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This is to certify that the

thesis entitled

THE IMMUNE RESPONSE TO SEPARATE AND/OR COMBINED
NEWCASTLE DISEASE AND INFECTIOUS BRONCHITIS
VACCINES IN CHICKENS

presented by

Maria N. Narimatsu

has been accepted towards fulfillment
of the requirements for

Master of Science
degree in

Department of Poultry Science

 

 

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LIBRARY
Michigan Stair:

University

OVERDUE FINES:
25¢ per day per item

RETURNING LIBRARY MATERIALS:
Place in book return to remove
charge from circulation records

 

 

THE IMMUNE RESPONSE TO SEPARATE AND/OR COMBINED
NEWCASTLE DISEASE AND INFECTIOUS
BRONCHITIS VACCINES IN CHICKENS

BY

Maria N. Narimatsu

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Poultry Science

1981

ABSTRACT

THE IMMUNE RESPONSE TO SEPARATE AND/OR COMBINED
NEWCASTLE DISEASE AND INFECTIOUS .
BRONCHITIS VACCINES IN CHICKENS
By

Maria N. Narimatsu

Infectious bronchitis and Newcastle disease have been
constant problems for the poultry industry throughout the
world for many years. Both infections spread with great
rapidity, causing serious economic losses. In laying flocks,
the major loss is decreased production and poor quality of
eggs. In young chickens there may be appreciable mortality,
particularly with Newcastle disease, and a loss in feed
efficiency resulting in lowered weight gains.

Efficiency in the immune response to combined Newcastle
disease and infectious bronchitis vaccine versus single
vaccines was investigated using the hemagglutination-
inhibition (HI) microtiter test to measure the specific
antibody concentration in the sera.

The effect of two factors, timing and method of vaccina-
tion on the production of immunity, was analyzed. Vaccination
of chicks at 10 days of age and 21 days of age via drinking
water, intraocular or by a combination of the two routes

(ND by eye drop and IB in the drinking water) did not show

.-

Maria N. Narimatsu
a difference in immune response between separate and combined
vaccines. Furthermore, vaccination at 10 days of age with
revaccination at 15 days of age elicited a better immune
response than one vaccination, either vaccination at 10 days

or 21 days of age.

DEDICATION
To my parents, Tosiyuki and Kimie Narimatsu,

sisters and brothers in appreciation of
their love, guidance and perseverance.

ii

 

 

ACKNOWLEDGEMENTS

The author wishes to express her sincere appreciation
to her major professor, Dr. T. 8. Chang, for his guidance
and assistance throughout the research experiments.

The author also wishes to thank Drs. G. R. Carter,

T. H. Coleman, and R. K. Ringer for their help and
encouragement.

A special thanks is expressed to Dr. Mario Nakano,
chief of the Poultry Disease Section, Instituto Biologico
de Sao Paulo, for his direction and generous support for
this training program.

Sincere appreciation is extended to all faculty and
staff members of the Department of Poultry Science for their
companionship and hospitality during the author's tenure at
Michigan State University.

Many thanks to all my friends, especially to Dr. Maria
Fuentes, Dr. R. K. Maes, Mr. A. Wayne Roberts, and Mr. Paul
Watkins, for their assistance and encouragement.

Thanks and acknowledgement are expressed to the Brazilian
Government, which provided through the Empresa Brasileira
de Pesquisa Agropecuaria (BMBRAPA) and the Instituto Biologico

de San Paulo the financial support for this research training

program.

iii

 

IN

LI

ll-‘xl

TABLE OF CONTENTS

Page

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW. 3
Newcastle Disease . 3
Infectious Bronchitis 4
Vaccine . . 5
Methods of Vaccination. 7

Age for Vaccination . 8

Effect of Passive Antibodies on Immune Response 9
Method to Detect Immunity . 10
MATERIALS AND METHODS. 13
Experimental Chickens 14
Experimental Design . l4
Vaccines. 17

Feed Formula. 17
Viruses . 17
Preparation of Antigen. 19

Enzyme Preparation. 21
Procedure . 21

Serological Procedure for Newcastle Disease

Virus and Infectious Bronchitis Antibodies 23

Chicken Erythrocytes. 25

Serum for Serology. 26
RESULTS. 27
Study I: Newcastle Disease . . 27

Study II: Infectious Bronchitis. 30
DISCUSSION . 37
Newcastle Disease 38
Infectious Bronchitis 38
Conclusion. 39
BIBLIOGRAPHY . 40

iv

Page

APPENDICES . . . . . . . . . . . . . . . . . . . . . . 47
.Appendix A. 47
Appendix B. 61
Appendix C. 65
Appendix D. 69
Appendix E. 73

Table

LIST OF TABLES

Vaccination protocols
MSU pullet starter 6148

Hemagglutination-inhibition (HI) titers to NDV
and IBV in prevaccination control birds

Means of HI titer to NDV of chickens vaccinated
at different ages with separate and combined
vaccines. . . . . . . . . . ...

Means of HI titers to NDV of chickens vaccina-
ted at different ages and by different methods.

Means of HI titers to IBV of chickens vaccina-
ted at different ages with separate and
combined vaccines

Means of HI titers to IBV of chickens vaccina-
ted at different ages and by different methods.

Appendices

Hemagglutination-inhibition (HI) titers in
group ”CONTROL" - unvaccinated birds.

Hemagglutination-inhibition (HI) titers in
group "NEWCASTLE" - vaccinated with a single
Newcastle disease vaccine or "BRONCHITIS" -
vaccinated with a single infectious bronchitis
vaccine . . . . . . . . . . . . . . .

Hemagglutination-inhibition (HI) titers in
group "COMB-WATER" - vaccinated with a combined
ND/IB vaccine . . . . . . . . . . . . . . . .

Hemagglutination-inhibition (HI) titers in
group "COMB-EYE" - vaccinated with a combined
ND/IB vaccine .

Hemagglutination-inhibition (HI) titers in

group "COMB-SEP" - vaccinated with a combined
ND/IB vaccine .

vi

Page
15
-18

22

29

31

33

36

48

49

50

51

52

Table

A-6

A-lO

A-ll

A-lZ

C-3

Hemagglutination-inhibition (HI) titers in
group "CONTROL” - unvaccinated chicks

Hemagglutination-inhibition (HI) titers in
group "NEWCASTLE" - vaccinated with a single
Newcastle disease vaccine or "BRONCHITIS" -
vaccinated with a single infectious bronchitis
vaccine

Hemagglutination-inhibition (HI) titers in
group "COMB-WATER" - vaccinated with a combined
ND/IB vaccine . . . . . . . . . . . . . .

Hemagglutination-inhibition (HI) titers in
group "COMB-EYE" - vaccinated with a combined
ND/IB vaccine . . . . . . . . .

Hemagglutination-inhibition (HI) titers in
group "COMB-SEP" - vaccinated with a combined
ND/IB vaccine . . . . . . . . ... .

Hemagglutination-inhibition (HI) titers in
group "CONTROL" - unvaccinated chicks

Hemagglutination-inhibition (HI) titers in
group "NEWCASTLE" - vaccinated with a single
Newcastle disease vaccine or "BRONCHITIS" -
vaccinated with a single infectious bronchitis
vaccine .

Hemagglutination-inhibition (HI) titers in
group "COMB-WATER" - vaccinated with a combined
ND/IB vaccine . . . . . . . . . . . . . .
Statistical analysis program.

Means and standard deviations - effect of time
and method of vaccination in the production of
antibody to Newcastle disease vaccine . .

Results of Z-factor analysis of variance with
repeated measures

Statistical analysis program.

Means and standard deviations - effect of time
and method of vaccination in the production of
antibody to Newcastle disease vaccine

Results of Z-factor analysis of variance with
repeated measures

vii

Page

53

54

55

56

S7

58

59

60

62

63

64

66

67

68

Table
D-l

D-Z

D-3

E-3

Statistical analysis program.

Means and standard deviations - effect of time
and method of vaccination in the production of
antibody to infectious bronchitis vaccine

Results of Z-factor analysis of variance with
repeated measures

Statistical analysis program.

Means and standard deviations - effects of time
and method of vaccination in the production of
antibody to infectious bronchitis vaccine

Results of Z-factor analysis of variance with
repeated measures . . . . . . . . . . . .

viii

Page

70

71

72

74

75

77

 

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OPPO

INTRODUCTION

Infectious bronchitis and Newcastle disease have been
a constant problem for the poultry industry throughout the
world for many years. Both infections spread with great
rapidity, causing serious economic losses. In laying flocks,
the major loss is decreased production and poor quality of
eggs. In young chickens there may be appreciable mortality,
particularly with Newcastle disease, and a loss in feed
efficiency resulting from lowered weight gains.

The purpose of this thesis was to compare the efficacy
of combined Newcastle disease and infectious bronchitis
(ND/1B) vaccines versus single vaccines. Two different
factors were considered--time of vaccination and method
of vaccination- -to analyze the effects of vaccination on
the production of antibody to Newcastle disease (ND) virus
in birds vaccinated with ND vaccine or IB vaccine alone or
in combination (ND/IB). The use of combined vaccine as
Opposed to two single vaccines would help to reduce the cost
of production and the stress on birds through less handling
and manipulation.

The immune response of individual chickens of different
ages exposed to different methods of vaccination against

Newcastle disease and infectious bronchitis was measured by

tflle~hemagglutination-inhibition (HI) test. This is a
1

2
convenient procedure for measuring the specific antibody
concentration in the sera, and the level of the latter test

is known to reflect the immune status of the bird to some

extent.

LITERATURE REVIEW

Newcastle Disease

 

Newcastle disease (ND) is an acute, highly contagious
and destructive disease of chickens and occasionally of
other fowls. It is characterized by respiratory distress
and encephalitis. Humans are susceptible and, when infected,
may deveIOp conjunctivitis (Hanson and Brandly, 1958;

Buxton and Fraser, 1977; Hanson, 1978).

The causative agent has been established as a RNA virus
of the paramyxo group of viruses (Lancaster, 1976). There
are several strains of the virus classified according to
virulence of the strains: lentogenic, mesogenic and velo-
genic. All three types cause losses in egg production in
laying birds (Hanson and Brandly, 1955; Grass, 1971;
Utterback and Schwartz, 1973). The strain of Newcastle
disease virus isolated in the current worldwide panzootic
and the 1971-1973 epizootic in California was classified as
velogenic viscerotropic (Utterback and Schwartz, 1973).

Nervous symptoms occur in some birds, especially young
ones. These symptoms include paralysis of the legs or wings,
and torticollis, resulting in a complete twisting of the
neck. In laying flocks the major loss is decreased produc-

tion of eggs and poor egg quality.

4

The incubation period is from two to five days. The
morbidity rate is high, and the mortality rate varies with
the age of chickens. The virus can be readily cultivated
in chicken embryos inoculated via the allantoic sac. The
virus has been grown in tissue cultures producing cytopathic
effects (Buxton and Fraser, 1977).

An important property of the Newcastle disease virus is
its capacity to agglutinate red cells. Avian erythrocytes
are commonly used for hemagglutination studies; however, red
cells of turkey and other avian Species can also be used.
Human, mouse, and guinea pig erythrocytes are also agglu-
tinated by the virus (Buxton and Fraser, 1977; Hanson, 1978).

The hemagglutinating activity of Newcastle disease
virus and the property of antiserum to specifically inhibit
such hemagglutination were first demonstrated by Burnet
(1942). The hemagglutination (HA) and the hemagglutination-
inhibition (HI) tests have since proved to be of great value

in diagnosis and research.

Infectious Bronchitis

Infectious bronchitis (IB) is an acute, highly contagious
viral reSpiratory disease of young and adult chickens and is
caused by the infectious bronchitis virus (IBV), a member
of the coronavirus group (Cunningham, 1975; Hofstad, 1978).
The disease is characterized by a bronchitis in young chickens
with characteristic gasping and a sudden drop in egg produc-
tion in layers. The disease was first identified in 1931 in

North Dakota by Shalk and Hawn and soon became widespread

(Hofstad, 1978).

5

Several distinct serotypes exist, such as Massachusetts,
Connecticut, Beaudette, JMK, Florida strain, etc. The
Massachusetts serotype is most common in poultry producing
areas and is used as seed virus for most IBV vaccine.
Antigenic variations among strains of bronchitis virus have
been described by Hofstad (1961). Despite some antigenic
difference among serotypes, they are closely related in
regard to immunogenicity.

The incubation period fiyrIB is from one to four days.
The morbidity rate is high, and the mortality rate varies
with the age group of chickens (Cunningham, 1952). Young
birds are considerably more susceptible. The virus grows
well in embryonating chicken eggs (Hofstad, 1978; Cunningham,
1975) and can be grown in cell cultures of the chicken
embryo (Hofstad, 1978) and in embryonic turkey kidney cells
(Coria and Peterson, 1971).

Normally the virus does not adsorb to the surface of
erythrocytes, but modification of the virus by enzymatic
treatment induces the hemagglutinating activity of the virus

(Corbo and Cunningham, 1959).

Vaccine

Vaccination has proved to be a practical method of
controlling Newcastle disease and infectious bronchitis
(Luginbuhl et al., 1955; Winterfield and Seadale, 1956;
Winterfield et al., 1957). Immunization has been carried
Ont since the development of vaccines in 1940 (Phillips,

1973). A variety of vaccines, vaccination programs and

 

p)

6
methods of administration have been introduced. It is
important for poultrymen to use the most efficient vaccina-
tion program.

Mass immunization of poultry against Newcastle disease
(ND) and infectious bronchitis (IB) either alone or in com-
bination has been reported by many investigators using tech-
niques such as aerosol or Spray (Crawley and Fahey, 1954;
Cough and Allan, 1973; Cough and Alexander, 1978; Yadin and
Orthel, 1978), dust (Markham et al., 1955), or by adding
vaccine to the drinking water (Luginbuhl et al., 1955;
Winterfield et al., 1957; Jordan and Nassar, 1973; Gough
et al., 1977).

Because of the necessity for vaccinating large numbers
of birds, and of the time and expense involved in repeated
vaccinations, the bronchitis vaccines have been combined
with Newcastle disease vaccines without interference in the
immune response from each vaccine (Markham et al., 1956).
However, there have been conflicting reports with regard to
these two viruses in certain combinations (Luginbuhl et al.,
1955). Raggi and Lee (1964) reported that the IBV component
of the vaccine interfered with the establishment of immunity
to Newcastle disease. Winterfield (1968) has found some
interference and a more prolonged reaction when bivalent
vaccines were used. Thornton and Muskett (1973) reported
a low rate of protection to artificial challenge with NDV
in chickens inoculated simultaneously with commercially
available monovalent ND and IB vaccines. Markham et a1.

(1956) showed an absence of interference when a combined

7
Newcastle disease and infectious bronchitis vaccine was
given to birds under optimal conditions. Zygraich et al.
(1973) reported no interference and no significant differ-
ences between birds vaccinated with the combined or the

separate vaccine.

Methods of Vaccination

 

Aerosol methods have been increasingly used for the
administration of Newcastle disease and infectious bronchitis
vaccines, either alone or in combination. In the aerosol
administration of the vaccine, a number of factors can
influence successful vaccination, such as the particle size
and distribution, virus concentration and stability
(Markham et al., 1955; Cough and Allan, 1973; Yadin and
Orthel, 1978).

Markham et al. (1955) reported that spray vaccine pre-
pared from the B1 strain of Newcastle disease virus and the
DG strain of infectious bronchitis virus, either alone or
in combination, could be successfully employed for mass
vaccination when dispersed as dusts over the heads of birds.
High titer of hemagglutination inhibition (HI) antibodies
and good protection have been obtained in the field (Price
et al., 1955). Gough and Allan (1973) have shown that the
aerosol route of administration can elicit protection within
three days in the absence of a detectable rise in antibody
titer. Gough and Alexander (1979) found no major difference
in the immune response following vaccination with live IB

vaccine by aerosol, intraocular and drinking water routes.

 

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The mass vaccination technique in the drinking water
has become a routine procedure with poultry farmers because
it is labor saving, causes less stress, and generally pro-
duces satisfactory results in controlling the Newcastle
disease and infectious bronchitis (Luginbuhl et al., 1955).
The drinking water method is simple, fast, inexpensive, and
handling of the birds is not required. It is an effective
way of administering vaccine to all birds in a flock. Lugin-
buhl et a1. (1955) demonstrated the practicability of immuniz-
ing chickens with IE and ND when these viruses were mixed

and added to the drinking water.
Age for Vaccination

Newcastle disease. Buxton and Fraser (1977) described
a standard program of immunization of replacement birds
against ND which gave maximum protection: first vaccination
at 21 days of age; revaccination at 8-10 weeks; again at
16-20 weeks; and every 5 months thereafter. Immunization of
chickens at one day of age always resulted in a poor immune’
response. Chickens are revaccinated when they are under 4
weeks of age to insure the production of an adequate level
of immunity. Allan (1973) reported that the vaccine is given
at 1 to 7 days of age and revaccination at 14 days of age

or later either by drinking water or aerosol.

Infectious bronchitis. The first vaccination in broilers
is recommended at an early age (4 to 5 days of age) and again

at 4 weeks. Replacement flocks should be vaccinated at 2 to

...L- c-

 

9
4 months (Hofstad, 1978). Davelaar and Kouwenhoven (1977)
reported that they vaccinated broilers at 6 to 14 days of
age either in the drinking water or by the spray method.

Effect of Passive Antibodies
on Immune Response

 

 

It has been stated by several authors that congenital
passive immunity may influence the immune response of young
chickens to vaccination (Lancaster, 1966; Allan, 1971, 1974;
Gough and Allan, 1976).

Brandly et a1. (1946) reported that passively conferred
immunity protected chicks against infection with ND virus
but interfered with active immunization. Bankowski and
Corstvet (1962) have shown that maternal immunity and
residual immunity at time of vaccination with B1 strain
vaccine can affect the immunity induced. Holmes (1979) also
found markedly suppressed antibody response when chickens
with passively acquired antibody were vaccinated with live
NDV vaccine. However, Raggi and Lee (1965) found that
passive antibodies did not materially influence immune
response to live virus vaccine as judged by challenge.
Davelaar and Kouwenhoven (1977) have demonstrated that immuni-
zation against 18 by vaccinating l-day-old birds by the
conjunctival and intranasal routes, despite the presence of
high levels of circulating maternal antibody, was as effective
as vaccination at an age of 15 days or later when passive

Protection has decreased.

10

Method to Detect Immunity

 

For determining flock immunity to Newcastle disease
(ND) the most commonly used serological methods are the
hemagglutination inhibition (HI) test (Allan and Gough,
1974; Spanoghe et al., 1977), and serum neutralization (SN)
test (Bankowski and Corstvet, 1962; Beard, 1971) by measure-
ment of antibody concentration in the sera. Another commonly
used device to determine immunity is the challenge test
(Spanoghe et al., 1977).

Under commercial conditions, high concentrations of
serum antibody are generally accepted as a reliable indica-
tor of flock immunity, but Levine and Fabricant (1950),
Beard and Easterday (1967), and Allan (1975) have shown a
lack of correlation between serum antibody concentration
and resistance of the respiratory tract to challenge.

The usual method of detecting immunity to IBV following
vaccination has been reported to be by serum neutralization
(SN) test (Cunningham, 1973; Gough and Alexander, 1978;
Hofstad, 1978), agar gel precipitin (AGP) tests (Gough and
Alexander, 1978) and challenge of vaccinated fowls 3 to 6
weeks after vaccination (Winterfield and Fadly, 1971;
Winterfield et al., 1972).

The serum neutralization (SN) test in eggs (Page and
Cunningham, 1962; Cunningham, 1973) has been the method used
most commonly, but it is time consuming, expensive, and it
is often difficult to determine accurate endpoint titers.

TPhe disadvantages of the SN test led to the development of

11
a HI test for the detection of antibodies to infectious
bronchitis virus.

Recently, many workers have shown the usefulness of the
HA and HI tests in serological studies (Corbo and Cunningham,
1959; Biswal et al., 1966; Bingham et al., 1975; Alexander
and Chettle, 1977; Bahl et al., 1977; Macpherson and Feest,
1978).

During the last few years several procedures for the
production of hemagglutinating virus and for the HA and HI
titrations for detecting antibodies to IBV have been reported.
This hemagglutinating activity of the virus has been induced
by enzymatic treatment of the virus or by chemical modifi-
cation of the erythrocyte surface (Corbo and Cunningham,

1959; Brown et al., 1962; Bingham et al., 1975; Alexander
et al., 1976; Alexander and Chettle, 1977; Bahl et al.,
1977).

Corbo and Cunningham (1959) described a hemagglutination
test for infectious bronchitis using a trypsin modified
virus, but the hemagglutination was not specifically inhibited
by immune serum. Recently Bingham et a1. (1975) have reported
that IBV Massachusetts, strain 41, when treated with phospho-
lipase C (type 1), will agglutinate chicken red blood cells
and that this hemagglutination (HA) could be inhibited by
specific antisera. Alexander et al. (1976), in a preliminary
examination of 9 strains of IBV, found 4 strains showing HA
activity after treatment with phospholipase C (type 1). It
was found that IBV M-4l strain possessed the best hemagglu-

tinating properties for use in the HI test and that results

12
compared well with the IBV SN test. Later, Alexander and
Chettle (1977) confirmed this work and developed a test
system which was as reproducible as were the HA and HI tests
for work with Newcastle disease (ND) virus.

Bahl et al. (1977) investigated the hemagglutinating
ability of 2 strains of infectious bronchitis virus after
the virus had been treated with phospholipase C (type 1)
and found that Beaudette strain caused no detectable hemag-
glutination. However, Massachusetts strain 41 agglutinated
chicken red blood cells (CRBC). This hemagglutination
(HA) would be specifically inhibited by antisera.

Alexander et al. (1976) and Bahl et al. (1977) have
shown the usefulness of the HI test for IBV as a rapid,
simple, inexpensive and highly reproducible method of

measuring antibodies against IBV.

MATERIALS AND METHODS

Time of vaccination was studied by comparing 3 groups:
(1) vaccination at 10 days of age and revaccination at 15
days of age; (2) vaccination at 10 days of age; and (3)
vaccination at 21 days of age. Groups (2) and (3) will
allow for a comparison of effectiveness of early versus
late vaccination, especially in view of the inhibiting
effect of maternal immunity of the chick, while group (1)
will allow for testing of the possibility to vaccinate
early yet, through revaccination, compensate for the inhi-
bitory effect of maternal immunity.

Method of vaccination was studied by comparing 4 groups
(plus 3 control groups): (1) CONTROL group, (a) bled at
10 days, (b) bled postvaccination, (c) bled pre- and post-
vaccination; (2a) NEWCASTLE vaccinated with ND vaccine in
the drinking water, (2b) BRONCHITIS vaccinated with IE
vaccine in the drinking water; (3) COMB-WATER vaccinated
with a combined (ND/1B) vaccine in the drinking water;
(4) COMB-EYE vaccinated with a combined (ND/1B) vaccine by
eye dr0p; (5) COMB-SEP vaccinated with a combined (ND/1B)
vaccine, ND vaccine by eye drop and IB vaccine in the drink-
ing water (the comparison between groups (1b and 1c) and
groups (2) through (5) was done to establish if in fact

antibody was produced in the latter groups, not to test
13

14
if vaccination produces antibody, which has been
established sufficiently [Hanson, 1978; Hofstad, 1978]).
The comparison between group (2) through (5) will test the
relative effectiveness of producing antibody from the dif-

ferent methods of vaccination.

Experimental Chickens

 

A total of 235 White Leghorn male chickens from the
same hatch were used. They were raised in the same
battery until vaccination in 2 sets of 135 and 100 chickens,

respectively.

Experimental Design

 

The objective of this study was to analyze the effect
of 2 factors on the production of immunity: time of vaccina-
tion and method of vaccination. Thus, the following groups

of animals were treated.

Factor 1. Time of vaccination was as follows: (1) vac-

 

cination at 10 days of age and revaccination at 15 days of
age; (2) vaccination at 10 days of age only; and (3) vaccina-
tion at 21 days of age. It should be noted that subjects

for groups (2) and (3) were taken from one set of a total of
135 animals, while group (1) was taken from a second set of

a total of 100 birds.

Factor 2. Method of vaccination protocols for the 3
groups of Factor 1 (above) are presented in Table l (a, b,

and c, respectively).

15

Table l. Vaccination protocols

 

Number of
Group birds Treatment Route

1A: Vaccination at 10 days and revaccination at 15 days of age

la "CONTROL" 10 unvaccinated (bled at 10 days)
1b "CONTROL" 15 unvaccinated (bled parallel to
treated groups)

2a "NEWCASTLE" 15 Newcastle disease D.W.*

2b "BRONCHITIS" 15 Infectious bronchitis D.W.

3 "COMB-WATER" 15 combined ND/IB D.W.

4 "COMB-EYE" 15 combined ND/IB I.O.**

5 "COMB-SEP" 15 combined ND/IB ND=I.O.
IB=D.W.

1B: Vaccination at 10 days of age

1c "CONTROL" 15 unvaccinated (bled at 10 days
and parallel to treated groups)

2a "NEWCASTLE" 15 Newcastle disease D.W.

2b "BRONCHITIS" 15 Infectious bronchitis D.W.

3 "COMB-WATER” 15 combined ND/IB D.W.

4 "COMB-EYE" 15 combined ND/IB 1.0.

5 "COMB-SE?" 15 combined ND/IB ND=I.O.
IB=D.W.

16

Table 1 (continued)

 

Number of

 

 

 

Group birds Treatment Route
1C: Vaccination at 21 days of age
2a "NEWCASTLE" 15 Newcastle disease D.W.
2b "BRONCHITIS" 15 Infectious bronchitis D.W.
3 "COMB-WATER" 15 combined ND/IB D.W.
.1. * .
D.W. = drinking water
a:
1.0. = intraocularly

17

Vaccines

Three commercially available vaccines, B1 type LaSota
strain live virus Newcastle disease; Massachusetts and
Connecticut strains live virus bronchitis; and combined
Newcastle-infectious bronchitis B1 type, LaSota strain -
Mass. 6 Conn. strains live virus recommended for primary
vaccination of fowls by the manufacturers, were used.

Each group of chickens was vaccinated with one of the
commercial vaccines administered by drinking water, eye

drOp or a combination of the two.

Feed Formula

 

The feed formula used to maintain the chicks is presented

in Table 2.

Viruses

Newcastle antigen LaSota strain (10 HAU/0.025 m1),
Newcastle disease virus antiserum (2/80 chicken), IBV
Massachusetts antiserum #041679, and normal chicken serum
(032880) were provided by USDA.*

The Massachusetts 41 (M-41) strain of infectious bron-

7.8

chitis virus (IBV) titer 10 per ml #081277 was supplied

by ASL.**

 

at
USDA - Veterinary Service Laboratory, Ames, Iowa.

**
ASL - The American Scientific Laboratories, Madison,
Wisconsin.

18

Table 2. MSU pullet starter 6148

 

 

Guaranteed Analysis 1/1/80
Crude protein not less than 20.0% Variation
Crude fat not less than 2.5 LNS
Crude fiber not more than 10.0

Ingredients: Grain products, plant protein products, animal
protein products, forage products, cane molasses, vitamin

B-12 supplement, ethoxyquin (a preservative), DL methionine,
choline chloride, niacin, folic acid, vitamin A supplement,
riboflavin supplement, vitamin E supplement, calcium panto-
thenate, D activated animal sterol, menadione sodium bisul-
fite (source of vitamin K activity), calcium carbonate,
defluorinated phosphate, magnesium sulfate, potassium sulfate,
salt, sodium selenite, manganous oxide, calcium iodate,

copper oxide, zinc oxide. AG-6148

DIRECTIONS

 

Feed as the sole ration to starting pullets according to
Michigan State University recommendations.
Manufactured by

Ralston Purina Co., Gen. Offices, St. Louis, MO 63188.

19
Preparation of Antigen

The infectious bronchitis antigen production was based
on the procedure described by Alexander and Chettle (1977)
and Bahl et al. (1977) using the M-41 strain of IBV as the
seed virus to provide the antigen for both hemagglutination
(HA) and the hemagglutination-inhibition (HI) tests, except
that the virus was stored at -20°C after phospholipase C
(PLC) treatment.

The Massachusetts 41 (M-4l) strain of infectious bron-
chitis virus was propagated in embryonated chicken eggs,
concentrated and treated with phospholipase C type 1 (PLC).

Ten-day-old embryonating chicken eggs were infected by
inoculating 100 EIDSO of M-41 in 0.1 ml into the allantoic
sac.

Infected eggs were incubated at 37°C for 72-96 hours.
Embryos that died up to 24 hours after inoculation were
discarded as non-specific. The remaining eggs were chilled
at 4°C overnight and the allantoic fluid was harvested. At
all times during harvesting and subsequent preparation for
enzyme treatment, the allantoic fluid was kept chilled in
an ice bath. The allantoic fluid was clarified by low speed
centrifugation. The virus was then centrifuged at 30,000 G
to concentrate lOO-fold by pelleting at 4°C for 1 hour in
the SW-27 rotor of a Sorvall-OTD-Z (DuPont Company, Instru-
ments Products, Biomedical Division, Newtown, CT 06470)
ultracentrifuge. The pellet was resuspended in 0.01M TRIS/
HCl buffer at pH 6.5. An equal volume of phospholipase C

' type 1 containing 1 unit of enzyme per ml was added to the

20
virus suspension and the mixture was incubated in a water
bath for 2 hours at 37°C. This antigen was titrated (HA)
and then dispensed into aliquots and stored at -20°C until
use.

The Newcastle disease antigen was produced by the method
described by Beard and Wilkes (1973) and modified by Schwartz
(1980) using the commercial LaSota strain as the seed virus.

Nine- to 10-day-old embryonated chicken eggs were

2 dilution of commercial LaSota strain

inoculated with 10-
vaccine in 0.1 m1 into allantoic sac. Infected eggs were
incubated at 37°C for 60 to 72 hours. Embryos that died up
to 24 hours after inoculation were discarded as non-specific.
The remaining eggs were chilled at 4°C overnight and the
allantoic fluid was harvested and frozen. The fluids were
thawed and 0.1% formalin added by volume, and held at 37°C
for 36 hours. The 2% (w/v) NaCl and 10% (w/v) polyethylene
glycol (molecular wt 6000) (all reagent grade chemicals)

were added and held at 4°C for 2 hours.

The virus was then centrifuged in a refrigerated (4°C)
Sorvall centrifuge at 4000 rpm for 1.5 hours, using a GSA I
head. The sediment (pellet) was reconstituted at 20X con-
centration in phosphate buffer. The concentrated antigen
was then sonicated for 2 to 3 minutes to disperse finely and
mix thoroughly. An equal volume of 100% glycerin was added
to the virus suspension; the antigen was checked for HA
titer and then dispensed in aliquots and diluted as needed,

using saline.

21

Enzyme Preparation

Phospholipase C type 1 from CZostridium perfringens
(C. welchii) (Sigma Chemical Company) was made up to contain
5 units per ml in phosphate buffered saline, pH 7.2 (PBS),
divided in vials, stored at -20°C and used to treat virus
in the manner described by Alexander et al. (1976),
Alexander and Chettle (1977) and Bahl et al. (1977) at a

final concentration of 1 unit of enzyme per m1.

Procedure

 

Control groups. A total of 40 birds served as

 

controls, as indicated in Table l (A and B). Control

group (1a) was bled at 10 days of age to establish maternal
immunity level at the time of vaccination for the respective
group (Lot 2); control group (1c) was bled at 10 days of age
to establish the maternal immunity level for Lot 1A and at
21 days of age to establish the maternal immunity level for
Lot 13, as presented in Table 3. Control groups (lb) and
(1c) were bled parallel to the experimental groups, 5 times,
in weekly intervals, beginning at 22 days of age and 20 days

of age,respectively.

Experimental groups. All chickens to be vaccinated
were deprived of water for 4 hours immediately before vaccina-
tion. The vaccine was given in quantities of water that
would be consumed in approximately 1 hour and at the manu-
facturer's recommended dose. After the drinking water
vaccine was consumed, the waterers were filled with fresh

water.

22

Table 3. Hemagglutination-inhibition (HI) titers to NDV
and IBV in prevaccination control birds

 

 

 

 

 

 

 

Control (la) Control (1;)

10 days of age 10 days ofiage 21 days of age
Bird # NDV IBV Bird—Y' NDV IBV NDV IBV
01 2* 512* 7777 0 8 0 - 2
02 2 128 7778 0 l6 0 2
03 2 128 7796 0 l6 0 8
04 2 128 7780 0 16 0 2
05 2 64 7783 0 8 0 2
06 2 64 7784 0 8 0 4
07 0 64 7785 0 16 O 8
08 0 64 7786 0 8 0 4
09 0 4 7787 0 16 O 4
10 0 4 7789 O 16 0 4
7790 0 8 O 2
7792 0 16 0 8
7793 0 l6 0 8
7794 0 16 0 8
7795 0 8 0 4

*
Titers expressed as the reciprocal of the serum

dilution.

23

The water used was sterile distilled. The waterers
were sterile plastic water cups.

Seven days after the revaccination (Lot 2) and 10 days
after vaccination for the other groups and at weekly intervals,
5 samples of serum were collected from the birds and tested
individually for specific antibodies for Newcastle disease
and infectious bronchitis (see Appendix A for raw data).

The immunity was evaluated by the average HI antibody status
measured weekly from serum samples as described by Cunningham
(1966) and Bingham 6t al. (1975)-

Serolggical Procedure for

Newcastle DISease Virus and

Infectious Bronchitis

Antibodies

Hemagglutination (HA) and hemagglutination-inhibition
(HI) tests. The immune response to infection was measured
by hemagglutination-inhibition (HI) test for IBV (Alexander
and Chettle, 1977; Bahl et al., 1977) using M-41 strain
treated with phospholipase C type 1 as antigen and for NDV
using LaSota strain as antigen (Beard and Wilkes, 1973;
Schwartz, 1980).

Hemagglutination and hemagglutination-inhibition titers
were carried out according to Cunningham (1966) and Bingham
et al. (1975) performed in Microtiter "U" bottom plates using

a manual 0.025 ml microtiter apparatus.* All dilutions of

 

*
Cooke Engineering Company, 900 Slater Lane,
Alexandria, Virginia.

24
virus or antisera were made in phosphate buffered saline,
pH 7.2 (PBS). The HI test was routinely carried out at

4°C.

Hemagglutination (HA) test. Twenty-five microliters
of virus suspension Inns serially diluted in 25 ul volume
of PBS and an equal amount of 0.5% suspension of chicken
erythrocytes was added to each well. The control contained
0.025 ml of PBS and 0.025 ml of RBC. The plate was shaken
gently and incubated at 4°C for 45 to 60 minutes. Hemag-
glutination was determined by observing the pattern formed
by the cells. Hemagglutination titers were expressed as
the reciprocal of the highest dilution of virus at which
100% of the area agglutinated (Bahl et al., 1977). The titer
of the antigen obtained was used to calculate the dilution
necessary to give a solution (in PBS) containing 4 HA units

in 0.025 ml for IE and 10 HA units in 0.025 ml for ND.

Hemagglutination-inhibition (HI) test. The beta-HI
test, which uses constant antigen and varying serum concen-
tration (Cunningham, 1966; Beard and Wilkes, 1973; Allan and
Gough, 1974; Bingham et al., 1975) was used with 4 HA units
.(M-41 strain) for infectious bronchitis and 10 HA units
(LaSota strain) for Newcastle disease as antigen dose.

Constant amounts of virus in 25 ul of antigen were
added to each dilution (decreasing concentration) of serum,
ranging from 1:2 through 1:2048. The serum-antigen mixture

was incubated at 4°C for 15 minutes before adding 0.5%

25
suspension of chicken erythrocytes followed by further incu-
bation at 4°C for 45 to 60 minutes. Individual HI titers
were expressed as the reciprocal of the highest serum
dilution (in 0.025 ml) causing a detectable inhibition of
the agglutination.

Analysis of variance was used to express the average of
each bleeding (HI titers) for the different groups of chickens,
in order to compare the immune response to the different vac-
cines used separately or as a combination. The analysis of
variance for repeated measures was performed using the BMDPZV
program (Dixon, 1977). A further test used was Tukey's test

to detect any difference between means, according to Gill (1978).

Chicken Egythrocytes

 

Blood was obtained from Single Comb White Leghorns by
vein puncture. Red blood cells were collected in sterile
Alsever's solution ("Manual of Microbiological Methods” in
Society of American Bacteriologists, McGraw-Hill Book Co.,
Inc., New York, 1957).

The blood was centrifuged and the supernatant fluid
removed. The cells were washed 3 times by centrifugation for
10 minutes at 1500 rpm in phosphate buffered saline (PBS).
After the last wash the erythrocytes were suspended in PBS at
a concentration of 0.5% for immediate use. A 0.5% cell sus-
pension in PBS was used for hemagglutination and hemagglu-

tination-inhibition tests.

26
Serum for Serology
Blood samples for serology were obtained by cardiac
puncture. They were allowed to clot at room temperature and
then stored overnight at 4°C, at which time the serum was
transferred to sterile tubes. Before testing, sera were

inactivated in a water bath at 56°C for 30 minutes prior to

use in the HI test.

RESULTS

Study 1: Newcastle Disease

 

Two different designs were followed to analyze the
effects of vaccination on the production of antibody to
Newcastle disease (ND) virus in birds vaccinated with ND
vaccine alone or in combination (ND/1B). Both designs pro-
vided for a 2-factor analysis of variance with repeated
measures, the 2 factors being (1) time of vaccination and

(2) mode of vaccination.

Design 1. In the first design the factor "time of
vaccination" was compared in 3 ways: "LOT 2" - 10 days
after hatching with revaccination on day 15; "LOT 1A" - 10
days after hatching; and "LOT 1B" - 21 days after hatching.
The second factor, "mode of vaccination", compared 2 dif-
ferent methods as follows: "NEWCASTLE" - vaccination with
ND vaccine alone, and "COMB-WATER" - vaccination with a com-
bined ND/IB vaccine in the drinking water. The dependent
variable, amount of antibody produced, was determined from
5 bleedings at intervals of 7 days each, beginning 7 days
after revaccination for "LOT 2" and 10 days after vaccination

in the cases of "LOT 1A" and ”Lot 1B."

27

28

The means for the amount of antibody are presented in
Table 4.

An analysis of variance for repeated measures was per-
formed using the BMDPZV program (Dixon, 1977). For the
complete program, see Appendix B. The results of this
analysis are presented in Table B3. The results indicated
that there was only one significant difference (P50.05) in
the amount of antibody produced, viz., on factor one, "time
of vaccination", and, as can be seen from Table 4, "LOT 2" -
vaccination on day 10 and revacCination on day 15 produced
the highest level of antibody response among the 3 groups
in contrast to "LOT 1A" - vaccinated at 10 days of age, and
"LOT 1B" - vaccinated at 21 days of age. No significant
difference over time was found; i.e., the relative amount

of antibody remained approximately the same (Table B3).

Design 2. In the second design, the animals were
vaccinated at 2 different times: "LOT 2" - 10 days of age
with revaccination at 15 days of age, and "LOT 1A" - vaccina-
ted at 10 days of age. Furthermore, 4 methods of vaccination
were contrasted: "NEWCASTLE" - vaccination with ND vaccine
alone in the drinking water; "COMB-WATER" - vaccination of
combined ND/IB vaccine in the drinking water; "COMB-EYE" -
vaccination of combined ND/IB vaccine intraocularly; and
"COMB-SEP"- the vaccination of ND vaccine by eye drop and
IB vaccine in the drinking water. As in Design 1, the

dependent variable, i.e., the amount of antibody produced,

was determined through 5 bleedings with intervals of 7 days

29

Table 4. Means of HI titer to NDV of chickens vaccinated
at different ages with separate or combined

vaccines

 

Treatment

Means Titer

 

Lot—2 ‘Lot 1A Lot 1B

Total
Means

 

Newcastle disease

vaccine

Combined ND/IB
vaccine

14.88

20.43

10.13
12.01

12.15
12.01

 

Total means

*
D.W. =
a,b

17.663

12.41

drinking water

Tukey's test.

Means not sharing the same letter are Signifi-
cantly different (PS0.05).

 

~\\\

30

each, beginning 7 days after revaccination for "LOT 2" and
10 days after vaccination in the cases of "LOT 1A" and
"LOT 1B."

The means for the amount of antibody are presented in
Table 5.

The results for analysis of variance for repeated
measures are presented in Table C3 (for complete program %
see Appendix C). There was a significant difference between [LI'

time of vaccination (P<0.05), in the sense that condition 5

"LOT 2", repeated vaccination, produced a higher level of

 

1 “nu '.m u
'efi-F? ,
5

antibody. Also, there was a significant effect for method
of vaccination and for the interaction method vs. time
(P<0.05). The means were compared within each lot. In

lot 2, "COMB-WATER" and "COMB-EYE" gave higher values than
"COMB-SEP"£uu1"NEWCASTLE" alone. However, no significant
difference was found between means when they were compared
using Tukey's test. In lot 1A, the mean values for the
different treatments were very similar and no significant
difference was detected. Again, no differences over time
were found; i.e., the amount of antibody detected during the

5 bleedings remained approximately the same.

§tudy II: Infectious Bronchitig

Two different designs were followed to analyze the
effects of vaccination on the production of antibody to
infectious bronchitis (IB) virus in birds vaccinated with
IR vaccine alone or in combination (ND/1B). Both designs

provided fer a 2-factor analysis of variance with repeated

31

 

 

 

Table 5. Means of H1 titers to NDV of chickens vaccinated
at different ages and by different methods
Means Titer
Treatment Method “Lot 2 Lot 1A7
Newcastle D.W.* 14.88:3.62 7.47:3.31
Combined ND/IB vaccine D.W. 20.43i3.62 8.52:3.31
Combined ND/IB vaccine I.O.** 23.64:3.62 6.32:3.31
Combined ND/IB vaccine ND=I.O. 16.50:3.62 6.93:3.31
IB=D.W.

drinking water

intraocularly

32
measures, the 2 factors being (1) time of vaccination and

(2) mode of vaccination.

Design 1. In the first design the factor "time of
vaccination" was compared in 3 ways: "LOT 2" - 10 days
after hatching with revaccination on day 15; "LOT 1A" - 10
days after hatching; and "LOT 1B" - 21 days after hatching.
The second factor, "mode of vaccination", compared 3
different methods as follows: "CONTROL" - unvaccinated
birds; "BRONCHITIS" - vaccination with IE vaccine alone in
the drinking water; and "COMB-WATER" - vaccination with a
combined ND/IB vaccine in the drinking water. The dependent
variable, i.e., the amount of antibody produced, was
determined from 5 bleedings at intervals of 7 days each,
beginning 7 days after revaccination for "LOT 2" and
"LOT 18."

The means for the amount of antibody are presented in
Table 6.

That vaccination, in comparison to non-vaccination,
will produce antibody is widely known (Hanson, 1978; Hofstad,
1978); thus, the reason for the introduction of the control
group, i.e., unvaccinated birds, was to show that vaccina-
tion had indeed taken place. The objective of this thesis
is to investigate whether time or method of vaccination
would make a difference in the production of antibody. For
this reason, the data from the control group are not included

in the analysis of variance that follows.

L-

33

Table 6. Means of HI titers to IBV of chickens vaccinated
at different ages with separate or combined

 

 

 

 

 

 

vaccines
Means Titer Total
Treatment Method Lot 2 Lot 1A Lot 1B Means
Control (unvac-
cinated) 3.93 1.55 1.34
Infectious bronchitis
vaccine D.W.* 33.92 13.06 17.13 21.37
:3.13
Combined ND/IB
vaccine D.W. 35.34 10.22 12.36 19.31
:3.13
Total Means 34.63a 11.64b 14.75b
$3.78 $3.78 23.78
*
D.W. = drinking water
a,b

Means not sharing the same letter are signifi-
cantly different (PS0.05). Tukey's test.

34

The results of this analysis of variance are presented
in Table D3 (for complete program, see Appendix D) and
indicate that there is only one significant difference
(P50.05), namely on factor one: "time of vaccination."
As may be noted from Table 6, "LOT 2", vaccination on day
10 and revaccination on day 15, produced the highest level
of antibody response among the 3 groups in comparison with
"LOT 1A", vaccinated at 10 days of age, and "LOT 1B",
vaccinated at 21 days of age. No differences over time
were found; i.e., the relative amount of antibody remained

approximately the same.

Design 2. In the second design, the animals were vac-
cinated at 2 different times: "LOT 2" - 10 days of age
with revaccination at 15 days of age, and "LOT 1A" -
vaccinated at 10 days of age. Furthermore, 5 methods of
vaccination were compared: ”CONTROL" - unvaccinated birds;
"BRONCHITIS" - vaccination with IB vaccine alone in the
drinking water; "COMB-WATER" - vaccination of combined ND/IB
vaccine in thedrinking water; "COMB-EYE" - vaccination of
combined ND/IB vaccine intraocularly; and "COMB-SEP" -
vaccination of ND vaccine by eye drOp and IB vaccine in
the drinking water. As in Design 1, the dependent variable,
the amount of antibody produced, was determined from 5
bleedings at intervals of 7 days each, beginning 7 days
after revaccination for "LOT 2" and 10 days after vaccina-

tion in the cases of "LOT 1A" and "LOT 1B."

35

The means for the amount of antibody are presented in
Table 7. Again, the control group (unvaccinated birds) was
not included in the analysis of variance.

The results of the analysis of variance for repeated
measures are presented in Table E3 (for complete program,
see Appendix E). As may be noted, there was only one sig-
nificant difference (P50.05), viz., on factor one, "time
of vaccination" and, as can be seen from Table 7, "LOT 2",
repeated vaccination, produced a higher level of antibody.
Again, no difference over time was found; i.e., the relative

amount of antibody remained approximately the same.

tauqm

 

36

Table 7. Means of H1 titers to IBV of chickens vaccinated
at different ages and by different methods

 

Means Titer

 

 

Treatment Method Lot 2 Lot 1A Total Means
Bronchitis D.W.* 33.92 13.06 23.49:4.92
Combined ND/IB vaccine D.W. 35.34 10.22 22.78i4.92

Combined ND/IB vaccine I.O.** 34.13 11.27 22.70:4.92

Combined ND/IB vaccine ND=I.O. 34.20 8.56 21.39:4.92
IB=D.W.

mm~m1 21 .

 

Total Means 34.40a 10.78b
£3.48 £3.48

 

*

D.W. = drinking water
**

1.0. = intraocularly
a,b

Means not sharing the same letter are signifi-
cantly different (PS0.0S). Tukey's test.

DISCUSSION

The assessment of immunity would have been best measured
by challenging vaccinated birds with an ND or IE virus of
known virulence. As stated above, this procedure was not
readily performable; for this reason, the immune response
was assessed by the titer of antibodies in the serum from 5
bleedings at 1-week intervals. These repeated tests allowed
for a more accurate assessment of the antibody levels which
to some extent reflect protection.

The objective of the present study was to analyze the
effect of two factors, (1) time and (2) method of vaccination,
on the production of antibody in chicks vaccinated against
Newcastle disease and infectious bronchitis vaccine, either
combined or separately.

The results reported above suggest that a combined
ND/IB vaccine administered in 10-day-old and 21-day-old
chicks via the drinking water, intraocularly, or by combining
two methods (ND by eye drop and IB in the drinking water)
will produce the same immune response as separate applied
vaccine, both ND and IB. Similar observations had previously
been made by Zygraich et a1. (1973).

Furthermore, vaccination at 10 days of age with revac-

cination at 15 days of age was found to produce better

37

38
immunity than either vaccination at 10 days or 21 days of

age.

Newcastle Disease

 

Considering the first factor studied, time of vaccina-
tion, it was found in both designs that vaccination at 10
days of age and revaccination at 15 days of age produced
the highest level of antibody, regardless of the method of
vaccination. When comparing this time of vaccination with
both vaccination at 10 days of age and at 21 days of age,
no difference between the two latter times was found. Fur-
thermore, the level of antibody produced was numerically
different over the time of the 5 successive bleedings.
However, there were no significant differences between the

repeated tests.

Infectious Bronchitis

 

Similar to the findings of ND, revaccination was found
to produce higher antibody levels than either vaccination
at 10 days of age or at 21 days of age. However, comparing
the latter two times, vaccination at 21 days of age indicated
a higher antibody titer, which may have been due to inter-
ference as a result of the very low level of maternal anti-
bodies at the time of vaccination. Similar observations
were made by Brandly et al. (1946), Levine and Fabricant
(1950) and Zygraich et al. (1973).

At any rate, the results suggest that it may be possible
to immunize the birds at a younger age, i.e., 10 days of

age, and thus to counteract the inhibitory effect of the

 

"

39
relatively higher maternal immunity level at this age with
revaccination at 15 days of age, rather than risk waiting
until 21 days of age for the first vaccination while still
producing less protection.

Given that revaccination appears to be preferable, a
combination of both ND and IB in the drinking water appears
to be the most effective method.

Even considering the results of study design two of
Newcastle disease, which produced a significant interaction
effect, the intraocular application was more effective for
revaccination, and drinking water application for vaccination
at 10 days of age. Also as a result of infectious bron-
chitis vaccination with further labor costs from large scale
with application, it may be argued that combined vaccine
in the drinking water application of ND and IB at 10 days
of age with revaccination at 15 days of age is the most

effective and efficient manner of vaccination.

Qpnclusion

Given the results of the present study, it may be
concluded that combined (ND/1B) vaccination, applied orally
via drinking water at 10 days of age with a revaccination
at 15 days of age, was the most effective procedure and

produced the higher level of antibody for the two diseases.

BIBLIOGRAPHY

BIBLIOGRAPHY

Alexander, D. J., Bracewell, C. D., and Gough, R. E. 1976.
Preliminary Evaluation of the Haemagglutination and
Haemagglutination Inhibition Tests for Avian Infec-
tious Bronchitis Virus. Avian Pathology, 5:125-134.

Alexander, D. J., and Chettle, N. J. 1977. Procedures for
the Haemagglutination and the Haemagglutination
Inhibition Tests for Avian Infectious Bronchitis
Virus. Avian Pathology, 9:9-17.

Allan, W. H. 1971. The Problem of Newcastle Disease.
Nature, 234:129-131.

Allan, W. H. 1973. The Effect of Neonatal Vaccination
Against Newcastle Disease in the Presence of Maternal
Antibody. Veterinary Record, 93:645-646.

Allan, W. H. 1974. Vaccination Against Newcastle Disease
with an Inactivated Oil Emulsion Vaccine at Day-Old
Followed by Aerosol Application of LaSota Vaccine
at Three weeks. Veterinary Record, 91:54.

Allan, W. H. 1975. Newcastle Disease. Developments in
Biological Standardisation, 23:445-450.

Allan, W. H., and Gough, R. E. 1974. A Standard Haemagglu-
tination Inhibition Test for Newcastle Disease. I.
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Avian Diseases, gz333-348.

40

41

Beard, C. W. 1971. Newcastle Disease Virus: Evaluation
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. LI “'20 15,-.9 '3‘ an Irv

 

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42

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43

Gough, R. E., and Alexander, D. J. 1979. Comparison of
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Avian Pathology, 9(2):131-142.

 

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89:11-19.

44

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577-583.

 

45

Price, R. J., Bottorff, C. A., Seeger, K., Sylstra, A. W.,
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‘Eflfifim

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46

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Infectious Bronchitis. Veterinary Record, 92:516-518.

 

APPENDICES

P.-

APPENDIX A

47

48
Hemagglutination-inhibition (HI) titers in

ted birds

unvaccina

"CONTROL"

group

Table A-1.

 

 

 

 

. . . r .
8422038420n422034 22094000M4000M42203242044200921004240042000M4220844ZOW4420

63111111181110..1111111111111111.111111111111511111‘111111‘11.511‘111111‘1111111111‘

o

680008400094400842000000084.20042000

100000000000000OWOOOOOOOOOOWOOOOOWO 0
0000000000000000 0000000000 00000 0 0 .
741357418374185741.357413573918374185741957413174185741857.»135741837418574135
0122301223012230122301223012230122301223012230122301223012230122301220.01223

 

44200%82004220023200“...0000220044200M320Aw

 

 

HI titer to NDV
;F=

S
k
C
.1
h
C
d
e
t
ca
n
..'-1
8C
nC
.la
dV
en
en
1
b.
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0
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605
b
m:
..u
DE
5 9
I 2
d
rt
.10
bL.
.. 3
AD

HI t

IBV

J-‘ter to

Tabl

49

Table A-2. Hemagglutination-inhibition (HI) titers in
group”NEWCASTLE"- vaccinated with a single
Newcastle disease vaccine or "BRONCHITIS" -
vaccinated with a single infectious bronchitis
vacc1ne

 

 

O
U
m
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z i .3
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79: 64
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A: birWs
. number; B = days of post-revaccinat' ' =
t 1on, C HI
3;:91‘ to NDV; Dn= Lot 2 - vaccinated at 10 and 15 days of
a E = group NEWCASTLE" or "BRONCHITIS" - vaccinated in

the drinking water; F = HI titer to IBV

50

-inhibition (HI) titers in group
- vaccinated with a combined ND/IB

n
O
.1
t
an
HR
.IE
tT
UAA
11W
06.
05B
auM
m0
eC
Hn

Table A-3.

vaccine

. 1

 

 

86669846266236 42626428423466842626826663236 42226422284262344223
9511 26131.13 1 6313163 632611 63131m311123 1 633316333 6313 6633
1 . 1 1

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1.11.11.11.1.1111111111111111.1111111.1.11.11.1.1111111111111111111111111111111111111...

62634623342436 64683.62. 68862684a¢2684626846263 22266626346226.62228424 68462633
13:1. 13 36 1. 161. 1.31 131 331 13.1 1.31 .33311151 . 1.33.1 333 361. 1.31. .

. 74 18574 18574135741857.41857419574185741857419374185741837419574133741857.4185

01223012230 12230abated-JoLat-‘30122301223012n¢3°1223°122301223012230122660.56‘230126‘3
. 11

    

53

 

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F = HI titer to IBV

o
’

water

51

ted with a combined ND/IB

V3CC11‘13.

Hemagglutination-inhibition (HI) titers in group

"COMB-EYE"
vaccine

Table A-4.

 

 

 

.
rgfi 11‘: I 1‘

24.6 25a .3 64 267:...269662 64 224 4 : 689346282292642328446236 65264382 4262 69824 2. 6
1.611313616313331 1131 6336 =11 2613 33 3163 3 6613 11 316 3 631312723635
1 . 11 T.

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.

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626115....1 «41.11 1.261 166.63 6.... 111 1613411.. 66311.61— 1 611 606 161.1 31—1 1

7” I 3574135741351. 419574195741357 9185~W4135741957 4135741957... 18574185741665.4135
30122301223012230122301223012230122301223012230122301223012230122301223

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44444445K. 555‘.5535555¢65l¢Qv5SSSCuSS—anu5%.55555‘.6435355-vnu5!.535I.64566666
Q 999999 9999 9 99999 99999999999999 9999999999999 9999 9 99999
7777777777777777777777777777777777777 77 777777 777777

7946 2
7946 2

A

 

 

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V

A - bird's number; B = days of post

titer to NDV; D = Lot 2 -
age; E = group "COMB-EYE" -

titer to IBV

52

in group

ND/IB

hibition (HI) titers

‘vaccinated with a combined

tion-1n

Hemafiglutina
"COM ~SEP"
vaccine

Table A-5.

 

 

F

4‘

C DE

'3.

 

662644226422286222626626

422926628
633 3113

5 55 555555555555555555555555555555555555555555555555555555555555555555555555

O
1111101‘.111111101111111111111111111111111.1116111151111111111‘1111111111111 1111

6268562638622666.693866863362636266326269886346266822668862362225364.688

131 131 1331111 11 1 131 1311 3131 .1311 3311 13 1:63.:— 161
.

741857418574185741957418574135741857418574185741857418574185741857418574185
O12230122301223012230122301223012230122301223012230122301223012230122301223

7777 444

     

 

 

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53

Table A-6. Hemagglutination-inhibition (HI) titers in group
"CONTROL” - unvaccinated chicks

 

 

(I)
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U
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nnr._n :thnnlnumnn -

 

A = bird's number; B = days of bleeding; C = HI titer to NDV;
D = Lot 1A; E = group "CONTROL" - unvaccinated chicks; F =
HI titer to IBV

54

Table A-7. Hemagglutination-inhibition (HI) titers in group
"NEWCASTLE" ~ vaccinated with a single Newcastle
disease vaccine or "BRONCHITIS" - vaccinated with
a single infectious bronchitis vaccine

 

 

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”HMNNNMMNMNNNN”MNMN”MM”NDNNMMN”NDNMMMNMMMNNNNNNNNNMN”NNMMMNNNNMNNMflflflfl”MN”N U
”MIDNNNNNNPJMOJ”MNNNNNNNNNNNN'JNN”MNKMJIJMNNMMNMNNIUMMNMNMNNNMNNNMMMMMNMNNMNM ”LINN” [:1

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to NDV; D = Lot 1A - vaccinated at 10 days of age; E = group
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F = HI titer to IBV

55

Table A-8. Hemagglutination-inhibition (HI) titers in group
"COMB-WATER" - vaccinated with a combined ND/IB

vaccine

 

 

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56

"COMB-EYE" - vaccinated with a combined ND/IB

Hemagglutination-inhibition (HI) titers in group
vaccine

Table A-9.

 

 

 

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57

"COMB-SEP”- vaccinated with a combined ND/IB

Hemagglutination-inhibition (HI) titers in group
vaccine

Table A-lO.

 

 

 

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group

Hemagglutination-inhibition (HI) titers in

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unvaccinated chicks

 

 

 

H

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59

vaccinated with
a single infectious bronchitis vaccine

Hemagglutination-inhibition (HI) titers in group
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disease vaccine or ”BRONCHITIS"

Table A-lZ.

 

 

 

 

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60

Table A-lS. Hemagglutination-inhibition (HI) titers in group
"COMB-WATER"-vaccinated with a combined ND/IB
vaccine

 

 

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to IBV

APPENDIX B

61

Statistical analysis pragram

Table B-1.

 

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MEASURES

INCLUUIHH RLFEATLD
CALIFORNIA

UNIVERSITY OF

CUVAFIAHCE

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IN IHIS‘VERSIUN Of

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181:

Means and standard dev

TableB—Z.

vacc1ne

158356

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APPENDIX C

65

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Table C-Z. Means and standard deviations

GROUP STRUCTUPE

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STANDARD DEVIATIONS FOR

HARGINAL
COUNT

LOTIA
COHHSEP

CONBOLHO

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th repeated measures

lance W1

Table C-3. Results of Z-factor analysis of var

 

H014 N021 NDPR NP35

N007

l-SI DEPENDENT VARIABLE

ANALYSIS OF VARIANCE FOP

LIYY

‘—
NC
<<
v-C

PRO

SOURCE

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68

 

time of vaccination

L = lot:

method of vaccination

group:

(; =

repetition

R =

APPENDIX D

69

7()

Table D-l. Statistical analysis program

 

aroezv - oneLvszs OF «ARInucfi AND COVARIANCE INCLUDING sEpEATED «ensues: , - ;
HEALYH SCIENCES DDMPDTIHG Each: TY :fififififi"0:§§’§‘29§$""ae"
UNIVERSITY :9 CALIFORNIA, Les AKGFLES '

COPYRIGHY (C) 1977, THE REGFNTS DE ’HE UVIVF?STTY OF CALIEDRNIA

IN THIS VEPSION CF uHcPZV "DISTINCT" "BRONCHITIS"

"COMB-WATER"

-- CCHPUTATIONS ARE PERFOPJ‘EC IN noquE oREnIsmu. "COMB-AQUA"

PQOGQAH CONTROL IN‘CRHATION

l==OBLEH TITLE 15 ”ANOV‘ BRONCHITIS 3 GflUPCS CC” 3 TFMPOS.0
[INPUT VARIABLES A3: 13.
' F0;NAT IS -(FH.O,57’F3QO,ZX,3‘2.393X,F300/5(3X,F3oc,9X,F3oc/)’-o

CASES ‘95 195.

IVSQIIBLE NAMES 0:5 SID,Nnn7,LOTF.GRUPC.IBOT.unlhglalthDZI.IEZI.NOZG,IBZ£,

"0352:3350

USE ‘PE LOOE'GPUPOQIBO7,1815,1320’1320918350
"I“ IS (2)3.2‘1.9‘0.
”03 IS (2’52"!3v3oq‘5200
BL‘N‘ IS "ISS- ~ '

[GROUP CCOF(3, IS 10293. _

' COPE09’ IS 2,3. ’.-
NAHE‘3’ :5 L072. LO'119LOTIB.

unwecu: Is DISTIncT.ccnsAcuA.
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DEFEND IS s.7.9.11.13. ~

LEVEL 15 5.
[END .

P’OELE" TI1LE o o o o o o O‘NO’A BRONCHITIS 3 GRUPOS 00H_3 TEHPOS.

 

NUHBEF DE vAEInaLEs To READ IN. . . . . . . .1. 13
names; DE JARIABLES ADDED 3v TRANSFORHATIONS. . 0
TOTAL NUMBEP cr VARIABLES . . . ... . . ... . . 13
NUHBEQ O: C0555 TU =E‘D IN. 0 o o o o o o o o o . 195
cass LABELING VARIABLES . . .‘. . . . . . . . .
LIHITS AND MISSING VALUE CHECKED BEFORE TaAnsrcaHITIcns
BLANKS APE. I O O O O O O C O ‘ 9 O O O O O O O O MISSING
INPUT UNIT NUMBER 0 o o o o o o o c o o o o o o 5
REHIND IKgUT UNIT szOR 70 REAOING. o 0.7.. o 0 N0
INPUT FORPIT ' , . »
‘pk039“x0F30392XO2FZO093x,:300/“‘RX'F30099x'FSQOI,, '
vAEIAaLEs To Be USED. ' ‘ A ‘
3 LoTE ‘ 0 stops 5 I807 7 131. T 9 1821
11 152! 13 1835 v -.
DESIGN SPECIFICATIONS
snoup . 3 a :' .
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BEFORE TanNSEDRMATIcu ' INTERVAL aANGE
vnnIneLE nIuInun aannun nzssruc DITEEGET cITEDDET GREATea LESS THLN
no. NAME LInIT LIHIT coo: cone nanE THnH on EOUAL To
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2.00000 LOTIA
3.00000 LOTIB

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NUHBER OF CAS€S REAB. a o o o o o a o o o o o 0 1‘2
CASES VITH DATA 'ISSING 0E BEYOND LIHITS . . 1!
EggthIHG "U”EER or CASES 0 o o o o o o o 153
CASES WITH GROUPING VALUES HOT USED. . . . . 00
9E"AINING Huflifia OF 3555: o a o o o o o o 65

1n the

ion

Jnat

Table D-Z. Means and standard deviations - effect of time and method of vacc

15 vacc1ne

bronchit

1005

production of antibody to infect

 

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lance W1

Table D-3. Results of Z-factor analysis of var

 

 

IP35

1021 R?“

1010

1H0?

l-SI DFPENDFNI VARIABLE

OF VARIANCE FOR

ANALYSIS

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}{ =

APPENDIX E

73

'74

Table E-l. Statistical analysis program

 

BFDpZV - ANALYSIS OF VA“: ANCE AKD COVARISNPE INCLUCING FEPEATE: MEASUQES
HEALTH 5 LIEVCES COHPUTING F'ACILITY '
UNIV‘RSITY 3F CALIFORNIA, LOS ANGE'ET PROGRAH REVISED NOVEHBEFT ’
‘CDPYRIGPT (C1 1977, THE REGENTS OF 7“" CHIV7°$TTY OF CALIFORNIA HANUAL DATE ' 1977 A

IN THIS VEQSTON CF oHDPZV

.. CCHPU'ATIDNS LEE PéPFORNEC IN DOUBLE DRSCISIDN.

P°OGQDP CONTPCL INEORNATTON ‘

IC°OELSH , TITLE IS “ANOVA BQOHCHITIS 5 GRUPCS CON 2 TFHPOS'
[INPUT VARIABLES AFB 1:.
FORHAT IS '(Fh.09~XgF3.3,ZX,:FZQCD3X,F3oC/5(3K,F3.a,9‘gF3oG/77-o
CASES ARE 195.
IVAPIgfiLE NAHES ARE SID.N0079LOTEOGRUPOCIEDT,N015933153NOZ191521ONDZDSIBZE!
NU3511339.
USE APE LOTEgGRUP031907.1815,13219182091835.
HIN IS (210,2‘199’0.
HAX IS (21520920599‘520o
BLANK IS ”135. - _
[GPOUp 600E131 IS 102. ' ' I -
CODECAT IS 213,895. '
NAFEC31 :5 L072 LOTIA.

NAHETB) IS DISTINCT ,CCHEAGUA,COHBOLHO,COHBSEP.
IDESIGN GQOUP IS 3".
- DEPEND IS 59799911913.

LEVEL 13 5.
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PROBLEM TITLE . . . . . . .ANOVA BRONCHI'IS S GRUFCS Gon_z TENPOS
NU"BE° OE VAPIABLES TO READ IN. . .'. . . . 13

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NUNEEE OE CASES TU PEAD IN. . . . . . . . . .. , 195 , ‘ "BRONCHITIS"
C‘SE L‘EELIMG VARIABLES o o o o o o o o o o - [I H =
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BLANKS NE. . . . . . . . . . . . . . . . . . . HISSING . COMB-WATER
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