HEMAGGLUTINATION-INHIBITION
ANTIBODIES IN EGG-YOLK FROM HEIS VACCINATED WITH
"LEUCOSIS-CONT MINATED" AND "LEUCOSIS-FREE" NEWCASTLE
DISEASE VACCINE

By
Richard Ellsworth_gacobs

A THESIS
Submitted to the Graduate School of Michigan State College
of Agriculture and Applied Science in partial ful-

fillment of the requirements for the degree of
MASTER OF SCIENCE
Department of Bacteriology and Public Health

1950

Tfiiblé

Acknowledgments

The author wishes to express his grati-
tude for the generous cooperation and guidance
given to him by Dr. Charles H. Cunningham,
Associate Professor of Bacteriology, Department
of Bacteriology and Public Health, Michigan State
College, and Dr. George E. Cottral, United States
Department of Agriculture, Regional Poultry

Laboratory, East Lansing, Michigan.

244574

TABLE OF CONTENTS

Object . . . . . . . . . . . . . . . . . . .

Review of Literature . . . . . . . . . . .
Newcastle Disease Virus . . . . . . . .
Avian Leucosis Complex . . . . . . . .
Interference Phenomenon . . . . . . . .
Hemagglutination Activity . . . . . . .

Experimental Procedure . . . . . . . . . . .
Vaccines . . . . . . . . . . . . . . . .

Newcastle Disease Virus Antigen for
Hemagglutination Tests . . . . . . . .

Egg-yolk for Hemagglutination-
inhibition Tests . . . . . . . . . . .

Hemagglutination and Hemagglutination-
inhibition Tests . . . . . . . . . . .

Calculation of Egg-yolk Hemagglutination-
inhibition Titer O O O O O O 0 0 O 0

Results and Discussion . . . . . . . . . . .
Results . . . . . . . . . . . . . . . .
Discussion . . . . . . . . . . . . . .

Summary . . . . . . . . . . . . . . . . . . .

Bibliography . . . . . . . . . . . . . . . .

Pa

0Q

(1)me

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55

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Table

Table

Table

Table

Table

Table

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Table

Figure

II

III

IV

VI

VII

VIII

IX

1

TABLES AND FIGURE

Procedure for Hemagglutination,
Virus dilution . . . . . . . .

Procedure for Hemagglutination-
inhibition, Virus dilution . .

Hemagglutination-inhibition tests
of yolk material at 22° C.
to 27° 0., Control Pen . . . .

Hemagglutination-inhibition tests
of yolk material at 22° C.
to 27° Co, Pen I o o o o o o o

Hemagglutination—inhibition tests
of yolk material at 22° C.
to 27° 0., Pen II . . . . . . .

Hemagglutination-inhibition tests
of yolk material at 22° C.
to 27° 0., Pen III . . . . . .

Hemagglutination-inhibition tests
of yolk material at 22° C.
to 27° 0., Pen IV . . . . . . .

Hemagglutination-inhibition tests
of yolk material at 22° C.
to 27° 0., Pen V . . . . . . .

Hemagglutination-inhibition tests
of yolk material at 22° C.
to 27° C., Summation of all
Pens O O O O O O O O 0 0 O O 0

Average weekly egg-yolk hemagglu-
tination-inhibition titers
for experimental period . . . .

Page

45

46

47

48

49

50

51

52

55

54

Object:

The purpose of this experiment was to determine
if there were any differences in the hemagglutination-
inhibition antibody titers present in yolk from eggs laid
by hens vaccinated with live-virus Newcastle disease vac-
cines prepared from "leucosis-contaminated" and from
"leucosis-free" embryonating chicken eggs.

This experiment was made possible through a co-
operative project of the Michigan Agricultural Experiment
Station and the United States Department of Agriculture,
Regional Poultry Research Laboratory, East Lansing,
Michigan, supported in part by a grant-in-aid from the
National Cancer Institute, in the possible transmission
of the agent of avian leucosis through embryonating
chicken eggs used in the production of avian virus vac-
cines. The experiment was designed as one phase of the
project since the eggs were available and afforded an
unusual opportunity to study the effect of the various

vaccines on the hemagglutination-inhibition antibodies.

REVIEW OF LITERATURE

Newcastle Disease Virus

 

What is now generally considered as Newcastle
disease was first reported by Kraneveldl in 1926 as a
highly diffusible and fatal infection of poultry preval-
ent in the Dutch East Indies.

Doylez in 1927 originally isolated the virus
of Newcastle disease during an epizootic amongst fowls
near Newcastle-on-Tyne, England. Doyle was successful
in demonstrating that the causative agent was a filtere-
ble virus. He reported that infected lung, liver,
spleen and brain which were dried in a dessicator over
CaCl2 in the cold were active at 40 and 100 days, but
the ovary was negative at both time intervals. Virus in
these various organs in a 50 per cent solution of
glycerine in physiologic saline was active after 197 days,
but was dead after 259 days. In physiologic saline, the
virus was active up to 86 days, but inactive at the
hundredth day. Virus in a spleen kept in a Petri dish
was active after 80 days, but was inactive after 150 days.
Blood in sealed pipettes was active after 109 days. In
the only experiments on the effects of temperature on the
virus, the virus was active after 48 hours at 37° C. and

inactive after 72 hours. Cross immunity tests proved

that the virus of Newcastle disease and fowl plague were

separate entities.

Burnett and Ferryzbindicated by comparative
filtration studies with virus-infected allantoic fluid
that Newcastle disease virus was from 80 to 120 mu and
fowl plague virus was 60 to 90 mu. They also showed
that Newcastle disease virus was more resistant to photo-
dynamio inactivation by methylene blue than was fowl
plague virus. These differences in conjunction with
known clinical and immunological differences pointed to a
complete etiological independence of the two diseases.

Stover4 identified a filterable virus as the
causative agent of a "nervous disorder" and "respiratory
nervous disorder" of chickens in California in 1942. The
"respiratory nervous disorder" described by Stover was
named "avian pneumoencephalitis" by Beach;5 He stated
that the disease had been present in California in 1955
or earlier. In 1944 Beachfishowed that ”avian pneumo-
encephalitis" was immunologically related to Newcastle
disease virus. The clinical picture of the disease
caused by the respective agents differed somewhat. In
this country Newcastle disease is associated with a com-
paratively low mortality rate in adult chickens and with
a high mortality rate in chicks. In other countries,
the disease is accompanied by a 100 per cent mortality

rate.

According to Beaudette7 Newcastle disease virus
may be found in the following material in the avian
species: saliva, feces, liver, spleen, lung, pancreas,
bone marrow, kidney, egg yolk, testes, intestines and
contents, bile, edematous material, crop contents, brain
and cord, blood and pericardial fluid.

Burnett8 reported that outbreaks of Newcastle
disease have been described chiefly from Asiatic countries.
The disease seems to be endemic in Japan, the Indies and
the Phillipine Islands.

Chu.2£.2£? reported that epizootic diseases of
fowls resembling Newcastle disease have been reported in
various parts of China and that Newcastle virus was
isolated and identified. They reported some minor clini-
cal differences in the Chungking strain and the Nanking
strain, and between the United States strains and those
isolated in other parts of the world.

In 1948, Osteen and Anderson10 reported that
Newcastle disease had been diagnosed in 42 states and the
District of Columbia.

CunhagtglflxL reported that Newcastle disease
virus was a complex of which approximately 67 per cent
was protein and approximately 27 per cent was lipid
material. A relatively small amount of nucleic acid,
some of which was of the desoxypentose type was also

present. They reported that the infectivity of the

purified material was such that lO-lo°72 grams of New-

castle disease virus constituted the 50 per cent end
point unit of infectivity for chick embryos.

Howitt §£_al12 found neutralizing antibodies
of Newcastle disease in the blood of various groups of
children and in some adults suffering from a mild central
nervous system illness with a short, non-fatal course.
These authors suggested the probability that poliomyelitis-
like, mild, central nervous system infections, especi-
ally in children, and influenza-like diseases in adults
may be contracted from chicks affected with Newcastle
disease.

Immunity conferred passively via the egg in
birds, in 33333 or via the colostrum in mammals, has
long interested immunologists.

During the Italian epizootics, Vianello
found that chicks which were the progeny of recovered
hens resisted Newcastle disease, whereas those hatched
from eggs from other sources died en masse.

Brandly 23 gl}5 revealed the transference of
Newcastle disease antibodies from immune hens to egg yolk
and the developing embryo and extra-embryonic tissues.
Significant quantities of specific antibodies were pre-
sent in extracts of embryo tissue and of the allantoic
and amnionic membranes after 6 to 9 or more days of in-

cubation. Antibodies were not detected in the serum of

embryos until the 15th day of incubation. Demonstration
of antibodies in blood serum paralleled maturation of the
developing embryo.

Rywosch16 and Polk at al;7 showed that the pre-
sence of antibodies was not due to the development of
complement or specific amboceptor in embryonating chicken
eggs. These authors were unable to demonstrate comple-
ment in embryo serum until the let day. Antibodies
could not be demonstrated until the 17th day in the young
chick and then only in a low concentration.

Brandly 23.3l;5 reported that complement was un-
necessary and exerted no appreciable effect or specific
neutralization of Newcastle disease virus by serum. An
antibody titer could be demonstrated for the first two
weeks of postnatal life. This was fellowed by a rather
rapid decline in serum antibodies and immunity. It was
shown that the antibody titer decreased with absorption of
yolk. Congenital passive immunity conferred a high de-
gree of refractivity to Newcastle disease virus. Congeni-
tal or passive immunity interfered with the active immunity
response of young chicks to vaccination. An egg-yolk
antibody titer of 102 or more neutralizing doses would sug-
gest a recent association of hens with Newcastle disease
virus. According to these authors, egg Yolk may be used
instead of blood serum for assessment of previous Newcastle

infection.

Schmittle and Millen18 described a technique
whereby they were able to detect the presence of anti-
bodies in unincubated eggs by means of the hemagglu-
tination-inhibition test. Egg yolk previously treated
with ethylene dichloride and ethyl ether was used in
place of serum in the test.

Prier gt alIg reported the presence of active
Newcastle virus in the yolks of 2 out of 50 eggs from
18 experimental vaccinated birds, 4 and 5 days, respec-
tively, after vaccination. Virus was not found in eggs
at various periods of l to 26 days after challenge ex-
posure of the vaccinated hens. Live virus was found in
one egg from a hen naturally affected with Newcastle
disease before she was experimentally inoculated with
live Newcastle virus vaccine. No virus was found in
eggs from hens that had resumed normal egg production
following vaccination.

Newcastle antibodies were found in eggs as
early as 19 days after vaccination, and were still pre-
sent in one group of eggs 109 days after vaccination.

De LayzO reported the isolation of Newcastle
virus by chick embryo culture from the yolk of 6 four-
day-old chicks and from embryos of the succeeding

hatch dead on the 15th day of incubation.

Avian Leucosis Complex

 

Caparini in 1896, according to Olson,21 was the
first to record leucosis in chickens.

Jungherrzg reported that the term leucosis sig-
nifies a group of diseases characterized by autonomous
proliferation of the precursors of leucocytes. It was
considered that the avian leucosis complex is comprised
of the fellowing: fowl leucosis, granulobastosis, lympho-
matosis, lymphoid tumors and neural lymphomatosis.
Vitamin E deficiency also caused a "lymphoblastoma"-like
condition in chicks. The genetic aspects in the trans-
mission of leucosis are highly complex. They are intim-
ately associated with the unsettled questions of
etiologic classification and gonadal transmission of the
avian leucosis complex. The filterable agent of
visceral lymphomatosis affects primarily young birds.
Immunological methods to differentiate interrelationships
among transmissible agents of the avian leucosis complex
are frequently impeded by resistant birds and by a lack
of immunizing prOperties of the agents.

Ellermann at 2&23 conducted extensive experi-
mental studies on the conditions of leucosis in the
common fowl. They recognized three general forms of
avian leucosis, namely, leucemic or aleucemic myeloid

leucosis, intravascular "lymphoid", i.e., erythroid

 

leucosis, and extravascular lymphatic leucosis, all of
which were considered to be transmissible and caused by
the same filterable virus.

Emmelg4 considered the following non-specific
factors, which may initiate blood changes, to be basic
in the development of avian leucosis complex: iron,
Salmonella toxins, vitamin A and K deficiencies and poor
ventilation.

25

Winton at al demonstrated that visceral
lymphomatosis might be induced by inoculation with a
filterable agent which is found in the tumor tissue,
and in the blood of chickens with the disease. The pre-
sence of the agent in certain chick embryos and in day-
old chicks was also demonstrated.

According to J’ungherrf3E3 the concept that the
cause of avian leucosis complex is unknown, is essen-
tially incorrect, because etiologic agents can be identi-
fied by methods peculiar to virology. Jungherr was of
the opinion that eggs could be carriers of the avian
leucosis complex. He also prOposed the possibility that
fowl pox vaccine produced from leucosis-contaminated
embryonating chicken eggs could be capable of transmit-
ting the avian leucosis complex when such vaccines were
used for immunization.

Doyle26 was the first to suggest that avian

lymphomatosis (fowl paralysis) could be transmitted

- 10 -

through the egg.

Van der Walle and Winkler-Junius in Holland in
192;?7 reported the first positive transmission of fowl
paralysis.

28.

Cottral st 31 reported the transmission of
lymphomatosis from parents to offspring through hatching
eggs by injecting embryonic and chick tissues into sus-
ceptible chickens. Only chicks and embryos that had no
gross abnormalities and which were from offspring of
"normal" sires and dams that had lived for 600 days free
of all clinical or gross evidences of lymphomatosis were
used. Chicks were inoculated with suspensions of pooled
liver tissue from 15-day embryos, 18-day embryos, newly
hatched chicks and with filtrates of liver tissue from
newly hatched chicks. After 300 days, the respective
groups of chickens that were inoculated had an incidence
of lymphomatosis as follows: 82.4 per cent, 100.0 per
cent, 76.5 per cent and 85.7 per cent as compared to an
incidence of 17.6 per cent in uninoculated controls.
These workers reported that only visceral lymphomatosis
was transmitted by these means.

Kisslingz9 reported positive results for leuco-
agglutination as a serological diagnosis for avian

lymphomatosis. Of 94 samples of chicken sera, 48 showed

the presence of lymphocyte agglutinins. 0f the 48

-11..

samples, 40 (83.3 per cent) showed lesions of the leucosis
complex.
25 ,

Winton‘etial have not obtained consistent re—
sults with the following diagnostic tests for the detec-
tion of lymphomatosis: (l) the hemagglutination of
chicken red cells with lipid antigens, (2) the Kahn test,
(5) heat coagulation of plasma and (4) the indirect com-

plement fixation test.

Interference Phenomenon

 

Hoskins reported50 that the presence of a cer—
tain strain of a virus in a susceptible tissue sometimes
prevented the growth of a related strain in the same
tissue; for example, inoculation of a monkey with both
virulent-pantropic and avirulent neurotropic strains of
yellow fever virus did not result in infection, provided
the neurotrOpic strain was injected within 20 hours after
the pantrOpic strain was administered. He also demon-
strated that interference could occur tetween agents, so
far as was known, that were antigenically unrelated.

Findlay and MacCallum31 studied the interfer-
ence between the virus of Rift Valley fever and the two
strains of yellow fever. Rift Valley fever was sero-
logically unrelated to yellow fever and no cross immunity
was induced. The neurotropic yellow fever virus pro-
tected mice against Rift Valley fever virus; and the Rift

Valley fever virus protected monkeys against pantrOpic

- 12 -

yellow fever virus. The authors proposed that manifesta-
tion of interference possibly resulted from the inability
of virus particles to invade cells which were occupied by
other actively multiplying virus particles.

Andrewes in 194233demonstrated interference be-
tween strains of influenza A virus in tissues. Inocula-
tion of 9 mice with 0.02 cc. of vaccine prepared from
W. S. strain of influenza A followed by inoculation with
0.02 cc. of a preparation of vaccine from a neurotropic
variant of the same virus strain resulted in the death of
only one mouse.

Sharples at 8—185 reported that the following
neurotropic viruses possessed oncolytic powers: Russian
Spring Summer virus, West Nile virus, Japanese B
encephalitis virus, St. Louis encephalitis virus and
louping ill virus. It was demonstrated that chickens
inoculated with tumor and with any of the above viruses
were usually rendered immune to further tumor challenge
even though the original inoculum did not produce a
palpable tumor.

Ziegler and Horsefa1134 demonstrated interfer-
ence between strains of the two serologic types of in-
fluenza A in chick embryos. These workers found that
embryos inoculated with either type of virus were re-

fractory to infection to the other type within 8 to 12

hours after inoculation. Likewise, the introduction of

- 15 -

a very large amount of one type caused interference with
the multiplication of the other type when injected
simultaneously. The interference between influenza B
strains did not render embryos insusceptible to infec-
tion from a large inoculum of influenza A strains.

Nenle and Henle,3 reported that the reciprocal
interference between strains of influenza A and B viruses
in the chick enbryo had been as readily demonstrable with
noninfective preparations as with active viruses; al-
though when a noninfective virus was employed, large
inocula were necessary to cause interference. A few
hours after the establishment of infection in the chick
embryo, additional multiplication of virus was inhibited
by the injection of a large amount of noninfective virus.
Preparations which showed interference caused by nonin4
fectious virus particles, "auto-interference", could be
produced by prolonged incubation of U18 inoculated chick
embryo, prolonged storage of infected allantoic fluid at
4° C. heating and ultraviolet irradiation.

2
36
Ziegler t al reported diet the capacity to

produce interference was inactivated by ultraviolet
irradiation or by heat at a less rapid rate than was the
property of infectiousness. Interference apparently was

caused by the altered virus particle itself. The pro-

perty could not be separated from the virus and was

-14-

specifically neutralized by the homologous type anti-
serum.

They also reported the well-established facts
that strains might not be successfully propagated if
serial passage was carried out with undiluted allantoic
fluid, and that higher virus titers were obtained when
dilute inocula were employed, might be attributed to
”auto-interference". It was the opinion of this group
that interference had been dependent upon the capacity
of virus particles to react with susceptible cells, and
that when quantitative saturation of such cells by
virus particles had been achieved, it was not possible
to establish infection with the same or other virus
strains, irrespective of their serologic type.

Henle and Henle57 stated that interference
was caused with great speed by the following: patho-
logical changes in the cell, exhaustion of metabolites
that are essential for an enzyme or amino acid pro-
duction, and production of antibodies.

Hirst58 reported that interference was
mediated by a specific cell receptor. Such a receptor
could have been reactive with both infective or nonin-
fective virus particles if the inactive virus particle
retained the particular or specific structure of
arrangement required for reception. Hirst concluded

that interference could have been quantitative

-15..

saturation of receptors by three means:

1. Multiplication of active virus (auto-inter-
ference)

2. Introduction of large amounts of active virus
against infectious virus present, a com-
petition for receptors

3. Saturation with noninfectious ultraviolet in-
activated virus particles.

Rivers39 indicated that genetic experiments on
the specific virus must be taken into account in seeking
an explanation for the interference phenomenon. He also
reported that closely related virus particles growing to-
gether freely in a single cell were indicative of the idea
of competitive growth. Since the initial rate of growth
of a particular virus in the cell was likely to be propor-
tional to the amount of that virus present, competition
for the substrate was sufficient to account for the ob-
servation that the virus which happened to get started
first might determine which member of the pair would grow.

Rivers postulated that the successful virus
swamped the synthetic mechanisms of the cell and under
these conditions, the less favored virus, after having
made an abortive attempt to grow, died. Evidence that
this virus would die had been derived from material com-
piled on genetic exchange. If there should be an ex-

change of parts of two unrelated viruses, most of the

hybrid products would not be expected to be viable.
Therefore, only the virus present in numerical excess
would survive. This idea was in accord with the mutual

exclusion and depressor effects of mixed infection.

Hemagglutination Activity

 

Hirst,4O in 1941, was the first to notice that
when allantoic fluid from chicken embryos previously in-
fected with influenza A virus was being removed, the red
cells coming from the ruptured vessels agglutinated in
the allantoic fluid. It was also demonstrated that in-
fluenza B and swine influenza viruses were able to agglu-
tinate red cells as well as influenza A. It was shown
that when infectious allantoic fluid, fresh or stored at
-72° C., from which the enbryonic red cells had been re-
moved by low-speed centrifugation, agglutinated normal
adult chicken red cells. The agglutinated cells formed a
ragged, granular pattern at the bottom of a test tube.

The pattern produced by a mixture of noninfectious
allantoic fluid and red cells was a sharp, round disc of
cells. Hirst41 later showed that the addition of specific
immune serum inhibited hemagglutination of red cells in
the presence of the homologous virus, but not when hetero-
logous strains were used.

Hirst41 prepared infectious allantoic fluid for
his studies by inoculating ll-day-old white Leghorn

embryos by the allantoic route with four different strains

- 17 -

of influenza virus: PR8 and V. S. strains of influenza A
virus, the Lee strain of influenza B virus and swine in-
fluenza virus. A 2.0 per cent saline suspension of adult
chicken red cells was used in this test. Titration of

the hemagglutinating ability of the viruses was determined
from serial 2-fold dilutions of the viruses in physio-
logic saline. Equal amounts of each virus dilution and
red cell suspension were combined. These mixtures were
immediately shaken and incubated at room temperature for
one hour.

Serum to be tested for the presence of hemag-
glutination-inhibition substances was diluted in B-fold
steps in saline. To 0.5 ml. of each serum dilution was
added 0.5 ml. of influenza virus-infected allantoic fluid
diluted from 10-3 to 10'.5 with physiologic saline. The
tests were incubated for one hour at room temperature.

In both the virus and the serum inhibition
titrations, readings were made by comparing the density of
the suspensions with the density of known suspensions of
red cells in saline. Hirst used the amount of red cell
sedimentation as indicated by the clearing of the supernate
as the index of the degree of hemagglutination rather than
the pattern of agglutinated cells in the bottom of a tube.

Standard suspensions of red cells for comparison
were prepared in concentrations of 1.0, 0.75, 0.50 and
0.30 per cent. The density of virus and red cell suspen-

sions which were between the 1.0 and 0.75 per cent density

- 18 -

of the standard were designated as %-, 0.75 and 0.50 per
cent ¢L7L, 0.50 and 0.50 per cent 7L747L, and less than
the 0.30 per cent 4747474.

In contrast with the small, sharply-outlined
disc in the bottom of the negative control, the size of
the disc in the bottom of the tube increased with increas-
ing degrees of hemagglutination. The margins of the discs
frequently had a characteristic irregular, lacy pattern
composed of clumps of cells in those tubes in which hemag-
glutination had occurred. This type of pattern was pre-
valent with only slight degrees of hemagglutination, and
there was no decrease in the density of the supernate.
This type of reaction was designated 1E.

The end-point of the titration of the hemagglu-
tinative ability of the virus or of the hemagglutination-
inhibition ability of the serum was arbitrarily taken as
the dilution where 7L+agglutination occurred. If {-74-
agglutination did not occur in any tube, the end-point
was assumed to be half way between two serial dilutions,
one showing more and the other less than %1+L agglutina-
tion.

Temperature differences between 27° to 57° 0.
resulted in no obvious differences in the end-point in

the agglutination test.

Hirst demonstrated that when almost all the
virus was active, the infectivity of the virus could be
destroyed without destroying the capacity to agglu-
tinate red blood cells. Infectivity was destroyed at
56° C. for 15 minutes and at room temperature for several
days. The fact that eggs inoculated with a number of
different influenza virus strains yielded allantoic
fluids with approximately the same hemagglutination titer
suggested that the virus particle concentrations were ap-
proximately the same and that these different virus
strains had similar hemagglutinative activity. He con-
cluded that the hemagglutination test provided a simple
way to determine the relative concentration of virus
particles independent of virulence.

Hirst and Pickles42 described a photo-elec-
tric densitometer which made possible a greater accuracy
in determining the degree of clearing of the red cell
suspensions.

Hirst43 proposed that the mechanism of agglu-
tination of chicken red cells by suspensions of influenza
virus was similar to the adsorption of bacteriophage onto
susceptible, specific receptors of bacterial cells.

After having attached themselves, the "phage" particles
multiplied, but there was no evidence that the influenza

virus multiplied on the chicken red cells. After a

-20-

suitable incubation period, the virus particles were
eluted from the red cells.

Hirst44 reported that the variations in serum
titers following influenza infection were due, to a con-
siderable measure, to the use of red blood cells from
different chickens. He noted that there were variations
in the serum inhibition with different preparations of
the same virus strains. He also showed that the agglu-
tination method was sensitive enough to measure small,
strain differences,and his findings correlated with re-
sults of neutralization tests.

Miller and Stanley45.made a study of several
variables encountered in chicken red blood cell agglu-
tination tests for the presence of influenza virus. The
best results were obtained when the ingredients had been
incubated at 24° C. The hydrogen ion activity had a
negligible effect on the titer between the range of
pH 6.0 to pH 8.0. Variation of results could also be due
to the specific behavior of the red blood cells from dif-
ferent chickens and to an instability of the red cells.

Salk‘fi6 reported that temperature had a marked
effect on the hemagglutination titer of influenza virus.
At 20° 0., higher titers resulted than at 35° 0. due to
the rapid rate at which the virus became dissociated from
the cells at the higher temperature. At 22° C. and 25° 0.

identical titers were obtained which were between the

-21-

titers at 2° c. and 35° c.

Salk described a simple method for titrating
the hemagglutinating capacity of influenza virus and the
corresponding antibody. A suspension of 0.25 per cent
red cells made from a 10.0 per cent stock suspension
which had been stored at 4° C. were used. Salk reported
that if the cells were kept under sterile conditions,
they could be used for more than a week. With complete
hemagglutination (4-), the clumps of agglutinated red
cells adhered to the bottom of the tube at the point of
settling and were characterized by a dispersed rather
than an aggregated pattern. A negative pattern (0) con-
sisted of a central, sharply outlined disc. Intermediate
reactions (f) appeared as irregular clumps. of cells
usually associated with a halo of finely aggregated or
unagglutinated cells. The highest dilution of the virus
which produced the-maximum agglutination represented the
end-point, and the titer was expressed as the reciprocal
of the final dilution of virus after the red cell suspen-
sion was added.

Brandly §t_§:? reported that from a serologi-
cal viewpoint, there were two procedures for hemagglu-
tination-inhibition tests for Newcastle disease: the
"alpha" and the "beta" methods. In the "alpha" method,
successive dilutions of virus were mixed with a fixed

amount of serum. In the "beta" method, successive

- 23 -

dilutions of serum were titrated against a predetermined
amount of virus. This amount, 10 hemagglutinating units
(HA units), was prepared from the greatest dilution of
virus in which at least 7L7Lhemagglutination occurred.
This dilution was considered to contain one HA unit. If
the end-point of the virus was 1:320, then 0.25 ml. of a
1:32 dilution of the virus was used as representing 10
HA units in the test. There was a 93 per cent agreement
between the two methods which seemed sufficiently satis-
factory to warrant the use of either one for diagnosis.

Storage of allanto-amniotic fluid virus in the
unfrozen state for intervals of several months did not
result in an appreciable loss of hemagglutination titer.
Cells from different chickens varied in their reaction
with Newcastle disease virus.

Burnet8 reported that a Victorian strain of
Newcastle disease virus was capable of agglutinating red
blood cells from the following species: chicken, man,
sheep I, guinea-pig, mouse, fowl, sparrow and frog.

Lush48 showed that the hemagglutination test
was applicable to Newcastle disease virus and to the
fowl plague virus. Hemagglutination-inhibition tests
showed that there was no serological relationship between
these two viruses.

Beach49 reported the application of the hemagglu-

tination-inhibition Newcastle disease test in the diagnosis

- 23 -

of avian pneumoencephalitis.

Brandly at also described a method for titrat-
ing the hemagglutinating ability of Newcastle disease virus
and its homologous antibody. For the hemagglutination test,
serial 2-fold dilutions of the virus were prepared in
saline, and to 0.25 ml. of each virus dilution was added
0.25 ml. of physiologic saline. The contents of the tubes
were thoroughly mixed and 0.25 ml. of a 0.5 per cent red
cell suspension was added and tubes were shaken. The test
was incubated at room temperature (23° to 27° 0.). Read-
ings were made at 15, 30 and 45 minute intervals.
Hemagglutination was indicated as 74(positive) and -
(negative).

For the hemagglutination-inhibition test, 0.25
ml. amounts of 1:5 diluted positive, negative and suspect
serums were added in place of the saline in the hemagglu-
tination test, and the results were read exactly like the
hemagglutination test. In this test, it was necessary to
include the hemagglutination test in order to determine
the titer of the virus. ' .

The hemagglutination titer of the virus was the
highest virus-dilution, before the addition of saline and
cells, in which a positive pattern was present. The
titer of the scrim was the lowest virus-dilution, before

the addition of serum and cells, in which a negative

—24-

pattern was present. To calculate the serum hemagglu-
tination-inhibition titer, he titer of the virus was

divided by the titer of the serum. multiplication of

the quotient by the dilution factor of the serum gave

the serum hemagglutination—inhibition titer.

These authors reported that formalinized virus
should be stored in the refrigerator. Virus which was
more than 60 days old and which showed a decrease in
hemagglutinating activity should not be used. In this
test, turkey serum could not be used satisfactorily with
chicken red cells because the serum of turkeys possessed
a normal agglutinin for chicken cells.

Burnet et_alSl demonstrated that red blood cells
which had been previously agglutinated by an excess of
Newcastle disease virus were "sensitized" by the virus.
"Sensitized" cells after washing twice with saline at
room temperature could be agglutinated by influenza A and
B, swine influenza, W. 5. strain of influenza A and vac-
cinia, but not by Newcastle disease virus.

These authors reported that the inhibition ef-
fect of human tears on hemagglutination by influenza
virus might be related to known non-specific inhibition
by certain normal sera and to inhibition by a component
of normal allantoic fluid studied by Rawlinson (unpub-
lished) in Burnet's laboratory.

Andersonsg showed that cells sensitized as

- 25 -

51
reported by Burnet were able to agglutinate normal

chicken red cells. Anderson's experiments indicated

that agglutination by sensitized cells was due to the re-
latively firm adsorption of Newcastle disease virus
particles on the surface of the cell. The cell sensitiz-
ing agent (GSA) which had been adsorbed on the surface of
the cells, even though thoroughly washed, retained its
infectivity for the chick embryo. Agglutinins on stable
cells were neutralized by Newcastle disease immune sera,
and the serum titers corresponded closely with the dir-
ect hemagglutination-inhibition titers.

55 indicated that the

Burnet and Anderson
changed character of sensitized red blood cells was due
to the adsorption to their surface of an agent other
than the virus produced during growth of Newcastle virus
in chick embryo cells.

They also reported that human red blood cells
treated with anniotic or allantoic fluid preparations of
Newcastle disease virus developed a new antigenic
character which allowed them to be agglutinated to a high
titer either by experimental Newcastle disease virus
immune serum or by most sera from recent cases of infec-
tious mononucleosis in man.

Osteen and Anderson in 194810 demonstrated

that diluting Newcastle disease positive serum with 9

parts of negative serum did not lower the hemagglutination-

- 25 -

inhibition titer. Diluting with 19 parts of negative
serum resulted in a lO-fold decrease in titer. These
workers believed that serious error because of dilution
was improbable. A hemagglutination-inhibition titer of
80 or more was considered to be diagnostic of prior
Newcastle infection. Diagnosis of Newcastle disease by
the hemagglutination-inhibition test was considered as
accurate and as reliable as the serum neutralization
test.
54

Hanson at 31 reported the presence of normal
hemagglutinins in the supernatant fluid of centrifuged
11- to 13-day yolk-sac membrane suspensions. The
reaction was indistinguishable from serum hemagglutina—
tion-inhibition. They observed that the normal hemag-
glutinins in both normal and Newcastle disease infected
yolk-sac membranes, completely "masked" the virus hemag-
glutinative activity and made the demonstration of the
latter impossible. These workers demonstrated that
normal chicken serum, added in the same manner and amount
as the immune serum in the virus hemagglutination-
inhibition test, inhibited the normal yolk-sac hemagglu-
tination reaction and the virus hemagglutinin so that it
could be demonstrated. Newcastle disease virus immune

serum inhibited both normal and virus hemagglutinative

activity.

- 27 -

49
Beach proposed two reasons for serums which

had the same virus-neutralizing titers, but which had a
wide hemagglutination-inhibition titer range: hemagglu-
tination-inhibiting and virus-neutralizing antibodies
might be independent substances but an independent sub-
stance which suppressed the hemagglutination-inhibition
reaction might be present in some serums. He also
stated that the hemagglutination-inhibition test alone
was considered inadequate to establish the presence of a
Newcastle disease in an area previously believed free of

Newcastle disease virus.

- 28 -
EXPERIMENTAL PROCEDURE

The presence of Newcastle disease antibodies
in unincubated egg-yolk from hens vaccinated with "leucosis-
contaminated" and "leucosis-free" Newcastle disease vaccine
was evaluated by means of the hemagglutination-inhibition
tests.

For a period of 10 consecutive weeks, begin-
ning on February 3, 1950, 5 eggs were collected weekly
at random from each of the 5 vaccinated groups of hens
and from the control group. These chickens had been
vaccinated from 19 to 25 weeks prior to the initial

collection of the eggs.

Vaccines
All eggs used for the production of the ex-

perimental live-virus Newcastle disease vaccines and all
chickens used for studies of the possible transmission
of the agent of avian leucosis through embryonating
chicken eggs used for the production of avian virus vac-
cine were supplied by the United States Department of
Agriculture, Regional Poultry Research Laboratory,
East Lansing, Michigan.

0 The vaccines were prepared in the Section of
Virology, Department of Bacteriology and Public Health
by Dr. Charles H. Cunningham who vaccinated the experi-

mental chickens in cooperation with Dr. George E.

- 29 -

Cottral, Regional Poultry Research Laboratory.

Live-virus Newcastle disease vaccines were pre-
pared from "leucosis-contaminated" and from "leucosis-
free" eggs. These vaccines were designated as No. 707
and No. 1435-1436, respectively.

No. 707 vaccines were prepared in embryonating
chicken eggs laid by hens which, through previous pro-
geny tests, had been shown to be transmitting the virus
of avian lymphomatosis through the ovarian route. The
eggs were considered to be "leucosis-contaminated".

No. 1435-1436 vaccines were prepared in embryo-
nating chicken eggs laid by hens of "leucosis-free" stock
of the Regional Poultry Research Laboratory. These hens
were from Inbred Line 15 which had shown a 0 to 20 per
cent incidence of lymphomatosis over a period of 10 years.

Three types of vaccines were prepared from Nos.
707 and 1435-1436. The first type consisted of 1/2 of
the liver from the Newcastle virus-infected embryo. The
second type consisted of the remaining 1/2 of the liver
and 1/2 of the extra-embryonic fluids and ground New-
castle disease virus-infected embryo. The third type con-
sisted of Newcastle disease virus-infected ground extra-
embryonic fluids, chorio-allantoic membrane and entire
embryo.

All vaccines were applied by the "wing-web or
stick method" to 30-day-old Single Comb White Leghorns,

28
Inbred Line 15. Cottral at El had previously

demonstrated by artificial infection that chickens from
Line 15 were susceptible to lymphomatosis.

The vaccinated chickens were housed in a build-
ing containing 5 isolation pens.

In Pen I, 51 females were vaccinated with
No. 707, Type 1 vaccine.

Pen II contained 50 females which were vac-
cinated with No. 707, Type 2 vaccine.

In Pen III, 25 chickens, the majority of which
were males, were inoculated with No. 707, Type 2 vaccine.
The remaining 25 males in Pen III were vaccinated with
No. 707, Type 3 vaccine.

Pen IV contained 50 females which were vac-
cinated with No. 1435-1436, Type 3 vaccine.

Pen V contained 50 chickens, the majority of
which were males, which had been vaccinated with
No. 1435-1436, Type 3 vaccine.

A total of approximately 50 males and 50 females
were not vaccinated and were kept at the Regional Poultry

Research Laboratory as controls.

Newcastle Disease Virus Antigen for Hemagglutination Tests

 

A strain of Newcastle disease virus (48-49-1105)
isolated in the Department of Bacteriology and Public
Health, Michigan State College on.Nay 31, 1949, from a
naturally infected chicken was used to inoculate embryo-

nating chicken eggs for the production of virus-infected

-.31 -

allantoic fluid to be used as the antigen in the hemag-
glutination tests. The virus had been originally
established and cultivated in the allantoic cavity of
embryonating chicken eggs through four passages and was
capable of killing all inoculated embryos by the end of
the second day following inoculation.

Single-Comb White Leghorn eggs obtained from
a commercial hatchery were used for prepagation of the
virus. The eggs were incubated at 99° F. in an electric,
forced-draft incubator. Embryos were lO-days-old at the
time of inoculation. The eggs were candled,and an area
of the chorioallantoic membrane void of large blood
vessels, which was a quarter turn distant from the
embryo and about 3 mm. below the base of the air cell,
was selected. At this point, a hole was drilled through
the shell, without piercing the shell membrane, by means
of a small drill which was attached to an electric motor.
A second hole was drilled through the shell over the top
of the air cell. Tincture of metaphen was painted over
the holes and allowed to dry. Before inoculating the
eggs, a teasing needle was flamed, and a small puncture
was made in the shell membrane exposed in the hole over
the air cell so as to prevent leakage of the inoculum
from the site of the injection because of the increased

hydrostatic pressure.

The eggs were inoculated with a B-D Yale, 1 cc.
tuberculin syringe fitted with a 27 gauge, 1/2 inch
needle. The needle was inserted through the hole in the
side of the egg to a depth of about l/4 inch, and the
inoculum was then injected. The holes in the shell were
sealed with melted paraffin and then returned to the in-
cubator.

The eggs were candled daily. Embryo mortality
during the first 24 hours was considered to be due to
non-specific causes and not to the action of the virus.
These embryos were discarded. Embryos which were living
on the second day following inoculation were selected
for collection of allantoic fluid. These embryos were
refrigerated overnight at 4° 0. prior to collection of
the fluid.

A small batch of eggs was removed from the re-
frigerator at a time for the collection of the infected
allantoic fluid. Tincture of metaphen was applied to
the shell in the area of the air cell and allowed to dry.
The shell over the air cell was cracked with sterile
forceps, and the shell was removed to within 5 or 10 mm.
of the base of the air cell. Sterile 5 cc. syringes
fitted with 20-gauge, l-inch needles, were employed for
the collection of the infected allantoic fluid. The

needle was inserted into the allantoic cavity, and the

fluid was aspirated into the syringe and expelled into

sterile 500 ml. Erlenmeyer flasks, which were chilled
with cracked ice. Only blood-free allantoic fluid was
collected. Fluid from the allantoic cavity of those
embryos which were not dead on the second day was placed
in a separate flask. The cotton plugs in the flasks
were sealed with Para-film before freezing the fluid at
-45° C. All fluids were bacteriologically sterile.

The fluids were frozen for at least 3 days and
then thawed at room temperature and dispensed in 20 ml.
amounts into sterile 30-ml. screw-cap vials. These vials
were then frozen until the fluid was needed. Before us-
ing, the infected fluid was thawed at room temperature
and activated with formalin to a final concentration of
0.1 per cent. Formalinized virus, as indicated in the
studies by Rached,55 was stored at 5° C. for 72 hours be-

fore using to allow the virus to be completely inactivated.

Egg-yolk for Hemagglutination-inhibition Tests

 

Newcastle disease hemagglutination-inhibition
antibodies were extracted by ethylene dichlorhie and
ether from unincubated yolk by the method described by

18
Schmittle and Killen.

At the time of collection of the egg-yolk, the
eggs were swabbed with tincture of metaphen which was
allowed to dry. The shells were cracked with sterile

forceps, and the egg contents were deposited in sterile

Petri dishes.

- 34 -

With the aid of a pipette, 1.5 ml. of the un-
incubated yolk material was transferred to a chemically
clean 30-ml. screw-cap vial, diluted with 6.0 m1. of
physiologic saline and thoroughly mixed. Two ml. of
chemically pure ethylene dichloride and 1.0 ml. of
reagent ether were added, and the contents of the vial
were thoroughly mixed. The mixture was incubated at
37° C. for approximately 12 hours and then centrifuged
at 1,000 r.p.m. for 10 minutes in an International
Clinical Centrifuge.

The ethylene dichloride fraction settled at the
bottom of the tube and was separated from the watery
supernatant solution by a fatty ring. The supernatant
fluid was removed with a chemically clean eye-dropper.
This fluid, approximately a 1:5 dilution of egg-yolk, was
substituted for the serum in the hemagglutination-
inhibition test for Newcastle disease outlined by the
U.S. Department of Agriculture.

Preliminary tests indicated that the egg-yolk
would have to be diluted approximately 1:160 with
physiologic saline in order to bring the hemagglutina-
tion-inhibition end-points of the various samples within

a readable range.

Hemagjlutination and Eemagglutination-inhibition Tests

 

Materials:

 

Virus The virus antigen consisted of the blood-free
pooled, Newcastle disease allantoic fluid.

Antibody The antibody was supplied in the extracted
egg-yolk.

Physiologic saline 0.85 per cent chemically pure

 

sodium chloride in distilled water.

Red blood cells Although it would have been desira-

 

ble to use the blood from one chicken throughout the ex-
periment in an endeavor to reduce variations in hemagglu-
tination titers obtained with cells from different donors,
as reported by Stuart-Harris,56 Hirst,42 and Miller and
Stanley,45 it became necessary to use two chickens because
the original donor became ill, and the red blood cells
were not suited for the test. The blood was collected

in a chemically clean, sterile test tube containing 1.0
m1. of a 2.0 per cent sodium citrate solution for each

9.0 m1. of blood. This mixture was then thoroughly
agitated to prevent clotting. The cells were washed three
times in 10 or more parts of physiologic saline, centri-
fuged twice at 1,600 r.p.m. for 8 minutes and once at 1,000
r.p.m. for 10 minutes. The supernatant fluid was removed,
and the washed cells were stored in the refrigerator. The

stored cells were found to be suitable for use for as long

as 5 to 7 days after collection. Immediately prior to use

_ 36 -

in the test, a 0.5 per cent physiologic saline suspension
of the cells was made.

Chemically clean, round bottom test tubes
(75 mm. X 10 mm.) were used in the tests. Serial Z-flald
dilutions of the virus from 1:5 through 1:5,280 were made
in physiologic saline in a row of tubes in a wire rack.
A 1:7% dilution was also prepared from which serially 2-

fold dilutions were made. This procedure provided Z-fold

 

dilutions of the virus with intermediate steps and

afforded a more accurate detection of the hemagglutina-
tion activity of the virus than the original Z—fold dilu-
tion of the virus. To a parallel row of tubes was
transferred 0.25 ml. of each virus dilution and 0.25 ml.
of physiologic saline except in the egg-yolk titration
tubes in which 0.25 ml. of extracted egg-yolk was added
instead of the saline. The tubes were shaken well and
0.25 ml. of a 0.5 per cent suSpension of red blood cells
was added. The egg-yolk control tubes contained 0.5 m1.
of yolk and 0.25 ml. of the cell suspension. The control
tube for the virus titration contained 0.5 ml. of
physiologic saline and 0.25 ml. of cells. The tubes were
shaken to mix all ingredients and were then incubated at
room temperature (22° C. to 27° 0.). Readings were made
at 15, 50, 45 and 60 minute intervals.

To facilitate the reading of the results, the

rack of tubes was placed under a fluorescent light, and

the pattern of the cells in the bottom of the tubes was
viewed with the aid of a mirror under the rack.

Complete hemagglutination was exhibited as a
uniform layer of agglutinated red blood cells covering
the bottom of the tube and was recorded as positive (71%
Where there was no hemagglutination, the sedimented cells
formed a central, sharply outlined disc in the bottom of
the tube and was classified as negative (-). Hemagglu- '
tination intermediate between positive and negative was
classified as partial hemagglutination (1f).

The titer of the virus was considered to be the
highest dilution of the virus which produced complete
hemagglutination. This dilution was considered to con-
tain one hemagglutinating unit, and the number of hemag-
glutinating units in the undiluted virus was considered

to be the reciprocal of the titer of the virus.

Calculation of Eggjyolk Hemagglutination-inhibition Titer

 

The titer of the virus was the highest virus-
dilutiOn before the addition of saline and cells in which
7Lorf hemagglutination occurred. The titer of the egg-
yolk was the lowest virus-dilution before the addition of
egg-yolk and cells in which —or ihemagglutination oc-
curred. To calculate the egg-yolk hemagglutination-
inhibition titer, the titer of the virus was divided by

the titer of the egg-yolk. Multiplication of the quotient

- 58-

by the dilution factor of the egg-yolk gave the egg-yolk

hemagglutination-inhibition titer.

Results

The results of the individual tests on egg-yolk
from unincubated eggs are shown in Tables III to VIII,
inclusive. The average titer per pen for each experi-
mental period and the average titer per pen for the
entire experiment are shown in Table IX.

n the Control Pen, the egg-yolk hemagglutina-
tion-inhibition titers were 10 or less except at the 5th
test when the titer was zero. The average hemagglutina-
tion-inhibition titer was 5 for the entire experiment.

In Pen I, the egg-yolk hemagglutination-inhibi-
tion titer for the initial test was 5,504. The titer de-
creased progressively to 2,560 for the 3rd test. The
titers for the 4th and 5th tests were 3,925 and 11,592,
respectively. The titer for the 6th test was 2,055. At
the 9th and 10th tests, the titers were 9,600 and 2,400.

In Pen II, the initial egg-yolk hemagglutina-
tion-inhibition titer was 2,432. The titers decreased
progressively until a minimum titer of 544 was obtained
for the 5th test. Beginning with the 6th test, the titer
gradually increased to 3,115 at the 8th test and de-
creased to 2,400 at the 10th test.

In Pen III, the egg-yolk hemagglutination-

inhibition titer for the first test was 2,816. The titer

- 4o -

of the 2nd test decreased to 2,688. No eggs were availa-
ble for the next 2 tests. The titer for the 5th test was
4,096. A titer of 2,548 was obtained for the 6th test
and 2,048 for the 7th test. The titer for the 8th was
5,333 followed by a decline to 720 for the 10th test.

In Pen IV, the egg-yolk hemagglutination-
inhibition titers showed greater variation than in the
other pens. An initial titer of 3,328 was obtained. For
the 2nd and 3rd tests, the titers were 1,920. A maximum
titer of 16,392 was obtained for the 5th test. With the
exception of the titer of 5,759 for the 8th test, there
was a decline in titers to 448 at the 9th test. The
titer for the 10th test was 1,496.

In Pen V, the initial egg-yolk hemagglutina-
tion-inhibition titer was 4,244. other than a titer of
6,080 for the 2nd test, there was a gradual decrease in
titer to 2,304 for the 5th test. A progressive increase
was recorded with a maximum of 7,328 reported for the 8th
test. A decrease followed with a titer of 1,712 for the

10th test.

Discussion

 

Eggs were tested for the presence of Newcastle
disease antibodies from February 3, 1950 to April 7, 1950,
and although there were fluctuations in the average weekly
egg-yolk titers during the period, the over—all trend was

a progressive decrease in titer.

- 41 -

Slight fluctuations or deviations from this
normal progressive decrease in the average weekly yolk
titers for the individual groups were considered to be
due to the random sampling method of obtaining eggs
from these groups for use in the experiment.

The sudden increases in the titers from the
normal progressive decrease with the occurrence of two
peaks, one at the 5th and the other at the 8th tests
with a gradual decrease at the 10th test, are illus-
trated in Figure 1. Since these increases occurred in
all pens, they could not be ascribed to differences in
vaccines. One possible reason might be the seasonal
effects on egg production.

The hemagglutination-inhibition test on the
yolk material from the non-vaccinated chickens in the
Control Pen gave an apparent diagnostic titer when the
dilution, 1:160, which was used fer the test on yolk
material from the other pens, was employed. But when a
dilution of 1:5 was used, the egg-yolk titers were not
significant.

The yolk titers in the Control Pen may be con-
sidered to be due to non-specific substances. Andersonsg
reported that Burnet and his associates previously
demonstrated that cells acted on by virus particles

develop the ability to be non-specifically agglutinated

- 43 -

by most normal human and animal sera. This non-specific
agglutination is associated with receptor destruction,

and it may also be produced by any bacterial enzymes which
can destroy receptors.

The yolk titer for the first test in Pen I,
5,504, was higher than the initial titers for any of the
pens. Pen I also had the highest average yolk titer for
the period of the test. All the chickens in Pens I to V
were vaccinated with a vaccine which was prepared by us-
ing the same strain and amount of live Newcastle virus,
but the source of the "leucosis-contaminated" vaccines
differed. Since all of the vaccinated chickens received
the same amount 0 Newcastle vaccine, the difference in
the yolk titers appears to be due to the presence of both
viruses in the same host.

In previous studies at the U.S. Department of
Agriculture, Regional Poultry Research Laboratory, the
highest incidence of lymphomatosis had been produced by
inoculating susceptible chickens with a vaccine which
had been prepared from infected embryo livers only. It
might be assumed that the vaccines prepared from liver
only for the present experiment contained the greatest
concentration of the leucosis virus. Since the chickens
in Pen I received "leucosis-contaminated" vaccine consist-
ing of liver only, and the highest hemagglutination-

inhibition titer was obtained in this pen, it seems

- 43 -

possible that the activity of the leucosis virus was at-
tenuated. Assuming that the livers contained the greatest
concentration of the leucosis virus, the use of large con-
centrations of inoculum might have either destroyed or
saturated all receptors ibr which they had an affinity.
The Newcastle disease virus would then have been free to
initiate growth unhampered by the leucosis virus.

If the high Newcastle yolk titer is to be taken
as an indication of the degree of suppression of the
leucosis virus, then the lowest incidence of lymphomatosis
would be expected from this group. Therefore, the extent
of growth of the leucosis virus and the extent of lympho-
matosis would possibly be to a degree inversely propor-
tional to the egg-yolk titer of the Newcastle virus.

Pen II received vaccine which contained New-
castle disease virus consisting of 1/2 embryo livers and
1/2 embryo fluids. The low average egg-yolk titer, 1,612,
in this pen would seem to indicate that the leucosis
virus had interfered with the growth of the Newcastle
disease virus. The lowered titer apparently indicated
that less growth had occurred as a result of competition
for metabolites. The assumption might possibly be made
that since active leucosis virus was present, the chickens
in this pen should show lymphomatosis. According to the
theory prOposed concerning the relationship of the yolk

titer of the Newcastle virus antibodies and the incidence

- 44 -

of lymphomatosis, the highest incidence of lympho-
matosis might be expected in this pen.

Pen III received commercial vaccine which con-
sisted of the entire extra-embryonic membranes and fluids
and the enbryo. The lowered yolk titer in this pen also
suggested interference with the growth of the Newcastle
disease virus by the leucosis virus. According to the
relationship which had been previously proposed for the
differences of egg-yolk titers and the incidence of lympho-
matosis, the average egg-yolk titer of 2,421 for Pen III,
which was slightly higher than the titer from Pen II,
would imply that the incidence of lymphomatosis in Pen III
should be slightly less.

Pens IV and V contained chickens vaccinated with
"leucosis-free" vaccine. The average egg-yolk titers were
4,097 and 3,498, respectively. These titers could be con-
sidered to be the normal for Newcastle virus as the vac-
cines were produced from "leucosis-free" eggs.

The incidence of lymphomatosis in these pens
would be expected to be the lowest of all the experimental
groups of chickens.

Data as to the actual incidence of lympho-
matosis in these several groups of chickens vaccinated
with "leucosis-free" and "leucosis-contaminated" vaccines
are not yet completed and available for comparative
studies of the possible relationship of the Newcastle dis-

ease virus egg-yolk titers and the agent of lymphomatosis.

-45-

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- 55 -
SUMMARY

Three types of live-virus Newcastle disease
vaccines prepared from "leucosis-contaminated" and
"leucosis-free" eggs were used for experimental vaccina-
tion of chickens. The presence of Newcastle disease
antibodies in the yolk of eggs from vaccinated hens was
evaluated by the hemagglutination-inhibition test.

The egg-yolk hemagglutination~inhibition
titers in the non-vaccinated Control Group ranged from
O to ll. These titers were considered to be the normal
range.

Eggs from chickens vaccinated with No. 707,‘
Type I "leucosis-contaminated" vaccine in Pen I had an
average egg-yolk hemagglutination-inhibition titer of
4,858.

Eggs from chickens vaccinated with No. 707,
Type II and No. 707, Type III "leucosis-contaminated"
vaccines in Pens II and III, had average egg-yolk
hemagglutination-inhibition titers of 1,801 and 2,042,
respectively.

Egg-yolk hemagglutination-inhibition titers
of 4,096 and 5,498 for eggs from chickens vaccinated
with No. 1435-1436, Type III "leucosis-free" vaccine
in Pens IV and V might be considered normal for New-
castle disease virus as these vaccines were produced

from "leucosis-free" eggs.

-56-

Fluctuations in average weekly egg-yolk titers
of eggs from the different pens during the course of the
experiment were considered to be due to the random
sampling method of obtaining eggs from each group.

The seasonal effect on egg production may have
contributed to certain variations in egg-yolk titers

during the experiment.

10.

ll.

-57..
EIBLIOGRAPHY

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12.

13.

14.

15.

16.

17.

18.

1‘3 0

20.

21.

-58-
TIBLIOGRAPHY (Continued)

Hewitt, B.F., Bishop, L.K. and Kissling, R.E., Pre-
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DeLay, P.D., Isolation of avian pneumoencephalitis
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22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

- 59 -
BIBLIOGRAPH (Continued)

Jungherr, E.L., The avian leukosis complex.In Dis-
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Ellermann, V., The leucosis of fowls and leucemia
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Emmel, M.W., Hemocytoblastosis in the chicken. Proc.
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Winton, B., Tenth Ann. Report Reg. Poultry Res. Lab.,
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Doyle, L.P., Neuritis or paralysis in chickens.
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Van der Walla, N. and Winkler-Junius, E., De Neuritis-
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Cottral, G.E., Burmester, B.R. and Waters, N.F., The
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Kissling, R.E., Leukoagglutination as a serological
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Hoskins, M., A protective action of neurotrOpic
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Sharples, G.R., Davies, M.C. and Cox,9 n .R.,
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34.

37.

38.

39.

40.

41.

43.

- 60 -
BIBLIOGRAPHY (Continued)

Ziegler, J.E. Jr. and Horsefall, F.L., Jr.,
Interference between the influenza viruses. I.
The effect of active virus upon the multiplica-
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Henle, W. and Henle, C., Interference between in-
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Ziegler, J.E., Jr., Lavin, G.I. and Horsefall,
F.L., Jr., Interference between the influenza
viruses. II. The effect of virus rendered non-
infective by ultraviolet radiation upon the
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Henle, W. and Henle, C., Interference of inactive
virus with the propagation of virus of influenza.
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Ibid., Studies of antigenic differences among strains
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Rivers, T.M., Viral and Rickettsial Infections of
Man. J.B. Lippincott Co., Philadelphia (1948):
158. ‘

Hirst, c.x., The agglutination of red cells by
allantoic fluid of chick embryos infected with in-
fluenza virus. Science, 94 (1941): 22-25.

Ibid., The quantitative determination of influenza
virus and antibodies by means of red cell agglu-
tination. J. Exptl. Med.,‘Z§ (1942): 49-64.

Hirst, G.K. and Pickels, E.G., A method for the
titration of influenza hemagglutinins and in-
fluenza antibodies with the aid of a photo-electric
densitometer. J. Immunol., 45 (1942): 275-285.

Ibid., Adsorption of influenza virus hemagglutinins
and virus by red blood cells. J. Exptl. Med., 26
(1942): 195-209.

44.

45.

46.

47.

48.

49.

50.

52.

53.

- 61 -
BIBLIOGRAPHY (Continued)

Hirst, G.K., Adsorption of influenza virus on cells
of respiratory tract. J. Exp. Med., 78 (1945):
99-109. '——

Miller, G.L. and Stanley, W.M., Quantitative aspects
of red blood cell agglutination test for influenza
virus. J. Exptl. Med., Zi,(1944)= 185-195.

Salk, J.E., A simplified procedure for titrating
hemagglutinating capacity of influenza-virus and
the corresponding antibody. J. Immunol., 49
(1944): 87-88. —

Brandly, C.A., Hanson, R.P., Lewis, S.B., Winslow,
N.S., Hoyt, H.H., Pritchard, W.R. and Nerlinger,
C.M., Variables and correlations in laboratory
procedures for Newcastle disease diagnosis.
Cornell Vet., 51 (1947): 524-556.

Lush, D., The chick red cell agglutination test
with the viruses of Newcastle disease and fowl
plague. J. Comp. Path. and Therap., 55 (1945):
157-160. “'

Beach, J.B., The application of the hemagglutination-
inhibition test in diagnosis of avian pneumo-
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Assoc.,l112 (1948): 85-91.

U.S. Dept. Agr., Agr. Res. Ad., Bureau Anim. Ind.,
Path. Divis., The hemagglutination and hemagglu-
tination-inhibition tests for the diagnosis of
Newcastle disease. Circular (1946): 1-4.

Burnet, F.M., Beverridge, W.I.B., McEwin, J. and
Boake, W.C., Studies on the Hirst hemagglutination
reaction with influenza and Newcastle disease
viruses. Aust. J. Exptl. Biol. and Med. Sci., 25
(1945): 177-192. ""

Anderson, S.G., The reaction between red cells and
viruses of the influenza group. Studies with New-
castle disease virus. Aust. J. Exptl. Biol. and
Med. Sci., 25 (1947): 164-174.,

Burnet, F.M. and Anderson, S.G., Modification of
human red cells by virus action. II. Agglutina-
tion of modified human red cells by sera from
cases of infectious mononucleosis. Brit. J.
Exptl. Path., 21 (1946): 256-244.

BIBLIOGRAPHY (Continued)

54. Hanson, R.P., Winston, N.S. and Brandly, C.A., In-
fluence of the route of inoculation of Newcastle
disease virus on selective infection of the
embryonating egg. Am. J. Vet. Res., 8 (1947):
416-420. "

55. Rached, S.H., Thesis submitted for degree of Ph.D.,
Mich. State College. The antibody response of
turkeys vaccinated with formalin inactivated New-

r7

castle disease virus (1948): a.

56. Stuart-Harris, C.H., Observations on the agglutina-
tion of fowl red cells by influenza virus. Brit.
' J. Exptl. Path., 24 (1945): 55-40.

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