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THESIS

This is to certify that the

thesis entitled

The Effect of Newcastle Disease

Virus on Susceptible and Resistant

Tissues Cultured from Chickens and Rabbits
presented by

Agnes Lenore Jones
has been accepted towards fulfillment

of the requirements for

Ph. D. degree in Bacteriology

Him-WK-

Major professor

Date \ S

07169

 

THE EFFECT OF NEWCASTLE DISEASE VIRUS 0N SUSCEPTIBLE AND
RESISTANT TISSUES CULTURED FROM CHICKENS AND RABBITS

BY

xfle~£3' Lenore {ghee

A THESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Appliefi Science \
in.partial fulfillment of the requirements
for the degree or

DOCTOR OF PEELOSOPHY

Department of Bacteriology and Public Health

1951»;

 

rte-teas

. \\\

 

”4715*

5
c 3
if,
1}.)
(I.
all.

In"

 

ACKNOWLEDGEMENTS

The author wishes to express her sincere thanks to
Dr. W. N. Mack, of the Department of Bacteriology and
Public Health, under whose guidance and supervision this
investigation was undertaken.

The author also wishes to eXpress appreciation to
Dr. H. J. Stafseth, of the Department of Bacteriology,
and to Dr. D. T. Ewing and Dr. C. A. Heppert of the Depart-
ment of Chemistry for their helpful suggestions and
criticisms of this thesis. ‘

Sincere appreciation is also extended to the typist,
Mrs. Ardeth Frisbey and to the fellow students and members
of the staff of the Departments of Bacteriology and
Pathology whose c00peration and encouragement made this

study possible.

 

 

779/6

THE EFFECT OF NEWCASTLE DISEASE VIRUS ON SUSCEPTIBLE AND
RESISTANT TISSUES CULTURED FROM CHICKENS AND RABBITS

3?

AfingU'Lenore/fonee

AN ABSTRACT

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

DOCTOR OF PHILOSOPHY

Department of Bacteriology and Public Health

1951;

Approved __L\AJ-fiY\"fiV\P\ou&/k‘:n_i

 

Lenore Jones

In these experiments a comparative study has been made
on the effect of Newcastle Disease Virus (N.D.V) on suscept-
ible and resistant tissues cultured from chickens and rabbits.

mbryonating chicken, adult chicken lung, liver and
spleen and adult rabbit lung, liver, spleen and lymph node
tissues were cultured. Both normal and immune adult chickens
and rabbits were used. Hemagglutination and infectivity
tests were carried out to determine the growth curves of
H'.D.V. in embryonating chicken tissues.

The growth cycle was composed of (a) a lag phase of 21;
hours, (b) a phase of rapid virus multiplication in which
peak infectivity titers were reached in 36 hours and peak
hemagglutination'titers in 36 to 14.8 hours, (c) a plateau
lasting 14.8 to 60 hours, (d) a sudden decrease in the rate
of virus production 96 hours after N.D.V. inoculation.

Growth curves prepared from hemagglutination titra-
tions on the tissues cultured from normal adult chickens
and rabbits were similar to those of the embryonating chicken
tissue but the peak hemagglutination titers occurred 36 to
96 hours after N.D.V. inoculation. This was also found in
the immune chicken lung and spleen cultures but not in the
liver cultures. In the immune chicken liver tissues and in
all the tissues of the immune rabbit there was a steep
decrease in infectivity titers from an LDSO of 10"7 on the
day of inoculation to 10"1 or 10"2 14.8 hours later. Hemagglu-
tination activity was recovered only in the undiluted fluids

 

 

 

 

——..-_ _.._

Lenore Jones

from.the immune chicken liver cultures.

Cytological degeneration was seen in the embryonating
chicken cultures 15 to h8 hours after N.D.V. inoculation.
The fibroblasts showed an increase in vacuolization of the
cytoplasm and a tendency in the fermation of giant cells
‘which contained 3 to 5 nuclei. The only degenerative changes
in the adult tissue cultures occurred in.norma1 adult
chicken liver and this did not appear until 7 or 8 days
after N.D.V. inoculation. There was a gradual degeneration
of the liver cells in which the cytOplasm.lost its staining
preperties and broke up into large granules and the cell
'wall became fragmented. The nucleus, however, was unaltered.
Neither the uninoculated cultures nor the cultures inocup
lated with heat-inactivated N.D.V. showed any degenerative

change.

 

 

 

TABLE OF CONTENTS

INTRODUCTION 0 O O O O O O O O O O O O O O O O O 0

I. The Use of Tissue Culture Techniques in
Studying Animal Viruses e e e e e e e e e e e

A. Methods of Demonstrating Virus Activity in
Tissufl Cultures. e e e e e e e e e e

1.
2.
3.
h.
5.

Animal Infectivity Tests. . .
Serological Tests . . . . . .
Hemagglutination Test . . . .
Hemagglutination Inhibition Test. . .
Growth Curves Illustrating the Multi-
plication of Viruses in Tissue Culture.
Abnormal Changes in Tissue Culture

Caused by Viruses . . . . . . . . . . .
Prepagation of Newcastle Disease Virus
in Tissue Cultures. e e e e e e e e e e

.0.
0..
O...
0 .0.
O O O O O

IIe Gh01ce Of V1PUC e e e e e e e e e e e e e e e

A. Description of Newcastle Disease Virus . .

B. The G.B. Strain of Newcastle Disease Virus

Etiology of Newcastle.Disease . . . . .
HOSt Range or N. D. V. e e e e e e

Description of Newcastle Disease in Chickens

.0...

.0...

O
0

Distribution of N. D. V. in Animal Tissues. .
Growth of N.D.V. in the Embryonating Egg. .

HistOpathology of Newcastle Disease in
Adult Chickens. e e e e e e e e e e e
Histological Changes in Embryonating

Chickens due to N. D. V. e e e e e e e e

MATERIAL ANDjMETHODS e e e e e_e e e e e e e e e e

A. Description of Procedure of the Experiment

1.

2.
3.
h.

Choice of Tissue Culture Technique for
Virus Pr0pagation e e e e e e e e e e e
Preparations for Tissue Cultures. . . .
Outline of the Experiment . . . . . . .
Description of Tissue Culture Technique

0

O...

O

O...

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TABLE OF CONTENTS Continued
Page

B. Methods Used for Determining N.D.V. Activity
in Infected Tissufi Cultures. e e e e e e e e e 2h
1. Infectivity Tests Using Embryonating
Chickennggs. e e e e e e e e e e e e e e e 22
2. Hemngglutination TOSUSe e e e e e e e e e 2
3. Methods Used for Animal Immunization. . . . 27

RESULTSe e e e e e e e e e e e e e e e e e e e e e e e 28
fie Thermolability Of the Virus. e e e e e e e e e 28

B. Results of Hemagglutination and Infectivity
TOBtIe e e e e e e e e e e e e e e e e e e e 29
l. Hemagglutination Tests with Tissue Cultures
Given Heat-inactivated N.D.V. . . . . . . . 29
2. Hemagglutination and Infectivity Tests
with Embryonating Chicken Tissue Culture. . 30
3. Hemagglutination and Infectivity Tests
with Rabbit and Chicken Tissue Culture. . . 32
Hemagglutination Tests. . . . . . . . . . . 32
IM°°t1V1tyTQat8eeeeeeeeeeeee 38

C. Changes in the Tissue Cultures Which Were
Infected with NoD.Ve e e e e e e e e e e e e e no

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . NB
SMARYO O O O O O 0 e 0 0 0 O 0 O O O 0 0 O O O 0 0 O 51'-
BIBLIOGRAPHY e e e e e e e e e e e e e e e e e e e e e 56

 

 

.’

 

 

 

INTRODUCTION

The purpose of this study was to determine whether or
not resistance and susceptibility of animals to Newcastle
Disease Virus (N.D.V.) could be attributed to specific
tissues or organs. The liver, lung and Spleen of adult,
normal and immunized chickens and rabbits, as well as
normal chicken embryo tissue were chosen for tissue culti-
vation. The chicken represented the naturally susceptible,
and the rabbit, the naturally resistant animals to N.D.V.
As soon as these tissues attained maximal growth in tissue
cultures they were infected with N.D.V. to study the
morphological changes and variation in their capacity to
support multiplication of the virus.

This was the specific purpose of the work but there
was also the more general purpose of seeking any evidence
which might contribute to a better understanding of
resistance and susceptibility of animal cells to a virus.

Virologists have been limited in many of their
practical studies because it has been impossible, in many
cases, to detect virus activity by any means other than
animal inoculation. Primary isolation of such viruses as
those of hog cholera, measles and infectious hepatitis,
may be accomplished in only the original host or a few large

 

animal species. This restricts laboratory investigations
greatly due to the high cost and the time involved in
maintaining these animals. Most of the recent studies
pertaining to the relationship of host cells and viruses
have been carried out with the purpose of finding a means of
increasing their host range so that small animals may be
used. In Spite of persistent research, no adequate explan-
ation has been found to account for the resistance of small
laboratory animals to these viruses.

On the other hand, there exists a group of viruses
which can be prepagated readily in small animals as well as
in the embryonating chicken egg. Consequently several
methods have been developed for determining virus activity.
For example, N.D.V., mumps and influenza viruses may be
propagated in the embryonating chicken egg; then, the
allantoic or amniotic fluids may be used as antigenic sus-
pensions for hemagglutinat ion, hemagglutination-inhibition
and complement fixation tests. In addition, N.D.V. may be
prepagated in most laboratory animals which may be easily
maintained for laboratory experimentation, e.g., chickens,
hamsters and mice. With these viruses, therefore, the
problem involves not an understanding of resistance but an
explanation of the factors responsible for the susceptibility
of these animals. If these factors could be detemined the
virologist might in turn find a means of lowering the

 

 

resistance of other small animals to the viruses of hog

cholera, infectious hepatitis, etc.

I. The Use of Tissue Culture Techniques in
Studying Animal Viruses

Viruses were first prepagated in tissue cultures when
Parker and Nye (1925) carried vaccinia virus through a
series of 11 cultures of rabbit testicular tissue. Since
that time many of the viruses known to cause disease in
animals have been grown in tissue cultures. This technique
has been applied to many types of virus studies such as:

(a) effects on the cells which viruses attack

(b) site of virus multiplication

(c) enumeration of conditions essential for virus pro-

pagation

(d) details of the mechanisms of natural and acquired

immunity caused by viruses

(e) changes in pathogenicity which.may occur as a

result of cultivation of the virus in tissue
cultures cepecially associated with the possibil-
ities of vaccine production

(f) determination of the effects of various chemical

compounds, including antibiotic agents, on the
multiplication of viruses

(g) attempts to adapt tissue culture techniques for

the purpose of rapid diagnosis of some virus

diseases.

a
.—

 

 

Ag‘uetheds of Demonstrating Virus Activity in TissuQACulturgs

 

Two methods have been used for demonstrating the multi-
plication of viruses in tissue culture. The first consists
of showing that the fluid withdrawn from a culture exhibits
activities characteristic of the virus in question; such as,
animal infectivity, hemagglutinating or complement fixing
activity. The second.method is the demonstration of
abnormal cellular changes or an alteration in the pH of
the surrounding fluid due to a decrease in tissue metabolism

caused by the virus.

1. Animal Infectivity Tests

Animal infectivity tests have proved to be the most
reliable for detecting virus multiplication and are necessary
to confirm the results of Serological and cytological
findings. With N.D.V., for example, serial dilutions of
the infected tissue culture fluids are inoculated into
embryonating chicken eggs and the fifty percent mortality
endpoint, LDSO, is determined according to the method of
Reed and.Muench (1938).

2. Serological Tests

Homagglutination and complement fixation tests provide
more rapid.means of demonstrating virus activity than do
animal infectivity tests. The basic principles of the
complement fixation.method are the same for viruses as for

bacteria. This test may be used.with.many viruses such as

e "t
1......

 

..._

 

 

 

those of the lympho-granuloma-psittacosis group, and the
mumps, influenza-N.D.V. group. The hemagglutination test
has the advantage of being more rapid but is limited to a
smaller number of viruses, namely the mumps-influenza-
N.D.V. group.

one utination Test: Hirst (l9h2a) and.McCle11and
and.Hare (l9h1), discovered that influenza viruses agglu-
tinate the red blood cells of fowl. Burnet (19112) reported
the agglutination of fowl cells by N.D.V.-infected fluids
of the embryonating chicken egg and.Levens and Enders (l9h5)
described.similar agglutination of sheep red blood cells by
mumps virus. The Bureau of Animal Industry (l9h6) stand-
ardized the hamagglutination test which is a modification
of the hemagglutination procedure described by Salk (l9hu).
This quantitative measurement has been described in the
following manner:

Two-fold serial dilutions of the virus suspensions
were made in physiological saline. To these
dilutions a suitable concentration of a red blood
cell suspension was added. After thorough shaking
the cells were allowed to settle for a period of
one to two hours. In the test tubes containing virus
the agglutinated red blood cells deposited in a
thin film covering the entire portion of the bottom
of the tube, while in the tubes containing no
detectable virus the cells settled out in the fomm
of a small sharply-outlined button. The endpoint
was considered to be the highest dilution of a
virus producing maximal hemagglutination and the
titer was expressed as the reciprocal of the final
dilution of virus. This titer has also been
designated as one hemagglutination unit. Thus if
one hemagglutination unit were represented by a
titer of 1600, two hemagglutinating units would
correspond to a titer of 800 and 8 hemagglutin-
ating units a titer of 200.

 

 

..._
.. m
“L.

u‘.

 

 

Two disadvantages in the use of the hemagglutination
test for this experiment have been reported. First, when
a N.D.V. suspension was treated by heating at 56° C for
1.0 minutes it was no longer infectious for embryonating
chicken eggs but the hemagglutinating activity was only
slightly decreased (Durusan, 19149). Thus the hemagglutinating
titer was found to be proportional to the sum of the in-
fections as well as the non-infectious virus particles.
Secondly, measurement by hemagglutination is restricted to
those suspensions in which large quantities of virus are
present. An N.D.V. suspension which will infect the
embryonating chicken when diluted 1,000, 000,000 times will
give maximal hemagglutination only up to a dilution of 1600.
Gordon (1952) showed that the multiplication of N.D.V. in
the embryonating chicken egg increased to a 10"6 titer
before there was any evidence of hemagglutination activity.
The hemagglutinating titer only then showed a proportional
increase as the infectivity titer in the embryonating
chicken egg increased from 10"6 to 10’9.

Hemaggutination Inhibition Test: Hemagglutination
has also been widely used for antibody measurement. This
adaptation of the test is called hemagglutination inhibition
and is based on the principle that the hemagglutinating
viruses, when mixed with homologous immune serum, lose their
ability to agglutinate red blood cells. The degree of
inhibition gives an estimate of the amount of antibody in

 

the serum. Two hemagglutination inhibition methods have
been used with N.D.V. In the "alpha" procedure, serial
dilutions of virus are admixed with a fixed amount of serum,
whereas in the "beta" procedure a definite amount of virus
suspension containing the hemagglutinating units is admixed
with an equal amount of serial serum dilutions before the
red blood cells are added. Brandly (19h?) reported that
there was a 93-percent agreement between the alpha and beta
procedures.
3. Growth Curves Illustrating the Multiplication of Viruses
in Tissue Culture

Animal infectivity tests and hemagglutination tests
have been used to prepare growth curves illustrating the
multiplication of viruses in tissue cultures. Of particular
interest are the growth curves established for poliomyelitis
virus by Salk (1952) and Syverton (1952), as well as those
for influenza and mumps viruses set up by Enders (1950).
Enders established growth curves for influenza and mumps
viruses in embryonating chicken tissue cultures using the
hemagglutination technique, whereas Salk and Syverton made
their determination for poliomyelitis virus in monkey
testicular tissue cultures using monkey infectivity tests.

Growth of these viruses occurred in a discrete cycle
characterized by:

(a) a lag phase - in which the virus required time for

infection and multiplication in the cell

 

 

(b) a phase of rapid virus multiplication.which reflects
the release of virus from within infected cells to
the extracellular fluid

(c) a plateau phase which represented a decrease in
virus production

(d) a stage in which the virus production was reduced
to zero. L

Influenza virus showed a very short lag phase, then

increased rapidly attaining a peak hemagglutination titer

at hB to 72 hours. Thereafter relatively little additional
virus was produced. Mumps virus increased relatively slowly
usually reaching maximum concentration in the fluid phase

on the twelfth day of virus cultivation. There was no
visual evidence of cytological changes in the tissue
cultures in which either mumps or influenza virus were
prepagated.

The duration of the growth cycle of poliomyelitis

virus was approximately 10 days. Significant amounts of
virus were not released into the liquid phase until the
fifth to seventh day. Occasionally, two successive cycles
of poliomyelitis virus production were observed in a single
culture. This was shown by a secondary rise in the quantity
of the virus released into the liquid from the eighth to

the eighteenth day of cultivation. By 30 days virus was not
detectable either in the tissue or liquid phase. The first

cycle was correlated with the destruction of cells which

 

 

 

 

1L

extended from explants while the second cycle occurred only
if there were a sufficient number of surviving host cells

‘within the explants.

h. Abnormal Changes in Tissue Culture Caused by Viruses
Viruses have been shown to cause degenerative changes
in tissue culture cells 38‘3611 as the formation of charac-
teristic intracellular inclusion bodies. Huang (19k3)
showed that the virus of equine encephalomyelitis completely
destroyed embryonating chicken cells within 72 hours. Salk
(1952) described a slower type of degeneration which was
produced in the fibrocytes of monkey testicular tissue
infected with pcliomyelitis virus. First, there occurred
various granular changes and vacuolization in the cytoplasm,
then a varying degree of cytolysis and a decrease in the
development of new fibrocytes surrounding the tissue explants
took place. Most of the nuclei were shrunken and pyknotic,
but a few appeared swollen and were stained less intensely.
Bland and Canti (1935). using the tissue culture technique,
found that the elementary bodies of psittacosis virus pass
through a definite cycle of develoPment and that the plaque
or inclusion body is essentially a colony of elementary bodies.
The effect of a virus on tissue metabolism may be
indicated by a decreased acid production in the tissue
culture. Huang (l9h3) noted that chicken embryonating
tissues from.h8-hour cultures of equine encephalomyelitis

virus were no longer capable of reducing methylene blue.

10

Endors'gthl. (19h9) found that acid production in flask
cultures of human embryonic brain, intestine, and skin-

‘muscle inoculated with poliomyelitis virus after variable
intervals, declined.more rapidly than in control cultures

without virus.

5. Prepagation of Newcastle Disease Virus in Tissue Cultures

Tepaccio (l93h) reported the first attempt to propagate
N.D.V. in tissue cultures. Embryonating chicken tissue
cultures were inoculated with a virus suSpension prepared
from the spleen of an infected chicken. Subcultures were
made every three to five days and 31 successive passages
were carried out. Virus was recovered from all passages.

Bankowski and Boynton (l9h8) also:reported the prepa-
gation of N.D.V. in tissue cultures. Embryonic chicken
livers, infected 36 hours previously, were cultured by the
suSpended cell method. After each tissue culture passage
portions of the suSpended cells were used for embryonating
chicken egg inoculation, so that every other passage was
an egg passage and not an uninterrupted series of tissue
culture passages. Subcultures were made every 72 hours
from the tissue culture passage to the embryonating egg and
at this time the.LD50 of the infected tissue culture ranged
from.10'h'to 10'7.

Scott (195h) described a simplified tissue culture pro-
cedure for the prepagation of the Roakin strain of N.D.V.
in the chorio-allantoic membranes of the embryonating

 

 

 

 

11

chicken. Growth curves were established using infectivity
tests as well as hemagglutination tests. It was found that
a peak LDSO titer of 10'"8 appeared in 2h.hours and a peak
hemagglutination titer of 128 appeared in 36 hours.

II. Choice of Virus

Newcastle'Disoaso Virus (N.D.V.) was chosen in this
experiment for the following reasons. First, N.D.V.
activity may be demonstrated by several laboratory pro-
cedures: by infecting the embryonating chicken egg, by the
complement fixation and the hemagglutination tests.
Secondly, this virus is relatively well known because of its
relationship to the mumps and.inf1uenza group of viruses.
A large part of recent investigations on host-cell-virus
relationships have‘boen carried out with tho mumps-influenza-
N.D.V. group of viruses, and, therefore, any additional
findings may be more easily fitted into an explanation of
the mechanisms of resistance and susceptibility. A third
reason for choosing N.D.V. is the fact that it is a rela-
tively thermolabile virus. jDurusan (l9h9) found that N.D.V.-
infocted allantoic fluids were no longer infectious for the
embryonating chicken egg after an incubation period of five
days at 37° C. This is a significant factor in tissue
culture studies because if the virus is not decreased by a
temperature of 37° C (the temperature of incubation) it is

difficult to prove that virus multiplication, not passive

 

12

transfer of the virus, has occurred. It follows, therefore,
that, if the fluids of the N.D.V.-infected cultures still
prove infectious after being incubated five days at 37° C,
multiplication of the virus must have taken place.

 

gmscription of Newcastle Disease Virus

1. Etiology of Newcastle Disease (synonym: avian pneumo-
enc ophal it i s )

Newcastle disease is an epornitic infection of poultry
characterized by a viromia and signs of involvement of the
respiratory, central nervous system, and gastro-intestinal
organs. Outbreaks of Newcastle disease due to immunologi-
cally identical strains have occurred in many parts of the
world; for example, in England, U.S.A., Asia, Africa and
Australia. Newcastle disease primarily affects chickens
and turkeys.

The etiological agentof Newcastle disease is a virus
of medium size. Burnot and Perry (1933) showed that the
particle size of N.D.V. was 80-120 mu. The virus particle
is filamentous (Bang, 1914.6) or spermlike (Cunha, _e_§ $1.,
19117). It is filterable through Berkefeld V. N and W
candles, Chamberland L3 and L5 filters, and Seitz pads
(ST-3). The virus may be preserved in 50 percent glycerine,
in the frozen state, or by lyophilization (Burnet and Perry,
1931+). Inactivation of the virus has been accomplished by
ultraviolet light when a clear suSponsion was irradiated by

 

13

1600-1800 A for 0.8-1.08 seconds. It may also be inacti-
vated by a 1/5000 dilution of formalin and by N/50 sodium
hydroxide in one hour (Brandly, et a1., 1946a).
. The maximal stability of N.D.V. lies between pH h to
pH 11 (Brandly, Moses and Jones, 19h6d). Miller and Stanley
(l9hh) found that the pH of the medium had only a minor
effect on tho hemagglutinating activity of the virus in a
range of pH 6 to pH 8.

Burnet (l9h5) found that, if the allanto-amniotic
fluid suspension of N.D.V. was held at 650 C for 15 minutes,
there was complete loss of infectivity but the hemagglutin-
ating activity was still 50 percent of the original titer.
Durusan (1949) also found that the hemagglutinating activity
of the N.D.V.-infected allantoic fluid was more thermostable
than the infectivity of the virus. The initial LD50 of the
virus was 10”?“32 and its hemagglutinating titer was 1280.
‘When the virus was stored for 13 weeks at no G the infectiv-
ity titer decreased to 10"l'62 whereas the hemagglutinating
titer remained constant. When held at room temperature
(22°-27° C) for 30 days the infectivity decreased to loss
than 10° but the hemagglutinating titer decreased only from
1280 to 6h0. Durusan also found that when the virus sus-
pension was hold at 62° C there was complete loss of
infectivity after 15 minutes but hemagglutinating activity

was not lost until after 25 minutes.

11L

The correlation of this study and Durusan's experiment
on.the thermolability of N.D.V. at 37° C has already been
mentioned. He found that when the virus suspension was
incubated at 37° 0 the initial LDSO of 10'9°32 dropped to
10"'5°5 after one day, to 10"3°62 after two days, to less
than 10‘3 after three days and to 100‘21 after four days.
iDuring the same period, however, the hemagglutination titer
changed only from 1280 to 6&0 and remained at 6&0 for more
than.eight days. For this reason it is evident that the
4 hemagglutination test is of value only when an increase in

hemagglutination titer occurs.

2. Host Range of N.D.V.

Newcastle disease occurs naturally in chickens,
turkeys, pheasants, guinea fowl, Sparrows, crows, franco-
lines and parrots (Beaudette, 19h2). Experimental infection
has been achieved in chick embryos, ducks, geese, pigeons
and several varieties of wild birds. Mice, hamsters, cotton
rats and monkeys may be infected experimentally with N.D.V.
resulting in a meningo encephalitis. Over 300 serial
passages in hamsters and 30 serial passages in mice have
been carried out (Reagan, 1952a, b). Several accidental
infections have occurred in human beings handling infected
fowl or working with the virus (Burnet, l9h3; Hungherr,‘g§
5;... 19119).

Rabbits, cows, horses, pigs, sheep, ferrets and guinea

pigs are considered to be resistant to N.D.V. (Beaudette,

 

15

l9u3). A meningo-encephalitis may occur in rabbits after
intracerebral inoculation but the virus has not been trans-

ferred from rabbit to rabbit.

3. Description of Newcastle Disease in Chickens

The present cpinion.is that the pneumonic and central
nervous system (CNS) symptoms doominate the American type
of Newcastle disease, while reapiratory and gastro-intestinal
symptoms are most prevalent in the Asian and European type
(Brandly,‘g§‘gl., 19h6a). In naturally-infected chickens
the incubation period is h to 11 days and is followed by
characteristic symptoms of pneumonia or central nervous

system involvement. Death may ensue after two to four days.

h. Distribution of N.D.V. in Animal Tissues

Karzon and Bang (1950) and.Hofstad (1951) reported the
following results from quantitative studies of the American
type of N.D.V. in tissues of experimentally-infected chickens.
‘When the virus was inoculated by the intranasal, intravenous
or intramuscular routes it was recovered from the blood,
spleen, kidneys, lung, brain, and trachial and intestinal
contents within 2h hours. Virus concentrations as high as
10'”5 and 10’7 (embryo lethal doses) were found by the fourth
day then gradually decreased until the virus could no longer
be isolated after the seventh day. Neutralizing antibodies.
against N.DtV. and hemagglutinin-inhibiting antibodies were

demonstrated five to seven days after N.D.V. inoculation;

 

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16

thus, the virus disappeared as soon as the antibody concen-
tration in the blood became relatively high. The virus
neutralization and the hemagglutination-inhibition tests
yielded parallel results early in the convalescent period
but later (22 to 100 days) the hemagglutinin-inhibiting
.activity of the serum showed a steep decrease.

Newcastle disease virus has been isolated from the
liver of infected chickens but never in sufficiently high

concentrations to yield consistent titers.

5. Growth of N.D.V. in the Embryonating Egg

When N.D.V. was inoculated into the allantoic cavity
of the embryonating chicken egg it was rapidly adsorbed by
the cells of the allantoic membrane, (Gordon, 1952). Within
the first hour the LDSO titer decreased from 10"“05 to
10‘1'6 and the hemagglutinating activity titer was lost.
The infectivity titer increased when tested every four hours,

-6 -8.3

until it reached.LD5 titers of 10 at 12 hours and 10

O
at 16 hours. The hemagglutinating activity did return after
12 hours; then, the titer increased from 10 to 1280 by the
thh hour. Meanwhile, the infectivity titer showed no

increase in the 16 to hB hour period.

6. HistOpathology of Newcastle Disease in Adult Chickens
Gressly the disease in fowl produced multiple focal

necrosis in the viscera, and hemorrhages, especially in

the respiratory and alimentary tracts; at times, an.inter-

stitial pneumonitis was observed (Brandly, 23 51., l9h6a, b).

l7

Cytologically, the exudative lesions in the lung con-
sisted of cellular and serous accumulations in the peri-
bronchial alveoli with extension into the lumen of the
regional tertiary branches. The exudate was composed of
various types of desquamated pulmonary cells including
swollen, vacuolated (foam) cells, and disintegrating hetero-
philes and erythrocytes. The central nervous systmm showed
perivascular infiltration, a honey-combed appearance of
degenerated neurons and gliosis near the altered neurons in
the lumbar cord, medulla and cerebellar nuclei (Brandly,'3§
‘31., l9h6b). In addition to focal necrosis in the liver,
pericarditis, and secondary inflammatory changes in the
abdmminal air sacs Fukushima (1932) found hyperplasia of
the intestinal lymphoid patches with occasional necrosis
and hyaline degeneration of the splenic follicles with
deve10pment of network-like homogeneous masses.

7. Histological Changes in Embryonating Chickens due to
N.D.V. '

Cytological changes in mmbryonating chicken eggs due
to N.D.V. were first described by Burnet (l9h2). 'When.the
virus was inoculated into the amniotic sac, multiple chorio-
allantoic hemorrhages, hemorrhagic encephalitis and respir-
atory lesions appeared within 36 hours. In sections of the
lung cellular changes were seen to be widespread. The

earliest change recOgnizable was a distortion of the nucleus

of epithelial cells giving a clear oval area within which

18

the chromatin was piled up into masses of various shapes

and distribution. In tracheal smears large vacuolated cells
‘were found arranged in agglutinated masses in which it was
impossible to pick out the outlines of individual cells.
Some cells showed a nucleus broken into many heavily stained
circular fragments as well as a great degree of cytOplasmic

degeneration.
B. The G.B. Strain of Newcastle Disease Virus

The G.B. strain of N.D.V. was chosen for this work
because it is a well-known field and laboratory strain and
it is highly pathogenic for chickens. This strain was
isolated.in May, 19h8 by Boney and Grumbles from an outbreak
of a severe nervous and respiratory infection of chickens
(Boney, 19h8). Chickens infected experimentally with the
virus show different symptoms depending upon the route of
inoculation. Those exposed by intramuscular injection
develOp a profuse diarrhea, weakness, and general paralysis
with tonoclonic symptoms. The same symptoms occur in
chickens exposed by the intranasal route but there is usually
an interstitial pneumonia as well. Morbidity and mortality
_up to 100 percent may occur in young susceptible chickens.

The LDBO for lo-day old embryonating chicken eggs is 10'9.

MATERIAL ANDTMETHODS
A.‘Desoription of Procedure of the Experiment

1. Choice of Tissue Culture Technique for Virus Prepagation

The simplest procedure which could be readily adapted
to large-scale tissue cultivation was chosen. The tissues
‘were minced, then.embedded in a plasma matrix in the bottom
of a Petri dish (9 cm.x 1.5 cm). When the tissues became
fixed in the plamma they were flooded with Hank's nutrient
solution. In this way it was possible to prepare and.main-
tain several hundred Petri dish cultures at one time.

This method.provided an additional advantage in that
representative cultures could be examined.microscopically
at a magnification of 100x to 970x. This was accomplished
by simply removing the nutrient fluid, covering the tissue
explant with a glass cover slip and lowering the objective
of the microscOpe into position directly over the tissue
growth. For comparative studies and photomicrographs
permanent preparations were made of the Petri dish cultures

by staining the tissue with Giemsa stain.

2. Preparations for Tissue Cultures

Heparinized plasma was used for the mat in which the
tissues were embedded. The plasma was filtered through
Seitz filter (St-3), heated in a water bath at 56° 0 for

20

30 minutes and tested for bacterial sterility as well as
hemagglutinating-inhibiting preperties. All the plasma used
showed a hemagglutinating-inhibition titer of less than
1A0 which according to the standards of the Bureau of
Animal Husbandry is considered to be within the normal range.
A quantity of 0.5 to 1.0 m1 of plasma was sufficient to
cover the bottom of a Petri dish.

Hank's nutrient medium, a balanced physiological
solution, recommended by Enders (1950) as a tissue culture
nutrient, was used for all cultures. Ten milliliters of
the nutrient solution was sufficient to cover each Petri
dish culture and to sustain growth of the cells for a period
of four to seven days. Phenol red, an indicator with a
color range between pH 6.898.h was incorporated in this
solution. The hydrogen ion concentration of the nutrient
was adjusted to a pH 7.2-7.h before it was added to the
cultures. The 0.3. strain of N.D.V. was used for both
tissue culture inoculation and animal immunization. The
cultures were inoculated by replacing the fluids with the
N.D.V.-infected allantoic fluid, diluted 1/200 in Hank's
nutrient solution. This dilution represented eight hemagglu-
tinating N.D.V. units. The succeeding fluid changes were
made with Hank's nutrient solution alone.

In the first two experiments all fluids were checked
for bacterial sterility using blood plates and N.I.H. this-
glycollate sterility-broth, but this was discontinued when

it was found that the Petri dish tissue cultures provided
a more sensitive test for bacterial growth. Often the
broth and blood plates failed to show bacterial contami-
nationuwhile the stained Petri dish cultures showed a few
‘bacteria.

Bacterial and mycotic contamination was reduced to
less than five percent by incorporating 100 units of peni-
cillin, 500 micrograms of streptomycin and two Candida
albicang units of endomycin” in each milliliter of Hank's
nutrient solution. Endomycin, a new antibiotic, was first
prepared by Gottlieb (1951). It inhibits the growth of a
‘wide variety of fungi pathogenic to plants and animals,
mmny gram positive and a few gram negative bacteria.

Chemically clean glassware was obtained by washing
‘with the detergent Tetra.D and then rinsing twice in tap
‘water and twice in distilled water.

3. Outline of the Experiment

Ten to twenty Petri dish cultures were prepared for
each "tissue culture group" in order to provide a sufficiently
large number of representative cultures for each virus
inoculum (Table I). The term ”tissue culture group" was
coined for convenience to designate five to ten Petri dish
cultures of one type of tissue embedded in either rabbit

or chicken plasma. Each group consisted of tissues

 

’The author is grateful to the Upjohn Company for
supplying the endomycin utilized in this experiment.

 

 

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e
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w
x
x
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a
.—
.x

1‘1...

w
M

 

FIGURE I

OUTLINE OF THE EXPERIMENT

The following is an outline of one complete experiment which com-
prised 27"tissue culture groups". Each tissue culture group con-
sisted of all the cultures prepared from one type of tissue embedded
in either chicken plasma or in rabbit plasma, for example, group 1
represented all the embryonating chicken tissues embedded in chicken
plasma, which included tissue receiving (a) heat-inactivated N.D.V.,
(b) active N.D.V., and (0) control cultures which were Hank's
nutrient solution instead of N.D.V. solution.

 

 

A. Cultures of tissues from embryonating chicken egg

1. Embryonating chicken tissue embedded in normal chicken plasma
2. Embryonating chicken tissue embedded in normal rabbit plasma

B. Cultures of tissues from normal adult chickens

3. Lung tissue embedded in normal chicken plasma
4. Lung tissue embedded in normal rabbit plasma

5. Liver tissue embedded in normal chicken plasma
6. Liver tissue embedded in normal rabbit plasma

7. Spleen tissue embedded in normal chicken plasma
8. Spleen tissue embedded in normal rabbit plasma
a C. Cultures of tissues from immune adult chickens

9. Lung tissue embedded in normal chicken plasma
10. Lung tissue embedded in normal rabbit plasma

11. Liver tissue embedded in normal chicken plasma
12. Liver tissue embedded in normal rabbit plasma

13. Spleen tissue embedded in normal chicken plasma
‘ 1h. Spleen tissue embedded in normal rabbit plasma

D Cultures of tissues from normal adult rabbits

15. Lung tissue embedded in normal chicken plasma
16. Lung tissue embedded in normal rabbit plasma

1?. Liver tissue embedded in normal chicken plasma
18. Liver tissue embedded in normal rabbit plasma

19. Spleen tissue embedded in normal chicken plasma
20. Spleen tissue embedded in normal rabbit plasma

E. Cultures of tissues from immune adult rabbits

21. Lung tissue embedded in normal chicken plasma
22. Lung tissue embedded in normal rabbit plasma

23. Liver tissue embedded in normal chicken plasma
2h. Liver tissue embedded in normal rabbit plasma

25. Spleen tissue embedded in normal chicken plasma
26. Spleen tissue embedded in normal rabbit plasma

2?. Lymph node tissue embedded in normal rabbit plasma

 

 

22

inoculated with active N.D.V., plus tissues inoculated with
heat-inactivated N.D.V., plus five to ten Petri dish cultures
of the non-infected tissue used for controls. Thus, the
first tissue culture group listed in Table I included the
cultures of normal adult chicken lung embedded in chicken
plasma which received active N.D.V. and those which received
heat-inactivated N.D.V. as well as the non-infected lung
tissue cultures which were used as controls. Five to ten
cultures were prepared for each tissue so that representative
cultures could be withdrawn at different intervals, to be
tested for N.D.V. activity and the tissue stained by the
Giemsa method for microscOpic examination and enough
cultures would be left for observation until the end of

the experiment. The tissues were embedded in both rabbit
and chicken plasma to determine if either plasma would
enhance or suppress multiplication of N.D.V., or if the
ability to support growth of the virus was a preperty of

the tissue alone.

An equal number of tissue cultures was maintained as
controls during the same period. These cultures were
subjected to the same conditions, and the same fluid changes,
with the exception that the N.D.V. suspension was not added,
In one eXperiment an additional set of controls was included,
in which a heat-inactivated N.D.V. suspension was added to each
type of tissue culture. This virus suspension was incubated

for seven days at 370 C so that it was no longer infectious

 

 

23

for the embryonating chicken egg but retained a hemagglup
tinating titer of 1/800.

h. Description of Tissue Culture Technique

The adult tissues used consisted of lung, liver and
spleen from normal, as well as N.D.V.-immune rabbits and
chickens. Lymph node tissue from normal and immune rabbits
'was also cultured. Embryonic tissue, consisting of chario-
allantoic membrane, brain and torso of 11-day embryonating
chicken eggs were used for all preliminary studies because
they were most easily obtained and gave consistently uniform
growth of fibrocytes and round cells.

Fragments of the tissues from animals were minced
'within one hour after removal. Each tissue was placed in
a Petri dish and flooded with warm nutrient solution, washed
free of extraneous blood and tissue fluids and then trans-
ferred to a fresh nutrient solution where it was cut with a
fine, curved scissors. The average fragment size approxi-
mated 0.1 mm in diameter.

These small fragments were embedded in the thin layer
of normal chicken or rabbit plasma covering the bottom of
a Petri dish and allowed to become fixed before Hankis
nutrient solution was added. Then the tissue cultures were
incubated for four days at 37° C. The nutrient fluid was
not changed during this period unless the medium became
acid. This occurred usually with embryonic and splenic

tissue. The four-day incubation period was sufficient to
assure the growth of even the most slowly develOping adult
tissues so that all cultures could be maintained simultan-
eously, be given the same virus inoculum, and be subjected
to the same eXperimental conditions.
_ On the fourth day the nutrient fluid was withdrawn
asceptically using separate pipettes for each culture to
avoid cross infection. The cultures were then flooded with
Hank's nutrient solution containing eight hemagglutinating
units of N.D.V. On the fourth, eighth, twelfth, and
eighteenth days after virus infection, the nutrient solution
'was removed and replaced with fresh Hank's nutrient fluid.
Samples of the fluid from each culture were withdrawn at
intervals of 2, 6, 12, 2h, 36, h8, 72 and 96 hours after
the N.D.V.-infected suSpension was added, as well as just
before and two hours after each subsequent fluid change.
Samples from each type of tissue culture were pooled for
each incubation period and stored at --200 C until they could
be tested for virus activity. Petri dish cultures represent-
ing similar incubation periods were stained by the Giemsa
method for microscOpic examination.
§:_¥§ggggscgiggr£:r Determining N.D.V. Activity in.Infected
l. Infectivity Tests Using Embryonating Chicken Eggs

All samples of the tissue culture fluids were tested
for N.D.V. activity by inoculating 0.2 ml of the fluid into

25

the allantoic cavity of lO-day embryonating chicken eggs.
Infectivity tests were also carried out on suspensions of
each type of tissue which were prepared at the time they
'were removed from the animals.

If N.D.V. was present in the tissue culture fluids or
in the suspensions of the animal tissues, death of the embryo
occurred within two to five days. At the time of death the
eggs were placed in the refrigerator (+h° C) for six hours
and then the allantoic fluids were withdrawn and tested for
bacterial sterility as well as hemagglutinating activity.

Quantitative infectivity tests were carried out with
the samples of tissue culture fluids which showed positive
infectivity in order to determine the 50 percent mortality
endpoint (LDSO)' Samples from culture fluids of embryon-
ating chicken tissue were tested at intervals of 2, 6, 12,
2h, hB, 72 and 96 hours after N.D.V. inoculation to deter-
mine the titer of virus. The LDSO titers of the fluids
removed from rabbit and chicken tissue cultures 96 hours
after virus inoculation were also determined.

The eggs which were used were received from‘w. S.
Hannah and Sons of Grand Rapids, Michigan. These eggs
were collected from flocks which had not received N.D.V.
vaccine.

In order to confirm the work of Durusan (1949), con-
cerning the thermolability of N.D.V. at +370 0, infectivity

tests were carried out with samples of an undiluted

 

26

N.D.upinfected allantoic suspension incubated at +370 C
from one to seven days. Serial dilutions were also set up
before incubation to determine if the diluted virus would
be more thermolabile than the undiluted virus. Hemaggluti-
nation tests as well as quantitative infectivity tests were
carried out with'samples of these solutions at 2h-hour

intervals up to seven days.

2. Hemagglutination Tests

Pooled samples from.each type of tissue culture were
tested for hemagglutinating activity, using the procedure
described by the Bureau of Animal Industry. In this method
serial two-fold dilutions of the virus suspension ranging
from l/lOO to 1/6h00 were prepared and to each test tube
an equal quantity of a 0.25 percent suspension of chicken
red blood cells was added. The test tubes were shaken
and allowed to stand at room temperature for two hours.
Results of the test were recorded after 30 minutes, one
hour, and two hours. The endpoint was considered to be
the highest dilution of the virus producing maximal hemp
agglutination, and the titer was expressed as the reciprocal
of the final dilution of the virus.

The hemagglutinating activity of the N.D.V.-infected
allantoic fluids incubated at +370 C from one to ten days
were also tested.

In order to determine if the tissues of the rabbit

and chicken possessed either hemagglutinating or

 

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27

hemagglutination-inhibiting activity before they were used
for tissue cultures, ten percent suSpensions of each type
of tissue were prepared, centrifuged and the supernatant
fluids were withdrawn for these tests. In the hemaggluti-
nation-inhibition test a modified B-procedure was used,

two units of N.D.V. suspension admixed with serial two-fold
dilutions of the tissue fluids ranging from the undiluted
fluid to a l/6h dilution.

3. Methods Used for Animal Immunization

Young adult rabbits and chickens were given four
0.5 milliliter doses of N.D.V.-infected allantoic fluid
intranasally at weekly intervals. For the first and second
inoculations, heat-inactivated virus was given. Active
N.D.V.-infected allantoic fluid was then used for the third
and fourth inoculations as well as for the challenging dose
which was given three weeks later. Three months after the
first inoculation the animals were bled and their sera
were tested for hemagglutinin-inhibiting activity. Those
animals showing a titer of at least 1/320 were killed and
their tissues removed for tissue cultivation. In this case
the standard Beta procedure for the hemagglutination-
inhibition test was used. A suspension containing 10 units
of N.D.V. was mixed with an equal quantity of serial two-
fold dilutions of serum, and incubated at room temperature
for 10 minutes before adding 0.25 percent suSpension of

chicken red blood cells.

 

RESULTS
A._Thermelability of the Virug

The undiluted solution of the G.B. strain of N.D.V.
showed a similar thenmolability when incubated at437° C as
did the strain of N.D.V. used by Durusan. All of the 0.3.

solutions which were diluted'beyend 10'1

showed a sharper‘
decrease in the LDSO titers within four days. After two
days the LDSO titers of the diluted solutions decreased to
10‘“, after three days.to l0"1 and after four days the virus
‘was completely inactivated. The hemagglutination titers
decreased from 1600 to 800 during the first 2h hours, then
remained at this titer for the seven-day incubation period.
Since the virus inoculum for the tissue cultures consisted
of a 1/200 solution these hemagglutination titers would
correspond to 16 and 8 respectively. It follows, therefore,
that if virus was recovered from.the tissue culture solution
which.showed an infectivity titer higher than 100 or a
hemagglutination titer higher than 8, multiplication of
the virus had taken place in the tissues.

Similarly, if the hemagglutination titer of the un-
diluted N.D.V.-infected allantoic fluid was 1600, this
fluid, when diluted to 1/200 to contain eight hemaggluti-

nating units represented a hemagglutinating titer of eight.

:r .. IF

. . ,.
_...-.__ ._-. --...-...._. .. .. -.._..
C C I I l \
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29

For example, the N.D.V. inoculum containing eight hemagglu-
tinating units could be diluted serially 1/2, l/h, 1/8 and

still produce maximal hemagglutination. It follows, then,

that when a titer higher than eight could be recovered from
a tissue culture fluid, that tissue had supported growth

of the virus.
B. Results of Hemggglutination and Infectivity Tests

Infectivity and hemagglutination tests were negative
on all the control tissues and tissue cultures, 1.9., in
samples of all the tissue removed from the annmal and in
all cultures of these tissues which were not given the
N.D.V. inoculation. Infectivity tests were negative also
on all tissues which received heat-inactivated N.D.V.

None of the tissue suspensions from the immune chicken or

rabbit showed hemagglutination-inhibiting activity.

1. Hemagglutination Tests with Tissue Cultures Given Heat-
Inactivated N.D.V.

When the heat-inactivated N.D.V. suspension was added
to the tissue cultures the initial hemagglutination titer
remained constant in all the cultures with the exception
of those of the lung and liver of the immune chicken and
the tissues cultured from the immune rabbit. The titer of
the fluids from the lung and liver of the immune chicken
and the lung and spleen of the immune rabbit drapped to a

titer of four within two hours, and remained constant

30

during the next 9h hours. The titer of the fluids removed
from the liver cultures of the immune rabbit also decreased
to four then to zero within 2h hours. None of the fluids
withdrawn from any of the tissue cultures after the first
nutrient change showed any hemagglutination activity (Figs.
II-VI).
2. Hemagglutination and Infectivity Tests with Embryonating
Chicken Tissue Culture

Embryonating chicken tissue cultures were used for all
the preliminary tests to determine the growth curve of
N.D.V. in tissue cultures (Fig. II). Within two hours
after virus inoculation the fluids from these tissue cultures
showed a rapid decrease of hemagglutinating activity from
eight to two due to adsorption of the virus by the tissue.
Within six hours hemagglutination occurred only in the
undiluted fluids and did not increase in titer until after
2h hours. Fluids from embryonating chicken tissues which
were embedded in chicken plasma thereafter showed a rapid
increase in hemagglutination titer reaching a peak titer
of 6k in 36 hours. This titer was constant from 36 to 96
hours, then disappeared for two to six hours after the first
fluid change. After eight days hemagglutinating activity V
appeared in the undiluted fluids but was lost completely
after the second fluid change.

In the fluids from the embryonating chicken tissues
which were embedded in rabbit plasma the hemagglutinating

 

 

 

 

 

 

 

Ir
6% .
’52-
IB'
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HA
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dasaaer ‘ _...-———-""““'_‘f
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'.o‘7 lo‘ (0' \o'

HEMAGGLUTINATION AND INFECTIVITY TITERS OF N. D.\/. IN EMBRYONATING

CHICKEN TISSUE CULTURES EMBEDDED IN CHICKEN AND IN RABBIT PLASMA

FIG. JI

N. D. V. TITERS IN CULTURES EMBEDDED IN CHICKEN PLASMA

“““““ N. D.\/. TITERS m CULTURES EMBEDDED IN RABBIT PLASMA

.‘N

 

 

32

activity increased more slowly reaching a peak titer of 32
‘within.h8 hours. After the first fluid change this titer
decreased from 32 to four but then rose again to eight on
the sixth day and remained at this titer until the eighth
day. After the third fluid change hemagglutinating activity
could still be recovered from the untiluted fluids of the
embryonating chicken tissues which had been cultured in a
rabbit plasma:matrix.

Fluids were withdrawn from these tissue cultures at
intervals of 2, an, AB, 72 and 96 hours after N.D.V. inocu-
latien for infectivity and hemagglutination tests. Since
the virus inoculum consisted of a 1/200 solution the LDBO
of the initial solution was 10‘7. This titer drapped to
10'5‘82 within an hours but rose to anZLDSO of 10“? within
36 hours, then decreased to 10'6'39 after 72 and 96 hours.
Fluids from.the mhbryonating chicken tissue embedded in
chicken plasma showed infectivity up to eight days while
those embedded in rabbit plasma were infectious for 12 days.
3. Hemagglutination and.Infectivity Tests with Rabbit and

Chicken Tissue Culture

There was little difference between the hemagglutination
titers of the tissues which were embedded in a rabbit
plasma and in chicken plasma matrix (Figs. III-VI).

Hemagglutinaticn Tests: When active N.D.V. was added to
the tissue cultures of the rabbit and chicken their hemp

agglutination titer decreased from eight to four or one

.
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_..._- __ __ __

 

33

within two to six hours (Figs. III-VI). This was followed
by a period of rapid virus production in which the titers
rose to 32 or 6h.within 36 to 96 hours in all the tissue
cultures except those of the liver (Figs. IIIb, IVb) and
spleen.(Figs. IIIc, IVc) of the immune chicken and in the
tissues from the immune rabbit (Figs. V, VI). -Fluids from
tissue cultures of spleen from the immune chicken showed

a hemagglutination titer or 16 within.h8 hours but did not
rise above this titer (Fig. IIIc, IVc). The most remarkable
loss of hemagglutinating activity was found in the fluids
removed from the immune chicken liver cultures (Figs. IIIb,
lVb) and in all the tissues cultured of the immune rabbit
(Figs. V, VI). In the period within two to 96 hours
following N.D.V. inoculation, hemagglutinating activity was
found only in the undiluted fluids from the immune chicken
liver cultures, and disappeared after the first fluid change.
In the immune rabbit tissue cultures the hemagglutination
titer decreased to one after two hours but disappeared
completely within 2h.hours.

It was found that the greatest variation in hemagglu-
tinating activity in the tissue cultures occurred during
the period after the first fluid change, 1.6., from four to
eight days after N.D.V. inoculation (Figs. III-VII). By
the end of this period all the normal rabbit and chicken
tissue cultures, with the exception of the normal chicken
lung, showed a hemagglutination titer of four. In the

normal chicken lung the hemagglutination titer increased

 

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from four to 16 in the cultures embedded in chicken plasma
(Fig. IIIa), and from four to eight in the cultures embedded
in rabbit plasma (Fig. IVs). In the immune chicken lung
cultures there was also a four-fold increase in the hemagglu-
tination titers. The imune chicken spleen cultures showed
hemagglutinating activity only in the undiluted fluids
(Figs. IIIc, IVs).

After the second and third fluid changes hemaggluti-
nating activity could not be found in the normal tissues
of the rabbit and chicken but reappeared within #8 to 96
hours in the undiluted fluids. Hemagglutinating activity
disappeared in most of the tissue cultures after the fourth
fluid change, 1.0., 18 days after N.D.V. inoculation.

gnfectivity Tests: Quantitative infectivity tests
were carried out with the fluids of all the rabbit and
chicken tissue cultures 72 hours after N.D.V. inoculation.
In the rabbit and chitken tissue cultures which showed.
hemagglutination titers of 32 to 6h the 12.1350 titers ranged
from 10"5 to 10"7 (Figs. III-VII). In the inmune chicken
liver cultures the hemagglutinating activity appeared only
in the undiluted fluids but an LD50 titer of 10'3 was found
(Fig. IIIb). Although the immune rabbit tissue cultures did
not show hemagglutinating activity, LDSO titers of 10-1 and
1.0-2 appeared within 72 hours after N.£D.V. inoculation
(Figs. V-VII).

35

Infectivity tests on the fluids of the normal rabbit
and chicken tissue cultures were positive up to 18 to 2k
days after N.D.V. inoculation. The immune chicken lung and
spleen.oultures also showed N.D.V. infectivity during this
period but the immune chicken liver and all the tissue
cultures of the immune rabbit failed-to show this activity
after the first fluid change, i.e., four days after N.D.V.

inoculation.“

0. Changes in the Tissue Cultures‘Which‘Were Infected with
N.ng.

The presence of phenol red in the nutrient medium made
it possible to check the pH of the fluids visually. The
hydrogen ion concentration of the fluids bathing the tissue
cudtures did not change after N.D.V. inoculation except in
the spleen tissue cultures. The N.D.V.-infected spleen

 

_H*Fluid samples from all the tissue cultures were
removed each day for four days after N.D.V. inoculation and
every-other day for the next 1h days for quantitativ
infectivity tests. These samples were stored at -20 C but
before the experiment was completed the freezing unit went
out of commission and the infectious activity of the fluids
‘was decreased so that quantitative results could not be
obtained. It was necesSary, therefore, to repeat the experi-
:ment and run only the quantitative infectivity tests which
‘were necessary to confirm the results of the hemaggluti-
nation tests. Thus quantitative infectivity tests were
carried out on the fluids removed from the embryonating
chicken cultures at intervals of 2, 2h, hB, 72 and 96 hours
after N.D.V. inoculation and on all the other tissue
cultures 72 hours after N.D.V. inoculation. The undiluted
fluids removed from these cultures 72 hours after each
nutrient change were tested for infectious activity but
quantitative tests were not carried out.

 

36

cultures showed acid production even after two fluid
changes but they were less acid than the non-infected
spleen controls.

The embryonating chicken tissue cultures showed a
decided cytological destruction after N.D.V. inoculation
(Plates I-II). The only cytological change in the adult
rabbit and chicken tissue cultures occurred in the liver
cultures of the normal chicken which were embedded in
chicken plasma and this did not take place until seven or
eight days after the N.D.V. inoculation. There was a gradual
degenerative change in these liver cells in which there was
a breaking up of the cytoplasm into large granules and a
fragmenting of the cell wall. The entire cytoplasm lost
its staining preperties but the nucleus remained unaltered.
The liver tissues of the normal chicken which were embedded
in rabbit plasma and the liver tissues of the immune chicken
as well as those of the normal and immune rabbit failed
to show any degeneration after N.D.V. inoculation (Plate IV).

None of the control tissue cultures or the tissue
cultures inoculated with heat-inactivated N.D.V. showed
any degenerative change.

In the embryonating chicken cultures which were embedded
in chicken plasma the new cells surrounding the tissue
transplant showed definite cytological changes within 15
hours after N.D.V. inoculation. There was an increase in

the vacuolization of the cytOplasm of the fibroblasts and

 

37

a tendency toward the formation of giant cells which con-
tained three to five nuclei (Pl. I, Figs. 2-3). Sometimes
it was difficult to determine whether these multinucleated
cells were giant cells or an agglomeration of fibroblasts
which had lost their individual cell boundaries and become
fused. It was necessary, therefore, to examine these
particular cells microscopically every few hours. When
several tissue culture fields were marked and examined in
this manner it could be seen that the multiple nuclei
appeared within a single cell. By h8 hours a pronounced
degeneration occurred in the fibroblasts surrounding the
tissue culture explants (Pl. II, Figs. 5-6). The cytOplasm
became more vacuolated and filled with fatty globules, the
cell membrane lost its continuity and most of the fibre-
blasts almost lost their staining preperties. In some
areas only shrunken pyknotic nuclei were left. Within
four or five days degeneration of all the fibroblasts
surrounding the tissue explant occurred leaving only a
fringe-like halo of faintly stained ghost cells. A few
scattered round cells remained.

The infected embryonating tissue cultures which were
embedded in rabbit plasma showed the same type of degen.
oration but this process took place at a slower rate,
lagging 2h to AS hours behind the degenerative changes
shown by the tissues embedded in chicken plasma (P1. II,
Figs. h, 6). The embryonating tissues which were embedded

in rabbit plasma seemed to be protected in some manner by
the rabbit plasma. Only about 50 percent of the fibro-
blasts surrounding the tissue explant showed degeneration,
and many of the remaining cells looked normal even 1h days
after N.D.V. inoculation. There was a higher proportion

of round cells produced by the embryonating tissues embedded
in rabbit plasma and these cells showed no cytological
change after exposure to the virus (P1. II, Fig. 6).

Multinucleated giant cells appeared also in these tissues

(P1. II, Fig. h). There were never more than two nuclei
found in any of the fibroblasts arising from the embryonp
ating chicken cultures which did not receive the N.D.V.
inoculum, or in the tissues which received the heat-

inactivated N.D.V. suspension.

 

EXPLANATION OF FIGURES III - VII

= hemagglutination titers
= nutrient fluid change

= period of hemagglutination titration

= hemagglutination or infectivity titers of
active N.D.V. in normal chicken or rabbit
tissue cultures

0
= hemagglutination or infectivity titers of
active N.D.V. in immune chicken or rabbit
tissue cultures

2 hemagglutination titers of heat-inactivated
N.D.V. in immune chicken or rabbit tissue
cultures

H.A. of heat-inactivated N.D.V. in normal
chicken and normal rabbit tissues were
omitted in the Figures because the titer
remained constant at 8 and decreased to
zero after the first nutrient fluid change

 

 

HA I?

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4—‘ I! J’
4-, F. — _.
2- 2_¥ I
I I I I
‘l, ‘ —'I—— ,_ J! ' b ,/‘. d ___,
dosaI“ _m \. . .A—m jLAA—-.“"."\. . . n “-157'U" . l ‘
N.D.V. inoeuIaI-‘en 22 24 2 4, e 2, ID a. I4- to (a 20 {a dc w :2 .4 «e la 20 29. d4
0 I____ to" >10" (0° 0 .. a
I0 o
ecfi iwIecIiviIr { ,
a
«O o >w 4 (0° .o°
IIIo. NORMAL AND IMMUNE CHICKEN LUNG CULTURES 'DIb NORMAL AND IMMUNE CHICKEN LIVER CULTURES He NORMAL AND IMMUNE CHICKEN SPLEEN

HEMAGGLUTINATION AND INFECTIVITY TITERS OF ACTIVE AND INACTIVE N. D.V. IN NORMAL AND IMMUNE CHICKEN TISSUES EMBEDDED IN

CHICKEN PLASMA

FIG. IEE

CULTURES

 

 

 

“A ‘i‘ // I :1 /
I’ % / .__+

 

 

 

 

 

 

 

 

 

 

 

 

 

   

. I
2' / I A ”I LP /
cIas q‘tfir , ‘ , 37—17% _:——l i L . .l/A ./\.
N.D.V. (ZecuIfl’I’n'oK a 2 I? 4 8 no .014 lfi I6 18 'LO 2'). 21‘ T 1 g 2 8 :0 IL ‘4 I4 ‘.
I .
I I
I IOEE' >10.4 Ibo 0 I [0‘7 710'4' >I0‘
KAI: C‘:Vt{ {
baa l E
J J 7|D'4 7(a"* ”3° 0 ‘ ,0 3 0
11a NORMAL AND IMMUNE CHICKEN LUNG CULTURES 11b NORMAL AND IMMUNE CHICKEN LIVER CULTURES EC

NORMAL AND IMMUNE CHICKEN SPLEEN CULTURES

HEMAGGLUTINATION AND INFECTIVITY TITERS OF ACTIVE AND INACTIVE N.D.V. IN NORMAL AND IMMUNE CHICKEN TISSUES

EMBEDDED IN RABBIT PLASMA

FIG. ISZ

 

 

 

 

 

I
(.4- ~
. ./
HA “a“
8
4- . I
2 / \v I
. ' I
4w \ ./L.../;\ .
my. tenth... , I a z '3 ;. .'1 .‘4 ;. .5 L. 2.1, £1—
E
I (0'5 >10”4 (0° 0
an lutchirl-Iy H
no"

la NORMAL AND IMMUNE RABBIT'LUNG CULTURES

HEMAGGLUTINATION AND. INFECTIVITY TITERS

 

 

 

-\-
; I .—— “’1 .
<' 1 c 3 .o (‘L m (c ,5 14: n. 2.4
I
I
I '07 >to"4 [0° 0
I 710"

1b NORMAL AND IMMUNE RABBIT LIVER CULTURES

OF ACTIVE AND

EMBEDDED

IN RABBIT PLASMA

FIG. I

INACTIVE N. D. V.

IN NORMAL

 

o
I
I
I
I

Ic

AND

5s
4"

f
/

_ __I
. A . : I/ L’ '. E" I I
1. 4 6 8 to 12, ,4. ‘2 IE

to" 7‘0”“ 10“

1° i1 24

210" o

NORMAL AND IMMUNE RABBIT SPLEEN CULTURES

IMMUNE RABBIT TISSUES

 

 

w" I '
adyufler I. I . _ X/Lr‘f’I

N.D.J- «‘npcu Ia’fn'ow < I). 4, 4 8 To 1.1 (q, u, I6 1'0 12 7.4

 

v

0
L

0

to!“ {nIeCIIVEIj {

IEO. NORMAL AND IMMUNE RABBIT LUNG CULTURES

HEMAGGLUTINATION AND INFECTIVITY TITERS

 

 

 

 

 

 

64’
6+
3% I I
.—. 32 __
I6- / \ ' I
16
8“ / I: i 8
4_\‘_ I_ __ 4 _ -._. ‘I
I \ I I
2 | \/\ I 1 \ / /__L l
o 2 4 s g I T I-I/I ' 44 -‘ 1 - - . T— T\ - - .
I o 7. :4 la \3 2a 11 1+ 7 1 4 5 8 W i?— I4 (6 I8 2,0 ’LL i4
. I
I v6 —| I
4 :0 ya: yo" 0 I to"
I >|o" O l 7.0-:
“N
Eb NORMAL AND IMMUNE RABBIT LIVER CULTURES EC NORMAL AND IMMUNE RABBIT SPLEEN CULTURES

OF ACTIVE AND INACTIVE N. D. V. IN NORMAL AND IMMUNE RABBIT TISSUES

EMBEDDED IN CHICKEN PLASMA

FIG. YI

 

 

FIG. CSZIE

6+

 

 

 

I
HA 31‘ . ‘1
I6- I
5‘
4' I /
2‘ V IL L
Jays aéI’er \ —'\
MOI], iuocuIaI'lon. "3 7. 4r 1: 8 to m, “I
I >154 10° 0
an“ infecI’leI] I
I 7ID' Io° o

 

HEMAGGLUTINATION AND INFECTIVITY TITERS IN NORMAL

AND IMMUNE RABBIT LYMPH NODE CULTURES EMBEDDED

IN RABBIT PLASMA

 

 

 

 

 

PLATE I

Results of Experiment with Embryonating Chicken Tissue
Cultures

F180 10

F180 2.

Fig. 3.

Normal embryonating chicken fibroblasts
embedded in chicken plasma matrix,
6-day culture.

Embryonating chicken fibroblasts embedded

in chicken plasma matrix, 5-day culture,

15 hours after N.D.V. inoculation, showing
many normal fibroblasts, a few multi-
nucleated fibroblasts and one multinucleated
giant cell .

Embryonating chicken-fibroblasts and round
cells embedded in chicken plasma matrix,
5-day culture, 2h hours after N.D.V.
inoculation, showing an increase in
vacuolization in cytOplasm of fibroblasts.

Magnification: 200x

 

 

 

 

PLATE I

 

F180 2

 

 

 

PLATE II

Results of Experiment with Embryonating Tissue Cultures

F180 I40

Fig. 50

Fig. 6.

Embryonating chicken fibroblasts and round
cells, embedded in rabbit plasma, 5-day

culture, 2h hours after N.D.V. inoculation,
showing a large multinucleated giant cell.

Embryonating chicken tissue culture,
embedded in chicken plasma, 6—day culture,
ha hours after N.D.V. inoculation,

showing complete degeneration of fibro-
blasts and abnormal round cells.

Embryonating chicken tissue culture,
embedded in rabbit plasma, 8-day culture,
96 hours after N.D.V. inoculation,
degeneration of fibroblasts not complete,
many normal round cells.

Magnification: 200:

PLATE I I

 

F180 5

 

F180 6

1+5

 

PLATE III
Cultures of Adult Chicken and Rabbit Tissue

Fig. 7. Normal adult rabbit lung, 8-day culture,
normal alveolar cells.

Fig. 8. Normal adult chicken lung, 8-day culture,
96 hours after N.D.V. inoculation shoving
normal-appearing alveolar cells.

Fig. 9. Normal adult rabbit spleen, 8-day culture,
96 hours after N.D.V. inoculation showing
normaloappearing spleen cells.

Magnification: 200x

 

PLATE III

 

F180 8

 

F180 9

15.6

 

 

 

 

 

 

PLATE N

Fig. 10. Normal rabbit liver, 8-day culture.

Fig, 11. Immune chicken liver, 8-day culture,
96 hours after N.D.V. inoculation showing
normal-appearing liver cells.

Magnification: 200x

 

 

 

PLATE IV

 

Fig. 1e

 

II?

 

 

DISCUSSION

The data from this study were compiled frOm hemagglu-
tination tests and microscOpic studies from three experi-
ments including cultures of embryonating chicken tissue,
normal chicken and rabbit tissue and immune chicken tissues
and from two experiments with cultures of immune rabbit
tissue. In the first experiment quantitative infectivity
tests and hemagglutination tests were carried out with the
fluids removed from the embryonating chicken cultures at
intervals of 2, 6, 12, 2h, 36, #8, 72 and 96 hours after
N.D.V. inoculation in order to prepare the N.D.V. growth
curves (Fig. II). At the same time it was possible to
determine the period in which the infectious activity and
hemagglutinating activity appeared in the tissue culture
fluids. In this eXperiment fluids from the cultures of
tissues from the normal chicken and rabbit and the immune
chicken were removed 72 hours after N.D.V. inoculation to
determine their LDSO titers also, and to compare these titers
with the hemagglutination titers. Quantitative infectivity
tests were also carried out with the fluids removed 12 hours
after N.D.V. inoculation from the tissues of the immune
rabbit, which were embedded in chicken plasma.

From the data obtained from the hemagglutination tests
and infectivity tests it was found that the growth curves

 

'49

of N.D.V. in the embryonating chicken tissues cultures
showed a cycle similar to that of influenza virus (Enders,
1950). This cycle is composed of (a) a lag phase which
occurred.within two to 2h hours after virus inoculation
when the virus was adsorbed by the tissue culture cells,
(b) a phase of rapid virus multiplication in which peak
infectivity titers were reached by AS hours and peak heme
agglutination titers within 72 hours, (c) a narrow plateau
lasting for an hours, and (d) a sudden decrease in virus
production. These growth curves of the G.B. strain of
N.D.V.'were similar to those determined for the Roakin
strain of N.D.V. in chorie-allantoic tissue cultures by
Scott (1953). The peak infectivity and hemagglutination
titers for the G.B. strain of N.D.V. in the embryonating
chicken tissue cultures were slightly lower. However,
lower titers were expected, for it has been shown that

the chorio-allantoic membrane yields a higher N.D.V.
content than embryo tissue when the embryonating chicken
egg is inoculated by the intravenous route (Hansen 23 g;.,
1952)., The appearance of hemagglutinating activity 21'.
hours later than infectious activity was also found by
Gordon (1952) when the embryonating chicken egg was inocu-
lated with N.D.V. The rate of multiplication of the virus
was slower and the hemagglutination and infectivity titers
were lower in the tissue cultures.

The fact that there was little difference in the peak

So

hemagglutination titers and infectivity titers found in
the fluids of tissue cultures embedded in rabbit and chicken
plasma indicates that these body fluids are not associated
with the resistance or susceptibility of the anmmal as far
as multiplication of N.D.V. is concerned. The rabbit plasma,
however, showed protection of the N.D.V.-infected liver ,
tissues of the normal chicken and prevented extensive degenp
eration of the embryonating chicken fibroblasts, although
it did not impair their ability to support virus growth.
Once the fibroblasts were destroyed in the embryonating
chicken cultures, which were embedded in chicken plasma,
there was a sharp decrease in virus multiplication and.the
virus activity found in these tissue culture fluids eight
days after N.D.V. inoculation could have arisen only from
a few viable'cells in the explant or the remaining round
cells. Since there were a considerable number of fibre-
blasts and round cells found in the embryonating chicken
tissues embedded in rabbit plasma 12 days after N.D.V.
inoculation, the rabbit plasma apparently spared these
cells so that the N.D.V. growth curve showed a slower de-
crease in virus multiplication as well as an extension of
the period of virus multiplication. If degenerative changes
did not occur in the tissue cultures of the normal chicken
and rabbit their capacity to support growth of the virus was
not exhausted until 12 to 18 days after N.D.V. inoculation.
The inhibition of growth of N.D.V. in the liver-tissue

 

 

51

cultures of the immune chicken and all the tissues cultured
from the immune rabbit was due evidently to a preperty
acquired by the tissues themselves during the period of
immunization because the fluids extracted from these tissues
did not show inhibition of the virus at the time they were
removed from the animal. The fact that the Spleen tissue
cultures of the immune chicken showed only slight inhibition
of virus multiplication may be explained on the basis that
the multiplication of the spleen cells continued to increase
at a much greater rate than that of the liver cells after
N.D.V. inoculation and there might have been a higher
percentage of new spleen cells which had not acquired the
N.D.V. inhibiting preperty. On the other hand, it could
have been due to the fact that the spleen of the immune
chicken does not play as important a role in the process
of immunization as does the liver.

Since all the tissues cultured from the immune rabbit
failed to support growth of N.D.V. to the same extent as
the tissues cultured from the normal chicken, and rabbit
as well as the lung and spleen tissues of the immune chicken
tissue the resistance of the rabbit to N.D.V. disease may
be due to the fact that the rabbit has many more tissues
which are capable of acquiring a N.D.V.-inhibiting prOperty
when the animal is exposed to the virus although the rabbit
may not be able to produce an antibody titer any higher
than that of the chicken. There is also the additional

52

possibility that when the rabbit is first exposed to N.D.V.
it may possess a plasma activity capable of protecting the
tissues so that degeneration of the cells may be prevented
until there are sufficient specific antibodies produced
against the virus to step the process. In this case multi—
plication of the virus in the rabbit tissues occurs without
production of disease.

Although the growth curve of N.D.V. in tissue cultures
was similar to that of influenza virus it is of interest
to note that influenza produced degeneration of the cells
of the embryonating chicken cultures (Enders, 1950).
Mumps virus did not produce cellular changes (Enders, 1952)
but this was not expected because this virus does not pro-
duce the same degree of damage in the embryonating chicken
egg as do influenza virus and N.D.V. Newcastle disease
virus, instead, seems to resemble poliomyelitis virus in
its ability to destroy tissue culture fibroblasts. The
production of multinucleated giant cells in embryonating
chicken cultures inoculated with N.D.V. may be of diagnostic
significance for these cells appear within 12 to 2h hours
after exposure to the virus. The possibility that the
tissue culture technique could provide a simple and rapid
means for identifying N.D.V. warrants further investigation.
The occurrence of multinucleated giant cells has often been
reported in tissue cultures of malignant tumors but never

in tissue cultures infected with a virus. Burnet (l9h2)

53

described the appearance of "large, vacuolated cells,
agglutinated in masses in which it is impossible to pick out
the outline of individual cells" in tracheal smears of the
N.D.V.-infected embryonating chicken egg. It is possible
that what would appear as an aggregation of superimposed
cells in a smear preparation could have been one single
multinucleated giant cell. Otherwise the production of
multinucleated cells has not been reported in connection
with N.D.V. When microscopic examination can be made on

a single layer of cells which can be obtained in tissue
culture preparations any changes in the isolated cells can

be followed more easily.

 

SUMMARY

In these experiments a comparative study has been.made
on the effect of N.D.V. on susceptible and resistant tissues
cultured from chickens and rabbits.

Embryonating chicken, adult chicken lung, liver and
spleen and adult rabbit lung, liver, spleen and lymph node
tissues were cultured. Both normal and immune adult chickens
and rabbits were used. Hemagglutination and infectivity
tests were carried out to determine the growth curves of
N.D.V. in embryonating chicken tissues.

The growth cycle was composed of (a) a lag phase of
2h hours, (b) a phase of rapid virus multiplication in which
peak infectivity titers were reached in 36 hours and peak
hemagglutination titers in 36 to h8 hours, (c) a plateau
lasting no to 60 hours, (d) a sudden decrease in the rate
of virus production 96 hours after N.D.V. inoculation.

Growth curves prepared from.hemagglutination titrations
. on the tissues cultured from normal adult chickens and
rabbits were similar to those of the embryonating chicken
tissue but the peak hemagglutination titers occurred 36 to
96 hours after N.D.V. inoculation. This was also found in
the immune chicken lung and spleen cultures but not in the
liver cultures. In the immune chicken liver tissues and in

all the tissues of the immune rabbit there was a steep

 

55

decrease in infectivity titers from an LD5O of 10'7 on the
day of inoculation to 10"1 or 10"2 hB hours later. Hem-
agglutination activity was recovered only in the undiluted
fluids from the immune chicken liver cultures.

Cytological degeneration was seen.in the embryonating
chicken cultures 15 to as hours after N.D.V. inoculation.
The fibroblasts showed an increase in vacuolization of the
cytoplasm.and a tendency in the formation of giant cells
which contained three to five nuclei. The only degenerative
changes in the adult tissue cultures occurred in normal
adult chicken liver and this did not appear until seven
or eight days after N.D.V. inoculation. There was a
gradual degeneration of the liver cells in which the cyto-
plasm lost its staining preperties and broke up into large
granules and the cell wall became fragmented. The nucleus,
however, was unaltered. Neither the uninoculated cultures
nor the cultures inoculated with heat-inactivated N.D.V.

showed any degenerative change.

 

 

 

 

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