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Date

This is to certifg that the

thesis entitled

Complement Fixation Reactions with
Newcastle Disease Virus.

presented bg

Henry Bloom

has been accepted towards fulfillment

of the requirements for

 

14.5. degree in Bacteriolog
/Maj0r professor
May 15.9 1351

 

 

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COi-zPIJSJ-LfiivT FDLATION REACTIONS

WITH NEWCASTLE DlSEASE VJBUS

by

Henry Harold gloom

A THESIS

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

Department of Bacteriologr
1951

THESlS

C 57/57

jugs-

ACKNOWLEDGENENT

This opportunity is taken for the expression of my sincere gratitude
to Dr. Walter N. Mick, Associate Professor of Bacteriology fer his constant

and effective guidance throughout this work.

Acknowledgement and thanks is also given by the author to Dr. H. J.
Stafseth, Professor and Head of the Department of Bacteriolog for his ad-
vice and criticism of the thesis.

255370

TABLE OF CONTEN TS

Introduction ......t...................................... 1
Review of Literature...................................... 2
Materials and Methods .................................... 8
Hemagglutination Titrations.........................l7
Complement Titrations ............................. 17
Antigen Titrations .....................s.......... 18
Sara Titrations ................................... 20
Results ..................................................21

D18CUBSiOD and CODClUSiODB 0.0000000000000000...0.0000000 24

Sum OOOOOOOOIOOOOCOOOOOOOOOOOOOOOOOOO0.000000000COOOO 26

BimogaPWOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.00.00.00.00. 27

-1-

INTRODUCTION

Until 1937 the complement fixation test for the detection of neuro-
tropic viruses was of little value. Since that time great strides have
been made in developing a specific complement fixation test not only for
this group of viruses, but for other viral and.rickettsial infections.
The factor causing the most trouble in setting up a complement fixation
test for neurotropic viruses has been, and still remains, to prepare an
antigen that is specific for a given homologous antibody; Neurotropic
viruses establish themselves in the central nervous system.and the great-
est source of antigen can be found in the brains of those species affected.
Normal brain material contains lipid materials. These substances are
capable of exhibiting anticomplementary action or non-specific fixation
in the complement fixation test. Mbch.work has been done to remove these
lipid substances from normal and infected brain material. Some of the
methods used to accomplish this extraction are long and tedious and.even

the "simplified" methods involve considerable time and effort.

It was found in this laboratory that the brains of Syrian hamsters
infected with Newcastle disease virus, did not exhibit any interference
reactions when used in the complement fixation test, together with specific
antibody. Since Syrian hamsters are readily obtained, easily kept, and
highly susceptible to hamster adapted Newcastle disease virus, an investi-
gation into the complement fixation reactions of Newcastle disease virus
was undertaken.

For brevity, Newcastle disease virus will, hereafter, in this paper,
he referred to as NDV,

.2-

REVIEW OF THE LITERATURE

An.infection of fowl caused by a filterable virus was reported by
Doyle (1926). This disease was given the name of NEW. From the Dutch
East Indies came a report of an infection of fowl which in later years
was shown to be similar to NDV (Kraneveld 1926). In 1940, poultry'raisers
in.California described a type of nervous disorder in their flocks. These
outbreaks revealed a respiratory disorder which was at first believed to
be due to infectious bronchitis. This disease was named "avian pneumoenp
cephalitis" (Beach 1912) . Later it was demonstrated that this new disease
in California was immunologically identical to Newcastle disease, the dif-_
ference between the California disease and that occurring elsewhere being
in the mortality rates. The disease in.most parts of the world is charact-
erized by high mortality, while in the United States the average mortality
is five to 10 percent (Beach 1942). In survivors, the egg production re-
mains low for a period of one to three months. The virus was first identi-
fied in the united States by Stover (1942). I

Newcastle disease affects the central nervous system as well as the
respiratory organs. Cases with involvement of the central nervous system
usually continue for a longer period of time than those which are essenp
tially respiratory. Nervous system.disorders are characterized by ataxia,
partial or complete paralysis of one or both legs, incoordination of the neck
muscles and tremor of the head (Beach 1942). All these symptoms are seldom

exhibited by any one bird but are usually randomly manifested in a flock.

-3-

There has been much discussion over the relationship of NDV either to
neurotropic or respiratory viruses. Burnet (1942) observed reactions common
to both Newcastle disease virus and influenza viruses. Both agglutinated
chicken red blood cells in similar titers and multiplied in the same manner.
Cunha, et a1 (1947) reported that "the resemblance to the influenza viruses
mmst.be extremely remote, from.the point of view of1morphology. With the
present knowledge, a valid basis for classifying the Newcastle virus with

the viruses of influenza is not obvious".

Bang (1948) was of the opinion that "Newcastle disease virus resembles
those of the encephalitis group in its ability to spread throughout the
developing egg and embryo, but it is similar to influenza virus in that
high concentrations are found in the allantoic fluid before death". Reagan
(1947) found that intracerebral inoculation of monkeys with hamster adapted
NDV resulted in.symptoms similar to the ones caused by poliomyelitis. He
found that egg adapted Newcastle virus upon intracerebral inoculation did
not produce any such symptoms. Wenner (1949) also reported a central nervous
system.disorder in monkeys intracerebrally inoculated with Newcastle disease
virus.

Newcastle disease virus also has its public health significance. An
epidemic of seventeen cases of conjunctivitis in Israel was reported (Yatom
1945). The infection almost exclusively attacked women who had contact
with fowl affected with Newcastle disease. No direct spread from.man to
man was noted. The epidemic stopped as soon as the diseased birds were re-

moved from the flock and destroyed. Ingalls (1949) reported two authentic

.4—

cases of Newcastle disease affecting the conjunctiva of man. In both cases
the individuals were exposed to fowl which had Newcastle disease. Identi-
fication was made by'hemagglutinationpinhibition tests and by virus isolap
tion through passage in embryonated chicken eggs. Hewitt, et a1 (1948)
gathered information on a mild central nervous system infection that took
place in Tennessee. All sera taken from the infected persons were tested
by the neutralization test against Eastern and'Western equine encephalomyel—
itis and St. Louis encephalomyelitis. No reactions with these viral anti-
gens were noted. Twenty out of twentybfive sera tested against NDV were
positive. Although no virus has been isolated, there seemed to be some
opinion that the Newcastle disease virus of fowl is the agent responsible
for many of the atypical central nervous system infections in man that have
been reported during the past few years. Like in the fowl, the manifesta-

tions are neurological in the young and influenzarlike in the adult.

The first successful application of the complement fixation test in
work with neurotropic viruses was accomplished by Howitt (1937). Essentially
her method of obtaining antigenic material for work with the neurotropic
viruses consisted of the following: brains of infected guinea pigs and mice
‘were 1ycphilized, extracted with ether, brought up to original volume with
ether, centrifuged at 2400 rpm for fifteen minutes and then dried again.
Physiological saline was used as a diluent for the antigen when tests were
to be made. Freezing and thawing five or six times in a.002-a1cohol bath
and again centrifuging at 2400 rpm for fifteen.minutes completed the pre-
paration. In this way Hewitt was able to differentiate the viruses used.

-5-

In these tests the sera were produced by experimentally infecting animals.
False positives resulting from Nassermann positive human sera did not affect

her results.

Casals (1941) objection to Howitt's (1937) work was that the resultant
antigen, although not anticomplementary was of very low titer. He reported
that "supernatants of brain emulsions, when centrifuged at 10,000 to 30,000
rpm for one hour and especially if filtered through a Seitz pad, showed no
anticomplementary action". Casals also found that the supernatant fluid
when filtered without first being centrifuged, was anticomplementary and
could not be used in the complement fixation test. An anticomplementary
antigen can be diluted to a point where it no longer interferes with the
test. However, since these neurotropic viruses have such low titers, it
is likely that the antigen would be diluted beyond usable antigenicity be-

fore the disappearance of anticomplementary activity.

Havens, et a1 (1943) stated that the clinical and pathological picture
of these neurotropic diseases were not distinct enough to warrant a diagnosis
of a specific disease. Serological studies are required for positive identi-
fication, except in the case of rabies and poliomyelitis. Havens' group
worked on the following viruses: St. Louis encephalomyelitis, Japanese B
encephalomyelitis, Eastern and‘Western equine encephalomyelitis, West Nile
and lymphocytic choriomeningitis. Syrian hamsters were found to be susceptible
to all but lymphocytic choriomeningitis. Centrifugation at high speeds was

used to remove anticomplementary material and other substances that gave non-

-6-

specific reactions in the complement fixation test. Immune serum.was pre—
pared by intraperitoneal inoculation of active virus into hamsters. None

of the antigens were anticomplementary, no antigens fixed complement in the
presence of normal inactivated serum, none of the antisera fixed complement
with antigens from.norma1 brain tissue after undergoing the same preparation
as did the infected brain material.

.A problem that arose in working with these viral agents was that there
was danger of the laboratory worker becoming infected. Casals (1942) used
ultraviolet light to inactivate these highly virulent viruses. The avirur
lent antigens he prepared had the same antigenicity, specificity, and lack
of anticomplementary effect as did the fresh antigens. Although specialized
standardized equipment must be used, the final product seems worth the effort.
This safe antigen may be lyophilized and stored for periods up to two years
without any decrease in titer. Casals (1945) also used heat to prepare avirup
lent antigens from.infected mouse brains. [A temperature of 60° for thirty
minutes rendered the material safe for routine laboratory usage. This method
does lower the titer of the antigen somewhat. It is, however, a simple pro-
cedure for producing avirulent antigens of a high enough titer for diagnostic

work.

There are other methods available for producing antigens that are not
anticomplementary, for work with these neurotropic viruses. Nolfe, et a1
(1946) prepared a rickettsial antigen from.embryonated chicken.yolk sacs.

DeBoer (1946) reported that this method is applicable to the preparation of

.17-

any infected tissue rioh in antigen. The preparation includes freezing,
drying, extraction with lipid solvents, drying and centrifugation and being
restored to volume before use. Espan;’(l948) modified.DeBoer's method.

This modification consists of using benzene for the lipid extractioh. The
antigen is lyophilized and upon rehydration.requires no centrifugation.
Casals (1949) recently prepared an antigen.by means of successive extractions
of infected brain tissue with acetone and ethyl ether. This antigen does

not react with'Wassermann positive sera.

Newcastle disease, a naturally occurring virus in fowl, was adapted
to the Syrian hamster by Reagan (1948). A California strain of this virus
was carried intracerebrally in hamsters, until the incubation period of the
disease decreased from two to six days to approximately twelve hours. After
twelve serial passages, the hamster adapted.virus was non-pathogenic for
chickens when inoculated intramuscularly or subcutaneously. Reagan (1949)
infected hamsters with NDV by intranasal and intracerebral routes. In.ham-
stars the incubation period is longer following intranasal instillation than
following intracerebral inoculation. Hamsters infected by either route showed
symptoms of irritability followed by involuntary motor reactions and paralysis.
The hamster is also susceptible to other types of neurotropic viruses (Len-
nette 1941) (Havens, et a1 1943). Sanders (1948) reported that hamsters,
intracerebrally inoculated with the Lansing strain of poliomyelitis virus,
showed signs of a polioelike infection but that poliomyelitis virus was not
recovered. The Syrian hamster is a good experimental animal for use in pro-

ducing neurotropic virus antigens.

-8-

MATERIALS AND METHODS

The Newcastle disease virus hamster-adapted strain was obtained from
Dr. R. Reagan, University of Maryland, College Park, Maryland. The virus
was from the 311th intracerebral serial hamster passage. Four additional
passages were made at this laboratory to provide a supply of antigen for

the serological tests that were made.

Newcastle disease infected allantoic fluid was obtained by inoculating
embryonating chicken eggs with an egg adapted strain of the virus. Upon

death of the embryo the allantoic fluid was harvested.
Pre ation o Hamste B n ti e

Hamsters were inoculated intracerebrally with 0.06 ml. of a 10 percent
suspension of Newcastle disease infected hamster brain. The incubation period
varied from three to seven days. Symptoms of the viral infection included
ruffled fur, conjunctivitis of one or both eyes, irratihility, spastic parar
lysis and death. The animals were sacrificed when in the acute stage of the
disease. The brains were aseptically removed, placed in sterile tubes, one
brain in each tube, sealed and stored at -40° C.

When required, each brain was triturated in a sterile mortar, using
alundum as an abrasive and physiological saline as the diluent. The resulting
10 percent suspension was then centrifuged in a horizontal centrifuge at 2,500
rpm for ten minutes. The supernatant fluid was removed from the sediment with

a sterile five m1. pipette attached to a rubber bulb. A sample of the super-

-9-
natant fluid was placed in thioglycholate medium for a sterility test.

The supernatant fluid was initially tested for viral activity by the
hemagglutination test. The technique used was that recommended by the

School of Public Health, University of Michigan:

Ten tubes were set up in a rack. One and eight tenths ml. of
physiological saline was placed in the first tube and 0.5 ml. in tubes two
to 10 inclusive. Two—tenths m1. of brain suspension was added to tube nump
her one, and the contents of the tube were thoroughly mixed. Five-tenths
ml. from tube number one was then transferred to tube number two and this
procedure repeated in all 10 tubes. Five-tenths ml. was discarded from the
last tube. The dilutions in tubes one through 10 were two-fold, ranging
from.1:20 to 135,120. The indicator system, consisting of 0.5 ml. of 0.5
percent chicken red blood cells, was added to each tube with the exception
of the first tube which was a dilution tube. The total volume in each tube
used in the test was one ml. The addition of blood cells produced virus
dilutions of 1:40 to 1:10,240. The tubes were shaken and put aside at’room

temperature for one hour.

The end-point was determined by the pattern formed by the cells
at the bottoms of the tubes. Positive agglutination of red cells produced
a thin diffuse layer of cells covering the bottom.of the tube. Negative
agglutination showed tightly packed discs of cells at the bottom of the tube.

Doubtful agglutination was reported when a ring of packed cells appeared at

-10-

the bottom of the tube. The end-point was reported as the greatest dilution

in which agglutination occurred.

Antigens prepared in this manner and possessing viral activity in
the hemagglutination test were then stored in sealed ampoules at - 0° C. until
used in the complement fixation test (Table I.). Normal hamster brain sus-

pensions were prepared in the same manner for controls.
Pre ation of Newca tle Dise e l i

The Shells of eight to 10 day old embryonating chicken eggs were
pierced using an electric drill. Care was taken not to puncture the shell
membrane. The holes were drilled above the air sac and alongside the allanp
toic cavity. Metaphen, l:10,000 dilution, was applied over the areas of
the shell that were drilled to prevent bacterial contamination of the embryos.
The shell membrane was then punctured with a sterile needle. Using a sterile
0.25 ml. syringe with a 27 gauge needle, 0.1 m1. of egg adapted NDV, cone
tained in allantoic fluid, was injected into the allantoic cavity. The
eggs were then returned to the incubator. All embryos dying in eighteen
hours or less following injection were discarded. Embryos dying fortyh
eight hours after inoculation were stored overnight at 498° C. before har-
vesting the allantoic fluid. .A sterile 10 ml. syringe with an 18 gauge
needle was used to harvest the allantoic fluid. The material was collected
under a.hood to prevent contamination. The yields of allantoic fluid ranged
from.five to nine ml. per egg. The allantoic fluid from.normal eight to 10

day old chicken embryos was collected as control antigen.

_]_1—

The hemagglutination test was used for the initial detection of NDV.
Four serial egg passages were required to increase the titer of the virus

to high hemagglutinating activity (Table I. ) . -

Prepggation of Antigerg in.Rabbitgg

Rice (1948) showed that chicken serum can not be used in the comple-
ment fixation test. Therefore, rabbits were used for the production of

antiserum.

Rabbits whose ages varied from.three months to one year were injected
with NDV hamster—adapted brain suspension, normal hamster brain suspension,
egg-adapted Newcastle virus allantoic fluid, and normal allantoic fluid
respectively. The rabbits were injected every third day intravenously,
using the marginal ear veins. Three injections each of 0.25, 0,50, 0.75,
and 1.0 ml. respectively were made using a one ml. syringe with a 24 gauge

needle.

Following the final injection the animals were allowed to rest for one
week'beforo bleeding. A preliminary bleeding was made to determine if the
rabbit serum.contained antibodies to the antigen. If the preliminary tit-
rations showed the serum to have low titers, further injections of one ml.
were made. The rabbits were bled by heart puncture, using a 20 ml. syringe
with an 18 gauge needle. Care was taken to prevent hemolysis of the blood
by first removing the needle and then slowly expelling the blood from the

syringe into the sterile tube used for collection. The tubes were slanted,

.12..

allowed to clot and left at room.temperature for thirty minutes. The serum
was removed from.the clot after rimming the clot and centrifuging at 2,500
rpm for fifteen minutes. The serum was inactivated at 56° C. for 20 minutes
and then stored at -l,0° C. Reinactivation at 56° C. for 10 minutes and
sterility tests, in thioglyoholate medium, were performed on all serum

prior to use in the complement fixation test.

The Complement Fixation Test

The methods employed were essentially those of Casals (1941). Pre-
paration and titration of the reagents are as follows:
Hemolytic System:
1) Saline
Eight and a half grams of dry chemically pure sodium chloride was dis-
solved in one liter of distilled water. The saline solution was then auto-
claved at fifteen pounds pressure for twenty minutes. This sterile solu-

tion was kept at 4-80 0. when not in use.

2) Sheep erythrocytes
Sheep blood was obtained from the Michigan Department of Health. Sterile
Alsever's solution was added in an amount equal to the volume of the blood
collected. The blood was stored at 4—8° C. for as long as three weeks with-
out the cells hemolyzing or becoming excessively fragile. The Alsever's

solution was made as follows:

.13.

Demose C...00.000.000.00000010025 game
SOdium Citrate 00000000000000. 40m gems
Sodium chloride .............. 2.10 grams
Citric acid................... 0.28 grams
Hater (distilled)............. 500 ml.
Autoclave five minutes at 10 pounds pressure.

The red cell suspension was washed three times using three volumes of
sterile saline solution and sedimentation of the cells at 1,200 rpm for ten
minutes. The final wash was centrifuged for twenty minutes to insure pack-
ing of the cells. The supernatant fluid of the final wash was always free
from color, the same being true for the diluent of the suspension of red

cells used in the test.

3) Antisheep hemolysis
Glycerinized antisheep hemolysin? in 1:100 dilution was called the
stock solution. This stock solution was prepared from the original pro-
duct by adding 0.1 m1. of glycerinized hemolysin to 9.9 ml. of sterile
saline. Storage of this diluted stock solution was at 4-80 0. for not

longer than seven days.

Hemolysin was titrated once a week. For the titration a dilution of
1:500 was made from the 1:100 stock solution, by diluting one ml. of the
stock solution with four ml. of sterile physiological saline. In a series
of 10 tubes higher dilutions were prepared as follows:

0.2 m1. hemOlYSin (1:500). o ooooooeoooeooooeeooeeeeo. 1:500
e salineoooooooooooo 131000
0 Salli-De 0.0.0.0.... 1:15m

m1
ml
ml. saline............ 132000
ml. 83111130000000.0000 132500

. hemolysin 1:500)

m1 :4
m1. hemolysin El:500) %
1111 1‘
m1. hemolysin (1:500) f

*Sharpi& Dhome, Philadelphia, Penn.

ml. hemolysin (1:1000) / 0 4.m1. saline....................l:3000
ml. hemolysin (1:1000) ,l 0 6 m1. saline....................1:3500
m1. hemOlySin (132000) ¥ 0.2 mlo saline“....c............c134000
ml. hemolysin (1:2000) ,4 o 3 ml. saline....................l:4500
m1. hemolysin (1:2500) ,4 o 2 ml. saline.....................1:5000
The contents of each tube were mixed thoroughly and 0.2 ml. of the diluted

hemolysin transferred to a clean tube.

,A 1:30 dilution of guinea pig complement was prepared with sterile
saline solution and 0.2 m1. added to the tube containing the diluted hemo—
lysin. Two-tenths ml. of a.two percent sheep«erythrocyte suspension and 0.4
ml. of physiological saline were also added to each tube. The tubes were
shaken and incubated in.a water bath at 57° 0. for one hour. One hemolysin
' unit was the highest dilution that exhibited complete hemolysis. Two units
of hemolysin'were used in all tests and the hemolysin was so diluted that
0.2 ml. contained two units. For example, if one unit equalled 0.2 ml. of
a 1:4000 dilution of hemolysin, two units, therefore, would equal 0.2 ml.

of a 1:2000 dilution.

4) Complement
The complement was obtained from guinea pigs. The guinea pfigs were
fasted for twelve hours prior to bleeding to prevent their sera from con-
taining excessive quantities of lipid materials. They were bled in groups
of 10 using a,10 m1. syringe and a 20 gauge needle. Five to 10 ml. of blood
was drawn from.each animal. Care was taken to prevent hemolysis by'removing
the needle from the syringe before expelling the blood into sterile tubes.

The blood from each guinea pig was placed in a separate tube and allowed to

.15..

clot at room temperature. After 30 minutes the clot was rimmed and centri-
fuged at 2500 rpm.for 10 minutes. .A 0.2 ml. sample of serum.was taken from
each tube and a screen titration was made to determine the complement titer.
Only serum.which titered 1:40 or higher was used in the final pooled comple-

ment.

During the early part of the work the complement was preserved with
Green's (1938) preservative. However, unsatisfactory complement resulted
and other methods of preservation were found more satisfactory. During the
greater part of the investigation one ml. volumes of complement were shell
frozen in.a Cozgalcohol bath, hermatically sealed in ampoules and stored at
-40° 0. No significant decrease in titer was observed in complement preser-

ved in this manner.

Complement was titrated each day a test was to be made according to the
following method: Dilutions of 1:10, 1:20, 1:30, 1:40, 1:50, and 1:60 of
complement were prepared. To 0.2 m1. of each dilution, 0.4.ml. of physio-
logical saline was added. The tubes were then incubated for one hour in a
37° 0. water bath. The sensitized cell system was made up 15 minutes prior
to its use. It consisted of equal volumes of a two percent sheep cell sus-
pension and two units of hemolysin, each contained in 0.2 ml., and were mixed
in independent tubes and incubated in the water bath. Four-tenths m1. of
the sensitized cells was added to each tube of the diluted complement.
Finalby, the tubes were read at the end of an additional 30 minute incuba-

tion period. The unit of complement was taken as the highest dilution that

-16-

showed complete hemolysis. Two units of complement were used in the
final complement fixation tests. For example, the highest dilution
showing complete hemolysis (1:40) was considered as one unit, than two
units would be contained in 0.2 ml. of 1:20 dilution.

A complement titration in the presence of each antigen used in the
investigation was made every two weeks. Two-tenths ml., containing two units
of antigen, 0.2 ml. of each complement dilution, and 0.2 ml. of physiological
saline were mixed together and the test conducted in the same manner as the'
daily titration. The daily complement titration was performed at the same
time. Anticomplementary or procomplementary activity of the antigen could
be demonstrated by comparison of the end points observed in the two titra-

tions. Table II shows the effect of the antigenpcomplement titrations.

ti e

Two units of antigen were used {h the complement fixation tests. In
order to determine this unit, the antigens were titrated as follows:
Two—fold dilutions of antigen, ranging from full strength to 1:128,
were made. Two tenths ml. of each antigen dilution was added to 0.2 ml.
of heated antiserum, diluted 1:10, and two units of complement contained in
0.2 ml.

A.hemolytic control of the serum consisted of 0.2 ml. of antiserum.and
0.4.m1. of physiological saline. Any hemolytic activity of antigen was demon-

strated by preparing 0.2 m1. of full strength antigen, 0.2 m1. of antiserum,

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-18-
and 0.2 ml. of physiological saline substituted for the complement. A red
cell control was made by adding 0.2 m1. of the two percent cell suspension
to 0.8 ml. of physiological saline. Four complement control tubes were pre-
pared as follows: Two-tenths ml. of two units of antigen used in the titra-
tion was added to each of the four tubes. Five-hundredths, 0.1, 0.15, and
0.2 ml. each of the two-unit complement dilution was added to each tube re-
spectively. Finally saline was added to bring the total volume to one ml.

in each of the tubes.

All tubes were incubated for one hour in a 37° 0. water bath. The sen-
sitized cell system.was made up 15 minutes prior to its use. It consisted
of equal volumes of a two percent sheep cell suspension and two units of
hemolysin, each contained in 0.2 ml., and were mixed in independent tubes
and incubated at 37° C. Four-tenths ml. of the sensitized system was added
to each tube of the diluted antigen. The tubes were read at the end of an
additional thirty minute incubation period. The unit of antigen was taken

as the highest dilution showing no hemolysis.

Antigen was also titrated in the presence of normal inactivated rabbit
serum to determine if any non-specific activity was present. The normal
rabbit serum was substituted for the heated antiserum and the test performed
as was outlined in the preceding paragraphs. Table III gives the results

obtained in the antigen titrations.

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.19-

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Two-fold dilutions of antiseru, ranging from full strength to 1:128,
were prepared. Two-tenths m1. of the serum dilutions, 0.2 ml. each of antigen
and complement, containing two units was added to a tube. A complete series
of control tubes was made with each titration. The tubes were incubated at
37° C. for one hour in a water bath. Four-tenths m1. of the sensitized cells,
previously incubated for fifteen minutes was added to each tube. All of the
tubes were incubated for an additional 30 minutes and read. The final end

point was taken at the highest dilution of serum showing no hemolysis.

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-2]:-
RESULTS

Hemagglutination tests on.uewcastle disease infected allantoic fluid
(Table I), showed that a high titer virus could be obtained by egg passages.
Hamster infected brain material did not produce comparable hemagglutination
titers. However, the titer of the hamster infected brain material was suf-
ficiently high to warrant its use in the complement fixation tests. The hema-
gglutination titers were of value in screening antigens. Hemagglutination-
inhibition tests were also found to be useful in screening antisera. The
materials exhibiting the highest titers were used in the complement fixation

teats.

In the complement fixation test it was necessary to titrate complement
to find the two-unit dilution that was used in the test. Too much or too
little complement will affect the sensitivity of the complement fixation titra-
tions. Complement was titrated in the presence of antigen to discover any
anticomplementary activity. None of the antigens used in the study exhibited
any anticomplementary action, when two units of antigen were used in performing
the titrations. Full strength normal allantoic antigen showed anticomplementary
action, however, at a dilution of 1:2 this activity was not demonstrated. All
the antigens, with the exception of Newcastle disease infected hamster brain,
produced an increase in the complement activity by one dilution tube. Newb
castle disease infected hamster brain material did not affect the complement

titration (Table II).

-22-

Four complement control tubes were set up in all antigen and serum
titrations. These controls were used to indicate if too much or too little
complement was used in the test. They also served as an indicator of anti-
complementary activity. Hemolysis was absent in the dilution containing
0.05 ml. of complement if the concentration of complement used in the test
was not too high. The second dilution, containing 0.1 ml. of complement was
partially or completely hemolyzed, indicating that enough complement was used
in the test. Finally, the dilutions containing 0.15 and 0.2 ml. of comple-
ment were completely hemolyzed. Since these complement control tubes con-
tained antigen and no antiserum, it was possible to detect anticomplementary
activity. Although two units of antigen were always used in the test proper,
full strength hamster brain suspensions, both infected and normal, were used
at various times, in the complement control tubes. At no time was the brain

suspension found to possess any anticomplementary activity.

Antigen titrations (Table III) were used to establish antigenicity and
also to determine the two unit dilution of antigen that was used in the tests.
High titers of Newcastle disease infected hamster brain and Newcastle disease
infected allantoic fluid were obtained, by their respective methods of preparer
tion. Antigens were tested in the presence of inactivated normal rabbit serum.
This was done as a control, since certain animal sera will show non-specific
reactions. 1“0 fixation of any significance was found between normal rabbit

serum and the antigens used throughout the work.

In the titration of the sera (Table IV), antigen.and antiserum prepared

-23.

from the same animal species were not used in the same titration. For exp
ample, Newcastle disease hamster infected brain antiserum.was titrated.with
Newcastle disease infected allantoic antigen. 'This was done to prevent organ
specific reactions taking place. Both brain material and allantoic material
are capable of eliciting an antibody response in rabbits. Since tests were
made for the presence of Newcastle virus antibodies, other antigenpantibody
reactions m was to be avoided. Adequate controls were made to demon-
strate the lack of non-specific reactions taking place in any of the titra-

ti one o

The complement fixing titers of Newcastle disease infected hamster brain
and Newcastle disease infected allantoic fluid were markedly lower than the
corresponding hemagglumination titers. Normal hamster brain was found to be-
a good antigen, while normal allantoic fluid was found to be a weak antigen.

A high titer was obtained with one normal allantoic antigen anvaewcastle
disease infected allantoic antiserum. This difference in titer between normal
allantoic antiserum.and the Newcastle disease allantoic antiserum.was pro-
bably due to the individual rabbits response to the allantoic fluids. A non-

specific reaction could have also taken place.

Throughout the study it was difficult to obtain enough rabbits to obtain
duplicate samples of antisera. Each animal's response to a particular antigen
is varied. A comparison of titers between sera of two or more animals injected

with the same antigen, would have been very useful in this type of work.

-24.
DISCUSSION & CONCLUSIONS

Learning the theory and technics of the complement fixation test was

the main purpose of this study. Upon reviewing the literature on complement
fixation.reactions of the neurotropic viruses,-difficulty with hamster brain
antigens was anticipated. Much.work has been published on the methods of
extracting anticomplementary substances from normal and infected brains of
different animal species, including the hamster. At no time was anticomple-
mentary activity observed during the course of this experimental work. No
special treatment of the brain material in any way was attempted. Perhaps
Havens et a1 (1943) and other workers assumed that because other animal brain
material (mice, guinea pigs, monkeys, and dogs) was found to be anticomple-
mentary, hamster brain should also be anticomplementary. In the:review of the
literature, no data were found on the extent of anticomplementary activity of
hamster brain materials. Since normal hamster brain material as well as the
Newcastle disease infected brain suspension did not exhibit anticomplementary
action, it appears that the NDV was not the agent responsible for the "dis-
appearance" of anticomplementary activity. The results obtained indicate
that by using hamster adapted neurotropic virus antigens, many of the problems

now encountered in the diagnosis of the encephalitides might be overcome.

The rabbit was shown to be a good animal for the production of Newcastle
virus antiserum. Normal rabbit serum did not>exhibit any non-specific action
with any of the antigens used. The complement fixation test is specific for
the antibodies of NDV when tested with allantoic and hamster brain adapted
NDV antigens.

-25.

The complement fixation test for the diagnosis of Newcastle disease is
not practical and the hemagglutinationpinhibition test now used, which is
an easier test to use, does an excellent job in the laboratory diagnosis of

this disease.

1)

2)

3)

-26-
SUMKARY

It was found that Newcastle disease virus infected allantoic fluid

and suspensions of infected hamster brain were good antigens for use in
the complement fixation test. The rabbit was found to be a good exper-
imental animal for the production of complement fixing Newcastle virus

antiserum.

No nonpspecific action was shown by normal rabbit serum.when used in

the complement fixation test with Newcastle disease infected allantoic
fluid, normal allantoic fluid, suspensions of Newcastle infected hamster
brain, and normal hamster brain.

No anticomplementary activity was exhibited by normal or infected
hamster brains.

1)

2)

3)

4)

5)

6)

7)

8)

9)

10)

ll)

12)

13)

-27.
BIBLIOGRAPHY

Doyle, T.M. 1927 A hitherto unrecorded disease of fowls due to a filter-
passing virus. Jour. Comp. Path. 8: Ther. 49: 144-169.

Kraneveld, F.D. 1926 Oven can in Ned.-Indib‘ heerschende Ziete onder het
Plumvee. Nederlandsch-Indische Bladen voor Diergeneeskunde; Deel
2g, Alf. 5: 448-450.

Beach, J .R. 1942 Avian Pneumoencephalitis. Proc. 46th Annual U.S. live-
stock Sanitary Assoc., 203-223.

Stover, D.E. 191.2 A respiratory-nervous disorder of chickens. Amer.
JOln‘. vet. Res.’ 2: 207-213.

Burnet, F.M. 191.2 The affinity of Newcastle disease virus to the in-
fluenza virus group. Austr. Jour. Exptl. Bio. & Med., £9 (2):
81-88.

Cunha, R., Neil, M. , & Beard, D. 1947 Purification and characters of
the Newcastle disease virus (California. strain). Jour. Immun.,
5; (1): 69—87.

Bang, F.B. 1948 Studies on Newcastle disease virus. Jour. Exptl. Med.,
fig (2): 233.2400

Reagan, R. 191.? Response of monkeys to poliomyelitis after injection
with Newcastle disease virus. Proc. 51st Annual Livestock Sani-
tary Assoc., 55-62.

Wanner, H.A. and Lash, B. 1949 Choriomeningitis encephalitis following
inoculation of Newcastle disease virus in Rhesus monkeys. Proc.
Soc. Exptl. Bio. & Med., 19: 263-265.

Yatom, J. 191.6 Conjunctivitis caused by virus of Newcastle disease.
Jour. Am. Med. Assoc., gig (8): 169 (in foreign new: letters).

Ingalls, W.L. & Mahoney, A. 1949 Isolation of the virus of Newcastle
disease from human beings. Amer. Jour. Pub. Health. , 22 (6): 737-740.

Howitt, Beatrice, Bishop, L.K., & Kissling, R.E. 194.8 Presence of neu-
tralizing antibodies of Newcastle disease virus in human sera.
Amer. Jour. Pub. Health., 18, (9): 1263-1272.

Howitt, B. 1937 Complement fixation reaction in experimental equine
encephalomyelitis, lymphocytic choriomeningitis 8: St. Louis
encephalomyelitis. Jour. Immun., 31: 235-250.

-28-

14) Casals, J. 8: Palacios, R. 1941 Complement fixation test in the diagnosis
of virus infections of the central nervous system. Jour. Exptl.
Med., 1‘; 409-4260

15) Havens, W.P., Watson, D.W., Green, R.E., Lavin, G.I., & Smadel, J.E.
1943 Complement fixation with the neurotropic viruses. J our.
Exptl. bbdq fl: 139-153.

16) Casals, J. 1942 Inactivated antigens for complement fixation tests with
central nervous system virus infection. hoc. Soc. Exptl. Bio. &
Med., 42: 501.

1'7) Casals, J. 1945 Heated avirulent antigens for complement fixation tests
with certain encephalitis viruses. Science., 192: 618.

18) DeBoer, C.J., & Cox, H.R. 1946 Specific complement fixing diagnostic
antigens for neurotropic virus diseases. J our. Bacty., 11;: 613.

19) Wolfe, Van der Scheer, Clancy 8: Cox 1946 A method for the preparation
of complement fixing antigens in a study of experimental Tsutsu—
pamshi disease (scrub typhus). Jour. Bacty., 21;: 247-253.

20) Espan-a, C. & Hammon, W. McD..l948 An improved benzene extracted com-
plement fixing antigen applied to the diagnosis of the arthropod-
borne virus encephalitides. J our. Immun., 22: 31-43.

21) Casals, J. 1949 Acetone-ether extracted antigens for complement fixation
with certain neurotropic viruses. Proc. Soc-Exptl. Bio. 8: Med.,
E (2)3 339-343.

22) Reagan, R., Reginold, L., Lillie, M., Hauser, J., & Brueckner, A. 1948
Response of the Syrian hamater to the virus of Newcastle disease.
Proc. Soc. Exptl. Bio. & Med., pg (2): 293-295.

23) Reagan, R. , Lillie, M., Smith, D., & Brueckner, A. 1949 COII'Ipa:I.':lson of
Newcastle disease virus in hamsters exposed by intracerebral in-
jection and intranasal instillation. Proc. Soc. Exptl. Bio. &
Med., 1; (2): 293-294.

24) Lenette, E. 1941 Susceptibility of Syrian hamsters to viruses of St. Louis
and Jap. B. encephalitis. Proc. Soc. Exptl. Bio. 8: Med., 4'1: 178-181.

25) Sanders, F.K. 1948 A poliomyelitis-like agent in hamsters inoculated
with Lansing virus. Fed. Proc., 1 (1): 309 (abstract).

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