and ulcers on the coronet and between the dibits.

Similar

sores and ulcers sometimes appeared on the face and head.
The first report in the United States of aphtha
or sore noutli was made by Hushworth (1899) (cited by Kohler,
1910).

It was an ailment usually seen in suckling lambs

although older sheep sometimes were severely affected.
Many supposed causes such as feeds, low vitality, un­
healthy environment, physical defects such as decayed
teeth were suggested as causes of the disease.
to be contagious as the udder and

It seemed

teats of the ewes be­

came affected from the lips of the lambs.
In 1900 in New Zealand, England and the United
States several articles were published ..re sum ably dealing
with this disease.

The editorial "The Diagnosis of Foot

and mouth Disease” (J. Comp. Path, and Ther., 1900) men­
tioned the differential diagnosis of sore mouth from foot
and mouth disease.

This inferred that sufficient knowledge

was obtained from field work to make possible the dif­
ferentiation of these diseases.

This article also indicated

the characteristic symptoms of this disease:
"The suspected flock number about 120,
and rather more them the half of them
had been bought in a public market at
Perth a fortnight previously.
While
these were being driven home it v/as
noticed that some of them were lame.
V/lien brought to the farm they were
put in the some field with about 50

11
other sheep v/hich had "been bought
from a neighbor.
About a week later
the disease began to appear among this
lot also, and at the time of If*.
Clark’s visit only about 20 animals
had not been attacked.
Without ex­
ception the animals that escaped
were over a year old, and all the
diseased ones wele lambs.
In every
case the disease affected the lipa
as well as the limbs.
The lesions on
the limbs extended from the top of
the hoof to the knee.
The foot v/as
tender at the top of the horn, and
in some cases there v/as a little se­
paration of the horn at the heel.
On
the head the lesions were confined to
the skin of the lips and nostrils,
and in no case did they involve the
mucous membrane of the mouth.
In both
positions the lesions took the form of
patches of dermatitis, in which the hair
was matted together with a partially
dried up exudate, or the place v/as cov­
ered v/ith a brown scab or crust.
Important points in the history were
that in spite of opportunities for in­
fection the cattle on the same form re­
mained unaffected, that the same disease
existed among the sheep on three neigh­
bouring farms, anti that on these also
it v/as confined to the sheep although
on one of them cattle were separated
from the affected sheep only by a wire
fence.”
This article mentioned not -only the character of
the disease but also that it was not necrobacillosis,
since cattle were not affected.
Gilruth (1900, 1906, 1907, 1908) (cited by ifchler,
1910) of IJew Zealand recorded a disease in sheep v/hich
was called acute facial eczema characterized by eruptions
on the face and ears of the lambs.

It v/as considered to

be due to eating rich feed such as rape or clover.

12
Clayton, Gilruth's assistant,

(cited by .ICohler, 1910)

further described this eruptive disease among a flock of
lambs.

A large _.ercentage v/ere affected; in some cases

massive scabs covered the v/hole face.

In others the lesions

were located only around the mouth and nostrils.
or leg lesions v/ere present.

Ho feet

Clayton could not see the

relationship between the disease and rich feed; neverthe­
less he believed it to be dietary in origin.
Gilrath (1906) (cited by Hohler, 1910) described
again an acute dermatitis of the face affecting the skin
of the mouth and nose of sheep in hew Zealand.

Ilia ex­

perimental work proved that the cause v/as a miero-organism.
In 1907 he reported a disease called acute stomatitis af­
fecting the lips and mouths of lambs, and found the cause
to be the came as the one causing the disease among sheep
described above,

nevertheless it was questionable whether

the actual lesions produced were the some.

He reported

that extensive ulcerative sores were found after docking
and earmarking at the tail stump and on the ears.
v/ere present in the region of the scrotum.

Ho lesions

The following

year it was proved to be a contagious disease by inoculat­
ing healthy ewes.
In the United States Lav/ (1900) (cited by Hohler, 1910
described an enzootic in lambs under the term of ulcerative
stomatitis.

He probably v/as the first to definitely

apply the term "contagious" to this disease.

H oy/ever, he

failed to demonstrate the causative agent, but believed it

13
v/as due to a microbe.
Berry (1901) (cited by hohler, 1910) described this
disease in the British Isles.

It v/as called contagious

pustular dermatitis, orf, and crusta labialis.

Iambs

v/ere more frequently infected, though sheep of all ages
v/ere susceptible.

The infectivity v/as so high in certain

flocks that all v/ere affected.

Affected sheep transmit

the disease to healthy ones v/ithin a fortnight.

Mortality

v/as low.
In France, Beano it (1901) (cited by J.lohler, 1910)
described a contagious ulcerative stomatitis of lambs and
goats.
Flook (1905) determined that the venereal form of
this disease v/as transmissible and that it affected not
only the vulva, bat also the mouth and nose as indicated
by ulcerative sores.

Symptoms v/ere shown among the healthy

animals about one week after exposure.
McFad.yean (1905) in Scotland, described a contagious
disease of the generative organs of sheep similar to lip
and leg ulceration.

In 1907, he succeeded in reproducing

the disease on the sheath of a susceptible ram.

He felt

that the disease was bacterial in nature and tried to iso­
late tiic causative organism but the results v/ere inconclusive.
He could not find any specific organism to substantiate
his observation, although he believed that the bucks which
v/ere newly introduced into the flock might v/ell be examined
for this infection before being used for breeding.

14
'Jilljams (1904) reported :aore cases of the disease
in England.

The symptoms v/hich he described v/ere similar

to those mentioned by the preceding authors.
Hutyra and Karek, in Hungary (1S06) (cited by Kohler,
1910) reported the presence of pustular stonatits on tv/o
imported bucks.

The native sheep showed symptoms and

lesions three days after the imported ones v/ere introduced
into the flocks.

Almost the entire flock displayed erup­

tions and ulcers on the lips, mouth and edge 'of the nostrils.
Hasenkamp (1908) (cited by Kohler, 1910) in Germany
observed a number of cases of ulcerative stomatitis in
slieop v/hich v/ere accompanied by foot rot.

He v/as able to

isolate E. necrophorus as the causative agent of the condi­
tion and observed necrotic lesions in the liver and lungs.
Koussu of France and Dollar of England (cited by
Hohler, 1910) described an ulcerative stomatitis of sheep.
The authors found the mortality of the animals affected
v/ith this disease to be as high as 15 per cent.
A year earlier (1907) (cited by Kohler, 1910) in
the United States Knov/les (1907-1903) reported infectious
lip and leg ulceration of sheep in Montana.

This v/as the

first reference to the infectious nature of this disease
in the United States.

The author'described the necrosis

bacillus as the cause and succeeded in transfering the
disease from infected sheep to healthy ones.

He described

it as having a distinctly seasonal occurx*ence during early
fall and winter.

He concluded, however, that poorly

15
nourished suffered to a greater degree than healthy animals.
■7ing

(cited "by Hohler, 1S10) in the publication

"Sheep Farming in .America" mentioned a disease contagious
in nature, similar to sore mouth which affected the teats
and udders of the ev/es.
The work of the Eureau of Animal Industry in the
suppression of lip and leg ulceration of sheep v/as intro­
duced by HeIvin and reported by Hohler (1910).

This pub­

lication v/as v/orthy although it followed all preceding
opinions v/hich retarded the necrosis bacillus as the sole
causative agent.

In their paper they intended to simplify,

under one name and one cause, the great variety of names
and causes previously given the disease.

They designated

as "necrobacillosis" those shin diseases v/hich v/ere caused
primarily by the necrosis bacillus and characterized by
the eruption,of vesicles or pustules on the feet, mouth,
prepuce, vulva, teats and udder.

They restricted the use

of the name to forms of the disease in v/hich the lesions
appeared in two or more parts of the body.

They distinguish­

ed it clearly from foot and mouth disease.

(There v/as a

foot and mouth disease outbreak in the eastern part of the
United States at that time).

Unfortunately they were not

aware of the possibility of any other virus.

They found

that the disease''rarely affected the v/ooly portion of the
body.

The authors knew it was contagious, and understood

that it "could have complications such as respiratory dis­
turbance and occasionally affected the liver 'and stomach.

16
They classified it into four forms:
leg

2) venereal

Z)

foot rot

namely, 1) lip and

4) sore mouth.

In the lip and leg form there v/ere two stages:
active or acute and inactive or chronic.

The former v/as

manifested "by lesions in various anatomical locations and
accompanied by inflammation, tumefaction, ulceration and
necrosis v/ith or without scab formation.
or less rapid destruction of tissue.

There v/as a more

The chronic type

v/as an example of inborn proliferation.

They described

the pustular formation as -"The lesions in the early stage usually
appear as an acute inflammation of the
skin on the outside of the lips.
The
pimple-like formation is attended v/ith
much inflammatory swelling v/ith a de­
cided tendency toward the formation of
pustules.
They dry and form crusts of
a dai’k grayish c o l o r
"
The sore mouth form v/as usually seen in lambs dur­
ing the fall of the year, although it did occur even earlier
in the season.

It v/as characterized by warty or pustular

patches on the lips v/hich v/ere covered v/ith a brown crust.
The victims presented nodules or patches on the lips,
mostly at the junction of the mucous membrane and the hairy
portion.
Tidsv/ell (Australia, 1910-1911) described a disease
similar to sore mouth with secondary infection.

According

to his classification, skin and subcutaneous tissue in­
volvement was termed foot-rot; localization about the
mouth v/as designated malignant stomatitis or aphtha; im­
plication of generative organs was v-ginitis, balanitis

17
or pizzle disease; and involvement of the digestive canal,
lungs or liver with abscesses or diphtheritic inflammation
was called metastatic.

He believed that the necrosis bacil­

lus was the sole cause.

However, the following excerpt

expressed the opinion that other complications v/ere necess­
ary to its promotion.
"There seems to be no room for doubt
that these lesions are due to the ne­
crosis bacilli itself, although, per­
haps, it could never give rise to them
failing the opportunity of entrance
afforded by some other lesions.
As a
consequence of its tendency to graft
on the other diseases, necrobacillus
is a frequent complication of such con­
dition as foot and mouth disease, swine
fever, which cause defects offering op­
portunities by the necro.horus bacillus."
According to this report,,. whether or not this was a virus
disease was at least being contemplated.
Ho are (1915) mentioned contagious pustular dermatitis
of sheep.
Zeller (1920) published on an investigation of a
variola-like form of the disease in the goat that occurred
in Southwest Africa.
From this time on, it seemed definitely established
that there existed some causative agent other than the
necrophorus bacillus.

In France (1925) Aynaud proved that

this disease - contagious pustular dermatitis or stomatitis was due to a filtrable virus.

He led in the correct con­

ception as to the etiological agent of the disease.
author related it closely to variola-vaccinia.

The

This v/as

18
very important transitional point in the study of the
disease.

This revolutionary discovery clarified the

etiology of this condition.
About the same time Blanc, et al (1922) v/ere also
able to demonstrate a filtrable virus in the lesions.
Horeover, they concluded that this disease v/as identical
in goat3 and sheep.
Since then a number of workers have added to the
information on this disease such as the incidence, reported
by Koussu (1924) in France; Jacotot (1924) in Annara; Lanfranci
(1925) in Italy; and Sedden and Belschner (1927-1928) in
Hew South '.Vales.

Cornell (1928) also reported the disease

in Tanganyika Territory.

Only after the finding of a

filtrable virus in the lesions was its characteristic nature
known and the close relationship to the pox disease group
established.
Newsom and Cross (1925) mentioned the occurrence of
sore mouth among feed lot lambs.
Glover (1928, 1950, 1955) ran a detailed experiment
and recorded the symptoms, immunity response and the inter­
relationship with pox.

He signified that five stages of

the disease occurred, namely;
pustule and scab.

macule, papule, vesicle;

He found that the virus had a rather

large particle size and noted by filtration its marked af­
finity for epithelial tissue.

This virus could pass through

a Berkefeld V filter, but not a Berkefeld IT nor Chamberland
Lg.

Histologically he described three stages;

(1) Papulo­

vesicle; which mainly showed a change of the outer layer

19
of epidermis.

The cells v/ere swollen to different de­

grees and the nuclei v/ere shrunken...
sizes appeared in the cytoplasm.

Vacuoles of various

The beginning of cellular

proliferation was also a matter of significance.(2) Vesiculo
pustule stage shov/ed more advanced degenerative changes
and proliferation.

Intracellular edema v/as so intensified

as to balloon the cells.

Leukocytic infiltration increased.

Vesicles became considerably larger and had the tendency
to be converted into pustules v/hich contained a few cocci.
(?) Subsequently the pustules erupted and crusts fox-med on
the lesions.

Both polymorphonuclear and lymihiocytic infil­

tration wex’e dominant.

Healing took place beneath the

ci’usts by regeneration of the epithelial cells and resolu­
tion of the cellular debris.

A similarity betv/een the

tissue changes in the thigh lesions anti those of the lips
v/as found after a comparative examination.

Subsequently

the cross-immunity and sei'ological test of this virus
with pox was repoi'ted.
,/elch (1926) stated that all names such as - sore
mouth of lambs, lip and leg ulceration of ewes, foot-rot,
venereal disease of bucks and even navel infection of young
lambs - designated different forms of the same disease.
Like others he attributed the cause of the disease to the
necrosis bacillus.

He sti'essed the weather, in that early

snow and cold seemed to provide favorable conditions fox*
the development of the disease.
Theiler (1928) desei’i' id ecthyma contagiosum of

sheep and goats in South Africa.

According to his de­

scription the condition seemed to be identical to that
reported by Glover (1928).
Howarth (1929) gave a detailed description of the
symptoms of the disease and the differential diagnosis
between lip and leg ulceration of sheep and diseases
caused by Bacillus necrophoru3:
Bacillus Hecrophorus

Filtrable Virus

Susceptibility
of various
animals

Horses, cattle, sheep,
Sheep and goats
goats, hogs, dogs, rab­
bits, chichens, mice,
guinea pigs, monheys,
hangaroos, reindeer, etc.

Ages

All ages

All ages, usually
young lambs and ewes
following lambing

Course and
duration

Slow and protracted,
several months or more,
individual sheep.
8
or 10 months to over a
year.

Hapid, individual
sheep approximately
G wechs; floch3,
2-5 months

Period of
incubation

Z to 10 days,

4 to 7 days

•

Parts affected

occasion­

ally 15 days
1

Shin, lips, mucous mem­ Exterior of lips, gums
brane of mouth and up­
palate, portion of the
per air-passages; lungs, face free of wool such
digestive tract, navel,
as the eyelids and in­
abdominal viscera, hoofs, side of ears; mammary
cartilage, bone, muscles glands, teats, poster!
and joints, mammary
part of abdomen and in
glands, teats; in fact no side of thighs which
tissue seems to be immune' are free from wool.
to the invasion of B.
necrophorus.
“

21
Bacillus Kecrophorus

Filtrable Virus

Lesions or
histopathology

Progressive tissue ne­
crosis characterized by
caseous degeneration
and invasion of t h e .
deeper tissue.
Diph­
theroid lesions of
raucous membranes.
Ab­
scess formation in the
connective tissue, liver,
or lungs.

Slight reddening and
elevation of the shin;
papules, vesicles, „ ,
pustules and scab
formation.

Intravenous
injection

Followed by m e tastatic
abscesses especially in
the liver and lungs.

No lesions produced

Immunity

None

Definite

He concluded that sheep and goats were the only animals
susceptible to this virus.

The filtrability of the virus

v/as inconsistent even when the same type of filter v/as
used.

Animals that had been ..exposed to this virus develop­

ed a high degree of immunity.

However the duration of im­

munity v/as not stated.
In the same year (1929) Hilandi and Styliamopoulo re­
ported worJc dealing with "1* ecthyma du mouton” in Greece
(cited by Boughton and Hardy, 1934).
Schmidt and Tunnicliff (1929) reported that the use
of a bacterin for immunisation against sore mouth v/as not
conclusive.

In an article by Bosv/orth and Glover (1931)

sore mouth v/as distinguished from diseases of venereal in­
fection by cross-immunity.
Newsom and Cross (1931) reported on the complications
of sore mouth.Ulcerative lesions v/ere present in the mouth,

22
on the tongue, in the omasum, reticulum, rumen and in
the small intestines.
lungs and liver.

Abscesses were observed in the

They attributed these complications to

ihvasion by Actihomyc e a neerophorus, although they believed
that the etiology of sore mouth v/as still questionable.
In 1934 another article v/as published that confirmed the
etiology of sore mcuth as a filtrable virus and in the
same year reported that this disease v/as transmissible to
man.
Carre (1952) described the duration of immunity
from the natural infection as being 2£ years and from the
artificial infection as one year.
Later ITev/som and Thorp (1938) included this disease
as one of feed lot diseases of lambs.
Erandenbnrgh (1932) in the paper "Lip and Leg Ul­
ceration in Sheep v/ith Report of Two Cases in L'an" failed
to describe clearly the nature of the disease.

He stated

that it v/as due to Actinomyces necrophorus, but v/as un­
successful in isolating it from the lesion found in man.
Seddon and McGrath (1931) reported v/ork on crossimmunity tests with the virus of infectious labial derma­
titis of sheep isolated from various anatomical locations.
Schmidt and Hardy (1932) wrote a detailed article
dealing v/ith immunity.

They found that "the goat virus

did not appear to be as virulent for the sheep as sheep
virus and vice versa."

The authors thought that this in­

dicated the existence of a sheep virus and a goat virus.

They did not determine whether a cross immunity existed
between the sheep and goat strains of virus.

The authors

realized that high degree of immunity was produced but
were not sure of the maximum duration;

They’ failed: to

pass the virus through Seitz and Handler filters.

Sus­

ceptibility in animals from the age of lambing to one
year was 90-95 per cent while in adult animals about 4-0-50
per cent were susceptible.
Boughton and Hardy (1954) declared that Handler
filtration would weaken the infectivity of the virus.

The

filtrability of the virus was irregularly demonstrated.
In 1955 they announced that the virus was filtrable through
Handler filters.

The susceptibility of the sheep to the

second exposure was 0.006 per cent.

Ho evidence could be

found that recovered animals acted as carriers of infection
from suckling to feed-lot lambs.
Newsom and Cross (1954) studied the virus and de­
clared it to be filtrable through Berkefeld V but not
through Handler normal filter.

They presented the case

of transmission of virus to man and produced "take" in
sheep by using the man’s vesicle fluid.
Tunnioliff and Hatischeck (1941) reported a viral
disease in the article "A Filtrable Virus Demonstrated To
Be The Infectious Agent In Ovine Balano-Posthitis".

How­

ever, they did not point out clearly whether other portions
of the body were susceptible.

They emphasized the pH value

of the viral suspension but did not state whether it would

24
play a part in the infectivity of the virus,

neither

did they state clearly how and under what conditions the
tissue should be ground.

In other words, the condition­

al factors in the treatment of the virus could b e o f im­
portance in relation to the infectivity of the virus.
question that still remained was --

The

could the virus which

they had investigated be a variation of sore mouth virus?
Bennettt Horgan, Hasecb (1944) published an article
in which the immunological relationship of sheep pox virus
and sore mouth virus was stated.

Cross-immunity tests

proved that goat-pox protected against sore mouth virus
in goats, but that there was

110

versa.

1. the two viruses therefore

They concluded that

protection afforded vice

closely related, goat-pox possessing at least one antigenic
factor that contagious pustular dermatitis lac3cs.
also claimed that there was

110

2. They

cross-immunity between sheep

pox and this disease.
Selbie (1S44) affirmed the transmissibility of the
virus from sheep to rabbits and compared the histopatholocical
♦

lesions of the disease in sheep with those of rabbits.

He

stated that the lesions produced by the virus in rabbits
were more proliferative than those usually found in sheep.
However, he pointed out that the histological picture was
somewhat different because of the hyperplasia of the hair
follicle was absent in Glover’s work since those lesions
were produced on the hairless shin of the thigh.

The rabbit

infeetion,was very similar to the chronic, papillomatous
form of the natural infection both in its course and histo-

logical characteristics.

«

Recently, Tunnicliff (1949) cleared the confusion
on this disease as follov/s:

"The posthitis, balanitis,

ulcerativevulvitis complex/ and lip and leg ulceration:
conditions are different manifestations of the same disease
for which the name ’ulcerative dermatosis of sheep’ is
proposed. ------

The virus of ulcerative dermatosis at­

tacks only the epidermal tissue, seldom, if ever, extend­
ing to the mucous membrane.

The virus of sore mouth has

a predisposition for the lips at the junction of the mucous
membrane and the epidermis, extending tc the face and interdigital space beneath the interdigital pouch. ------ lactcriologically the lesions of ulcerative dermatosis are
quite free from the neorophorus organism and all ordinary
pus-producing bacteria.

Foot rot and sore mouth lesions

both contain quite a collection of miscellaneous bacteria,
and the necrophorus organism is usually present.

It is

certainly a secondary invader in the case of sore mouth,
and, as yet its role is not entirely determined in the
case of foot rot."

:.:aiiriais aid :-1t::gds

Varioas tissues of different animal species were
employed in an effort to produce lesions following inocula­
tion of contagious ecthyma virus.
Sheep, rabbits, guinea, pics, chorioallantoic mem­
branes of the dcvelopinc chick embryos and sheep skin
crafts on the chorioaliantoic nembr-nes v:ere used in this
s t tidy.
Exposure of the skin of sheep and rabbits to the
virus.

Commercial ovine-ecthyma vaccine* prepared aceord-

inc to directions for field ve.ccination was used to inoculate
shin on the inner t.’.ich of sheep and coo.ts.

The same type

of inoculum was used to infect the scarified sJ:in of the
lips of sheep.
The inner side of the flan;: area was used for inocula­
tion.

It was necessary to clear, off the crease oj>d dirt

before inoculation.

The area v;as washed in alcohol and

after the shin v/as dry it vrs scarified with a razor blade
to s..c

a decree that blood and lymph .just began to o o z e out.

The virus suspension was applied and the animal was held
still for several mina.ti-s to avoid spreading the virus
to other parts of the bod;
the virus "tone11.

end to increase the chance for

Shin biopsies were taken from the sheep

after inoculation at 6, 12, 24, £6 hours; 2, Z ,
S, 9, 11, 1C and 20 days.

4, 5, 6, 7,

Skin biopsies were taken from

*
Fort Douce laboratories, Fort Dodge, Iowa.

the goats after inoculation at 6, 7 and 8 da,; s.
Obviously it v/as impossible to secure all the material
from one sheep, although it v/culd have been the ideal
■■•method -of— collection.- ■■■-Several- sheopaand goats v/ere u s e d .

to obtain the various decrees of pathulo_ical chances,

ho

biopsy material v/as tahen from the lip lesions.
A similar!type of inoculum v/as applied to the shin
on thebachs of rabbits (6).

The hair v/as removed by olippin

and shaving before exposure to the vaccine.
Rabbits (8) and guinea pigs (8) v/ei\ exposed to
the

vaccine by dropping the suspension in the eyes.
Rabbits Y/ere also inoculated, by the intraocular

and intrater ticular (?:) route.

(8)

Zenhcrs solution v/as used

for fixing the shin and other tissues.

The usual methods

of ...roces: ing sectioning, mounting and staining v/ith heraotoxylin and eosi/i v/ere used.
Exposure of the chorioal 1 antoic membrane and sheep
shin graft on choriallantoic membrane to virus.
Commercial ovine ecthyma vaccine v/as diluted 1:100
v/ith nutrient broth.

One thousand units each of penicillin

raid streptomycin v/ere added to each ml. of diluted virus
suspension in order to combat bacterial contamination.
The embryos v/ere eleven days old v/hen used for
chori oal-antoi c membrane inoculation.

Twelve day old

embryos v/ere used for allantoic sac inoculation.

Sight

to nine day old embryos v/ere used for intravenous and intra-

28
cerebral inoculations (Beveridge and Burnett, 1946).
Bor skin grafting the nine-da# old cmbrpos v/ere
also used.

The eleven-da: old

~ensymatie- stud#.-

enbr.yoswore used for

The amount ■of-inoculum usedv/es-as-

the
fol-■•----■■

lows:
Chorioallantoic membrane

inoculation

0.1 cc.

Chorioallantoic membrane

plus skin craft

0.1 cc.

Intra-allantoic sac inoculation

0.2 cc.

Intravenous inoculation

0.05 cc.

Intracerebral inoculation

0.02 cc.

The embryos v/ere incubated at 07° C for varying
lengths of t.ne after inoculation.
Fixation and process!/:,;-; of tissues.
1.

The inoculated eggs v/ere kept in the original posi­
tion (artificial air sac upward).

2.

The shell vvrs opened

so f fieri o j if! y t o

<■

area of inoculation.
0.

Three to five nl. of Kelly’s solution v/as dropped
through the opening onto the chorioallantoic M e m ­
brane end allowed to remain for 1 hr.

4.

The chorioallantoic membrane v/as cut along the edge
in order that the whole inoculated area could be
removed.

5.

The membranes were further fixed in nelly’s solution
for an additional 1-1-1 hr.

6.

They were washed in running water for 8-12 hrs.

7.

The membranes v/ere trimmed and passed through grading

29
concentrations of alcohol for dehydration.
In 80fj alcohol - 24 hr. .
In 9hf> alcohol - no longer t' an 6 hr.
In lOOfj alcohol - no longer tiian 1-1?; hr.
8.

They were kept in cedar wood

oil

for 12 hr. (less

tine v;as preferable for thedelicate
9.
10.

ncmbrai.es)

Two changes of parofiin.
Embedded in paraffin.

The advantages of this nodifled nethod w e r e ;
1.

L'inimiu. disturbance of the chorioallantoic menbraiic.

2.

The chance of infection and spread of the viral agent
was minimised.

2,

The chorioallantoic membrane was nore easilyremoved.

4

Before fixation s. culture of each membrane v/as

mr.de

on agar to chech for sterility.
The usual procedures for socti.ning and mounting
were employed.
hothod of Staining:
Hematoxylin and eosin gave satisfactory results.
HacCollun1s’modified Goodpasture stain v/as used
as a further check for bacterial contamination since local­
ised bacterial infection may escape detection by culture.
Some sections showed necrotic areas due to bacterial con­
tamination.
Safranin-Light Green stain was used for tisso.es
which v/ere to be examined for inclusion bodies (Cawdry, 1948)
The method of fixation, mounting and staining of

50
sJ:in grafts v/as the same as mentioned previously under
shin biopsy.

Seven, 9 and 10 day infected shin grafts

..’ere harvested and sectioned.

The .Method of grafting

basically folldv/ed Goodpast',.re,:'s techniouo for human s h i n .
(1958).

Virus v/ac introduced immediately after grafting.

Penicillin and streptomycin v/ere used to hold dov/n the
bacterial infection.

Full thiejvnes: of sheep shin v/as

used except that the loose connective tissue v/as carefully
removed.

(Goodpasture et al, 1955)

The method for enzymatic activity study.
Oxidase;

This method v/as used by homeis and Mallory

(Lillie, 1948).

Two solutions v/erc prepared:

Solution A:
Alphanaphthcl

0.5 g m .

Distilled water

50 cc.

heated until a.lpiiruiaphthol started to melt, 85^ potassium
hydroxide added drop by drop until the alghanaphthol
completely dissolved.
Solution 3:
Dimethyl parr.pheny 1 enediamine
Distilled v/ater

0.5 gm.

50 cc.

Botii solutions should be bcpt in the darh, even though
they v/ere g od only for a fev/ v/eehs.

The procedure general­

ly used v/as as follows:
1.

Air dried smears of chorioallantoic membranes v/ere fixed
for 3 hr. in formalin-alcohol solution.
with 4 parts of 95fj alcohol.

1 part formalin

ri
2.

Stained two to five minutes in an cruel volume mixture
oi’ cclution A and B.

S.

Rinsed in distilled water end mounted in glycerol.

4.

The oxidr.so granules were dark blue in color.

Acid Phosphatase Hethod:
This raetiiod v/as ./olf'1s modification (./oil’ e_t a l ,
1942) oi G-oruori1s method (1941).

It was based on tne

idea of changing the organic phosphate into inorganic
phosphate.
The chorioallantoic tissues v/ere dehydrated and
fixed in anhydrous acetone and hept in the ice box for
twenty-four hours, followed by three changes of acetone
at rccn ter.per at are within two hoars.

This v/as done the

3ame way as in alkaline phosphatase preparations, so that
the routine work of fixation would bo simplified.

The

solution of substrate v/as composed of lend nitrate and
sodium bcta-glycerophosphate with the buffer at pH 4.7
C//5 acetic acid 9 parts with '.75 sodium acetate 11 parts).
Ac cording to Gormori (1941), the parafriii infiltration
process could last no longer then, three hours, because
the heat would affect the enzyme.

Sections were cut at

7 microns in thickness, and were mounted with glycorolalbunin.

Then treated as usual to remove the paraffin

and passed through the alcohols to water.

Thereafter the

procedure was as outlined:
1.

The slides were rinsed with distilled water.

2.

f ey v/ere then incubated in the following solution for
12-18 hr. at 27°G.

Acetate buffer at

pH 4.7

12 parts

lead nitrate (0.1

L')

10 parts

1:: stilled water

74 parts

Sodium - teta-ipl^xerqpiiosph te. (Z.2*4),.. .......

4 parts

Total

100 parts

5.

The: v/ere rinsed in distilled water.

4.

Then rinsed in Z ‘/> acetic acid.

5.

Followed with a thorough rinse in distilled water.

6.

Immersed in yellow ammonium sulfide (1:50 dilution)
for 1-2 minutes.

7.

./ashed v/ith tap water.

S.

Counter stain if desired.

(nenatonylin-eosin was not

satisfactory, because it v/oula overshadow the lead
sulfide granules if they were not in lar^o quantity.)
Site of phosphatase activity was shown by brown or
dark deposits of lead sulfide.
Control sections should be used.

Sodium flouride could

be used as an inhibitor.
Alkaline phosphatase method:
The original idea (Gormori, 1989) of the alkaline
phosphatase was based on the formation of calcium-phosphate
which was precipitated from the calcium-£lycerophosphate
by ensynatic reaction.

The calcium-phosphatae yjrecipitatc

was practically' insoluble at a pH 8-10.

The optimum pH

of the alkaline phosphatase v/as a little above pH 9.

A

silver compound was used to replace part of the salt into
silvei* salt, tP.cn it would be reduced by the liyht into

33
metallic silver which would a..pear as black or blackbrovmish granules in the cell.

The tissue v/as fixed in

acetone for dehydration and treated as mentioned previously
,ir;, the, acid phosphatase ...staining...metie.d*.,-- After..-the- tissue
was dehydrated it v/as processed in the following manner:
1.

Hardened in absolute alcohol for 24 hours.

2.

Cleared in iCylene, lensene or Toluene.

3.

embedded in paraffin.

4.

Sectioned and mounted.

5.

Passed through two changes each of xylene and abso­
lute alcohol.

6.

Then soaked in etin r-alcoiiol (equal parts) solution
of collodion (0.5-1/-) for five minutes.

7.

Drained for 1-2 minutes and hardened in 80/- alcohol
for five minutes, then transferred into the water.

8.

Incubated at 87°C overnight (8-14 hours).

Calcium

control sections were put into 0.1/ calcium nitrate
solution.

Phosphatase sections were incubated in

the following solution:
Sodium beta-glycerophosphate (5.2/)

6 parts

Calcium nitrate (Ca(hCc Jg'd-HgC) (2/)

9 part3

Sodium barbitol (10/)

6 parts

I.Iagnesium sulfate ( H g S O ^ ^ H g O ) (0.1 k)

6 parts

Distilled water

33 parts

This solution had a pH of 9.0.
9.

All slides were rinsed in distilled water after in­
cubation.

They were treated under a strong light

v/ith 5'j silver nitrate for 50 minutes.
(100 v/att b\lb at 1.5 ft.)
10.

Acetone for dehydration and removal of the colloidin
film.

.\ .'.' .
'.“................^ .' ■

Alkaline phosphatase v/as siiov/n as a black or black-brownish
deposit.

*

RESULTS

llo significant lesions resalted from inoculation
of the virus on rabbit shin nor instillation in the eyes
of rabbits or guinea jigs.

likewise no lesions resulted

from intraocular or introtesticular inoculation of the
virus in rabbits.
V/hen the virus was introduced into chick embryos
by the intraallantoic sac, intravenous or intracerebral
routes no significant lesions were observed.

I ll bTOPATIIOLOGY

(1)

Histopathological study of the shin in contagious

ecthyma.
Biopsied specimens of shin which had "been inoculated
v/ith the virus of contagious ecthyma were examined.

The

various stages in the pathogenesis were observed from
six hours after inoculation to 21 days at which time the
area was well healed.

The tissue response to the viral

infection v/as observed as early as 12 hours (Pig. 7) after
the virus v/as applied by the scarification method.

The

histopathological changes were limited primarily to the
epidermis, but secondarily the dermis v/as involved due to
the continued action of the virus mid reaction of the
tissues.

The gross pathology did not exactly coincide

with the histopathology.

For instance, the stage of macule

which was seen clinically proved by microscopic examination
to be an early stage of typical vesicle formation or even
that of a pustule (Fig. 4).

The material having a gross

hemorrhagic appearance was not revealed to be hemorrhagic
microscopically.

At the pustule stage the hemorrhages in

the upper and lower corium were not seen macroscopically.
The first sign of the micro-pathologicaJL changes of this
disease was the disturbance of the nucleus of the epithelium.
This indicated the part of the cell which v/as first at­
tached by the virus.

Either the number or the size of

nucleoli, or the sise and shape of the nucleus v/as changed

from slender and oval to large and round.

Lost frequently

both changes appeared simultaneously v/hicli made it difficult
to decide which occurred first.

ITo inclusion bodies either

cytoplasmic or intranuclear v/ere found.

The nucleoli were

enlarged and tooJc a heavier Jhexatoxylin stain.

Chromatin

material was reduced and marginated peripherally to the
nuclear membrane.

Along with the increase in the number

of the nucleoli and the enlargement of the nuclei, the
epithelial cells had proliferated so that the epidermal
layer was frequently composed of 5-10 layers of irregularly
arranged prichle cells, which resulted in various degrees
of acanthosis.

(Fig. 7)

The tonofibrils were not too

clear, although the cells v/ere still well cemented to­
gether.

The arrangement of basal eell3 lost its regularity.

Hitosis appeared more often than normal.

At the periphery

of the nucleus, immediately between the cytoplasm and
the nuclear membrane, a clear zone usually appeared sug­
gestive of a tendency of the nucleus and cytoplasm to separ­
ate.

In some areas the cells showed a peculiar lengthen-

ing of their nuclei with a more granular appearance along
the inside margin.

However, this did not occur often.

In most of the areas the epithelial cells simply became
large and round, and lost their customary arrangement.
oj*
This was true especially^the basal cells.
Inflammatory
reactions v/ere indicated by distended lymphatics and blood
vessels with extravasation of lymphocytes and red blood
cells at the lower layer of the corium.

The latter change

occurred after the vesicle stage (Fig. 11) “but before
pustule formation (Fig. 12).

The severity of the inflamma­

tory reactions vtiried greatly with the stage at which the
vesicle was examined.
often was seen.

Slight edema '"beneath the epidermis

In most cases just a lymphocytic infil­

tration existed in the upper part of the corium during the
early stage of infection.

The continued action of the

virus within the cell produced further tissue reaction.
ICeratinization (Fig. 3) v/as increased a little at the sur­
face.

These cells became .keratinize 30 rapidly that most

of the nuclei and outlines of the cell3 still existed.
Therefore a layer of parakeratinized epithelial cells v/as
first formed under the regular keratin.

This layer of

paraJceratinized epithelial cells was first formed under
the regular keratin.

This layer of parakeratinization

was composed of the outer layer of epidermal cells with
a vacuolization of the cytoplasm, with or without a nucleus.
The nucleus when present had no definite shape or detail.
The cells v/ere well cemented together and showed no loosen­
ing between cell membranes.

They wore arranged horizontally

parallel with the surface line of the skin.
The formation of the vesicle v/as the result of the
changes in both the nucleus and the cytoplasm.

In general,

these changes became deereasingly severe from the keratinized
layer toward the basal layer.

The nuclei enlarged in size

and displayed reduced chromatin, and v/ere swollen first
to a round shape v/ith light homogenous hematoxylin stain,
in contrast to the basophilic staining 7/roperty it had

formerly.

Dinappearance of chromatin occurred but the

nucleoli remained as a bluish round shape.

Subsequently

it submerged v/ith the ballooning degenerative process.
....
'

(Fig. 9)

'... ..... ..... ..... ..... ~ .. .....

The cytoplasm became granular and took a lighter
and less uniform stain.

The vacuoles which v/ere present

in the cytoplasm v/ere of various 3izes and usually round
in shape.

From their shape it v/as noted that several small

vacuoles coalesced to form a large irregular one (Fig. 10).
A network remained in the cytoplasm making a mesh work be­
tween the vacuoles.

many cells underwent liquefication

or burst due to the tension of the accumulated fluid.
The spaces then became filled v/ith the accumulated fluid.
Various types of cells as well as coagulable material v/as
found in the vesicular content (Fig. 11).

Heanv/hile other

cells swelled due to intracellular edema and eventually
underwent ballooning (hydropic degeneration).

These cells

were characterized bp a clear space formed around the
nucleus which extended peripherally so that the cytoplasm
was reduced to a thin network.

The cells of the outer

layer of the epidermis showed more ballooning than the
inner layer of the cells.

The basal cell layer was hardly

involved in the hydropic degeneration at all unless the
vesicle formation v/as excessive.
the nucleus lost its fine detail.

As the cell wa3 ballooned,
Usually just a rather

homogenous fine granular lighter basically stained outline
of it remained.
nature.

The cells consequently lost their prickle

The arrangement of the basal cells suggested a

40
tendency toward highly active prolifcration.

As the

proliferating and "ballooning continued these cells soon
"became fused together and formed the so called intraepidermal
vesicle.
Under natural conditions, the lesions v/ere so ex­
posed that the secondary invaders, mainly pyogenic "bacteria,
easily gained their way among the impaired epithelial
cells.

The vesicles, almost without exception, "became

pustules "by this invasion v/ith the accumulation of debris
of resistant cells and polymorphonuclear leukocytes.

If

the "bacterial contamination occurred early and pus formation
proceeded the incubation period of the virus, then the
vesicle filled v/ith pus and pi’oduced a pustule (Fig. 14).
In this instance, vesicle formation could not have been
present.

The histopathological change was manifested

primarily by a pustule (Fig. 12).

At this stage, before

the pus accumulated, it was easily mistaken as a macule
clinically because only redness and localised swelling
wa3 observed.
In striking contrast was the infiltrations of lympho­
cytes and polymorphonuclear leukocytes.

lymphocytic in-

filtra.tion seldom went beyond the basal layer of the
epidermis (Fig. IS) unless it was destroyed in the ad­
vanced pustular stage,

lymphocytic infiltration occurred
-fibers
in the upper layer of the coriura among the collngen^iust

beneath the epidermis, where the virus had invaded, some­
times even earlier than the proliferation and swelling

41
of the epithelial cells.

Practically no polymorphonuclear

leukocytes v/ere found intlie early stage of the lesion.
However, soon after the bacterial invasion many migrated
through the epidermal layer.

Blood vessels and lymphatics

v/ere distended greatly; free red blood cells appeared in
the nearby areas.
fied.

Edema of the upper corium v/e.s intensi­

It v/as not uncommon that extensive hemorrhage in

both upper and lov/er layers of the corium v/as 3ecn v/hen
pustular format! on 'v/as at a high rate.

These spaces, as

a result of reticular degeneration and colliquative vesiculation, v/ere filled by pus to form the intraepidermal "pock11
of various sizes(Fig. 17).

After lymphocytic infiltration

occurred on the dermal side the pustule eventually burst
and dried out.

A crust v/as formed by cellular debris on

top of the pustule.

7/hile the pus v/as drying off, the crust

v/as getting larger and thicker and soon covered the whole
pustule.

The formation of the papillomatous growths of

the epidermal cells appeared as the crust formed.

They

projected finger-like processes into the corium to form
various sizes of papillae. The basal cell layer always
existed along the papillae,

At the same time, the cells

of the sebaceous glands (Fig. 15) began to show the de­
generative changes, consisting of reticular degeneraton
of the cytoplasm, shrinking of the nuclei, and a decrease
in the chromatin material.

Eventually the sebaceous glands

appeared to contain abnormal hollow lunina.

If the crust

formation was too heavy the cells of the sweat glands (Fig.
16) were affected with vacuole formation in the cytoplasm.

The nuclei of the cells v/ere pushed toward t e lumen and
reversed the ordinary arrangement of apocrine gland struc­
ture.

They v/ere arranged in such way that there seemed

to be two layers of lining cells, thesuperimposed layer
consisting of expelled nuclei and part of the cytoplasm
and the basal layer consisting of cell membrane, vacuoles
and the rest of the cytoplasm.

The subepidermal collagen

v/as heavily infiltrated v/ith lymphocytes.

The epidermis

v/as thickened and the epidermal cells, formed a lattice
work around the intraepidermal "pocks".

The papillae

forme.tion v/as greatly increased as the crust hardened
(Fig. 19).

This made the pustule firm.

The intraepidermal

"pocks" v/ere either encapsulated by the epithelial cells
which conseouehtly became organised (Fig. 18), or gradually
the cell debris v/as resorbed by the penetration of blood
vessels.

In the healing process, capillary formation

v/as prominent in the lesion.

The subepidermal lymphocytic

infiltration in varying degrees v/as manifested guite often
in all the stages:

even three weeks after the infect *on

and the skin appeared normal.
gradually.

The papilla*disappeared

Thus the. skin was left even and smooth without

any sign of previous infection.

Occasionally, a cicatrix

might be formed due to the bacterial infection.
The histopathological changes overlapped each other,
and it was difficult to divide them into different stages.
io

Clinically these stages could be referredAas macule, papule
(Fig. 5), vesicle, pustule (Fig. 4), or scab; but in the

microscopic study there was no clear division between
the stages, unless arbitrarjlijdefined.

For this reason,

the lesions were described as a continuous series.

As

a result the continuity of the pathological changes v/as
not interrupted. .
(2)

Ilistopathology of the shin graft.
Only sheep shin was

grafts were collected

used in fchis study.The shin

six, nine, and ten days after in­

oculation of the virus (Pigs. 22, 25, and 24).
virus v/as applied into the
of observation.

The

six-day graft afterthree days

The nine or ten day shin grafts received

the virus immediately after grafting.

The fusion of the

ectoderm of the chorioallantoic membrane with the lower
layer of the corium of grafted shin (Pig. 25) showed good
interlacing in all living grafts.

If fusion of the graft

with the ectoderm tooh place successfully, there would be
no separation by fibrin, hemorrhage or other exudates.
The ectoderm of the chorioallantoic membrane disappeared
completely.

However, in certain cases it remained in

traces as "epithelial pearls" (Fig. 26) in the mesodermal
layer.

Sntodermal cells were affected by intracellular

edema, ballooning and a light degree of proliferation
(Pig. 26) v/ith a tendency to give simple finger-lihe pro­
jections.

Congestion of blood vessels and distended lymphatic

ducts v/ere common in mesoderm especially in these areas
where edematous change was lirominent.

ITo inclusion bodies

either intraplasmic or intranuclear were found.

Frolifera-

44
tion of epithelial cells of crafted skin showed the same
change as in the natural inoculation of the sheep skin.
The changes of the nucleus were sometimes even more strik­
ing .

Thecharacteristics as described previously were

not only the enlargement of the nucleus and the disappear­
ance of chromn.tin materials, but also the nucleoli,which
uieve
usually two in number and of the same size,stained more
deeply.

Intracellular edema, vacuolization and ballooning

degenerations al30 were shown in epidermal cellular changes.
Ho clear vesicle v/as found.
pock uoas present.

Ho pustule or intraepidermal

Hoticeable hyperkerntinization was rarely

shown Art those areas in which evident cellular reactions
had existed.

However, occasionally the parakeratinization

was located in some areas on the epidermis (Fig. 24).

The

stratified epithelium was proliferated.
The sebaceous glands v/ere either shrinking .in si.ze
or undergoing a degenerative process. A hollow space could
usually by observed (Fig. 24).

The sweat glands were oc­

casionally encircled by connective tissue which indicated
that the reaction was inflammatory in nature.

This con­

nective tissue sometime invaded the glandular portion and
replaced the glandular cells.

There was hyalin-like material

in the lumina of the sweat glands which might be the result
of flooding of red blood cells while cutting and manipulating
the tissue.
Edema of the upper corium just beneath the epidermal
layer and the mesoderm of chorioallantoic membrane were

45
very prominent in both controls and infected grq-fts
(Figs. 21 & 26).

Infiltration of eosinophilic granulocytes

in the edematous fluid v/as usually present.

Occasionally

some degree of the chichen red blood cell -extravasation
v/as seen in the dermal layer.

Blood vessels of the shin

grafts v/ere filled v/ith the nucleated red blood cells.
It indicated that the blood circulation of the shin graft
had had good connection v/ith chich embryo membrane.

If

any of the original red blood cells of the shin v/ere left,,
it usually v/as in a hyalinized state.

In some of the material,

small isolated necrotic foci v/ere found, frequently located
at the border between the shin graft and the chorioallantoic
membrane.

These v/ere the result of bacterial invasion and

seemed to have no effect on the viral changes.

These foci

showed typical necrotic change.
(5)

Histopathology of the chorioallantoic membrane of the

chich embryo.
The infected chorioallantoic membranes v/ere harvested
from one to seven days after the viral suspension had been
applied.

The gross change between control and infected

membrane could hardly be distinguished (Fig. 27).

The

thichness of the mesoderm was tremendously increased due
to edema.

This v/as common for all the sections including

those of control membranes.

The ectoderm proliferated to

more than fifteen layers of epithelial cells.

This prolifer­

ative reaction of ectodermal cells could be divided into

46
three different types, namely; traumatic, viral, and mi:-;ed.
Traumatic proliferation (Fig* SI) produced a characteristic
reaction to the mesenchymal cells shown by radiation of
the mesenchymal cells toward the area ...where the trauma
occurred.

This was different from that of the prolifera­

tion produced b^ the virus (Fie. 32) which was shown by
margination of the mesenchymal cells in lines parallel
to the ectoderm.

Usually a scab formed on top of the trau-

matically produced proliferated area. as contrast to the
viral one which produced no scab.
There was hardly any difference of intracel. ular
changes in the proliferated ectodermal cells between that
of the traumatic type and of the viral type in the early
stage (less than forty-eight hours after inoculation).
After this time, in the viral type there was disappearance
of chromatin in the nucleus, uneven staining in the cyto­
plasm, vacuolation and ballooning degeneration (Fig. 37).
These were not shown in the traumatic type.

Furthermore,

especially after seventy-two hour incubation, the viral
type often showed granular hyalin-lihe degeneration of
the cytoplasm (Fig. 34), and quite often the entoderinal
cells were involved as shown by a light degree of prolifer­
ation, vacuolation and ballooning degeneration (Fig. 36).
The hyalin-lihe degeneration of the cells as the result
of viral action had a rather peculiar appearance.

It v/as

manifested by different sizes of round granules with homo­
genous eosinophilic staining.

Some large ones occupied

47
the v/hole apace of the cytoplasm; while some small ones,
a do sen or more, v/ere scattered in the cytoplasm as stippliugs.
At this stage, it v/as usually nixed v/ith hydropic degeneration.
The, nucleus often disappeared (Fig. 59).

The,outline o f .

the entoderm ‘became saw-edged instead of a smooth line
(Fig. 58).

Tiie nucleoli of the entodermal cells v/ere pushed

aside into a creseent shape a3 the nucloi were swelling.
There v/as no evidence of these changes in the traumatic type.
Occasionally the hyalin-lihc granules could be seem in
the entodermal cells.
Epithelial "islets” or "pearls" of various sijses
were observed in the mesenchymal layer.

An archipelago

of epithelial islets formed as out shirt 3 of the prolif cro.ted
ectodermal area.

These islets also occur in bacterial in­

fected chorioallantoic membranes.

Sometimes, this archipelago

was formed by congested blood vessels (Fig. 52) instead of
the epithelial islets.
The mixed form of proliferation showed both the
characteristics of traumatic and viral jjroliforations, but
eventually the latter would overshov/ the former v/ith its
characteristic changes of the specific tissue reactions as
described above.
The capillaries, particularly after five days of
incubation, often increased in number in the subectoderm
whether it had proliferated or not.

The ni-mber of the tiny

vessels v/as so great that they were usually mistaken to
be hemoiwhages.

Sdema.tous reaction of the mesoderm reduced

48
greatly after five days of inoculation (?ig. £8), after
which time it was even hardly to be seen.

Infiltration

of eosinophilic and lieterophilic granulocytes in the mesen­
chymal layer were not ^micommQ.n^.pia’ti.cularly at the., site of
proliferation.
The lesions shown in the control chorioallantoic
membranes were edema; traumatic proliferation, occasional
infiltration of granulocytes and epithelial islets and
pearls.
(4)

Histopathology of Snzymatic Activity Study.
Three hinds of enzymes were studied, namely alhaline

phosphatase, acid xdiosphatase and oxidase.

Alhaline

phosphatase was studied with buffer at pH 9.0 and acid
phosphata.se with buffer at pH 4.7.

Oxidase study wa3 after

Romeis and Ilallory’s method and failed to give conclusive
results.
ing.

The acid phosphatase study also was not encourag­

However, there were some clues obtained in alhaline

phosphatase sections.

The infected cells, especially the

proliferated ectodermal cells, showed an increase in intensity
of silver desposition v/hich indicated that the alhaline
phosphatase activity was increased (Fig. 41).

It was seen

both in the nucleus and cytoplasm particularly along the
cell membrane.

In the infected nuclei there were more and

larger granular depositions than in the normal controls.
In the mesoderm the alhaline phosphatase was also increased
along with the increased number of the mesenchymal cells.
Ho significant amount of changes existed in the edematous
area and the blood vessels.

DlSCUSSICiT
(1)

The shin lesion under natural conditions:
In,thu,discussion of the histopathplo^^ oi’ contagious

ecthyma of sheep, the terminology of this disease was first
considered.

Although it was customarily called "dontagious

ecthyma1', it seemed that this term did not express specifical­
ly the characteristics of the disease.

Having considered

its specific pathological changes and course this disease
v/as found to be primarily a vesicular rather than a pustular
type.

"Ecthyma1' originated from the Greek word "edthyma"

which meant a pustule.

Contagious ecthyma showed a pustule

forming clinically and histopathologically under the
natural conditions, however, the significant thing v/as
that it was not a specific lesion of the virus.

It v/as

believed that viral infection would not give pus formation
unless there were pyogenic bacteria involved.

So in this

co.se, it was bet er to call this condition of the skin
"contagious vesiculo-pustular dermatitis".

This v/as more

expressive and descriptive in regard to both the nature
and the symptoms of the disease.
The mode of entrance of the virus into the cell
v/as very interesting.

Generally it v/as stated that the

cell membrane was semipermeable membrane,

This v/as .true

and could be applied to highly ionizable elements or simple
groups of atoms.

However, it could not easily be explained

in the case of protein and nucleoprotein molecules v/hichh

v/ere ordinarily nondiaJLizable.

It might have "been ex­

plained that protein got into the cell due to its hydrolysis
to amino acids which passed through the cells and resynthesi
into protein.,

Evidently this w: s not true in the case of a

viral invasion "because the virus itself v/as nucleoprotein
in nature and must remain intact.

How these non-dialisable

nucleoprotein molecules got through the semipermeable mem­
brane has not been well explained to date.

It has been

suggested that selective permeability might account for such
a phenomenon.

But the "how11 and "why" of the inechanism of

selectivity of the membrane remained unsolved and called
for further v/orjc of a physico-chemical nature.
According to the histopathological changes, the
causative agent --

virus

wasnot only dermatropic

in nature but strictly stated v/as epidermotropic.

The

epithelial cell of the epidermis was the location attached
by the viru.3.

Since the first significant change of the

cell was the swollen nucleus, the site of invasion v/as ex­
pected to be there,

nevertheless it was hard to state

definitely that the virus did penetrate the nucleus; al­
though this statement could be substantiated by the swollen
portion itself.

It was not impossible that the virus pro­

pagated within the nucleus so that the volume was increased.
Further, the enlargement of the nucleoli was another point
that indicated two possible explanations.

First, the

virus might propagate on "the nucleoli", sotthat the nucleoli
v/ere enlarged and stained much more deeply than normal.

51
The nucleo protein nature intensified the basophilic
property of the nucleoli if the virus were quantitatively
increased.
nucleus.

Increased volume would cause enlargement of the
Second, the propagation of the virus stimulated

cell division.

It was a normal phenomenon that the nucleus

and nucleoli increase in size while cell-division was tak­
ing place.

It was found that the number of cells (epithelial

cells) did increase (epithelial proliferation) later on.
Therefore, it was not uhreasonable to say that the virus
attacked the nucleus first.

This v/as substantiated by the

heavier basophilic staining of the nucleus at an early stage.
Moreover, the cytoplasm did not show any visible change while
the nucleus was swelling and the nucleoli v/ere enlarging.
The cytoplasm showed no change until there was a clear zone
around the nucleus which formed after the swelling of the
nucleus.
Decrease of chromatin material began with a margination
that could indicate the viral agitation "in" the nucleus
v/as not compatible with the existence of the chromatin.

It

•also might have been "used up" by the viral catabolic action,
or it might have been a normal phenomenon of some fast
growing epithelial cells.
Once the clear zone around the nucleus appeared, two
things happened simultaneously:

the shrinking of the nucleus

with heavier basophilic staining property and the hydropic
degeneration of the cytoplasm with edema.

The shrinking

of the nucleus could have been due either to the result

52
of

damage done by the virus or to the increase of intra­

cellular pressure of the cytoplasm.

The clear zone dif­

fused outward and caused the cytoplasm to take on a light
degree of retlculo-degenerative appearance.

Thia meant

that the cytoplasm lost its fine granular eosinophilic
staining and became a light staining mesh work.

This

might have been the re 3 u.1 t of the increase of "water"
content in the cytoplasm to such a degree that the hydro­
philic

30

I lost its equilibrium of solvation and reached

a critical seta-potential of the colloidal particles whicji
underwent agglomeration.

The reticular degenerative change

began at the clear zone around the nucleus and extended
outwardly.

This was shown by the staining property of

the cytoplasm which, in this case, was unstained around
the nucleus and stained at the periphery; the deepest stain­
ing, of course, was near the cell membrane.

This kind of

change initiated the ballooning degeneration and the un­
stained area in the cytoplasm became larger and larger until
the whole cytoplasm was affected.

The vesicle formation

was simply due to the bursting of the ballooned cells
mixed with lymphatic fluid.
Formation of the intracytoplasmic vacuoles might
indicate a loss of equilibrium of the solvation of the
colloidal system in the cell, since it is known that the
cytoplasm is hydrophilic sol which is delicately hydrated
by the solvent,

normally conditions are such that all

of the water is uniformly held.

If water was in excess

of the adsorbing power of the colloidal particles, two

things might happen to maintain the equilibrium of solva­
tion, namely:

(1) an increase of the zeta-potential of

the colloidal particles, so it coaid tahe ap the excess
amount of solvent; or (2) a decrease of the amount of
the solvent to maintain the equilibrium of the hydrophilic
colloidal system.

However, in this case the colloid

cell enclosed in a membrane —
known.

cell - whose nature is u n ­

The cell has a relatively definite volume, but it

also has

n > some

capacity for changing its volume.

ingly, if the volume of the disperse medium —

Accord­

water —

is increased for some reason (a viral interference here)
the cell might compensate by increase either the degree
of solvation or the number of the colloidal particles.
this study both probably occurred.

In

The growth of the virus

would increase the amount of colloid in the cell.

Con­

currently cells became enlarged which involved an increase
in the amount of disperse medium —

water — .

This phenomenon

which was observed under the microscope, was generallycalled
intracellular edema.
The continuation of the edematous process would
lead to a condition described as "ballooning degeneration".
Eventually cells night burst causing reticular degeneration
with the formation of vesicles.
Increase in keratinization was the result of the
dying out of high proliferative action of the epithelial
cell itself and the stimulating nature of the virus.

The

hornified cells first became parakeratinized and then wholly
keratinized.

In this case, the material was pseudo-keratin

54
v/hicli was different from that of hair, nail, and so forth
which '..j-.s eujcoratin in nature.
The lymphocytic infiltration no doubt played a
very important part in the localizing of the virus.

Be­

cause from the very beginning to the end of the course of
pathological change, the lymphocytic infiltration was
always dominant either in the upper dermal layer or from
the upper dermal layer extending to the lower layer.

It

might have been a reason why this virus was epidermotropic
in nature.

It could have been a well defined line built

b y the lymphocytic infiltration at the dermal layer.

The

failure of-the blood to transmit this disease was very
likely due to the fact that there was no virus present.
Quoting Boughton and Hardy))19?5,

"A3 reported b^ Glover

(1928) and confirmed by the writers*experiments, intra­
venous and subcutaneous inoculation of susceptible animals
with the virus of contagious ecthyma fail to produce lesions
of any sort, nor are an: general systematic symptoms mani­
fested".

The virus possibly was barred from the blood by

the lymphocytic infiltrate, especially the epidermal lympho­
cytic infiltrate.

The lymphocytic infiltration of the

lower layer of the corium was always persisting whether
the pustule v/as forming or whether recovery was tailing place.
Strangely enough, the lymphocytic infiltration did not
extend through the basal cell lay ex1 into the I.ralpighian
layer as did the polymorphonuclear leukocytes.

If the

basal cell layer v/as destx'oyed, there were many lymphocytes

mixed with polymorphonuclear leukocytes at the pustular
stage.

Jhether the basal cell layer had a mechanism to

fence off the lymphocytic migration or the environmental
condition of the epithelial layer v/as not suitable for the
lymphocytes remained in doubt,

./hntever the mechanism v/as,

in this case the lymphocytic infiltrate v/as apparently
the specific reaction of the tissue to fence out the viral
infection in protecting the body.
The other question about lymphocytic infiltration
v/as the manner in v/hich the virus initiated this type of
cellular reaction.

According to Ilenkin (19<10), the inflam­

matory reaction v/as initiated by a little known substance
called "Leukotaxin".

Further v/orJ: is necessary to establish

whether or not "leuhotaxin" played a part in this viral
infection v/hich elicited only lymphocytic reaction.
The papillomatous hyperplasia of the epidermal
layer usually appeared after the crust (scab) formed on
the pustules.

This hyperplasia extended into the corium

with a v/ell defined basal cell layer. , It seemed iihely
that these papillomatous projections v/ere the result of
the formation of the crust v/hich exerted pressure and ad­
hered tightly to the sin, so that this organ had no ex­
pansion elasticity.

As long as the virus was still active

in the cells, proliferation continued and the cells ac­
cumulated and bulged out.

Due to the limiting factor on

the pustule and the fast growing nature of the cells them­
selves, the i)roliferation v/as forced mechanically downward

56
to the loose and soft layer - the corium.

Therefore it

tool: on a finger-like papillomatous form or saceote pro­
jection.

However, if enough keratinized material accumulated

above the area of proliferation ""'to""'exert sufficient pres­
sure the finger-like projections would be formed even
without a scab.

In this disease both of these situations

occurred.
Fewer changes occurred in lesions on goat skin.
Vesicle formation and proliferation of the epithelium
could not be demonstrated.

The result v/as inconclusive

due either to too small a number of goats used or to the
differences in individual species.

Further work was necess­

ary to substantiate the histopathological changes.
Rabbits gave no reaction to this viral infection,
although various inoculations had been given.

This con­

tradicted the work of Selbie (194-4) and Glover (1953)
who were able to propagate the virus on rabbit skin.
Howarth (1925) also was not able to propagate this virus
experimentally on rabbits.

Therefore, the individuality

and breed of the rabbits might have played a part in
this discrepancy of results.
(2)

The lesion of the artificial skin graft onto the chorio­

allantoic membrane of the chicken embryo.
The application of skin graft onto the chorioallantoic
membrane of the chicken embryo was handicapped by the fact
that the incubation period of the virus was relatively long

and the period of hatchability of the egg relatively short.
The available length of tine used in chorioallantoic mem­
brane grafting v/as usually only 8-10 days.

Eecause of this

9-day old chichen embryos v/ere used rather than the embryos*
of 10-12 days.

The shin graft v/as harvested before the

19th day of incubation.

Goodpasture (1938) had reported

that undesirable changes occurred in shin grafts after
18th day of embryo.

He stated:

"Heterologous grafts of

this hind became embedded and vascularized, and grew rapidly
and continuously under these conditions until about the
18th or 19th day of incubation.

At that tine there seemed

to be a sudden change v/hcreby the grafts were caused to
undergo rapid regression and degeneration".

Shin treated

by scarification before it v/as grafted cut down the required
length of adaption and propagation.

Implanting impaired

the shin and contamination detracted from the ability to
graft.

Streptomycin and penicillin were”used in attempts

to eliminate bacterial contamination.

The use of duch or

goose eggs might have proved more successful since the
available length of usable time v/ould have been longer.
The edematous change of the corium and chorioallantoic
membrane possibly were non-specific tissue reactions.
The full thichness of the shin might have been responsible
for that reaction.

This night have been the reason why

Goodpasture (1938) did not use the full thichness of sJcin.
Unfortunately adequate equipment to perform the splitting
of shin between epidermis and dermis v/as 1 aching.

The

58
sheep skin v/hich v/as used for grafting v/as comparatively
thin, however.

Attempts v/ere made to cut off as many of

the loose connective tissue strands as possible in order
to decrease4the thichness of the shin.

The difficulty of ~

adapting the shin onto the chorioallantoic membrane was
in direct relation to its thickness*

However, the least

possible manipulation of the shin decreased the chance for
bacterial contamination.

The inflammatory reaction in the

grafts could have been due to too much manipulation.
Microscopically the cellular reactions of the
epithelial cells showed similarity with these produced in
natural infection.

The swelling and clear zone, around the

nucleus, the intracellular edema, the ballooning degenera­
tion and the proliferation of epithelial cells were very
likely the specific reactions of viral infection as com­
pared with material infection and the controls.
I'any newly formed blood vessels penetrated the shin
graft.

This rapid generation of blood vessels was important

for the survival of the graft.

The unification or anastomosis

between the newly penetrated blood vessels with the original
vessels of the skin could not be located.

Whether the

original vessels were anastumised by the new ones or the
new ones formed a system by themselves remained to be in­
vestigated.
ICeratinization and p-raJceritinization seen in some
locations in the sections nay have been due simply to
lack of desquamation, so that when accumulated it seemed to

59
be a hind of "hyperherutinization".
The epithelial "pearls" which always appeared in
the mesodermal layer seemed to be the accidental formation
fron the ectoderm cells”.’ ’’’'Evidently"'it was "not a specific
reaction since it was found in both infected and non in­
fected tissue.

It might have been an segregation of some

Q.f the ectodermal cells by the chcnotaxic mechanism.
(T )

The Infected eaorioallantoic mei.ibra.ne.
The cellular reactions of t :e ect:: dermal and ento-

dermal cel. s left
tie virus.

110

d ubt of the s. ecific response to

Sut both the ectoderm and entoderm we e in­

volved whereas in the shir, the epidermis was only involved.
This was due perhaps to the delicate nature of the chorio­
allantoic membrane.

Hyalin degeneration which was never

seen in the cells of natural shin lesions v/as shown in
the chorioallantoic membrane, especially in these membranes
which were harvested three or more days after the virus
was applied.

It appeared as eosinop .ilic homogenous granule:

of various sizes in the cytoplasm and war called hyalin
degeneration v/hich might have been the result of the dis­
turbance of the colloidal system, manifested by coagulation
of the particles.

Hydrophilic protein colloids easily lose

the equilibrium of hydration by adding the electrolytes.
The coagulation of the particles might be considered as
an example of coacervation.

The electrostatic force of

the colloidal particles tended to pull them together and

60
caused coalescence or agglutination of the particles.
But these forces were opposed by the elasticity cf the
water shells.

Virus could he considered as contribwting'to

’"the electi’olyte*value bf the cytoplasm

It propagated

intracellularly, and may therefore cause a lowering of
the electrohinetic potential of the protein particles and
rendered t .em more hydrophobic.

The protein particles

agglutinated and water v;as set free.

The "clumps" v/hich

were eosinophilic stained appeared as hyalin degeneration,
.hater separated in vorious degrees designated as hydropic
degeneration, vacuolization or ballooning.
As for the proliferation of the ectccornal cerls,
the arrangement of mesenchymal colls and the structure of
the ectoderm occurred in a different manner from that in
the shin lesion.

In the shin there v/as a germinative layer

from v/hich the epithelial cells generated.

In the chorio­

allantoic membrane the ectoderm had only one layer of
cells - simple scuamcus epithelium.

The thichening of

the ectoderm was due to viral irritation.

The accumulation

of the mesenchymal cells on the* ectoderm might bo due to a
positive chemotaxis mechanism,

./hereever proliforation

of the ectodermal area v/as seen there v/as some degree of
increase in the number of mesenchymal cells just beneath
or nearby.

Especially

the actively proliferating area,

the arrangement of the mesenchymal cells afforded a con­
vincing phenomenon.

The arrangement of the mesenchymal

cells was such that those closest to the ectoderm v/ere

61
Greatest in density.

Evidently the mesenchymal cells "ad­

hered" to the ectoderm.
The formation

of the epithelial "islets" or "pearls"

'In'the mesenchyme (mesoderm) could he due to the mcchanism
of "negative chemotaxis".

Somehow the ectodermal cells

detached themselves from the ectoderm or the newly formed
ectodermal cells did not become at'-ached to the ectoderm,
and were left alone to form the "islet", or if organised,
the "pearl" formed,

So far there v/as no available evidence

of having made a connection with the virus, because it
occurred wherever the chorioallantoic membrane had some
agent applied to it (in this case; saline, broth, virus
or bacteria).
Eosinophilic and hetorophilic granulocytic infil­
tration happened whenever there v/as a stimulant or foreign
material which produced a degree of infiltration.

ITo lympho­

cytic reaction to the viral infection was seen as in shin
infection.

Moreover, microscopically it was not so easy

to distinguish these two hinds of cells in embryonic blood,
whether or not by this infiltrative reaction, these cells
played the same function as the lymphocytes did in the shin
lesion remained in doubt.
The edematous reaction of the chorioallantoic mem­
brane was greatly reduced five days after t’e inoculation.
This was due to the course of the edema v/hich lusted only
a short time because of absorption and growth of the embryo.
As the chich develops v, especially ofter Id-or 15 day’s, the

water content v/.is reduced.

The materials used in this

3tudy were collected from 5-7 days after inoculation of
the 11-day old embryo.

Consequently the embryos, 16-18

days old, 'would under normal conditions, have a reduced
water content.

Therefore the disappearance of edema of

the chorioallantoic membrane v/as the result of normal
phy 3 i o1 o£i cal pro cesses.
Through all those changes of the chorioallantoic
membrane it was evident that cone of the tissue reactions
were not specific to the virus studied; sue": as edema of
the mesoderm, formation of epithelial “islets" or "pearls"
and simple proliferation of ectoderm.

bven heterophilic

and eosinophilic granulocytic infiltration could fail into
this category.

This infiltrative reaction was seen in

all these tissues iuc.hu&ing "controls".

Also this reaction

was observed in those v/hich had bacterial contamination
or in those v/hich v/ere harvested aft..r virus inoculation.
The only difference between these mentioned above v/as the
degree of the infiltration,

bacteria contaminated material

had the most conspicuous infiltration;

viral material the

next, and the "control" section showed the least reaction.
Theiincreasing numb cm* of capillaries under the ecto­
derm which was collected 5-7 days after inoculation also
raised doubt as to whether or not it was a specific tissue
reaction to the virus invasion.

The chorioallantoic membrane

functioned in a respiratory manner for the embryo; as the
embryo grew, the oxygen requirement increased,

llo doubt

the capillaries would be increased to meet the demand for
oxygen.

It was also true that the older chorioallantoic

membrane had more blood vessel a, u.S*. low t they were more
conspicuous than the younger ones.

Panohackoff (1917)

had sJiovm that the ectodermal layer of the chorioallantoic
membrane at 10 or 12 day old was a thin highly vasicular
membrane in which a rich capillary network lay beneath
or within the delicate epithelial layer.

However the num­

ber of capillaries seen was tremendously 'high, much higher
than in the control.
one another.

The capi-larios were ccntigenous to

It appeared as to ..hether or not this was

connected with the virus infection.

Further investigation

is needed to clarify this point.
(4)

The Fnsymatic Study.
Sns,.untie study of tissue sections have been made

for sc.me time,

hobinson (1925) demonstrated phosphatase

in bone tissue.

Gomori (1929, 1941) systematically studied

the distribution of enzymes in biological material.

Ibac-

farlane and Salaman (1958) and Hoagland et al (1942) applied
it to virus work.

I.abut and Furth (1941) started a series

of histoohemical studios on tissue enzymes.

Baurne and

J.IacICinnon (1945-1944) discussed the normal distribution of
alkaline phosp.ntase i:. normal tissues.
(1947) worked on human skin.

?1 slier and Click

Bncouraged b; all thc-oe

workers the writer did this part of the investigation on
contagious ecthyma virus with the chorioallantoic membrane.

64
It is imov/n tiiat virus is nucleo-protein in nature
and possesses no independent metabolism.

The propagation

and "reproduction" of virus requires enzymic activity
v/hich is definitely provided by the host cells - the sane
as cello use in carrying on their ov/n metabolism.

Therefore

the virus may be loohed upon as an intracellular parasite.
Sased on the intracellular activity of virus, on intimate
relationship betv/een the enzymatic activity and the site
of the virus might be expected.

The location of enzymes

as shown by silver deposition v/as tahen as an indication of
the site of the virus or as the site of the enzymatic activity
v/hich v;as influenced by the viral invasion.

Since there

v/ere available methods to indicate the location of the enzyme
in the cell qualitatively or quantitatively, (Gomori, 1959,
1941; nab at and Furth, 1941, 1945; Lillie, 1948) further in­
formation about contagious ecthyma virus could be expected
by this a.proach.
In this investigation, measurement of oxidase and
acid phosphatase activity did not give any significant values.
These enzymes v/ere either non existent or the quantity v/as so
cma.ll tiiat no evidence v/as visible under the microscope.

Ilcre

the failure to demonstrate acid phosphatase activity might
have been due to the substrate used.

Sodium-beta glycero­

phosphate (./olf, 1945) v/as substituted for sodium-alphaglycerophosphate.

The solubility of the beta form of gly­

cerophosphate is relatively low and this might o.t least
in this part account for the negligible phosphatase activity.

65
little is hnown about the enzymes of the chorio­
allantoic membi’ane.

nevertheless some alhaline phosphatase

activity v/hich is widely distributed (Ilrugelis, 1946)
could be e j ected*

The demonstration of such activity

by the deposition of silver would contribute to the under­
standing of contagious ecthyma virus.

Alhaline phosphatase

activity was found to increase quantitatively in ectodermal
cells and mesenchymal cells, both in the cytoplasm and in
the nucleus.

Pathological changes, such as enlargement of

the nucleus and proliferation of the cells were accompanied
by an increase of alhaline phosphatase.

Evidently the

activity of the enzyme is intimately related to the activity
of the cell.

Panielli (1945-194-6) mentioned that the amount

of enzyme increased proportionally with the activity of the
coll.

The proliferative reaction and the intranuclear re­

actions v/ere indicative of high activity of the cell.

How­

ever the question could be raised as to whether the site of
silver deposition coincides with the localization of the
enzyme.

In support of an affirmative answer two bits of

circumstantial evidence nay be presented.

In the first

place, the demonstration of the alhaline phosphatase v/as
dependent on its action with a substrate —
phosphate —

the organic

in presence of calcium ions which form calcium

phosphate at the site of the enzyme.

This was followed

by the replacement of calcium by silver.

Then silver compound

v/as reduced by light, metallic silver deposits being readily
seen under the rnici'oscope.

Therefore, it was reasonable to

66
conclude that the location of the silver deposit was the
place where the enzyme was localized.

Secondly, if a

change of enzyme site did occur, it would most likely hap,s~pen-i»*H;he-fixing- procedure, used... i n .preparing„,.the secjbions. ,.
It is unlikely that such a change would take place during
the staining procedure which was carried out after the tissue
was fixed.

The evidence, therefore, strongly supports the

view that the test as carried out for alkaline phosphatase
activity very li3:ely indicated the original physiological
position of the enzyme,

nevertheless, due to the limited

amount of work done in this field of investigation (Stafford
and Atkinson et al, 1948; kmmel, 1946) no positive conclusion
can be drawn.

SUI.IIARY
The iiistopathology of the chin of sheoiD infected
by contagious ecthyma, virus v/as studied.

Liopsy material

v;as tahen from infected areas at six hours and at intervals
up to t-./enty days after infection.
pathological response v/as:

The characteristic

proliferation, vacuolation,

and ‘ballooning degeneration of the epidermal cells.

Ho

inclusion todies either intranuclear or inti’aplasnic v/ere
found.
Various routes of inoculation of ciiicJ: embryos v/ith
the virus v/ere used.

Inoculation of the chorioallantoic

membrane produced a lesion basically similar to that of
the shin of infected sheep.

This established the epidermo-

tropic nature of the virus.
Graftings of sheep shin onto chorioallantoic membranes
v/ere made and the pathological changes in the grafts v/ere
almost the same as in the naturally occurring disease.
Enzymatic preparations of the chorioallantoic mem­
brane v/ere studied,

neither oxidase nor acid phosphatase

activity v/ere demonstrable.

However, alhaline phosphatase

activity v/as clearly shown to increase in both the cyto­
plasm and in the nucleus of the infected cells.
Rabbits and guinea pigs proved to be refractory to
this virus.

Goats failed to produce typical pathological

changes.
Clinically, the disease in sheep passed through five
stages, namely, the formation progressively of macules, papules,
vesicles, pustules and scabs.

Fig. ..«
Sheep Ho. 18.

Sore mouth lesion produced experimentally eight days after
scarification showing pustules
with crust formation.

68

- .i.

Pig. 1

Fig. 2.
Sheep Ho. 18.

Ventral view of Fig.

69

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Pi^. Z ,
Sheep Ho. 12.

Pour days after scarification
showing the papule stage.
(inner thigh)

70

FiC» £

Fig. 4.
Sheep Ho. 15.

Six days after inoculation
showing well-developed pus­
tules on the inner thigh.

71

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Kormal epidermis of sheep shin
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720 X.

72

Fig. 5

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780 X.

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135 X.

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Fig. 8

Fig. 9.

High magnification of the "bal­
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780 X.

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Proliferation and ballooning
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Degenerated holocrine cell of
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IS5 X.

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Organized pustular "pocK"
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720 X.

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specific reaction
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onto chorioallantoic
days after graft.

88

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Proliferation end ballooning of
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Fig. 25.

Ballooning degeneration v/ith
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Ballooning degeneration of
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gland3.
1?5 X.

91

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The fusion of chorioallantoic
membrane with the shin graft.
Entoderm at loft.
IE5 X ,

92

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155 X.

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The appearance of normal chorio­
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three day3 after inoculation.

94

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Fig. 28.

Cystic edema of infected chorio­
allantoic membrane•

95

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Fig. 29

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the trauma was made.
70 X.

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Archipelago of blood vessels •
in the. mesoderm beneath the
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two days after inoculation.
155 X.

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70 X.

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720 X.

106

Fig. 39

Fig. 40.

Alkaline phosphatase activity
shown by the deposits of silver
in the cells of normal chorio­
allantoic membrane as control.
It. is <pro&uceiL under the identic­
al* condition as Pig. 41.

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BIBLIOGRAPHY
Aynaud, L‘.
La Stomatitis irustuleu.se Contagieuse des Cvines
(Chancre du L'outon). Ann. de I ’Inst. Past., 57,
.,,48.9,,,1925
(Abstracted in ,Trop.^Vet .■■»■■■■•B u l l .
64, 1924)
—
Bennett, S. C. J., E. S. Korean and K. A. Hasecb
The Pox Diseases ol’ Sheep ar.d Goats.
J. Comp.
Path. & Ther., j54_:151, 1944Beveridee,
I. B. arid P. K. Burnet
The Cultivation of Viruses and Kickittsiae in
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Series 256, His Majesty's Stationary Office,
London.
1946
Blanc, G. , C. L'elanidi and T. Caminopetros
Recherches Experimentals sur une Haladie Eruptine
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Bosivorth, T. J. and R. 3. Glover
Some Observations on the Mature of Certain Diseases
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Bought on, I. B. and ./. T. Hardy
Contagious Ecthyma (Sore mouth) in Sheep and Goats.
J. An. Vet. Med. Assn., 84:n.s. 58, 150, 1954
Boughton, t , p, and J. T. Hardy
Immunisation of Sheep and Goats Against Sore Mouth
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Tex. Agr. Exp. Sta. Bull.,
504:16, 1955
Bourne, G. and II. MacKinnon
The Distribution of Alkaline Phosphatase in Various
Tissues.
Quart. J. Exp. Physiol., 52:1, 1945-1944
Brandenburgh, T . 0.
Lip-and-Leg Ulceration in Sheep with Report of
Tv/o Cases in Hon.
J. Am. Vet. Ked. Assn., 81:
n.s.
54, 818, 1952
Carre, M.
Some Diseases of Sheep.

Vet. Rec., 12:225, 1952

•

110
Cornell, R. L.
Ecthyma Contagiooum of Sheep and Goats.
Ann.
Rept. Vet. Path., Tanganyika Territory, 1928
Cov/dry, h • 1/«
Laboratory Technique in Biology and I'edicine.
— ed.■■■■•2.-- The: ./illiams and V/ilkins*,- Co., Baltimore. ■■■:1948
Danchakoff, V.
The Position of Respiratory Vascular Hot in the
Allantois of the Chick.
Am. J. Anat., 21:407, 1917
Danielli, J. F.
A Critical Study of Techniques for Determining*
the Cytological Position of Alkaline Phosphatase.
J. Exp. Liol., 22:110, 1945-1946
Emmel, V. XI.
The Intracel_ular Distribution of Alkaline Phos­
phatase Activity Following Various llethods of
Histologic Fixation,
/mat. Rec., £5:159, 1946
Fisher, I. and D. Glick
Histochemistry XXX: Localisation of Alkaline
Phosphatase in normal and Pathological Human
Skin. Proc. Soc. Exp. Eio1. & Hed., 66:14, 1947
Flo ok,

H.
Ail Outbreak of Veneral Disease Among Sheep.
Comp. Path. & Ther., 16:274, 1905

Glover, R. E.
Contagious Pustular Dermatitis of Sheep.
Comp. Path. & Ther., 41:518, 1928

J.

J.

Glover, R. E.
Contagious Pustular Dermatitis of the Sheep.
Rept. of Director, Inst, of An. Path., Univ.
of Cambrdige, 129, 1950
Glover, R. E.
Contagious Pustular Dermatitis; Cross-immunity
Experiment with Various Strains and Seriological
Tests.
Third Rept. of the Director, Inst, of
An. Path., Univ. of Cambridge, 1955Go raori , G.
Hicrotechnical Demonstration of Phosphatase in
Tissue Sections. Proc. Soc. Exp. Biol. & lied.,
42:23, 1929

Ill
Goraori, G.
The Distribution of Phosphatr.se in normal
Organs and Tissues.
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17:71, 1941
Gomori, G.
Distribution of the Acid Phosphatase in the Tissue
under normal and under Pathological Conditions.
Arch. Path. £2:189, 1941
Goodpasture, S. J. and G. J. Buddingh
The Preparation of Antismallpox Vaccine by Culture
of the Virus in the Chorio-allantoic membrane of
Chich Embryo, and Its Use in Human Immunization.
Am. J. H y g., 21:519, 1955
Goodpasture, E.
, B. Douglas and K. Anderson
A Study of Human Shin Grafted upon the Chorioallantois of Cliich Embryo.
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1958
Hatziolos, II.
Observations sue une epizootie a 1ecthyma contagious
des levres.
Rev. Gen. de Med. Vet., 59:265, 1950
Hoagland, C. L. et al
Constituents of Elementary of Vaccinia; VI.
Studies
on the Mature of the Enzymes Associated v/ith the
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Ho are,

E.
Contagious Pustular Dermatitis of Sheen.
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System

Hov/arth, J. A.
Infectious Pustular Dermatitis of Sheep and Goals.
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Special Pathology and Therapeutics of the Diseases
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Jacotot, H.
L 1ecthyma Contagieux des levres en Annam.
Inst. Past., 40:49, 1926

Ann.

Ilabat, E. A. and J. Furth
A Histochemical Study of the Distribution of
Alhaline Phosphatase in Various normal and Heoplastic Tissues.
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Hnov/les, II. G.
Infectious Lip-and-Leg Ulceration of Sheep.
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112
Krugelis, E. J.
Distribution and Properties of Intracellular
Alkaline phcsphe.tase. Eiol. Bull., 90:220, 1946
Lanfranchi, A.
Di recerche sperimentale su la stomatite _uietulo. contagiosa degli ovine ,^uovaHVet.-r 1 , 1925
Lillie, R. D.
Histopathologic Technic.
Philadelphia, 1948

The Lla.chiston Co.,

k'acfarlar.e, LI. G. and H. K. Salaman
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k'elonidi, G. paid LI. Stylicmopoulo
L*ecthyma du Bouton en Greece.
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Henkin, V.
l.Iechanism of Leukocytosis with Inflammations.
Arch. Path., 50:565, 1940
Hohler, J. R.
Lip-aaid-Lcg Ulceration of Sheep: The Treatment
and Cause. E. A. I., U. S. D. A., Cir. 160, 1910
The following individuals were cited by Hohler
as resorting information on Contagious Ecthyma,
williams (1394)
Rushwood (1899)
Gilruth (1900, 1906, 1907, 1908)
Clayton (1906)
Law (1900)
Berry (1901)
Lesnoit (1901)
Hutyra and Harek (1906)
Hasenkarap (1908)
V/ing
IIoussu, G.
L* ecthyma contagious: des levres ches le mouton.
I.Ied. Vet., 100:9, 270, 1924 (Cited by Glover,
J. Comp. PathV d Ther., 41:518)

115
Hewsom, I. E. and F. Cross
Diseases of Colorado Feeding Lambs.
Agr. Exp. Sta. Lull. 305, 1925

Color.

newsom, I. E. and F. Cross
Some Complications of Sore Louth in Lambs.
J.
- Am. Yet. Led.- Assnvr 78 fn.-ar.-Si? 559', " 1 9 5 1 ■

IT e v ; 30 in, I. E. and F. Cros3
Sore Louth in Feeder Lambs duo tc a Filtrable
Virus.
J. Am. Vet. Led. Assn., 84:n.3. 57, 233,
1934
—
Hewsom, I. E. and F. Cross
Sore Louth in Sheep Transmissible to Lon.
J.
Am. Vet. Led. Assn., G4:n.s. 57, 799, 19l‘4a
Hewsom, I. E. and F. Thorp, Jr.
Lamb Diseases in Colorado Feedlots.
Exp. Sta. Lull. 448, 1953

Colo. Agr.

Robinson , K.
The j r 0 3 . i i b l e Significance of TTexosephosphoric
Esters in Ossification. Biochem. J . , 17:286, 1923
Schmidt, II. and E. A. Tunnieliff
Soremouth of Lambs end Lid3.
Agr. Exp. Sta., 12, 1929

Ann.'Rept. Tex.

Schmidt, H. and ./. T. Hardy
Sore Louth (Contagious Ecthyma) in Sheep and
Goats.
Tex. Agr. Exp. Sta. Lull. 457, 1952
Seddon, I. and IT. Lelochner
Infectious Labial Dermatitis or "Scabby Louth"
in Sheep and Goats.
Vet. Res. Rept., Dept. Agr.,
Hew South Jales, Australia. j3:56, 1927-1928
Sedaon, I{. and T. LcGrath
Cross-immunity Tests with the Virus of Infectious
Labial Dermatitis of Sheep. Vet. Res. Rept.,
Dept. Agr. , Hew South ./ales, Australia. _6:109, 1951
Selbie,

?. R.
Experiments on the Transmission to the Rabbit of
the Virus of Contagious Pustular Dermatitis of
the Sheep.
J. Comp. Lath., 54:161, 1944

Stafford, R. C. and './. L. Atkinson
Effect of Acetone and Alcohol fixation and
paraffin Embedding on Activity of Acid and Alkaline
Phosphatases in Rat Tissue.
Science, 109:279, 1948

114
Sumner, J. E. and G. I. Somers
Chemistr;. and Methods of Enzymes.
Academic Press, Inc., 11. Y. , 1947
The H e r ,
—

—

ed. 2.

A.
Ecthyma Contagiosum of Sheep and Goats.
Sept.
15 & 14. Dept. Agr. Union of S. Africa, Onderstepoort, 1:7, 1928

Tidsv/ell, P.
On i/ecrobacillus with Special Reference to lalanitis
(Sheath Disease) in Sheep.
Rept. Govt. Bureau
of Microbiology, new South ./ales, Australia,
2:52, 1910-1911
Ttumicliff, E. A. and 1. H. MatischecJc
A Filtrable Virus Demonstrated to be the Infectious
Agent in Ovine Ealano-Posthitis.
Science, 94:283,
1941
Tunnicliff, E. A.
Ulcerative Dermatitis of Sheep.'
10:240, 1949

Am. J. Vet. Res.

i/alley, T.
Contagious Dermatitis, "Orf" in Sheep.
Path. & Ther., 3:4, 353, 1890

J. Comp.

Velch, H.
Soremouth in Lambs and Other P o m s of Ilccrobacillosis in Sheep.
Montana Aar. Exp. Sta. Bull.,
1 8 6 , 1926
Villiams, G. H.
Contagious Disease Affecting the Genital Organs
of Sheep.
J. Comp. Path. & Ther., 1_7:64, 1904
Volf, A., S. A. Kab a t ,and V. new nan
Histochemical Studies on Tissue Enzymes:
III.
A Study of the Distribution of Acid Phosphatases
with the Special Reference to nervous System.
Am. J. Path., 19:197, 1945
Voodruff, A. 1.'. and E. V. Goodpasture
The Susceptibility of the Chorio-allantoic Mem­
brane of ChicJc Embryo to Infection with Fowlpox
Virus.
Am. J. Path., _7:<209, 1951
Zeller, H.
liber Pochen bei zeigen Sudwest Africa.
ans dem Reich. 102:501, 1920

Arbeiten

"

115
LITERATURE HEAD BUT EOT CITED
Bodansky, 0.
The Effect of Alpha-amino-acids and magnesium on
the Activity of Kidney and Intestinal Phosphatases.
J. Biol. Chem., 115:101, 1956 f" ^
" 'J....
Bodansky, 0.
Are the Phosphatases of Bone, Kidney, Intestine
said Seram Identical? The Use of Bile Acids in
their Differentiation.
J. Eiol. Chem., 110:541,
1937-------------------------------------- --Danielli, J. P.
Establishment of Cytochemical Technioae Nature,
157:755, 1946
Dov/nie, A. V/.
Study of lesions Produced Experimentally by Cov/pox
Virus.
J. Path. Bnct., 48:561, 1939
Elias, II. 3.1.
An Outline of the Histology of Domestic Animals
to be used in connection with the textbook of
Human Histology for Students of Veterinary Science,
Zoology and for Veterinary Pathologists.
School
of Vet. Sci., .Middlesex Univ., .Yaltham, Hass.
Findlay, J. D. and S. H. Yang
Capillary Distribution in C oy; Shin.
1948
Gamhles, R. M.
Bluetongue of Sheep in Cyprus.
Ther., 59:176, 1949

Science, 1 6 1 :10,

J. Comp. Path. &

Gould, B. S.
The Action of "Alkaline Phosphatase", J. Biol.
Chem., 156:365, 1944Irons, J. V. et al
Comparative Susceptibility of Embryonic Chicks,
Chorio-nllantoic J.'embrane and Rabbit's Eye to
Variola Virus.
J. Bact., 41:55, 1941
Klander, J. V. and II. Brown
Experimental Studies in Eczema.
& Syphil., 15, 1927

Arch. Dermat.

ICyes, P.
Normal Leukocytes Content of Bird's Blood.
Rec., 93:197, 1929

Anat.

116
lauffer, M. A. C.
Viruses. The 20th Ann. Priestley lectures,
Pennsylvania State College, State College, Pa.

1947

lever, V/. P.
Diseases Caused By Virus, Kistopathology of the
Skin, 200, j r B t lippincott C o P h i l a d e l p h i a .
1949
Kclaughlin, G. D. and E. R. Thers
Hotes on Animal Skin Composition.
Chem. Assn., 428, 1924Mi cholson, II. 3. and K. Ikeda
Microscopic Changes in Variola.
& Syphil., 15:128, 1927

J. Am. leather

Arch. Dermat.

Moog, P.
The Physiological Significance of the Phosphomonoesterases.
Biol. Rev., 21:41, 1946
I'Tewman, V/. at al
Histochemical Studies on Tissue Enzymes:
IV.
Distribution of Some Enzyme Systems which liberate
Phosphatase at pH 9.2 as Determined with Various
Substrates and Inhibitors; Demonstration of Three
Groups of Enzymes.
Am. J. Path., 26:257, 1950
R a o , R. S.
Cultivation of Sheep pox Virus on Chorioallantoic
Membrane of Chick Embryo.
Indian J. Med. Res.,
26:497, 1928
Rosenthal, T.
J. Am. leather Chem. Assn., 11:462, 1916
Sumner, J. B. and G. I. Somers
Chemistry and Methodsoof Enzymes,
Academic Press, Inc., IT. Y. 1947

ed. 2.

1

Urbach, E.
Metabolism and Biochemistry of the Skin.
Skin
Diseases nutrition and Metabolism, 11, Grune &
Stratton, Inc., IT. Y . , 1949