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THE EFFECT OF BETA-PROPIQLACTONE
0N SOME OF THE {NFECTIOUS AGENT S
THAT PRODUCE DISEASES OF CHICKENS ‘

THESIS FOR we: DEGREE '0‘? PH. 23.
momsm STATE UNIVERSITY
' 'ACHIT CHOTISEN'
1955

0-169

This is to certify that the
thesis entitled
The Effect of Beta-propiolactone

on Some of the Infectious Agents that
Produce Diseases of Chickens.

presented by
Achit Chotisen

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Microbiology

DJ -W\~'VV\.M&C/

Major professor

 

August 12, 1955
Date

 

 

THE EFFECT OF BETA-PROPIOLACTONE ON SOME OF THE INFECTIOUS
AGENTS THAT PRODUCE DISEASES OF CHICKENS

by
Achit Qhotisen

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health
1955

Achit Chotisen

Beta-propiolactone in 0.025 per cent concentration in
Newcastle disease infected allantoic fluid destroyed infec-
tivity without destroying antigenicity of the virus. A
vaccine against Newcastle disease, using this concentration
of beta-propiolactone, prevented infection in experimental
chickens. or R3 vaccinated birds, none showed symptoms of
Newcastle disease after being challenged with a virulent
strain of Newcastle disease virus 16 days following vaccina-
tion. In a group of 39 non-vaccinated birds, 38 birds (97.h%)
developed symptoms or died from the challenging dose of virus.
One bird in this group remained well. Serological tests on
paired sera showed increases in both hemagglutination inhibi-
tion and neutralizing antibodies in the vaccinated group. In
contrast, the non-vaccinated group did not demonstrate an
increase in antibodies. A concentration of 0.025 per cent
beta-propiolactone also destroyed the infectivity of infec-
tious bronchitis and infectious laryngotracheitis virus con-
tained in allantoic fluid but not completely fialmgnella
pullgzum. The results suggest the possibility of making a
multiple virus vaccine for prevention of these diseases in
chickens. In 0.1 per cent concentration of beta-propiolac-
tone, figlggnella pgllgzumgwas killed but the antigenicity
of Newcastle disease virus was destroyed. All chickens
vaccinated with 0.1 per cent beta-propiolactone in Newcastle
disease virus allantoic fluid were susceptible to infection

111

Achit Chotisen

by challenging with Newcastle disease virus. This type of
inactivated vaccine is preferable to live virus vaccine
because of its high antigenicity and ability to destroy
infectivity of the virus.

iv

To

LUANG‘VIPACHANA SUTHAPOCHANA
(Arch Chotisen)

and ’

NANG VIPACHANA SUTHAPOCHANA
(Huang Chotisen)

my beloved father and mother

ACKNOWLEDGEMENTS

The author wishes to eXpress his sincere thanks to Dr.
‘U. N. Mack, under whose constant supervision and technical
assistance this investigation was undertaken and to whom the
results are herewith dedicated.

Grateful acknowledgement is also given to many fellow

graduate students for their help and assistance.

vi

TABLE OF CONTENTS

I. INTRODUCTION . . . . . . . . . .
II. HISTORY . . . . . . . . . . . .
1. Newcastle Disease . . . . .

B. Immunization . . . . . . .

1. Killed Virus Vaccine .

2. Live (Attenuated) Virus Vaccine

C. Serological Tests . . . . . . . . . . . .

l. Hemagglutination (HA) and Hemagglutina-

tion Inhibition (HI) Tests

2. Serum Neutralization Tests .

III. MATERIALS AND METHODS . . . . .

A. Newcastle Disease Virus . .

B. Preparation of Virus Suspension .

C. Preliminary EXperiment . .

D. Preparation of Newcastle Disease Vaccine

E O vacc1nation O O O O O O O O O O O O O O

F. The Bactericidal Activity of Beta-Propio-

lactone (B.P.L.) on fialmgnellg pullorum
l. The Action of 0.025 per cent B.P.L. on

Salmonella pullorum mixed with New-

castle Disease Virus Suspension . .

2. The Action of Different Concentrations

of B.P.L. on Salmonella pulloggm .

vii

PAGE

O\O\WUJl-'

10
13

1h
23
27
27
27
30
33
33

35

35

37

3. The Effect of 0.1 per cent B.P.L. on
the Antigenicity of Newcastle Disease
Virus . . . . . . . . . . . . . . . .
G. The Effect of B.P.L. on Infectious Bron-
chitis Virus . . . . . . . . . . . . . . .
H. The Effect of B.P.L. on Infectious
Laryngotracheitis Virus . . . . . . . . .
(I. Serological Tests . . . . . . . . . . . . .
1. Hemagglutination (HA) and Hemagglutina-
tion Inhibition (HI) Tests . . . . . .
2. Serum Neutralization Tests . . . . . .
IV. RESULTS . . . . . . . . . . . . . . . . . . . . .
DISCUSSION . . . . . . . . . . . . . . . . . . .
VI. SUMMARY . . . . . . . . . . . . . . . . . . . . .
VII. LITERATURE CITED . . . . . . . . . . . . . . . .

.4

viii

PAGE

.h9
. 73

ThBLE

II.

III.

VI.

VII.

VIII.

LIST OF TABLES

‘Virucidal Effect of a Beta-propiolactone on
Newcastle Disease Virus Infectivity Test in
Eggs.....................

Egg Infectivity of Newcastle Disease Virus
Before and After Incubation for Two Hours at

Hemagglutination Test of Newcastle Disease Virus
Treated with Beta-propiolactone . . . . . . .

Bactericidal Effect of B.P.L. on.§glmgg§;l§,

10 um . . . . . . . . . . . . . . . . . . .

Virucidal Effect of B.P.L. on Infectious
Bronchitis'Virus . . . . . . . . . . . . . . .

Virucidal Effect of B.P.L. on Infectious
Laryngotracheitis Virus . . . . . . . . . . .

Hemagglutination and Hemagglutination-Inhibition

Tests Procedure . . . . . . . . . . . . . . .

Results of Immunity Challenged of Vaccinated

Chickens (0.025% of B.P.L. Vaccine) 0.2 ml.
VirusMuscularly...............

Results of Virus Challenge of NondVaccinated
Control Chickens 0.2 m1. Virus Intra-

musculary O O O O O O C O O O O O O O O O O 0

ix

PAGE

31

31

32

37

MO

#2

H6

57

58

TABLE PAGE
X. Results of Hemagglutination Inhibition Tests
on Sera from‘Vaccinated Chickens Group 1
(Vaccine 0.5 ml.) . . . . . . . . . . . . . . . 59
II. Results of Hemagglutination Inhibition Tests
on Sera from'Vaccinated Chickens Group 2
(Vaccine 1.0 ml.) . . . . . . . . . . . . . . . 6O
XII. Results of Hemagglutination Inhibition Tests
on Sera from‘Vaccinated Chickens Group 3
(Vaccine 1.5 m1.) . . . . . . . . . . . . . . . 61
XIII. Results of Hemagglutination Inhibition Tests
on Sera from'Vaccinated Chickens Group N
(Vaccine 2.0 m1.) . . . . . . . . . . . . . . . 62
XIV. Results of Hemagglutination Inhibition Tests
on Sera from NonJVaccinated Control Chickens . 63
XV. Results of Hemagglutination Inhibition Tests
on Sera from Chickens Vaccinated with 0.5 ml.
NOV-0.025% B.P.L. Vaccine Containing figlmgnellg
pullozgm. . . . . . . . .'. . . . . . . . . . . 6h
XVI. Results of Serum Neutralization Tests in.Eggs
from‘Vaccinated Chickens Group 1 (Vaccine 0.5
ml.) . . . . . . . . . . . . . . . . . . . . . 65
XVII. Results of Serum Neutralization Tests in Eggs
from‘Vaccinated Chickens Group 2 (Vaccine 1

ml.) . . . . . . . . . . . . . . . . . . . . . 66

xi

TABLE PAGE
XVIII. Results of Serum.Neutralization Tests in Eggs
from Vaccinated Chickens Group 3 (Vaccine 1.5
ml). 67
XIX. Results of Serum Neutralization Tests in Eggs
from Vaccinated Chickens Group N (Vaccine 2.0
ml) 68
XX. Results of Hemagglutionation Inhibition Tests
on the Sera from Vaccinated Birds . . . . . . 69
XXI. Immunity Challenged of Vaccinated Chickens (0.5
ml. of 0.1% B.P.L. Vaccine) 0.2 ml. Virus
Intramuscularly............... 70'
XXII. Results of Hemagglutination Inhibition Tests on
,Sera from‘Vaccinated Chickens 0.1% B.P.L.
Vaccine (Amount 0.5 ml.) . . . . . . . . . . . 71
XXIII. Results of Serum.Neutralization Tests in Eggs from
Vaccinated Chickens'Vaccine Contained 0.1%
B.P.L. (Amount 0.5 ml.) . . . . . . . . . . . 72

I. INTRODUCTION

Beta-propiolactone (B.P.L.) was found to be an effective
virucide and of low toxicity by Mangun gt_§l, (1951). Hartman
25,51, (1951) also reported that a number of viruses as well
as bacteria were killed by the action of B.P.L. Beta-propio-
lactone mixed with Newcastle disease virus suspension held at
37°C for 1 to 2 hours effectively destroyed the virus.

Newcastle disease is an infectious, highly contagious
and destructive virus infection of poultry and wild birds.
Certain mammals, including man, have been reported to be sus-
ceptible to this disease. Newcastle disease is caused by a
virus which resembles in some respects the viruses of influ-
enza and mumps.

Newcastle disease in poultry causes serious economic
losses. High mortality rates among young chickens are reported
throughout the world (Beaudette, 19h3, 19H8, 1951). Chicks
that survive an outbreak are sometimes impaired with respect
to growth and efficiency of feed utilization. The decrease
in egg production after an attack of the disease, together
with the serious damage to egg quality is of sufficient magni-
tude to be of economic importance (Berg gth§1., l9h7; Knox,
1950).

The object of this study was to produce a satisfactory
vaccine against Newcastle disease, with the following qualities:

1

(l) Incapable of producing the disease in susceptible animals;
(2) Capable of conferring protection against Newcastle disease
for an adequate period of time; (3) Non toxic; (h) Free of

other infectious agents.

II. HISTORY

A. Nggcastle piggasg

Newcastle disease (NOD) was first known as a highly fatal
infection of poultry prevalent in the Dutch.East Indies. The
disease was first reported by Kraneveld in 1926 (Beaudette,
19%3). Kraneveld demonstrated that the causative agent was
present in the secretion from the respiratory tract. He elim-
inated the possibility that the cause was bacterial in nature
by Berkefeld filtration. The disease was also reported to
cause an outbreak in poultry near Newcastle on the Tyne in
England by‘Doyle (1927). ‘Dcyle demonstrated that the cause
is a filtrable virus and is present in the body fluids, organs
and excretions of affected birds. The disease resembles fowl
plague, but it can be differentiated by serological tests.
Doyle found that there is no cross immunity between NCD and
fowl plague and that each virus may be completely neutralized
by the homologous immune serum. 'Doyle proposed the name New-
castle disease in describing the disease.

During the decade following its recognition, Newcastle
disease was reported from India, the Philippine Islands, Korea,
Japan, Australia, Ceylon, Kenya, Palestine and Syria; in fact,
the disease is widely spread throughout the world (Beaudette,
l9h3, l9h8, 1951). In the initial appearances of the malady,

the disease was seldom recognized as NCD, therefore, it was
3

given many names, such as pseudo-fowl pest, pseudovogelpest,
avian pest, avian distemper, Ranikhet, Madras fowl pest, Chosen
disease and so forth (Brandly gt gl., 19H6 a). The similarity
in symptoms of these diseases reported from the orient and
England brought about an exchange of viruses for comparison.
It was found that regardless of name and minor differences in
symptoms, the viruses from the Dutch East Indies, India, Phil-
ippine Islands and England were immunologically identical.
Later, it was shown by Japanese workers that their viruses
were identical with the Philippine virus (Brandly gt gl., 19h6
a). Thereafter most of the various names under which the dis-
ease had been known were discontinued and NCO has been recog-
nized as the proper name of this disease.

In the United States this disease was described in Cali-
fornia by Beach in l9hl as a nervous disorder occurring after
or towards the end of outbreaks of a disease which.was thought
to be chicken bronchitis. He also suggested that the disease
might be due to a virus or to some dietary disturbances. Later
Stover (l9k2 a, b) described this disease as "a respiratory
nervous disorder" of chickens that could be transmitted to
other chickens by contact. The filtrability of the infective
material was also demonstrated, and, by cross immunity experi-
ment, it was shown that the virus of the respiratory disorder
‘was distinct from the virus of infectious bronchitis and from
infectious laryngotracheitis. Beach (19h2) proposed the name

"Avian Pneumoencephalitis" for this disease. The unusually

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(a
. i

mild character and low mortality rate which characterized the
malady upon its initial appearance in America allayed suspicion
that it might be NCD. Later Beach (l9hh b) observed the highly
virulent nature of the cultured pneumoencephalitis virus and
suspected that it might be related to the virus of NCD. Small
amounts of anti Newcastle and anti pneumoencephalitis sera were
used for in 21512 neutralization of pneumoencephalitis virus.
The results indicated that pneumoencephalitis virus was immuno-
logically identical with Newcastle disease virus (NDV).

Burnet and Perry (193M) found that NDV is easily propagated
in embryonating chicken eggs and reaches high titer (108 to 109
infectious units per ml.) in the chorioallantoic fluid. By
ultrafiltration experiments they estimated the size of NDV

particles to be 80-120 mu.
The virus can pass through Berkefeld.V, N,‘w, Chamberland

candles L3 and L5 and asbestos discs (Doyle, 1927; Stover, 19h2
a, b; Beaudette, 19h3). Bang (l9h6, 19%?) observed the shape
of NDV by electron microscope and found it to be spherical in
shape in allantoic fluid, but this form could be converted into
filamentous forms if the virus was concentrated by ultracentrif-
ugation and suspended in water. Cunha £5 £1, (19%?) concentrated
NDV from chorioallantoic fluid and described it as sperm shaped.
The head measured 70 x 180 mu. and tail 500 mu. long.

NDV produces disease in chickens, turkeys, pheasants,
Guinea fowl, sparrows, crows, francolines and parrots (Beaudette,

1903). Experimental infection has been achieved in chick

embryos, ducks, geese, pigeons and several varieties of wild
birds. Mice, hamsters, cotton rats and monkeys may be in-
fected experimentally with NDV, resulting in a meningo-
encephalitis.

Bowen

Earlier efforts to develop a satisfactory vaccine against
NCD were by physical means or chemical agents. In the process
of attenuation or inactivation of the virus, the results were
often irregular and unsatisfactory (Beaudette, l9h3). The
procedure for attenuation or inactivation of the virus was
responsible for inadequate or excessive denaturation of the
virus protein. The irregularities in initial virus content

of infected tissues added to the variable and unsatisfactory

results.

1. 1 ed ru ac in

Topacio and Coronel (1939) failed to immunize birds with
a vaccine made by using infected tissue or "crop virus" treated
with chloroform, formalin, lactic acid or merthiolate.

Haddow and Idnani (19k1) developed a formalin treated
vaccine from infected tissues by adsorption of the virus on
aluminium hydroxide gel (adjuvant). The principle involved
here was concentration of the virus by the adsorption process
trollowed by inactivation of the virus with dilute formalin.

.It was presumed that after injection of the vaccine, the ad-

sorbed virus would be gradually released in minimal quantities

into the birds' systems and would thus produce immunity with
no apparent symptoms. The vaccinated birds resisted a chal-
lenge inoculation of 10 - 100 or more minimum lethal doses of
virus between the eighth and the twenty-first day after vac-
cination.~

Iyer and Dobson (l9hl) prepared a vaccine from infected
chicken embryos by suspending_the embryos in a buffer solution
and treated them with gentian violet. The vaccine was placed
at 37°C for 72 hours to complete the inactivation. The results
of immunization tests in chickens were irregular and unsatis-
factory. Later Iyer (19h3) reported that satisfactory results
were obtained from this same type of vaccine. At this time he
also reported that his tests proved that formalin had no value
in the preparation of a suitable vaccine from egg produced
virus.

Beach (l9h2) reported encouraging results obtained in
preliminary experiments in immunization of young chickens
with formalized saline suspensions of infected chicken embryos.
His results showed that a high degree of protection against
avian pneumoencephalitis (NCD) could be obtained by this vac--
cine. The vaccine contained 0.1 or 0.2 per cent formalin.

Two doses of vaccine were more effective than a single dose
when tested in chickens of more than 7 days of-age. Chickens
vaccinated at 2 or 3 days of age were inadequately protected

'by a single or double injection of vaccine.

Later Beach (19% a) reported that two injections of
formolized chicken embryo vaccine yielded a valuable, although
incomplete, degree of protection against the natural infection
in chickens. The chickens were able to withstand a severe
natural outbreak of NCD with relatively low mortality and only
a slight decrease in egg production. An effective vaccine
could be made with an embryo content varying from 12.5 to 50
per cent. It had to be used from 1+0 to 509 days after prepa-
ration.

Brandly g1; 31,. (191+6 c) reported that formalin inactivated
vaccine produced higher protection than ultraviolet irradiation
(1600-1800 A0) inactivated vaccine. Vaccines containing 0.25
Per cent formalin were found to possess excellent keeping quality.
Formalin inactivated vaccines retained their immunogenicity well
for a period up to l+71 days under a pH range from 5 to 9. The
best formalin fixed vaccines were obtained by using high titer
Virua. The minimal virus titer for maximum vaccine potency
had to be 107 embryo lethal doses per 0.05 ml. of inoculum from
egg material. For vaccine production, eggs produced by NCD
immunie hens, and containing the yolk sac with content, were
inferior to vaccines made from the same eggs but without the
yolk sac. Addition of the yolk material from the eggs of
sus{Baptible hens did not alter the potency of the vaccine.
Formarlin inactivated vaccines produced a temporary immunity.
Some immunity was present up to 1t months in some cases but in

Others only slight or negligible refractory was found 3 weeks

after vaccination. The addition of certain adjuvants, such as
lanolin, mineral oils and alumina gel into the formalized
vaccine increased the duration of resistance without producing
unduly severe tissue reaction. When killed mcobacterig
butnicum was employed, severe inflammatory reaction and tuber-
culin sensitivity developed. .

Brandly 93; 21. (191+6 d) reported the presence of specific
antiviral activity in the yolk material of eggs laid by the
Newcastle disease vaccinated hens. This was true also for eggs
from hens recovered from NCD. Embryonic tissues and allantoam-
nionic membranes also possessed the property of virus neutral-
ization. Vaccines made from eggs that contained this antiviral
activity appeared to be inferior to vaccine made from the eggs
from susceptible hens. Congenital passive immunity was found
1' or about two weeks after hatching. This immunity interfered
with active immune response to vaccination.

Acevedo and Mendoza (191+?) reported satisfactory results
from chicken embryo tissue vaccine inactivated by 0.05 per cent
formalin and crystal violet. Another type of vaccine contain-
ing 0.5 per cent potassium alum, 0.7 per cent sodium borate
and 0.1 per cent formalin was also found to enhance the anti-
b°dy production. Later Coronel (l9l+7) announced a successful
”merino using chicken embryo tissue suspended in alum gel and
maciiivated with 0.1 per cent formalin.

Adler gt 3;. (1951) stated that formalinized tissue vac-

°1ne did not prevent the spread of the disease as was shown

10

by isolation of NDV from vaccinated birds exposed to the
disease. Vaccination, however, prevented serious loss.
Doll gt_§;, (1951) confirmed that commercially killed
NCD vaccine did not protect against NCD infection. They
described paralytic and fatal infection after challenging

exposure to virulent NDV.

2. Live Attenuate Virus accin

Immunization of poultry against NCD with a killed virus
had met with little success. However, chickens recovered from
naturally occurring infection, or challenged with a potent
virus developed an immunity which appeared to be permanent.
Attenuated strains of NDV have been developed by numerous
workers for the production of durable immunity in chickens.

‘ Iyer and Dobson (19RO) reported that serial passage of
the Hertfordshire strain of Newcastle disease virus on the
chorioallantoic membrane of embryonating eggs attenuated the
virus. This attenuated virus was satisfactory for a vaccine.
From 1% to 33 serial passages were found to be necessary for
attenuation of the virus. This cultivated virus did not
regain its original virulence by passages through chickens.
Inoculation of chickens with this attenuated virus protected
them against a challenging dose of 108 lethal doses. Later
Van Roekel gt al, (l9h8) found that an attenuated strain of
NDV could immunize chickens when applied by the cutaneous

"stick" method in the web of the wing. A general systemic

11

:reaction was noticed in some birds in the form of a slight
decrease in activity and appetite. In laying birds, egg pro-
duction and egg quality were adversely affected, as is the
case in a natural outbreak.

Komarov and Goldsmit (l9h6) reported that after ten intra-
cerebral passages of virulent fowl virus through ducklings, an
attenuation of NDV developed. Fowls inoculated with this
attenuated NDV did not develop symptoms of NCD and were immune
to challenge. 'Later duck embryo adapted strains of NDV were
reported by Markham gt 31. (19”?) and Clancy gt 2;. (l9h9).
The vaccine was applied by the wing web method. Some of the
b weeks old birds showed slight depression but continued to
feed and grow normally.

Hitchner and Johnson (l9h8) recommended the use of B 1
strain of NDV, which was of low virulence and could be used
to vaccinate chickens of all ages including day-old chicks.
Vaccination was accomplished by intranasal instillation of
0.05 ml. of the vaccine. All vaccinated birds were fully
protected from the disease when challenged 2 days after vac-
cination. Doll 2; 2;, (1950) reported that, as indicated by
hemagglutination inhibition test, the immunizing action of
the B l vaccine was more efficient by intranasal instillation
than by intramuscular injection. After vaccination, reSpira-
tory-symptoms could be seen in birds of all ages. Reduction
in egg production, from 20 to 50 per cent for a period of 2
to h weeks, and 10 percent mortality in day-old chicks made
this type of vaccine unsuitable.

l2

Bankowski and Boynton (19h8) found that NDV could be
propagated ig_gitgg in modified Simm and Sander's medium
containing bovine serum ultra filtrate. The virus propagated
by this method seemed to be reduced in virulence when inocu-
lated into chickens. Later Bankowski (1950) confirmed that
by 50 serial passages in Simm and Sander's medium, the viru-
lence was decreased and could be used as an immunizing agent
for chickens by atomizing the vaccine. This method of virus
production maintained antigenicity as indicated by the hemag-
glutination inhibition (HI) tests on sera of chickens receiving
this vaccine and a solid immunity in the vaccinated chickens
to a challenge dose of 2 x 105 minimum infective doses of
virulent virus. Although no respiratory symptoms were observed,
some mortality (6.6%) and nervous symptoms were seen in
chickens.

The successful intracerebral transmission of NDV through
Syrian hamsters was reported by Reagan gt,gl, (l9h7). This
hamster brain virus could be neutralized by immune chicken
serum as shown by tests in embryonating eggs. Later the same
authors (19h8) reported that continued intracerebral hamster
passage of the virus had resulted in decreasing pathogenicity
for chickens. The twenty-ninth intracerebral hamster passage
virus produced typical NCD in approximately 10 per cent of the
vaccinated birds. Less than 2 per cent of the birds showed
symptoms when inoculated with the forty-ninth virus passage.

13

Beaudette (l9h8) described the disadvantages in using
the attenuated live virus vaccine. Some systemic reaction
and some mortality were observed following their use. The
possibility of transferring other infectious agents from the
incubated eggs, which.were used in.vaccine production, should
also be taken into consideration. Mbreover the use of atten-
'uated live virus vaccine produced a mild infection in the
flock which might be considered as a source of infection for
non-vaccinated birds.

Zagar and Pomeroy (1950) found that following vaccination
with commercial NCD live vaccine, NDV could be isolated from
yolk material up to 10 days after vaccination. In the same
vaccinated group they found a drop in production, and off-

color, malformed and soft-shelled eggs.

QW

The evaluation of the efficiency of the NCD vaccines was
done by immunization test. The birds were vaccinated.with
varying amounts of NCD vaccine and after 16 days were challenged
with virulent NDV. At the time of the immunization test, sero-
logical tests were also performed to measure the antibody con-
tent of the sera. .

There are two methods of measuring the content of anti-
bodies against NCD in sera: the hemagglutination inhibition
test and the serum neutralization test. Comparison was made

of the antibody content in sera of birds taken before

lb

vaccination, in sera of birds taken after vaccination and also
of the antibody content in the sera of the non-vaccinated
control birds. The efficiency of the vaccines was then eval-
uated by: (1) The protective effect of the vaccine against
the challenging dose of virulent NDV, and (2) The increase in
the antibody content of the vaccinated group.

1. :em:;; uti atio ‘; a d zzma;;lu inat cc nhibitio i

Hirst (19h1) found that when allantoic fluid infected
with influenza virus was mixed with washed, normal chicken red
blood cell suspensions, agglutination of the cells occurred
within 5 to 20 minutes. The red blood cells sedimented rapidly
and formed a characteristic ragged, granular pattern on the
bottom of the tube. In contrast, when cells were mixed with
uninfected allantoic fluid no hemagglutination occurred and the
cells settled out slowly forming a sharp, round disc at the
bottom of the tube. He also reported that hemagglutination was
inhibited with influenza antiserum, and that the inhibition‘was
specific for the homologous strain of virus.

The same phenomenon was discovered also by McClelland and
Hare (19h1), who showed that human and guinea pig red blood
cells were also agglutinated by influenza virus.

Burnet (19h2) reported the ability of NDV to agglutinate
chicken red blood cells in a manner similar to that shown by
influenza virus. A modification of the Hirst test, as suggested
by LMSh (19h3), was used in reading the results. He also

15

reported the agglutinability of NDV on red blood cells of man,
the guinea pig, mouse, sparrow and frog. Hemagglutination
inhibition by specific NCD immune serum was also demonstrated.

Lush (19103) found that the reading, as described by Hirst
(191+2), was not satisfactory when used with NDV, because the
Virus affected only a small proportion of the red blood cells
and did not reduce the density of the suspension sufficiently
to permit the use of Hirst's technique. Therefore, the pattern
01‘ the agglutinated cells was used instead. One twenty-five
hundredth of a milliliter of virus suspension with an equal
Volume of 2 per cent red blood cells suspension was used in
the test. The results were read after incubation at room
temperature for 2 hours and were recorded as 1t K, 3 K, 2 K,

1 f, t, and 0, representing maximum to no agglutination. She
also reported that by using the hemagglutination inhibition
test, it was shown that no serological relationship between
fowl plague and NDV is existed.

Salk (l9hh) described a simplified procedure for titrating
the hemagglutinating capacity of influenza virus and the cor-
responding antibody. He used a 0.25 per cent red blood cell
suspension made from-a 10 per cent stock preparation stored
at We. Five-tenths milliliter of virus dilution was added
to 0.5 m1. of 0.25 per cent red blood cell suSpension. The
I‘eadings were made after I} to 2 hours incubation at room
temperature. In complete agglutination (K) the clumps of
cells settled to the bottom of the tube with a dispersed

l6

pattern rather than a compact pattern. A negative pattern
(-) consisted of a central, sharply demarcated disc. Irreg-
ular clumps of cells associated with a halo of finely aggre-
gated or unagglutinated cells represented an intermediate
reaction ()5). The end-point was the highest dilution of the
virus producing maximal hemagglutination and the titer was
eXpressed as the reciprocal of the final dilution of virus
after the addition of red cells suspension. In the HI test
the serum dilutions were prepared directly in the red blood
cell suspension. Two hemagglutinin units of virus in 0.5 m1.
of virus suspension were mixed with 0.5 ml. of red cell-serum
dilution. The readings were made as in the HA tests. The HI
titer of the serum was the highest dilution of serum producing
complete inhibition of hemagglutination. Salk also noted that
the titers of the agglutinin of several samples of infected
allantoic fluid were essentially the same when tested with
different batches of red cells, provided that the cells were
carefully washed and suspended in the same way.

Burnet g_t_ a_l_. (19%") showed that there was a difference
between destruction of infectivity and hemagglutination activ-
ity of NDV. After heating at 60°C for 15 minutes the infec-
ti.Vity titer of the virus was about 1 to 108 of the original
tlter, whereas the hemagglutination titer was still 50 per
cent of the original titer. At 65°C for 15 minutes the hemag-
glutination activity was destroyed completely. The addition

01‘ formalin in concentration of 0.1 to 0.25 per cent of the

17

virus destroyed its infectivity without changing the HA titer.
It was shown by filtration that smaller particles or those
less readily adsorbed by the filter than the virus, were re-
sponsible for a small but definite portion of agglutinating
activity. Sensitization of chicken red blood cells by either
NDV or influenza virus made the cells unagglutinable by NDV.
The viruses in the influenza group could be arranged in a
linear series, so that red blood cells sensitized by a given
Virus were agglutinated by the succeeding virus but not by
the preceding virus. The series, in abbreviated form, was
mumps, Newcastle disease, most influenza A strains, influenza
B and swine influenza. Further work by Burnet §_t_ g1. (19%)
demonstrated that this was also true for human type 0 red
blood cells.

Burnet gt 2;. (191+6) and Burnet and Anderson (191+6) re-
ported that red blood cells treated with amniotic or allantoic
fluid preparations of NDV developed a new antigenic property.
This new property produced agglutination in high titer, either
by experimental NCD immune serum or by most sera from recent
Cases of infectious mononucleosis in man. They explained that
an agent other than the virus (cell sensitizing agent), pro-
duced during the growth of NDV in chick embryos, was reaponsible
for the changed character of the cells by adsorption to their
S"Arface.

Brandly _e_1_:_ g1. (191+6, b) applied the Hirst phenomenon, as
InC>dified by Lush (l9’+3) to identify NDV in infected amnioallantoic

18

fluid and found indifferent results. Satisfactory results
were obtained after the red cell suspension was reduced from

2 to l per cent or less and incubated at room temperature
(25°C) rather than at ice box temperature (6°C). The readings
were taken at 15, 30 and NS minutes instead of at 2 hours.
They further reported that the difference between NDV and

fowl plague virus could be determined by the HA test on various
specific erythrocytes. NDV agglutinated swine red cells only
slightly but plague virus clumped them completely. Rabbit red
cells were agglutinated by NDV from one-fourth to one-half the
degree obtained with chicken red cells but plague virus agglu-
tinated these cells to a titer comparable with that of other
Species tested. It was found also that the HA titer of the
NDV was retained well after treatment with heat, ultra violet
ray and formalin sufficient to destroy infectivity.

Cunha gt,§l, (19%?) reported that the intensity of the
reaction was too low when the Lush's modification of the test
was used. Satisfactory results could be obtained when the
tests were carried at 23° to 27°C. The dilutions of virus
were made in buffered saline (pH 7.h) and a final red blood
cell concentration of 1 per cent was used. The end point
dilution was the tube that matched the standard tube of 0.63
per cent cell concentration. They also reported that the
hemagglutinative capacity of NDV was closely associated either
with the virus itself, as indicated by infectivity measurements,

or with material consisting of particles of sedimentative

19

character closely resembling those of the virus. A factor in
normal allantoic fluid was found to have an inhibitory action
on the HA test. Variation in the ratio of the hemagglutinative
activity and infectivity was greater than in influenza virus.
This variation was due to the relatively low capacity of NDV
to agglutinate red blood cells and greater difficulty in con-
trolling the conditions of the reaction.

Florman (19“?) showed that HA test with.NDV was markedly
affected by temperature. At room temperature the reactions
were difficult to read, especially with low dilutions of virus.
This effect resembled a prozone reaction and disappeared when
the tests were carried out at #00. Florman thought that the
low HA titer was due to rapid elution of the virus at room
temperature so that agglutination was masked in lower dilutions
of virus. At M°C NDV appeared to be adsorbed more completely
by and to elute less rapidly from chicken red cells than at
room temperature. Although the end-point of hemagglutination
at #00 was lower than at 37°C. Anderson (191+7) explained that
higher titer of virus at 37°C was considered to be due to the
disruption of virus aggregates in contact with red blood cells
with ultimate liberation of single virus particles.

Anderson (19%?) reported the hemagglutinability of the
fresh red blood cells by the NDV stabilized red blood cells
as described by Burnet and Anderson (19h6). This agglutina-
tion was caused by an agglutinin on stable cells. He also

stated that all manifestations of NDV action on red blood

20

cells were essentially due to the virus particles pg; _§,
NDV in infected embryonic fluid was found to be nonhomogenous
and larger units of NDV can be segregated. After hemagglutina-
tion, elution occurred by the partial disruption of the aggre-
gates and one virus particle remained attached to the red blood
cell. This remaining particle could leave the red cell only by
transfer to another cell, not by elution into the supernatant
fluid. Its presence on the red cells rendered the cells sus-
ceptible to agglutination by NCD immune serum and functioned
as the retained hemagglutinin. The agglutinin on stable cells
was neutralized by NCD immune serum. The serum titers corre-
sponded closely with the direct HI titers. In NCD immune serum,
the same antibody molecule seemed to function either as an
anti-hemagglutinin or as agglutinin for sensitized cells.
Infectious mononucleosis immune serum, which caused hemagglu-
tination of NDV sensitized human cells, had no action as an
anti-hemagglutinin.

The influence of the route of inoculation of NDV on
development of virus hemagglutinin and of virus LD 50 titer
for embryo had been reported by Hansen.g£fl_l. (19“7).
Hemagglutinin developed most rapidly in embryonating eggs
inoculated by intravenous route, yolk sac and allantoic route
respectively. Materials from embryos inoculated upon the
chorioallantoic membrane failed to show HA titer until 2%
hours after inoculation. HA titer showed a progressive in-

crease from the first appearance of the hemagglutinins until

21

the death of the embryo. Normal hemagglutinin was found in
both normal and infected yolk sac membranes, and thus masked
the virus hemagglutinative activity. This normal hemagglutinin
was inhibited by the addition of either normal serum or NCD
immune serum. They also reported that virus suspensions having
an embryo ED 50 titer of 10‘5 or less failed to produce demon-
strable hemagglutination. Suspensions with an embryo titer of
10"6 had corresponding hemagglutinin titers ranging from un-
diluted or 1:5 to 1:160. with an embryo titer of 10'8 the
agglutination.range was from 1:160 to 131,280. The embryo

ED 50 titration was reported more sensitive in detecting small
quantities of virus than the HA test.

Brandly £3,5l, (19%7) reported that the storage of allan-
toamniotic fluid infected with.NDV in the unfrozen state for
an interval of several months had not been found to result in
appreciable loss of HA titer. They confirmed the variations
in sensitivity among the cells of different birds. The varia-
tion in hydrogen ion concentrations within the range of pH
6 to pH 8 did not cause an alteration in agglutination, vel-
ocity, pattern or titers. Comparisons were made between the
"alpha" procedure wherein successive dilutions of virus are
admixed with a fixed amount of serum and the "beta" procedure
wherein serum is titrated by dilutions against a selected
small quantity of virus.- The "alpha" procedure had been
found somewhat more economical than the "beta" procedure

from the standpoint of equipment, materials and manipulations.

22

The latter, however, was more satisfactory for quantitative
titrations. The agreement of 93 Per cent between the results
of the "alpha" and "beta" procedure, indicated that relatively
high accuracy could be obtained by each procedure. A HI titer
of 80 for "alpha" procedure was considered to be positive.
All sera which by the "beta" procedure failed to inhibit 10
HA units when diluted 1 in 5 were considered to be negative.
They further stated that during the ascending period, generally,
the first 3 weeks following a simulated natural infection or
following vaccination, a good agreement of H1 and serum neutral—
izing titers might be obtained. During the descending phase,
these titers might show either correlation or divergence. The
EH antibodies were observed to disappear first. Revaccination
of birds increased both serum neutralizing and HI antibody.
Osteen and Anderson (19MB) reported that a diagnostic HI
titer was obtained from the test birds at an earlier time than
was obtained by the serum neutralizing method. This was true
when.using a diagnostic titer of 80 or more for the HI test
and 1,000 or more neutralizing doses for the serum neutralizing
test. Dilution of positive serum with negative serum in a
ratio of 1:9 failed to lower the serum neutralizing titer, but
with 1:19 lowered the tenfold titer to the next measurable
level. He further mentioned that serious error because of
dilution with serum from non infected bird was improbable due
to the high morbidity in NCD. Therefore, a composite sample
of 10 birds in a flock of 1,000 in number could be used for

23

serum neutralization test. The HI test was equally accurate
and reliable as the serum neutralization test if the test was
conducted carefully.

Beach (19H8) described the application of HI test in the
diagnosis of avian pneumoencephalitis (NCD). “Beta" procedure
was used and the tests were read from the pattern of agglutina-
tion as described by Lush (l9h3) by placing the rack above the
mirror. He found that HI titers were extremely variable but
ranged higher with sera of birds which had been hyperimmunized
or had survived seven field infection. The titer, however,
was not always correlated with the severity of exposure to the
virus or with the degree of the disease. Sera from chickens
which had been vaccinated against pneumoencephalitis were nega-
tive to the test, suggesting that active virus might be required
for the production of HI antibodies. The specificity of the
HI tests had been confirmed. .1 close relation was found be-
tween the ability of serum to inhibit the agglutination of red
blood cells and to neutralize the virus as tested by chicken
inoculation. Sera having the same virus neutralizing titer,

however, had a wide range of HI titer.

2. fierum fleutralization Tests
Doyle (1927) first demonstrated that serum from NCD im-

mune chickens would neutralize or inactivate the NDV as shown

by chicken inoculation.
Burnet and Ferry (193%) reported that NDV multiplied

raPidly in embryonated chicken eggs. Using the chorioallantoic

2L:

membrane method the virus caused death invariably within h8
.hours. The developing embryos could, therefore, be used as
an indicator of the presence or absence of a lethal dose of
virus. Later Burnet (19%2) reported that NDV could be prOpa-
gated in eggs by various methods. Inoculation of the virus
into the allantoic cavity of the embryonating eggs, the virus
multiplied freely and caused specific death of the embryos.
.Although the characteristics of NDV resembled those of the
influenza virus, i,g, hemagglutination and multiplication in
eggs, there was no serological relation between these two
viruses.

Keogh (1937) found that NDV could be neutralized by its
corresponding immune serum. This was shown by mixing undiluted
serum with the virus in zitgg and inoculating it upon the
Chorioallantoic membrane of embryonating eggs. The serum
‘protected the embryos against 1,000 embryo lethal doses of
Virus. ‘When the inoculation of virus was done prior to injec-
tion of the serum, the eggs invariably died,even.when the serum
‘Was much more than sufficient to neutralize the virus, though
‘the eggs survived longer than the controls with minimal infec-
‘tive dose of virus without serum. Keogh also observed that
‘there was no significant difference by either egg or chicken
inoculation.

Beach (l9h2) demonstrated the neutralizing effect of NCD
immune serum. An equal part of immune serum was mixed with

100 minimum infective doses of virus. The mixture was inoculated

 

25

into chickens after # hours at refrigerator temperature. The
chickens inoculated with serum-virus mixture were not infected,
while the control birds succumbed. The less severe infection
produced in chickens by inoculating with a mixture of serum
diluted 1 in 10 and virus suspension indicated partial neutral-
ization of virus had occurred. Undiluted normal serum or more
diluted immune serum could not neutralize the infectivity of
the virus.

Minard and Jungherr (l9hk) developed a method for the
quantitative neutralization test of avian pneumoencephalitis
virus in 8 to 10 days embryonating eggs. The method consisted
of mixing equal part of dilutions of virus suspensions in broth
and undiluted serum. The mixture was incubated at 37°C for 1
hour and 0.1 ml. of the mixture was inoculated into the allan-
toic cavity of embryonating eggs. The eggs were reincubated
for h days. The majority of the infected embryos were found
dead within #8 to 72 hours after inoculation. Embryos sur-
viving this period were usually found to live and hatch normal-
.ly. The neutralizing doses were expressed as the reciprocal
«of the difference between the titers of the control and the
serum-virus mixtures. The dilution at which one-half of the
:1noculated embryos survived was taken as the critical titer.
fThey also stated that parallel titrations of 2 sera by intra-
Imuscular injection in chicks and allantoic cavity injection

1n.embryos gave similar results.

26

In 19M5 the Committee on Transmissible Diseases of Poul-
try of the U. S. Livestock Sanitary Association considered
the serum neutralization test to be diagnostic if the tested
serum neutralized at least 1,000 embryo lethal doses of NDV.

Brandly g§__;. (19H7, b) reported that neutralization
tests in embryonating eggs were satisfactory and reproducible
within tenfold titer limits. The standard procedure was adding
undiluted serum to an equal quantity of various dilutions of
virus to the estimated and point infective titer. Control
titrations of the virus were always made in parallel. The
mixtures were incubated for 30 to 90 minutes at room tempera-
ture prior to injection. There was no significant difference
in titer within 2% hours and between the temperature range
from 23° to 38°C. The results were evaluated by 50 per cent
mortality during a subsequent % days' incubation period of
‘the eggs. It was found that virus titers in chickens and in
eggs via several routes are virtually identical. The highest
neutralization.values were obtained in chickens, somewhat
lower titers were obtained with the allantoic chamber. The
:neutralization titers of the serum did not change when the
serum was treated with 0.02 to 0.01 per cent of Merthiolate

<3r l per cent phenol.

III. MATERIALS AND METHODS

A. flewcagtle Qisease Virus

NDV strain 5h-55-18 was isolated in the Department of
Microbiology and Public Health, Michigan State University on
June 22, 195% from a naturally infected chicken. The brain
from an infected bird was ground with sand and suspended in
2 ml. broth, with 10,000 units of penicillin and 10,000 mu.
of streptomycin per ml. This suspension was inoculated into
the allantoic sac of 3 ten-day embryonating eggs and reincu-
bated at 37°C. The virus killed 2 embryos within 72 hours
after inoculation and the other one at 96 hours. The allan-

toic fluid was harvested from the embryos dead at 72 hours.

B. Ereparation gf lirug §uspensign

White Leghorn eggs obtained from a flock free of NCD were
used in all studies. The embryos were from 8 to 10 days old,
but in each test all embryos employed were of the same age.
{The eggs were incubated in an electric, forced draft incubator
iat.37°C. Infected allantoic fluid was passed through 2 sets
<>f 20 embryonating eggs serially by the allantoic route and
Ireincubated at 37°C. The virus killed all the embryos within
‘+8 hours after inoculation. The pooled allantoic fluid was
kept frozen at -2S°C for use for further passages.

27

28

One hundred lO-day embryonating eggs were used for the
propagation of the stock virus. The eggs were transilluminated
for selection of an area on the chorioallantoic membrane that
was free from large blood vessels. A small hole was drilled
through the shell without piercing the shell membrane with a
small drill attached to the chuck of an electric motor. An-
other hole was drilled through the shell over the top of the
air cell. Before inoculation, tincture of metaphen was applied
over the holes and allowed to dry. The shell membrane over
the air cell was punctured with a sterile dissecting needle
to allow equalization of pressure within the egg when the in-
oculum was injected into the allantoic cavity and to prevent
leakage of the inoculum from the site of injection.

The eggs were inoculated via the allantoic cavity with a
tuberculin syringe fitted with a 27-gauge, one-half inch needle
lflnrough the hole on the side of the egg. One-tenth ml. undi-
luted infected allantoic fluid, from the third egg passage,
was used for each egg. The holes in the shells were sealed
'with melted paraffin and the eggs returned to the incubator.
A11.eggs were candled every 2% hours to determine death of
embryos. Death of embryos in the first 21+ hours were consid-
ered to be due to injury. The embryos dead after 21: hours
were removed from the incubator and chilled in the refrigerator
at MOC for h to 16 hours to diminish possible hemorrhage from

ruptured vessels when the allantoic fluid was collected.

29

To harvest the fluid the shell over the air cell was
swabbed with tincture of metaphen, allowed to dry and the
shell was cracked and removed with sterile forceps. Sterile
scissors and forceps were used to cut the shell membrane and
allantoic membrane underneath the air cell. Allantoic fluid
was then removed. The allantoic fluid from h to 5 eggs was
pooled in the same test tube and tested for bacterial sterility
in thioglycollate medium. The allantoic fluids were then
frozen. Tubes showing bacterial contamination were discarded.
The contents of the bacteria-free tubes were pooled in a sterile
Erlenmeyer flask and then distributed in 10 ml. amounts into
glass vials and sealed. The vials were then stored at -25°C
until needed. This NDV suspension.was labeled Sh-SS-lB P h.

Titration for virus infectivity was done by inoculation
of serial tenfold dilutions of virus suspension into the allan-
toic sac of 8-to 10-day embryonating eggs as previously de-
scribed, 5 eggs per dilution were used with 0.1 m1. inoculum
Der egg. The eggs were incubated for 7 days after inoculation.
The mortality rates were recorded and the LD 50 was determined
by the method of Reed and Muench (1938). The titer of NDV
suspension Sh-55-18 P h was found to be 10“9°5 LD 50, which
means that the virus suspension contained 109-5 embryo lethal

doses (LD 50) in 0.1 ml. The HA titer of the virus was 1:320.

30
C. i in erime t

B.P.L. used throughout these experiments was supplied by
Dr. Thomas L. Grestiam of the B. F. Goodrich Company, Breck-
ville, Ohio. To facilitate the measurement of B.P.L., espe-
cially when small amounts of infected allantoic fluid were
used, B.P.L. was diluted 1 in 10 with sterile distilled water
before use in every experiment. The percentage of B.P.L in
the experiment was the percentage of concentrated B.P.L. in
the final virus mixture.

Varying amounts of B.P.L. (0.025, 0.05, 0.1, 0.2 and 0.3%)
by volume were sprayed on the virus suspension in a Petri dish.
This was done by drawing a drop of the B.P.L. solution into a
one-fourth ml. syringe with a 27-gauge needle. The content of
the syringe was expelled and then the plunger of the syringe
*was rapidly forced back and forth to produce a fine spray of
B.P.L. The dish was rotated continually. The mixture was
transferred to a test tube and incubated at 37°C for 2 hours
together with a virus suSpension not containing B.P.L. as
control. After the end of the two-hour period, the mixtures
were then inoculated into the allantoic sac of lO-day embryo-
nating eggs, 0.1 ml. per inoculum and 5 eggs per B.P.L. dilu-
tion. The eggs were incubated for 7 days after inoculation
before being discarded. The infectivity test of the control
virus was done to determine the titer of the virus after in—

cubation at 37°C for 2 hours.

 

31

The B.P.L.-virus mixtures were then tested for hemagglu-
tinability to determine if B.P.L. changed that characteristic.

TABLE I

VIRICIDAL EFFECT OF BETA-PROPIOLACTONE 0N NEWCASTLE DISEASE
VIRUS INFECTIVITY TEST IN EGGS

 

% B.P.L. Mertality Rate #
343? 3%
0:1 0
0.2 0/h
0.3 0/5
0.0 Control 5/5

#’ ume at r o. d ad
Denominator No. nject

TABLE II

EGG INFECTIVITY or newcasrzs DISEASE VIRUS BEFORE AND AFTER
INCUBATION FOR TWO HOURS 1r 37°C

Virus
Virus‘Dilutions Titer

und. 10"6 10'7 10"8 10'9 10"10 LD 50

 

Before

Incubation 5/5 5/5 5/5 5/5 3/5 2/5 9.5

After
Incubation 5/5 5/5 5/5 5/5 2/% 2/5 9.h3

flumerato; NoI dead

Denominator No. injected

32

nonponapoammoaon oz .
:oapmnapsawmmsmm \

 

owdua
ONmua
ommaa
ommafl
Ommud
ONmaH

m.o
«.0
H.o
mo.o
mmo.o

Og0m0m oz
Honpnoo

‘L‘k‘k‘k‘k

\‘k‘k‘k‘k‘k
\‘k‘k‘k‘k‘k
\‘k‘k‘k‘k‘k
\‘k‘k‘k‘k‘k
‘k‘k‘k‘k‘k‘k
\‘k‘k‘k‘k‘k
‘L‘L‘k‘k‘k‘k

 

noose 4m o:o\H omm\a ooH\H om\a o:\a cm\a OH\H mxa .onn .a.a.m a x anaa>

 

ozoaoaquaoma:<amm maHz
cayenne npmH> mmamnHo aaamaosmz no some zonaazeaoaooazmm

HHH mqmda

33
D. e ation of ewcastle i as e V n

The frozen NCD infected allantoic fluids were thawed
and centrifuged at 2,000 r.p.m. in horizontal centrifuge
just prior to use to remove any gross particles. From
several preliminary experiments it was found by inoculation
of embryonating eggs that 0.025 per cent by volume of B.P.L.
destroyed infectivity of NDV without changing the hemagglu-
tination titer. Two hundred ml. of allantoic fluid were
transferred into a sterile 500 m1. Erlenmeyer flask with
rubber stopper. Five-tenths m1. of an aqueous solution
(1:10) of B.P.L. was added by sterile serological pipette
to the virus suspension drOp by drop with constant shaking.
The final concentration of B.P.L. in the virus suspension
was 0.025 per cent. After addition of B.P.L. the mixture
was incubated at 37°C for 2 hours. The vaccine thus prepared,
was used for inoculation of the experimental chickens imme-

diately after incubation.

E. flaccination

White Leghorn chickens about 10 months old were used in
these experiments. All birds were obtained from an apparently
disease-free flock and were kept for observation for the in-
dication of any disease for about 10 days prior to the eXperi-
ment. The birds were all apparently healthy at the beginning
of the experiment. 0f the 82 birds obtained, #3 birds were

3%

used for vaccination and 39 birds were used as non-vaccinated
controls. The two groups of birds were separated in 2 isola-
tion units. Prior to the experiment the birds were marked
individually by wing band and were bled from the heart. The
sera were separated and kept frozen as prevaccination sera for
the vaccinated group and normal sera for the control group.

The birds receiving vaccine were divided into M groups.
Each group‘was inoculated.with the same vaccine but in differ-
ent dosage. Group 1 consisted of 13 birds and received 0.5
m1. of vaccine. Groups 2, 3 and h consisted of 10 birds each
and received 1.0, 1.5 and 2 m1. of vaccine respectively. All
injections were intramuscular into pectoral muscle, using 2
ml. syringe with a l-inch 22-gauge needle. The control birds
received no injections. .

None of the vaccinated birds showed symptoms of NCD or
other untoward effects of injection of the B.P.L. inactivated
NCD vaccine. All the vaccinated birds were bled from the
heart again on the sixteenth day after vaccination just before
challenging with NDV. The sera were separated and kept
frozen as postvaccination sera.

Both vaccinated and non-vaccinated control birds were
challenged with the same lot of virus as that used to make
the vaccine (5H-55-18 P h). The amount of the virus intro-
duced into each bird was equal to 2 x 109'5 embryo lethal
doses. Two-tenths milliliter of full strength virus was
inoculated into the pectoral muscle of each bird.

35

F. e a t icidal ct vit 0 on almonella ullorum

The incidence of fialmonella pullgzpm_in eggs from infected
hens had been demonstrated by Mallman and Moore (1936). There-
fore, the transmission of this organism.would be possible if
eggs from pullorum-infected hens were used in vaccine produc-
tion. The purpose of this experiment was to determine if
§a1mgnellg pullozum would be killed by B.P.L. in the concentra-
tion that did not alter the antigenicity of NDV as shown in
the vaccination experiment.

figlmonella pullorum used in this experiment was the strain
5h-55-552, isolated from a naturally infected chicken in the
Department of Microbiology and Public Health, Michigan State
University. This experiment was done in three parts.

1. The fictiot
mxe wi . WWW- o
fialmonella pullorum was grown on a nutrient agar slant for

    

: ca-t e o_

 

   

#8 hours at 37°C. The growth was removed from the agar slant

with 3 ml. of sterile saline solution and was transferred into

a small test tube. The bacterial suspension was shaken vigor-
ously for several minutes to distribute bacteria evenly in the
suspension. The standard plate count method was used in deter-
mining the number of fialmonella pullorum in the bacterial sus-
pension and it was found to contain 2,000,000,000 organisms in
0.1 ml. One-tenth milliliter of fialmonglla pgllorum suspension
was added to a 10 ml. suspension of NDV lot 5h-55-18 P M. An

 

36

aqueous solution of B.P.L. (1:10) was added to make a final
concentration of 0.025 per cent. The mixture was made in

the same way as described under the preparation of NCD vaccine.
The mixture then was shaken for a few minutes and incubated

at 37°C for 2 hours. The vaccine was used immediately after
incubation. I

The vaccine was tested for sterility. One loopful of
the vaccine was transferred into a tube of nutrient broth
and incubated for #8 hours at 37°C. After #8 hours' incuba-
tion, the growth in the nutrient broth was observed and was
transferred to Kligler's medium and carbohydrate media.

After #8 hours' incubation of the differential tests, Sal:
22921.13 W was confirmed.

Ten White Leghorn chickens were used in this eXperiment
and were obtained from the same source and were kept under
the same conditions as described in the preceding experiment.
The birds were bled from the heart before vaccination and
the sera separated and frozen for serological test to be
done later. Five-tenths milliliter of NCD vaccine containing
figlmgggllg_pgllggugpwas inoculated into the pectoral muscle
of each bird. No symptoms of either N00 or pullorum infection
were observed during the observation period. Seventeen days
after vaccination, all birds were bled from the heart. The
sera were separated and kept frozen as postvaccination sera.
The birds were killed; post mortem examination revealed no

gross change indicating either NCD or pullorum infection.

 

2. The Action of Different Concentrations of B.P.L. on §al-
monella pullorum

figlmonella pullorum was seeded into 200 ml. nutrient broth
in a 500 ml. Erlenmeyer flask and incubated at 37°C for 18
hours. Standard plate count showed that the number of organ-
isms was 150,000,000 organisms per ml. At the end of 18 hours,
10 m1. of the culture was distributed into each of 8 tubes.
An aqueous solution of B.P.L. (1:10) was added into each tube

 

so that the final concentration of B.P.L. varied from 0.025,

'Iz—‘fifl-a —-———

0.05, 0.1, 0.2, 0.3, 0.h and 0.5 per cent respectively. One
tube was used as a control and contained no B.P.L. The tubes
were then incubated at 37°C for 2 hours. To dilute the B.P.L.
0.1 ml. of each tube was transferred into a tube of nutrient
broth and incubated for 15 days at 37°C. The bactericidal
effect of B.P.L. was shown by the absence of growth in the

subcultured tube after 15 days' incubation.

TABLE IV
BACTERICIDAL EFFECT or B.P.L. ON sgpggngppg_gpppgggu

_ _ _‘_*

 

%B.P.L. Growth
0.025 K
0.05 K
0.1 -
0.2 -
0. -
0.3 -
0.5 -
Control no B.P.L. /

— No growth in nutrient broth after 15 days'incubation.
/ Growth after 15 days'incubation.

 

38

3. e ff ct of 0.1 e ent B on the ti enicit of
newcastle Disease Virus

The information given from the experiment on the action
of different concentration of B.P.L. showed that salmonella
pullorum was killed when mixed with B.P.L. at the final con-
centration of 0.1 per cent. This experiment was to determine
whether or not B.P.L. at 0.1 per cent concentration would
destroy the antigenicity of NDV.

Ten m1. of NDV suspension (lot 5h-55-18 P h) were mixed
with 0.1 m1. of aqueous B.P.L. (1:10) as previously described.
The concentration of B.P.L. was 0.1 per cent. The B.P.L.-virus
mixture was incubated for 2 hours at 37°C and was immediately
thereafter inoculated into 5 chickens.

Five‘White Leghorn chickens about 1 year of age were used
in this experiment and each was kept separately in a wire cage.
The prevaccination sera were obtained as in the previous ex-
periment. One-half milliliter of the vaccine was injected
into the pectoral muscle of each bird. All birds remained
apparently healthy after the vaccination. The post-vaccination
sera were obtained on the fifteenth day following vaccination
and were stored in a frozen state. The birds were then chal-
lenged with 0.2 m1. of undiluted Newcastle disease virus sus-
pension (lot 5h-55-18 P h) intramuscularly. The challenging
virus dose was from the same lot and amount as used to chal-

lenge the original vaccine test.

39
G. e o cti u c ti 1 u

Infectious bronchitis virus (strain V 11% D) was used in
this experiment. One-tenth of a milliliter of infected allan-
toic fluid was inoculated into the allantoic cavity of each of
twenty lO-day embryonating eggs and reincubated at 37°C. All
embryos were dead within 2h hours after inoculation. The eggs
were chilled and the allantoic fluids were harvested as de-
scribed in the preparation of NDV suspensions. The allantoic
fluids were pooled together and kept frozen for further use.
Five milliliters of virus suspension were distributed into
each of 6 test tubes. An aqueous solution of B.P.L. (1:10)
was added to 5 of the 6 tubes so that the final concentration
of B.P.L. in each tube was 0.025, 0.05, 0.1, 0.2 and 0.3 per
cent respectively. The sixth tube received no B.P.L. and was
the virus control. The mixtures then.were incubated for 2
hours at 37°C together with the control virus. After incuba-
tion the mixtures were immediately inoculated into allantoic
cavity of lO-day embryonating eggs, using 0.1 ml. per egg and
5 eggs per dilution. The inoculated eggs were reincubated for
7 days. The mortality of the embryos dead within 12 hours
after inoculation were regarded as non specific.

The infectivity titer of infectious bronchitis virus was
determined by the same method as described for NDV and was

found to be 106:12 LD 50 per 0.1 ml. of inoculum.“

MO

TABLE V
VIRUCIDAL EFFECT OF B.P.L. ON INFECTIOUS BRONCHITIS VIRUS

‘—

 

 

 

% B.P.L. mortality Rate #
0.025 0/5 r-
3325 8&5
0: 0/3 3‘
0.3 0/5 2
- Control No B.P.L. 5/5 (
# Egbrzg gegd L

Embryos inoculated :_

H. The Effect of B.P.L. on Infectigug ngygggtracheitis Virus

Infectious laryngotracheitis virus (strain 11-3-5“) in
chorioallantoic membrane was used as the source of the virus.
The membrane was a pooled harvest from 12-day egg passages.
The infected membranes were ground with sterile sand in a
mortar with pestle and suspended with a small amount of nutri-
ent broth. Penicillin (10,000 units) and streptomycin (10,000
mu. per ml. of nutrient broth) were added to the suspension to
prevent and inhibit bacterial growth. The final snapension
was deposited on the chorioallantoic membrane in the artificial
air cell of 12-day embryonating eggs as described by Beveridge
and Burnet (19h6). TWO-tenths of a milliliter were used to
inoculate each egg. The eggs were reincubated at 37°C and
candled at 2h-hour intervals after inoculation. Embryo mor-
tality during 2% hours after inoculation.was considered as

non specific. 0n the fourth day after inoculation, the eggs

M1

were candled and infected chorioallantoic membranes were
removed. The harvested membrances were stored in a frozen
state at -25°C.

The infectious laryngotracheitis virus suspension was
prepared as described above. Five milliliters of virus sus- to
pension were distributed into each of 6 test tubes. An aqueous
solution of B.P.L. (1:10) was then added in different quantities
to the first 5 tubes to make the final concentration of B.P.L.

(71.. a 'vag-ni- .as

0.025, 0.05, 0.1, 0.2 and 0.3 per cent respectively. The mix-
tures, together with the sixth control tube of virus suspension
without B.P.L., were incubated at 37°C for 2 hours. After in-
cubation, two-tenths milliliter of the mixture from each tube
was deposited on the chorioallantoic membrane in the artificial
air cell of 30 twelve-day embryonating eggs, using 5 eggs per
tube. On the fourth day after inoculation, the chorioallantoic
membranes were harvested and examined for the grayish, thicken-
ing, yellow plaque as the criterion of growth of the infectious
laryngotracheitis virus. Infectious laryngotracheitis virus
generally does not kill the embryo.

#2

TABLE VI
VIRUCIDAL EFFECT OF B.P.L. ON INFECTIOUS LARXNGOTRACHEITIS VIRUS

 

 

 

 

% B.P.L. Lesions

0.025 .. Fa
0.05 _ 5;

0.1 - E

0.2 - i

0.3 - 5
Control No B.P.L. / I,“
_. _t 9— j f
- No thickening of the chorioallantoic membrane. Li

/ Thickening of the chorioallantoic membrane, characteristic
lesions of Infectious Laryngotracheitis virus.

I. Serolggical Tests

Blood samples were drawn with aseptic precaution. Pre-vac-
cination bleedings were done just prior to the vaccination.
Post-vaccination bleedings were done prior to the challenging
by untreated virulent virus. About 15 ml. of blood were drawn
from each bird by cardiac puncture, using li-inch 18-gauge
needle attached to a lO-ml. glass syringe. The blood was
allowed to clot for about one-half hour and then the tubes
were wrung with sterile wood applicators. The tubes were
stored in a refrigerator at MOC for M to 5 hours. Finally
the tubes were centrifuged in a horizontal centrifuge at 2,000
r.p.m. for 10 minutes to separate the sera. The sera were
removed from the clot and stored in a freezer at -25°C.

Some of the Newcastle disease vaccinated birds were bled

and sacrificed at the end of the second and third months after

 

43

the challenging virus dose. The birds were then examined for
evidence of Newcastle disease. The blood sera collected from
these birds were frozen and held for serological examination.
The paired sera samples were tested for the antibody con—
tent by:
1. Hemagglutination-inhibition tests.

2. Serum-neutralization tests in embryonating eggs.

l._fiemagglutination (HA) and Hemagglutination Inhibition (H1)
22.21:.

(1) Virus: Blood-free Newcastle disease virus suspension
(lot 5h-55-18 P h), which had been previously prepared and kept
frozen at -25°C were used throughout these tests.

(2) Red blood cells: Blood was drawn by cardiac puncture
from chickens. The tubes receiving the blood contained 1 ml.
of 2 per cent sodium citrate in saline solution for each 9 ml.
of blood. The blood was mixed with the anticoagulant thoroughly
to prevent clotting. The cells were then sedimented by centri-
fugation at 1600 r.p.m. for 8 minutes and the supernatant plasma
removed. The cells were then washed 3 times with 10 volumes of
saline solution. The final centrifugation to pack the cells
was for 10 minutes at 1,000 r.p.m. After washing, the packed
cells were stored in the refrigerator at HOG. The cells were
found satisfactory for use in the test for as long as 6 days
(Hirst, 19%2; U.S.D.A., 19h6). A 0.5 per cent suspension of
red blood cells in saline solution.was freshly prepared imme-

diately prior to use in the tests.

 

 

1+1:

(3) Saline solution: Sterile chemically pure 0.85 per
cent sodium chloride solution was used throughout the tests.

(h) Serum: The serum to be tested was diluted 1 in 5
with saline solution or was diluted as indicated in the result.

The tubes used in the test were chemically clean, round
bottom glass tubes (75 x 10 mm.). The virus suspension was

thawed and centrifuged in a horizontal centrifuge to remove

' " L‘s"; fAIx.:lY‘-4~eml.-. I

the insoluble substances at 2500 r.p.m. for 20 minutes. The

supernatant fluid was removed and used for making virus dilu-

!

tion. From the preliminary experiment it was found that New-
castle disease virus suspension 5H-55-l8 P H produced maximum
hemagglutination in a dilution of 1:320. Progressive two-fold
dilutions of the virus from 1:5 through 1:640 were made in 0.85
per cent saline solution. Twenty-five-hundredths milliliter
of diluted (1:5) serum was distributed into each tube of the
hemagglutination inhibition test. In the hemagglutination
test 0.25 ml. of saline solution was used instead of diluted
serum. Twenty-five hundredths milliliter of virus dilution
was transferred into parallel rows of tubes containing diluted
serum or saline solution according to the test. The control
tubes contained either saline solution and cells or serum and
cells for the respective tests. The proctocols of the proce-
dure used in hemagglutination inhibition and hemagglutination
test are given in Table VII, page h6. The tubes were shaken
and 0.25 ml.J0f a 0.5 per cent red blood cell suspension was

added to each tube. The tubes were shaken again and were

 

l+5

incubated at room temperature of from 22°C to 27°C. Readings,
the result of the tests, were taken at 15, 30 and #5 minutes.
The earlier readings were made to detect hemagglutination
which might occur in the low-dilution tubes and disappear
before the final readings (E5 minutes) were made. The reading
was facilitated by viewing the tubes from the bottom of the
'rack through a mirror placed under the rack.

Maximum hemagglutination was exhibited as a uniform layer
of agglutinated cells covering the bottom of the tube and was
recorded as positive (I). In the control tube and in the
tubes where there was no agglutination, the cells sedimented
into a central, sharply demarcated red disc in the bottom of
the tube and was recorded as negative (-). Irregular clumps
of cells associated with a ring of finely aggregated cells
were recorded as partial or slight agglutination (é).

Virus hemagglutination (HA) titer was the highest dilution
of the virus which produced complete hemagglutination. This
dilution was considered to contain 1 hemagglutinin unit. The
number of hemagglutinating units in 0.25 ml. of the undiluted
virus then was the reciprocal of the dilution of the virus.

The serum titer was the lowest dilution of the virus in
which hemagglutination was completely inhibited. This dilution
was considered to contain hemagglutination inhibition units
equal to the number of hemagglutinin units in that dilution.
The hemagglutination inhibition units in 0.25 ml. of undiluted

serum were then computed and were expressed as the hemagglutination

h." *_

“mu—t... .c

#6

:inhibition titer of that serum. For convenience in calculation
of the hemagglutination inhibition titer of the serum, the
:following formula could be used:

fieciprocal of virus titer x dilution of serum = HI titer of
Reciproca 0 serum ti er serum

A HI titer of 80 or more was considered to be diagnostic

of previous vaccination or infection with Newcastle disease. 3

TABLE VII

HEMAGGLUTINATION AND HEMAGGLUTINATION-INHIBITION TESTS
PROCEDURE

Material Und. 1/5 1/10 1/20 l/hO 1/80 1/160 1/320 1/6h0 Control

HA Test
‘Virus(dilution ----- 0.25 ml. per tube ----- 0
ml.
0.85% Saline ----- 0.25 ml. per tube ----- 0.5
solution (ml.)
0.5% Suspension ----- 0.25 ml. per tube ----- 0.25

r.b.c. (ml.)

HI Test

'Virus(dilution ----- 0.25 ml. per tube ----- 0
ml.
Serum(dilution ----- 0.25 ml. per tube ----- 0.5
ml. '
0.5% Suspension ----- 0.25 ml. per tube ----- 0.25
r.b.c. (ml.)

 

“7

2. Serum Neutralization Tests

Newcastle disease virus suSpension (5h-55-18 P h) was used
in performing serum neutralization throughout this study. Prior
to use the virus suspension was thawed and centrifuged at 2500
r.p.m. for 20 minutes in a horizontal centrifuge to remove the
insoluble precipitate formed by freezing. One thousand units
of penicillin per m1. of nutrient broth were used in making
virus dilutions to prevent possible contamination from either
virus suspension or serum. Greater concentrations of penicil-
lin have been used by Brandly e3_gl, (19%6, b) in serum neu-
tralization tests without alteration of titers. From prelim-
inary experiment it was found that the LD 50 titer of virus
suspension was 10'9'5. Serial ten-fold dilutions of allantoic
fluid containing the virus were prepared in nutrient broth
from 10"1 to 10'10. Separate pipettes were used in preparing
each dilution, Equal parts of undiluted serum and diluted
virus were mixed in separate tubes for each dilution of virus.
To compensate for the increased dilution of the virus in the
serum-virus mixture, the control virus dilution was mixed with
an equal volume of broth. The serum-virus mixtures were incu-
bated at room temperature for 30 minutes before being inoculated
into eggs. The virus dilutions were freshly prepared prior to
the test and the infectivity test in eggs was done in parallel.

One-tenth milliliter of the serum-virus mixture was inocu-
lated into the allantoic sac of 8-to-10-day embryonating eggs
by using a tuberculin syringe attached to a 27-gauge, fi-inch

 

 

M8

needle. Five eggs were inoculated from each serum and virus
dilution. The preparation of eggs was the same as described
under the preparation of the virus suspension. A single
syringe was used for each serum sample. Bacterial sterility
tests were done on each preparation. After the eggs were
inoculated, the holes in the shell were sealed with melted
paraffin and reincubated for 7 days at 37°C. The eggs were
candled daily. The criterion of viral infectivity was the
mortality of the embryos. Mortality within the first 2% hours
after inoculation was considered as non specific and was not
included in the calculation of mortality rates.

The virus titer was the highest dilution of virus in
which 50 per cent mortality of the embryos occurred and was
considered as 1 embryo lethal dose. The number of embryo
lethal doses in 0.05 ml. of the undiluted virus suspension
was the antilogarithm of the reciprocal of the dilution.

The serum titer was considered to be the lowest dilution
of virus in which the serum neutralized the virus to the ex-
tent that 50 per cent of the embryos survived. The difference
between the logarithm of virus titer and logarithm of serum
titer is the neutralization index, the antilog of which is
the number of neutralizing doses in 0.05 ml. of undiluted
serum.

For convenience in demonstrating the results both virus
and serum titers were expressed as the negative logarithms of

their respective values throughout this study.

I.” -
L “'3‘.

‘sm AA.- .AAA_ 1

 

“9

IV. RESULTS

Preliminary tests indicated that beta—propiolactone
(B.P.L.) is capable of destroying the infectivity of Newcastle
disease virus. ‘When concentrations of 0.025 through 0.3 per
cent B.P.L. were added to the virus (Table I, page 31) and
this mixture was then injected into embryonating eggs, no
death due to infectivity occurred. It was also shown that
the B.P.L. in these concentrations, was not toxic to the
embryos. To make certain that the virus was not destroyed
by the incubation period of 37°C for 2 hours, a control sample
of virus alone was incubated at the same temperature for 2
hours. The results of this eXperiment are shown in Table II,
page 31. The virus suspension contained LD 50 per cent titer
of 9.50 before the inoculation period and dropped to 9.h3
following the incubation treatment. No significant virus
alteration was found to result from this treatment.

The effect of varying the concentration of B.P.L. on the
hemagglutination property of Newcastle disease virus is illus-
trated in Table III, page 32. No alteration in the hemagglu-
tination titer could be detected until 0.3 per cent concentra-
tion of the B.P.L. was used. The control material contained
virus only and the virus with B.P.L. concentration through 0.2
per cent agglutinated erythrocytes at a 1:320 dilution. A 0.3

per cent concentration of B.P.L. in the virus suspension

50

reduced the hemagglutination titer to 1:160 which is within
the accuracy of a tube test.

' To determine what effect the B.P.L. would have upon the
antigenicity of Newcastle disease virus, 5 chickens were vac-
cinated with Newcastle disease virus which had been treated
with B.P.L. The concentration of B.P.L. was 0.1 per cent and
the vaccine prepared in the same manner as has already been
described. Sera were collected from the 5 birds prior to
vaccination and 15 days after vaccination. The antigenic
stimulation produced by the vaccine in these 5 chickens is
indicated by the results of the serum hemagglutination inhibi-
tion tests given in Table XXII, page 71. In birds 85 and 86
there was no rise in the HI titer. In bird 87 there was a
doubtful rise. In 2 birds 88 and 89 the HI titer gave a
significant rise in titer but the results were not outstanding.
From the results of the HI tests as given in Table XXII it
appeared that the concentration of B.P.L. was such that the
antigenicity of the virus was altered below a satisfactory
level.

Serum neutralization tests were done using embryonating
chicken eggs to detect infective virus. The results of these
tests are given in Table XXII. None of the 5 birds vaccinated
with this vaccine showed a significant difference between the
prevaccination and postvaccination neutralizing index. The
group combined had an average prevaccination neutralizing

index of 1.h8 and a postvaccination average of 1.72 with a

51

difference of 0.2%. The results of the neutralization tests
are in agreement with the hemagglutination inhibition tests
in that, there was not a significant increase in antibodies
between the prevaccination and postvaccination sera.

Fifteen days following vaccination the 5 birds received
a challenging injection of 0.2 ml. of untreated virus intra-
muscularly. The result of the virus injection is shown in
Table XXI, page 70. Two birds, 85 and 86, died from Newcastle
disease, the remaining birds although developing symptoms of
the disease, recovered after prolonged illness.

The next step was that of adjusting the concentration of
the B.P.L. in a vaccine to a point which would retain a large
margin of safety for virus pathogenicity, however, retaining
antigenicity. Returning to Table 1, page 31, it was determined
that as little as 0.025 per cent B.P.L. would destroy the path-
ogenicity of the virus. Therefore, a vaccine was prepared
using 0.025 per cent B.P.L. Eighty-two chickens, all of the
same age and source were divided into 2 lots of H3 and 39
respectively. The first lot of #3 birds was divided into M
groups of 13, 10, 10 and 10 birds and received 0.5, 1.0, 1.5
and 2 m1. of the vaccine respectively. The second lot of 23
birds did not receive the vaccine.

Just prior to vaccinating the chickens, all birds were
bled; their sera were held in a frozen state to await the

collection of postvaccination sera.

52

The vaccinated chickens were observed daily for 15 days
and none gave evidence of illness. 0n the sixteenth day fol-
lowing vaccination the test birds (those receiving the vaccine)
were again bled and the sera were separated from the clots and
stored in a frozen state.

A challenging injection of 0.2 ml. of virus (L050 2 2 x
1095) was given on the sixteenth day to both the vaccinated
and control birds. Again none of the vaccinated chickens gave
evidence of disease while the non-vaccinated group began to show
evidence of Newcastle disease on the fourth day following the
challenge injection of virus. The final results of virus in-
jection in the non-vaccinated birds are given in Table IX.
page 58. These birds showed dullness which rapidly developed
into a marked depression accompanied by progressive weakness.
Cyanosis of the comb and diarrhea were the prominent symptoms.
Figure 1, page 53, illustrates the vaccinated and non-vaccinated
chickens on the seventh day following the challenging injection
of live virus. Deaths in the non-vaccinated birds began on the
fifth day following virus injection and by the ninth day 38 of
the 39 birds (97.h%) had become ill and died. Only one control
bird did not develop Newcastle disease. The disease was fatal
to 33 of the 39 (8%.6%) chickens.

Hemagglutination inhibition and neutralization tests were
done on the paired sera from the vaccinated chickens. The
results of HI tests on the N groups of vaccinated chickens are

given in Tables X through.XIII, pages 59-62. Considering the

53

.25.? mucous nape—wean: unease?
n33 wuwwncfimne nevus hoe £525» on» no 3:35
nnoxeuno covenaoempnno: was named A.A.m.m meodv
caucus» canon? 032252 :33 meanness» 383.30

 

 

’1‘!
(D
“‘1
:21?

H:

i A
”‘CSE

51+

results, as given in these h tables collectively, there was
no evidence that the injection of 0.5, 1.0, 1.5 or 2 ml. of
vaccine produced different results. Of the M3 chickens re-
ceiving the vaccine, all showed a substantial increase in H1
titer when the prevaccination and postvaccination sera were
compared. The range of this increase was from 10 to 80 (bird
#011) and 5 to 1600 (bird #009). The average prevaccination
HI titer was 10 and the postvaccination 320.

The results of the serum neutralization test in embryo-
nating chicken eggs on the same paired sera are shown in Tables
XVI througthIX, pages 65-68. Only representative samples of
serum, chosen at random, were tested for neutralizing anti-
bodies. Considering the results given in these h tables c01-
lectively, again in every instance there was a marked increase
in the antibody titer as seen in the prevaccination and post-
vaccination indexes. The average prevaccination serum neutral-
izing index of the h groups was 1.19. The average postvaccina-
tion index was h.08. The latter being approximately h times
greater than the former. Indexes of the prechallenge.sera are
above those recommended for the diagnosis of Newcastle disease
by Osteen and Anderson (19h8).

The hemagglutination inhibition tests were done on blood
sera from 23 of the non-vaccinated chickens. The results of
these tests are given in Table XIV, page 63. Briefly, only
one chicken's (189) serum showed a 1:“0 HI titer. Referring
to Table IX, page 58, it will be seen that, for unknown

55

reasons, this bird was the only one of 39 birds in the controls
that did not show evidence of disease following challenging
with live virus. 'Whether or not the existing 1:h0 HI titer

in chicken 189 was responsible for its protection against
Newcastle disease virus is unknown.

In Table XX, page 69, the HI serum titers of the h groups
of vaccinated chickens are summarized as well as the titers
tested at 2 and 3 months after challenging with untreated
virus. The sera were from those birds withstanding the bleed-
ing procedures several times. The majority of sera show a
marked increase in HI antibody titer, as would be expected.

To demonstrate that a vaccine containing 0.025 per cent
B.P.L. and Newcastle disease virus in allantoic fluid was in-
capable of producing Newcastle disease, 20 ml. of the vaccine
‘was injected intraperitoneally into a chicken. The chicken
receiving this massive dose of vaccine was from the same source
as all other chickens in this work. Injection of this amount
<of Newcastle disease virus vaccine did not produce illness in
'the bird. This amount of vaccine (20 m1.) represents 10 times
'the amount of virus that was used in the largest vaccine dose
in this study. -

Chicken eggs may contain Salmonella pullorum in various
nlllmbers. It, therefore, seemed advisable to determine the
eIfect of various concentrations of B.P.L. upon the bacterial
c’ell. Table IV, page 37, gives the results of testing various

c’Cmcentrations of B.P.L. on 2,000,000,000 figlmonella pullorum

56

organisms contained in 10 ml. of Newcastle disease infected
allantoic fluid. It was found that 0.025 and 0.05 per cent
B.P.L. did not destroy the organisms. A 0.1 per cent concen-
tration of B.P.L. was found to destroy the bacterial cells in
the allantoic fluid. This concentration of B.P.L., however,
has been shown to destroy the antigenicity of the Newcastle
disease virus.

A vaccine containing 0.025 per cent B.P.L., Newcastle
disease virus and Salmonella pullorum.was made and 0.5 ml. of
this mixture was injected into 10 chickens. Sera from these
10 birds all showed an increase in the HI antibody titer
(Table.XV, page 6%) for Newcastle disease virus but failed
to demonstrate blood agglutinins to Sglmgnella pullorum. All
10 chickens were destroyed at the end of this experiment and
no lesion of Newcastle disease or pullorum infection could be
determined.

The effects of increasing concentration of B.P.L. on the
viruses of infectious laryngotracheitis and bronchitis are
given in Tables VI and V, pages #2 and NO. ‘With both of these
viruses which are infectious to the chicken, 0.025 per cent
B.P.L. was found to destroy the infectiveness of the agent as
indicated by the results of testing in embryonating chicken

eggs.

57

TABLE‘VIII

RESULTS OF IMMUNITY CHALLENGED OF VACCINATED CHICKENS
(0.025% B.P.L. VACCINE) 0.2 ML. VIRUS INTRAMUSCULARLY

 

 

Number of Amount of Date Date
Birds Vaccine Vaccinated Challenged Result*
(m1. )
13 0.5 10-26-5h 11-12-5h -
10 1.0 10-26-5h 11-12-5h -
10 1.5 10-26-5h 11-12-5h -
10 2.0 10-26-5h 11-12-5h -

* - No reaction

58

TABLEIX

RESULTS OF VIRUS CHALLENGE 0! NON-VACCINATED CONTROL
CHICKENS 0.2 ML. VIRUS INTRMUSCULARY

g ‘ A M _
w .—

Birds Symptoms Death Birds Symptoms Death
Number Days After 'Days After Number ‘Days After Days After
Challenged Challenged Challenged Challenged

‘__— h—k k A A—.

161: l: 5 195 l: 9
l1083 '+ 5 I+077 1. 9
173 h 5 l1091: l: 9
1:082 1+ 5 1:007 I: 9
l1096 l: 5 152 I: 9
167 ’4' 5 172 1+ Killed
166 ‘4' 5 “-037 ‘0 Killed
175 h 5 #0 1 1: Killed
M378 1: 5 1:089 I: Killed
179 h 5 170 11' Killed
1:085 1: 5 l#088 1: Recovered
171 10 6 182 1: Recovered
’+O95 1+ 6 t108$: 1: Recovered
186 l: 6 h091 1: Recovered
181: I+ 6 190 l: Recovered
#090 1+ 6 189 None 0
\3098 l: 7
193 h 7
169 l: 7
I1:086 h 7
11-100 It 7
151 ‘1 7
l+092 k 7

59

TABLE.X

RESULTS OF HEMAGGLUTINATION INHIBITION TESTS ON SERA FROM
VACCIRATED CHICKENS GROUP 1 (VACCINE 0.5 ML.)

_ A Q ‘_ A; _

 

 

 

Number Virus;Dilution Serum
Chicken HI
Serum Und. 1/5 1/10 1/20 1A0 1/80 1/160 1/320 1/6l+0 Titer
102A K K K K K K - - - 10
1023 K - - - - - - - - 320
128A K K K K K K - A - - 10
128B K - - - - - - - - 320
inn K K K K K - - - - 20
151:}: K - - - - - - - - 320
hoioi K K K K K K - - - 10
noise K - - - - - - - - 320
159A K K K K K K K - - 5
1593 K - — - - - - - - 320
156: K K K K - - - - - Mo
1563 K K - - - - - - - 160
157A K K K K - - - - - no
1573 K - - - - - - - - 320
1M3: K K K K K K - - - 10
lh3B - - - - - - - - - 1600
126A K K K K K K K - - 5
126B K - - - - - - - - 320
101A K K K K K K - - - 10
101B - - - - - - - - - 1600
129: K K K K K K - - - 10
129B K - - - - - - - - 320
PA K K K K K - - - - 20
PB K - - - - - - - - 320
139A K K K K K K - - - 10
1393 K - - - - - - - - 320
A : Prevaccinated serum K : Hemagglutination
B 8 Postvaccinated serum - No hemagglutination

Virus control HA titer 1:320

60

TABLEAXI

RESULTS OF HEMAGGLUTINATION INHIBTION TESTS ON SERA FROM
VACCINATED CHICKENS GROUP 2 (VACCINE 1.0 ML.)

— —

 

 

Number Iims pilut ions Serum
Chicken. HI
Serum. Und. 1/5 1/10 1/20 1/h0 1/80 1/160 1/320 1/6h0 Titer
160A K K K K K K K - - 5
1608 - - - - - - - - - 1600
153: K K K K - - - - - Mo
1533 - - - - - - - - - 1600
163: K K K K K K K - - 5
1633 K K - - - - - - - 160
h011A K K K K K K - - - 10
M0113 K K K - - - - - - 80
165A K K K K K - - - - 20
165B K - - - - - - - - 320
168A K K K K K K - - - 10
168B K K - - - - - - -- 160
inoi K K K K K - - - - 20
lhOB K - - - - - - - - 320
131: K K K K - - - - no
131B K - - - - - - - - 320
135: K K K K K K K - — 5
1353 K - - - - - - - - 320
now K K K K K K - - - 10
h01hB K - - - - - - - - 320
A : Prevaccinated serum
B : Postvaccinated serum
K Hemagglutination
- No hemagglutination
Virus control HA titer 1:320

61

TABLEIXII

RESULTS OF HEMAGGLUTINATION INHIBITION TESTS ON SERA FROM
VACCINATED CHICKENS GROUP 3 (VACCINE 1.5 ML.)

w

 

Number Virus 211nt102§ Serum
Chicken HI
Serum Und. 1/5 1/10 1/20 1/h0 1/80 1/160 1/320 1/6h0 Titer
157A K K K K K K K - - 5
1k7B K K K - - - - - - 180
1381 K K K K K K K - - 5
1383 - - - - - - - - - 1600
127A K K K K K K - - - 10
1273 K - - - - - - - - 320
#0101 K K K K K - - - - 20
u01oa . 320
ix» K K K K K - - - - 20
1308 K - - - - - - - - 320
162A K K K K K K - - - 10
1623 - - - - - - - - 1600
h009A K K K K K K K - - 5
H0098 - - - - - - - - - 1600
1MB; K K K K K K K - - 5
1h83 K - - - - - - - - 320
1871 K K K K - - - - - #0
1873 K - - - - - - - - 320
159A K K K K K K - - - 10
159B K - - - - - - - - 320

A I Prevaccinated serum

B = Postvaccinated serum
K Hemagglutination

- No hemagglutination
Virus control HA titer 1:320

62

TABLE XIII

RESULTS OF REMAGGLUTINATION INHIBITION TESTS 0N SERA FROM
VACCINATED CHICKENS GROUP H (VACCINE 2.0 ML.)

Number i s 11 ion Serum

Chicken HI
Serum Und. 1/5 1/10 1/20 1A0 1/80 1/160 1/320 1/6h0 Titer

11:61 K K K K K K - - 5
11:63 K - - - - - - - - 320
1:093A K K K K - - - - - 1+0
l+0933 K - - - - - - - - 320
z+0991 K K K K K K - - - 10
l+0993 K - - - - - - - - 320
#0121 K K K K K K - - - 10
1.0123 K - - - ‘- - - - - 320
1951 K K K K - - - ~ - Mo
11:53 - - - - - - - - - 1600
1811 K K K K K - - - - 20
1813 - - - - — - - - - 1600
1331 K K K K K K - - - 10
1333 - - - - - - ~ - - 1600
1|+11 K K K K K - - - 20
1313 - - - - - - - - - 1600
1611 K K K K K K - - - 10
1613 K - - - - - - - - 320
1581 K K K K K - - - - 20
1583 K - - - - - - - - 320

—_

A Prevaccinated serum
B Postvaccinated serum
K Hemagglutination

V

No hemagglutination
irus control HA titer 1:320

63

TABLE XIV

RESULTS OF REMAGGLUTINATION INHIBITION TESTS ON SERA FROM
NON-VACCINATED CONTROL CHICICENS

.—

Chicken Eigus pllut igg Virus

Serum HI
Number Und. 1/5 .1/10 1/20 1/#0 1/80 1/160 1/320 1/6h0 Titer

#08# K K K K K K - - - 10
#089 K K K K K - - - - 20
#091 K K K K K K K - - 5
#080 K K K K K - - - - 20
189 K K K K - - - - - #0
186 K K K K K K K - - 5
#097 K K K K K K - - - 10
151 K K K K K K K - - 5
#083 K K K K K - - - - 20
195 K K K K K K - - - 10
#077 K K K K K - - - - 20
179 K K K K K K K - - 5
193 K K K K K K - - - 10
#007 K K K K K - - - - 20
170 K K K K K K - - - 10
#086 K K K K K K K - - 5
#088 K K K K K K - - - 10
#096 K K K K K K K - - 5
169 K K K K K K - - - 10
#090 K K K K K K - - - 10
16# K K K K K K - - - 10
182 K K K K K K K - - 5
172 K K K K K K K - - 5

r
l
3

K Hemagglutination
- No hemagglutination
Virus control HA titer 1:320

6h

TABLE XV

RESULTS OF HEMAGGLUTINATION INHIBITION TESTS ON SERA FROM
CHICKENS VACCINATED‘WITH 0.5 ML. NDV-0.025% B.P.L.

VACCINE CONTAINING WA W

__ __

 

 

Number vuus nilug ion Serum
Chicken HI
Serum and. 1/5 1/10 1/20 1/#0 1/80 1/160 1/320 1/6#0 Titer
901 K K K K K K K - - 5
903 K - - - - - - - - 320
91: K K K K K K K - - 5
913 K K K K - - - - - #0
931 K K K K K - - - - 20
93B - - - - - - - - - 1600
9#a K K K K K K - - - 10
983 K K - - - - - - - 160
95: K K K K K K K - - 5
95B K - - - - - - - - 320
96A K K K K K K - - - 10
963 K K - - - - - - - 160
971 K K K K K K K - - 5
973 K - - - - - ~ - - 320
98: K K K K K K K - - 5
988 K - - - - - - - - 320
991 K K K K K K K - - 5
993 K K K - - - - - - 80
100A K K K K K K K - - 5
100B K - - - - - -- - - 320
A = Prevaccinated serum
B = Postvaccinated serum
K Hemagglutination
- No Hemagglutination
Virus control HA titer 1:320

65

TABLE XVI

RESULTS OF SERUM NEUTRALIZATION TESTS IN EGGS FROM
VACCINATED CHICKENS GROUP 1 (VACCINE 0.5 ML.)

Vlrus Qilut 10;; 5221.13;
Chicken Neutral-

izing
10’3 10'“ 10'5 10"6 10'7 10"8 10'9 Titer Index

Serum Virus
Number Titer

_‘ ._

 

 

Numerator numbe dead
DenomInator numBer Injected

1021 # 9.62 - - - 3/5 #/5 2/5 0/5 7.6# 1.98
1023 9.62 - 5/5 5/5 /5 0/5 - - 6.37 3.25
1281 9.62 - - - 5/5 5/5 1/# 0/5 7.66 1.96
1283 9.62 5/5 2/5 1/5 0/5 - - - 3.66 5.96
15#A 9.118 - - - 5/5 5/5 3/5 1/5 8.32 1.16
15#3 9.#8 5/5 5/5 11/5 0/5 - - - 5.37 “.11
#016: 9.h8 - - - 5/5 5/5 3/5 0/5 8-16 1-52
#0163 9.#8 5/5 5/5 1/5 0/5 - - - 5.#8 #.0

159A 9.#8 - - - 5/5 5/5 5/5 0/5 8.5 0.98
1593 9.#8 5/5 5/5 5/5 3/5 - - - 6.16 3.32
156A 9.62 - - - 5/5 573 #/# 0/5 8.5 2.12
1563 9.62 - - 5/5 5/5 3 0/5 - 7.33 2.29
157A 9.#5 - - - 5/5 #/5 2/# 0/# 7.78 1.67
1573 9.#5 h/# 5/5 5/5 3/5 - - - 6.16 3.29
11:31 9.#8 - - - 5/5 5/5 ‘+/5 2/5 8.6 0-85
1#33 9.#8 5/5 3/5 0/5 0/5 - - - #.66 #.82
1261 8.#9 - - - 5/5 3/# 0/# 0/5 7.33 1.16
1263 8.#9 5/5 h/h 3/5 1/5 - - - 5.32 3.17
Average prevaccination Neutralizing index l.g7

Average postvaccination Neutralizing index 3. 0

# A Prevaccinated serum
B Postvaccinated serum

66

TABLE XVII

RESULTS OF SERUM NEUTRALIZATION TESTS IN EGGS FROM
VACCINATED CHICKENS GROUP 2 (VACCINE 1 ML.)

Klaus pilutigg 5.22...“
Chicken Neutral-
Serum ‘Virus izing

Titer Index

_

Number Titer 10'3 10°“ 10'5 10"6 10'7 10‘8 10'9

-_~

 

 

160A #' 9.N8 - - - 5/5 5/5 2/5 0/5 7.83 1.65
1603 9.#8 3/5 2/5 0/5 0/5 - - - 3.5 5.9
1531 9.38 - - - 5/5 3/5 2/5 0/5 7.h8 2.0
1533 9.#8 #.5 3/5 2/5 0/5 - - - “.33 5.15
1631 9.h5 - - - 5/ 5/5 5/5 2/5 8.82 0.63
163B 9.h5 5/5 5/5 3/5 2 - - - 5.5 3.89
#0111 8.16 - - - 5/5 5/5 #/5 0/5 7.62 0.5%
h011B 8.16 5/5 5/5 5/5 3/5 - - - 6.16 2.0
165A 8.16 - - - 5/5 5/5 0/5 0/5 7.5 0.66
1653 8.16 5/5 5/5 2/5 1/5 - - - 5.0 3.16
168A 8.16 - - - 5/5 5/5 0/5 0/5 7.5 0.66
1683 8.16 5/5 5/5 3/5 0/5 - - - 5.16 3.0
Average prevaccination.Neutralizing index 1.023
Average postvaccination Neutralizing index 3.85
#’ A Prevaccinated serum
B Postvaccinated serum

Numera or umber dead
Denominafor numBer Injected

67

TABLE XVIII

RESULTS OF SERUM NEUTRALIZATION TESTS IN EGGS FROM
VACCINATED CHICKENS GROUP 3 (VACCINE 1.5 ML.)

__ L .___ h—

irus ilution S2223
Chicken Neutral-
Serum Virus izing

Number Titer 10’3 10'“ 10'5 10"6 10‘7 10"8 10"9 Titer Index

* A

J

 

1#7A # 8.#9 - - - 5/5 #/5 2/# 0/5 7.78 0.71
1878 8.#9 5/5 5/5 5/5 3/5 - -- - 6.16 2.33
138A 8.#9 - - - 5/5 3/5 0/5 0/5 7.16 1.33
1383 8.h9 3/5 1/5 0/5 0/5 - - - 3.32 5.17
127A 8.89 - - - 5/5 5/5 2/5 0/5 7.83 0.66
1273 8.#9 #Kh 5/5 5/5 1/5 - - - 5.62 2.87

#0101 9.83 - - - 5/5 5/5 5/5 0/5 8.50 1.
#0103 9.50 5/5 5/5 #/5 2/5 - - - 5.6# 3.86
130A 9.88 - - - 5/5 5/5 5/5 0/5 8.50 0.98
1303 9.#8 5/5 5/5 3/5 0/5 - - - 5.16 #.32
162A 8.#9 - - - 5/5 3/5 2/5 0/5 7.38 1.01
1623 8.119 5/5 #/5 1/5 0/5 - - - #.#9 #.00
Average prevaccination Neutralizing index 1.00

Average postvaccination Neutralizing index 3.75

# A Prevaccinated serum
B Postvaccinated serum

numeratog number deag
Denomina or num or n ected

68

TABLE XIX

RESULTS OF SERUM NEUTRALIZATION TESTS IN EGGS FROM
VACCINATED CHICKENS GROUP ’+ (VACCINE 2.0 ML.)

‘—__ M

Ilzus pllutign Serum
Chicken Neutral-
Serum ‘Virus izing

Number Titer 10-3 10-5 10-5 10'6 10'7 10"8 10"9 Titer Index

~— ~ A‘. _____

 

1#61 # 9.83 - - - 5/5 5/5 5/5 3/5 9.16 0.67
1#63 9.83 5/5 #/5 0/5 0/5 - - - #.83 5.#5
#0931 9.83 - - - 5/5 5/5 5/5 0/5 8.50 1.83
1*0933 9.83 5/5 3/8 0/5 0/5 - - - “.33 5.50
”099A 9.h8 - - - 5/5 5/5 2/5 0/5 7.83 1.65
#0993 9.#8 5/5 5/5 1/5 0/5 - - - 5.83 3.56
#0121. 8.#9 - ‘- - 5/5’ 5/5 0/5 0/5 7.50 0.99
h012B 8.#9 5/5 5/5 2/5 0/5 - - - h.83 3.56
1#51 9.#8 - - - 5/5 5/5 2/5 1/5 8.0 1.#8
185B 9.M8 N/5 2/5 0/5 0/5 - - - 3.78 5.70
181A 9.#8 - - - 5/5 5/5 2/5 0/5 7.83 1.65
1813 9305 3/‘1 2/5 0/5 0/5 - - - 3.60 5.85

Average prevaccination Neutralizing index 1.378

Average postvaccination Neutralizing index .936

A‘ A_._.__ h; _.

1

# A Prevaccinated serum
B Postvaccinated serum

ume at nume dad
DenomInanr number ingected

69
TABLE.XX

RESULTS OF HEMAGGLUTINATION INHIBITION TESTS ON SERA
FROM VACCINATED BIRDS

 

Postchallenged

 

m1. Prevac- Postvac-
Bird Vaccine cination cination HI Titer*
Number Received HI Titer HI Titer 2 months 3 months
102 0.5 10 320 1280 -
13 0.5 10 320 - 1280
12 0.5 10 230 2560 -
101 0.5 10 l 0 - 2560
129 0.5 10 320 - 2560
15# 0.5 20 320 - -
“2%? 8'3 12 338 15:0 256°
156 0.5 #0 160 - 1280
137 0.5 #0 20 - 2560
12 0*; 12 1323 no 2...
2 0 e '-
P 0.5 20 320 - 2560
168 1.0 10 160 2560 -
#011 1.0 10 80 2560 -
1#0 1.0 20 320 2560 -
#01# 1.0 10 320 - 2560
135 1.0 5 320 2560 -
121 1.0 #0 320 6#0 -
1.; 13 29,- 323 a. 256°
160 1.0 5 1600 - 1280
153 1.0 #0 1600 - 1280
#009 1.5 5 1600 2560 -
1#7 1.5 5 180 2560 -
l#8 1.5 5 320 - -
187 1.5 #0 20 2560 -
138 1.5 5 l 00 - 2560
l 9 1.5 10 320 - 2560
127 1.5 10 320 2560 -
#010 1.5 20 320 - 2560
1 2 1.5 10 1 00 - 2560
132 2.0 10 320 - 2560
#693 3'8 #6 333 2328 '
1:833 3° 18 338 513328 '
.0 -
1#1 2.0 20 1600 2560 -
l#5 2.0 #0 1600 - 2560
161 2.0 10 230 - 2560
181 2.0 20 1 0 -- -
158 2.0

20 320 - -

* Sera diluted l:#0
- Hemagglutination-inhibition test not done

7O

TABLE‘XXI

IMMUNITY CHALLENGED OF VACCINATED CHICKENS (0.5 ML. 0?
1x B.P.L. VACCINE) 0.2 ML. VIRUS INTRAMUSCULARLY

‘ k h

 

Birds ‘Date Date
Number Vaccinated Challenged Result
85 2-2-55 2-17-55' Died on 2-22-55, Newcastle
disease
86 2-2-55 2-17-55 ‘Died on 2-22-55, Newcastle
disease

87 2-2-55 2-17-55 Off teed depression, sneezing
and gaSping no nervous
symptoms. Necovered.

88 2-2-55 2-17-55 Symptoms of Newcastle disease
as above and recovered.

89 2-2-55 2-17-55 Symptoms of Newcastle disease

as above and recovered.

71

TABLE XXII

RESULTS OF HEMAGGLUTINATION INHEBITION TESTS ON SERA FROM
VACCINATED CHICKENS 0.1% B.P.L. VACCINE
(AMOUNT 0.5 ML.)

Number vuug pilut ion Serum
Chicken HI

Serum und. 1/5 1/10 1/20 1/h0 1/80 1/160 1/320 1/6h0 Titer

A
__ g.‘

BSA K K K K K - - - - 20
853 K K K K K - - - - 20
86A K K K - - - - - - 80
863 K K K - - - - - - 80
87; K K K K K - - - - 20
878 K K K K - - - - #0
88A K K K K K K - - - 10
883 K - - - - - - - - 320
89A K K K K K K K - - 5
891 K K - - - - - - - 160

___—_

A Prevaccinated serum
B Postvaccinated serum
K Hemagglutination

V

N0 hemagglutination
irus control HA titer 1:320

72

TTBLE XXIII

RESULTS OF SERUM NEUTRALIZATION TESTS IN EGGS FROM
VACCINATED CBICKENS VACCINE CONTAINED 0.1%
B.P.L. (AMOUNT 0.5 ML.)

Eirug Dilution Serum
Chicken Neutral-

Serum ‘Virus _ _ izing
Number Titer 10'3 10'“ 10'5 10'6 10'7 10 8 10 9 Titer Index

 

BSA # 9.50 - - - 5/5 5/5 5/5 3/5 9.16 0.3%
853 8.83 - - - 5/5 5/5 1/5 0/5 7.62 1.21
861 9.50 - - - 5/5 5/5 3/5 0/5 8.16 1.3“
863 8.83 - - - 5/5 5/5 1/5 0/5 7.62 1.21
871 9.50 - - - 5/5 5/5 2/5 0/5 7.83 1.67
87B 8.83 - - - 5/5 5/5 2/5 0/5 7.83 1.00
881 9.50 - - - 5/5 5/5 1/5 0/5 7.62 1.88
883 9.50 - - 5/5 3/5 2/5 1/5 0/5 6.66 2.8%
891 9.50 - - - / 3/5 0/5 0/5 7.16 2.18
893 9.50 - - “/5 3/3 3/5 0/5 0/5 7.16 2.3“

Average prevaccination Neutralizing index 1.h8

Average postvaccination Neutralizing index 1.72

I

# A Prevaccinated serum
B Postvaccinated serum

umer tor numbe de d
DenoEInagor numBer inflected

V. DISCUSSION

The results give evidence that beta-propiolactone is an
excellent compound to alter Newcastle disease virus. ‘Hhen
0.025 per cent B.P.L. was used.with the virus, the pathogen-
icity-was so altered that infection'was not observed. Although
the infectivity was destroyed the antigenicity of the virus
was retained. Challenging amounts of virus failed to produce
disease in chickens vaccinated by the method described. The
serological evidence of antibody stimulation was found by both
the serum neutralization and hemagglutination inhibition tests.
This evidence was marked in every chicken tested although the
time between vaccination and challenging virus was comparative
short.

Advantages of this vaccine are many: Other virus infec-
tions (excluding lymphomatosis), such as infectious bronchitis
and laryngotracheitis viruses were destroyed by the concentra-
tion of B.P.L. best suited to produce a potent vaccine. Al-
though the number of pullorum organisms used in this experiment
was probably in gross excess of that naturally occurring in
infected eggs, there was evidence of bactericidal effect upon
the organisms. No agglutinins for pullorum*were found in the
sera of those birds receiving the vaccine containing the

organisms.

73

7h

There would be small likelihood of dissemination of the
Newcastle disease virus in a vaccine of this type because of
the absence of infectivity. Evidence supporting this is the
fact that the very sensitive embryonating eggs did not show
existence of living virus; and when a massive dose was admin-
istered to a chicken, no symptoms of illness resulted. A
properly made vaccine would be incapable of Ifield break."

A 0.025 per cent concentration of B.P.L. destroyed the
infectivity of infectious bronchitis and laryngotracheitis
viruses. It is, therefore, possible to incorporate these
viruses, providing their antigenicity is not altered, into a
vaccine. If effective, a triple virus vaccine of this nature
would be very desirable and convenient.

There remain other questions to be answered before B.P.L.
can be used extensively with Newcastle disease. For instance,
‘what effect does B.P.L. have on other strains of Newcastle,
lymphomatosis, infectious bronchitis and laryngotracheitis
‘viruses? What is the duration of solid resistance to the
«disease considering the volume of vaccine used? However,
studies of pathogenicity and antigenicity, showed that the
use of B.P.L. treated Newcastle disease virus is basically

.sound.

VI. SUMMARY

1. The virucidal effect of beta-propiolactone (B.P.L.)
upon.Newcastle disease, infectious bronchitis and laryngotra-
cheitis viruses was determined.

2. It was found that 0.025 per cent B.P.L. completely
destroyed the infectious properties of Newcastle disease virus
but did not destroy the antigenicity.

3. A vaccine made by adding 0.025 per cent B.P.L. to New-
castle virus was shown to completely protect chickens against
a challenging injection of live virus. Control chickens not
receiving the vaccine but challenged with live virus showed
typical symptoms of Newcastle disease and the majority of
birds died from the infection.

h. Paired sera collected from the chickens before and
after vaccination showed a marked rise in neutralizing and
hemagglutination inhibition antibodies.

5. The same concentration of B.P.L. (0.025 per cent) as
teas used to make the Newcastle disease vaccine, was shown to
«Testroy the infectivity of infectious bronchitis and laryngotra-
Cheitis viruses.

6. Large numbers of fialmoggllg,pulloz§m‘were not destroyed
by 0.025 per cent B.P.L. A vaccine made for Newcastle disease
and containing fi. pullorum was injected into chickens. The

Chickens did not show evidence of either Newcastle disease or
75

76

pullorum infection and at the end of this experiment these
birds were sacrificed and no evidence of bacterial infection
could be found. The sera from these birds did not give
evidence of pullorum agglutinins at the close of the experiment.

7. Advantages of this type of vaccine are discussed.

(1)

(2)

(3)

(N)

('5)

(6)

(8)

(57/

(10) Be.

(1)

(2)

(3)

(h)

(5)

(6)

(7)

(8)

(9)

(10)

VII. LITERATURE CITED

Acevedo, R. A. and Mendoza, I. L.
19h7. Chicken embryo vaccine against Newcastle disease.
Amer. J. veto ROS. £391-1020

Adler . 3., Willers B. H. and Campbell, J.
1951. Newcastle disease (Avian Pneumoencephalitis) in
Hawaii. Amer. J. Vet. Res. Rake-1&7.

Anderson, S. G.
19H7. The reaction between red cells and viruses of
the influenza group. Studies with Newcastle
disease virus. Aus. J. Exp. Biol. Med. Sci.

25,: 163-175 .

Bang P. B.
#6. Filamentous forms of Newcastle disease virus.

Proc. Soc. Exp. Biol. fled. 6833-5.

Bang . B.
19H7. Newcastle virus: Conversion of spherical form
to filamentous form. Froc. Soc. Biol. Med. Qk:

Bankowski, R. A. and Boynton,‘w. H.
19h8. Preliminary report on the propagation of avian
pneumoence halitis (Newcastle disease) ig,vitgo.
veto mde J: 305.306.

Bankowski, R. A.
1950. Further studies on in vit cultivated pneumo-
encephalitis (Newcagfle disease) virus and its
use as a vaccine. Vet. Nbd. fiie322-327.

Beach, J. B.
19h1. A nervous disorder in chickens. Nu-Laid News.

E315.

Beach, J. H.
19h2. Avian pneumoencephalitis. Proc. hbth Ann. Meet.

Beach, J. R.
l9hha.‘Vaccination of pneumoencephalitis. Proc. N8th.

Ann. Meet. U.S. Livestock San. Assn. 177-18”.

77

(11)

(12)

(13)

(1“)

(15)

(16)

(17)

(18)

(19)

(20)

78

Beach J. R.
l9ahb. The neutralization in vitrg of avian pneumo-
encephalitis virus Sy'Newcastle disease immune
serum. Science. 1903361-362.

Beach, J. H.
19MB. The application of the hemagglutination-
inhibition test in the diagnosis of avian
pneumoencephalitis. J. Amer. vet. Med. Assn.

”£385‘910

Berg L. R., Bearse G.‘B. and Hamilton, C. M.
l9k7. Effect of Newcastle disease on egg production
and egg quality. Poultry Sci. 29:616-622.

Beaudette, P. R.
l9h3. A review of the literature on Newcastle
disease. Proc. H7th Ann. Meet. U.S. Livestock
sane Assn. 122-177 a

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