AN ABSTRACT OF THE THESIS OF
Ferhat Abbas for the degree of Master of Science in Veterinary
Science presented on October 28, 1992.
Title: PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST INFECTIOUS
LARYNGOTRACHEITIS VIRUS OF CHICKENS AND THEIR USE IN AN
INDIRECT IMMUNOFLUORESCENCT DIAGNOSTIC TEST.
Abstract approved:
Redacted for Privacy
7).7ames R.
Monoclonal
antibodies
Andreasen Jr(J
were
developed
against
USDA
challenge strain of infectious laryngotracheitis virus (ILTV).
Indirect immunofluorescence test was used to detect antibodies
in supernatants of hybridomas. Hybridoma cells were developed
by fusing Sp 2/0 myeloma cells with spleen cells obtained from
mice
immunized
four
times with partially purified USDA
challenge strain of infectious laryngotracheitis virus.
The
supernatant of three hybridomas, designated as 2D1D8, 2E11G2,
2C6C7 were found positive for antibody activity against USDA
challege strain of ILTV. Hybridomas producing antibodies were
cloned by the limiting dilution method.
All
three
monoclonal
antibodies
reacted with
USDA
challege strain of ILTV, S 88 00224 strain of ILTV, and 86
1169 strain of ILTV in an indirect immunofluorescence test.
None
of
adenovirus
the
301
monoclonal
or
parrot
antibodies
herpes
reacted
virus
in
with
an
avian
indirect
immunofluorescence
test.
The monoclonal antibodies were
isotyped, and all three monoclonal antibodies were found to be
IgM.
PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST
INFECTIOUS LARYNGOTRACHEITIS VIRUS OF CHICKENS AND
THEIR USE IN AN INDIRECT IMMUNOFLUORESCENCT
DIAGNOSTIC TEST.
by
Ferhat Abbas
A THESIS
Submitted to
Oregon State University
in partial fulfilment of
the requirements for the
degree of
Master of Science
in Veterinary Medicine
Completed October 28, 1992.
Commencement June 1993.
APPROVED:
(Redacted for Privacy
Assilant Professor of Veterinary(Xedicine in charge of Major
Redacted for Privacy
Dean, College of VetL-inary Medicine
Redacted for Privacy
Dean of Graduate
Date thesis is presented October 28, 1992.
Typed by (Ferhat abbas).
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude and appreciation
to my major professor, Dr. James R. Andreasen Jr;
for his
great guidance, constant encouragement, attention and help.
I would also like to thank Dr. Stephen Kaattari and Dr.
Janet Andrews
for their advice and help throughout this
thesis.
I would like to thank Dr.
Donald
Mattson, and Rocky
Baker for their constant help and guidance.
Finally, my special thanks to my father Haji Najaf Ali
Khan and my other family members for their love, emotional
support, understanding, encouragement, and patience.
TABLE OF CONTENTS
page
Chapter I
Literature Review
Infectious laryngotracheitis(ILTV)
1
1
History
1
Classification
2
Morphology
3
Virus replication
3
Resistance to chemical and physical agents
4
Strain classification
5
Laboratory host system
6
Genome of ILTV
7
ILTV proteins and glycoproteins
8
Pathogenesis
9
Immunity
11
Diagnosis
13
Treatment, prevention and control
15
Monoclonal antibodies
References
17
21
Chapter II
Production of monoclonal antibodies against USDA
challenge strain of infectious laryngotracheitis
30
virus
Introduction
31
Materials and method
33
Source of virus
33
Virus growth and purification
33
Mouse immunization
36
Fusion and cloning
37
Production of cloned antibodies
39
Screening by using indirect
Immunofluorescence test
40
Immunoglobulin subclass
41
Results
Monoclonal antibodies
42
Isotypic characterization
42
Indirect immunofluorescence assay
42
Discussion
References
Bibliography
42
48
51
53
LIST OF FIGURES
page
Figures
Chapter II
1. Indirect immunofluorescence staining of cells
infected with USDA Challenge strain of ILTV
with 2D1D8 monoclonal antibody
46
2. Indirect immunofluorescence staining of cells
infected with avian adenovirus 301 with 2D1D8
monoclonal antibody
47
LIST OF TABLES
page
Tables
1. Isotypes of monoclonal antibodies secreted by
the three cloned hybridomas producing ILTV
-specific antibodies
44
2. Reaction of monoclonal antibodies with different
strains of ILTV and some other viruses as
determined by indirect immunofluorescence
45
PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST
INFECTIOUS LARYNGOTRACHEITIS VIRUS OF CHICKENS AND
THEIR USE IN AN INDIRECT IMMUNOFLUORESCENCT DIAGNOSTIC
TEST.
CHAPTER 1
LITERATURE REVIEW
Infectious Laryngotracheitis
Infectious laryngotracheitis (ILT) is an acute disease of
chickens characterized by signs of respiratory depression,
gasping, and expectoration of bloody exudate.
Although the
disease affects all ages, the most characteristic signs are
observed in adult birds.
History
Infectious laryngotracheitis was first described in 1925,
and the outbreak on which that report was based occurred in
October and November of 1923
(May & Tittsler, 1925).
Some
reports speculate that the disease may have existed earlier,
as an unusual high mortality among live poultry was observed.
Investigations were made and finally it was concluded that the
losses
were due to a disease which, if not new had not been
previously extensively studied (Beach, 1926).
In some poultry periodicals, it has been stated that ILT
was introduced into the United States by show birds from
Canada in 1921 (Beach, 1926).
The first authentic report of
2
its occurrence in the United States, involved a Rhode Island
flock in November, 1923 (May & Tittsler, 1925).
and it was thought that
of the disease was undetermined,
mortality could be due to
1925).
The etiology
exposure to cold (May & Tittsler,
The disease was transmitted to healthy birds by means
of a throat swab from dead birds (Graham, et al., 1930; May &
Tittsler, 1925).
Old
names
for
include
ILT
infectious
bronchitis,
infectious tracheitis, tracheo-laryngitis, chicken "flu" and
The term laryngotracheitis was
Canadian "flu" (Beach, 1926).
used as early as 1930 (Beach, 1930; Graham, et al., 1930) and
the name infectious laryngotracheitis was adopted in 1931 by
the Special Committee on Poultry Diseases of the American
Veterinary Medical Association.
A mild form of ILT was
reported and designated as the subacute or chronic form of the
disease (Graham, et al., 1930).
The cause of ILT was first
shown to be a filterable virus in 1930 (Beaudette, 1930).
Classification
Infectious laryngotracheitis has been identified in most
countries.
It is a serious disease of chickens
States, Europe, and Australia.
virus
has
Herpesviridae.
all
the
Infectious laryngotracheitis
characteristics
The virus
is
in the United
cuboidal,
of
enveloped,
sensitive, and contains a core composed of DNA.
member of the subfamily Alphaherpesvirinae
the
family
ether
ILTV is
a
(Roizman, 1982).
In the last 50 years, more than 80 distinct herpesviruses have
3
These
been isolated from a wide variety of animal species.
herpesviruses
share
structural
1982).
(Roizman,
features
"Herpes" is a Greek word which means "creep".
The term herpes
was used in medicine to describe a variety of skin conditions
and diseases for at least 25 centuries (Roizman, 1982).
In
recent years the known membership of the herpes group has
increased considerably.
share the
Twenty or more members
following properties (a) a genome of double stranded DNA (b)
a regular icosahedral capsid made of 162 capsomeres and often
surrounded
by
a
lipid-containing
and
envelope,
(c)
intranuclear assembly of the capsid around the genome.
Morphology
icosahedral
Electron microscopic examination reveals
viral particles in infected chicken embryo cells.
particles are similar in
These
structure to herpes simplex virus.
There are
The hexagonal virion is 80-100 nm in diameter.
three main structural components: the core, the capsid, and
the envelope.
The envelope is 195-250 nm in diameter and has
fine projections at the periphery of its membrane
et al., 1963).
The capsid
(Watrach,
has icosahedral symmetry and is
composed of 162 elongated hollow capsomeres.
The diameter of
a complete virus particle is 195-250 nm (Cruickshank, et al.,
1963; Watrach, et al., 1963).
Virus Replication
Replication of ILTV is similar to that of herpes simplex
and pseudorabies virus.
Virus
enters
into
the
cell by
4
pinocytosis after being adsorbed on the cell surface.
Rate of
adsorption depends on the volume of inoculum and cell system.
Enveloped particles adsorb more readily than naked particles
(Holmes & Watson, 1963).
Once inside the cells, host enzymes disrupt the envelope
and capsid of ILTV and its DNA is released.
migrates
to
the
cell
nucleus.
After
components can be observed in the nucleus.
10-12
Viral DNA
hours
ILTV
A sharp rise in
intracellular virus content occurs after 12 hours of infection
and a maximum virus titer is found between 30 and 36 hours of
infection.
titer gradually diminishes
Virus
thereafter
(Reynolds, et al., 1968).
The viral envelope is formed as the nucleocapsid migrates
through the cell's nuclear membrane.
The enveloped virus
particles accumulate in a vacuolar membrane in the cytoplasm.
Then the vacuoles containing virus particles migrate to the
plasma membrane and
are released after
cell membrane (Nii, et al., 1968).
disruption of the
A maximum titer of ILTV
reaching 106 plaque forming units/ml of tracheal exudate is
found on the third day after inoculation of 8-week-old white
leghorn chickens.
Virus may also be isolated from the lungs
and livers of infected chickens but virus titers are low in
those organs (Chang, et al.,1973).
Resistance to Chemical and Physical Agents
Infectious laryngotrachetis virus particles are sensitive
to lipolytic agents, heat, and various disinfectants.
5
A solution of 3% cresol or 1% lye can inactivate LTV in less
than
ILTV can survive
min.
1
for
a
long period when
lyophilized or kept at -20 to -60 C (Goldhaft, 1961).
Strain Classification
Most of the ILTV strains appear antigenically similar
virus neutralization
based on
(VN)
or immunofluorescence
tests (IFT) using reference-specific antiserum. Some strains,
however,
have
been
neutralized poorly by
(Burnet, 1936; Pulsford & Stokes, 1953).
such antisera
Some other workers
have also suggested there is minor antigenic variation among
the strains of ILTV (Russell & Turner, 1983; Shibley, et al.,
1962).
In attempts to find useful techniques for differentiating
the
antigenically-similar
strains
of
ILTV,
restriction
endonucleases have been used to study the viral DNA.
basis
of
classified.
these
findings
some
ILTV
strains
have
On the
been
Differences in DNA fragment patterns have been
found between an Australian vaccine strain and a field strain
by this method (Kotiw, et al., 1982), and other workers have
found differences between American and European ILTV strains
and between an American field strain and a vaccine strain
(Leib,
et al., 1986).
Differences in DNA patterns between
live ILTV vaccines, reference strains of ILTV, and ILTV field
isolates from North Carolina have also been studied by using
restriction endonucleases (Guy, et al., 1989).
Differences
between DNA patterns of vaccine strains and Georgia field
6
isolates of ILTV were also found (Andreasen, et al., 1990).
Differences among restriction endonuclease DNA fingerprints
of Pennsylvania field isolates, vaccine strains, and challenge
strains of ILTV have also been reported
(Keller,
et al.,
The use of cloned DNA fragments in reciprocal DNA:DNA
1992).
hybridization also appears promising for differentiating ILTV
strains (Kotiw, et al., 1986).
Monoclonal antibodies can be used to study the antigenic
relationship among virus isolates and other isolates of the
same
family.
been done
Work has
to
observe antigenic
relationship in paramyxoviruses, and different strains have
been classified by using monoclonal antibodies in reactions
such as the indirect immunoperoxidase test, haemagglutination
inhibition
test,
and
the
binding pattern
of monoclonal
antibodies with the infected cells (Alexander, et al., 1989;
Collins,
et al.,
1989).
Monoclonal antibodies potentially
could be used to distinguish strains of ILTV.
Laboratory Host System
Infectious laryngotracheitis virus can be propagated in
embryonating chicken eggs.
opaque
plaques
on
the
The virus causes formation of
chorio-allantoic
membrane
resulting from proliferative and necrotic lesions.
deaths occur in 2-12 days postinoculation (Brandly,
(CAM)
Embryo
1935;
Brandly, 1937).
Infectious laryngotrachetis virus can also be grown in
chicken embryonic liver (CEL), chicken embryo kidney (CEK),
7
and chicken kidney (CK) cell cultures (Atherton & Anderson,
1957).
Chicken embryonic liver and CK are the preferred
culture systems (Hughes & Jones, 1988).
the avian
The virus can also grow in cultures of cells from
Chicken
immune system, but only to extremely low titers.
leukocytes (buffy
coat) can also be used in vitro for viral
Viral growth at low titer has
growth (Chang, et al., 1977).
been observed in macrophages cultured from bone marrow and
spleen (Bulow & Klasen, 1983).
Susceptibility of macrophages
in vitro does not correlate with susceptibility of infection
with ILTV (Louduvaris, et al., 1991).
Genome of ILTV
A comparative study of DNA density and behavior in tissue
culture of 14 different herpesviruses
found that the nucleic
acid core of ILTV is DNA, with a density of 1.704 g/ml, which
is
consistent
with
DNA
values
herpesviruses (Plummer, et al.,
of
1969).
some
of
the
other
A molecular weight
estimation of ILTV DNA was made by summation of restriction
endonuclease fragments cleaved with Bam HI (102.1 x 106) and
Hind III (97.35 x 106).
Difference between the estimates may
indicate the presence of submolar fragments.
indicate
that
the
molecular
weight
of
These findings
ILTV
DNA
is
approximately 100 x 106, with the genome having two isomeric
forms (Kotiw, et al., 1982).
Other workers also investigated
genome isomerism of ILTV
using restriction endonuclease
analysis, and ILTV DNA was found to have two isomers with a
8
molecular weight of 109 x 106 (Leib, et al., 1986).
Clones of
ILTV have been used in hybridization experiments by workers
and it was found that the genome is 155 kilobase pairs (Kbp)
and is comprised of a long unique sequence of 120 Kbp and a
short unique sequence of 17 Kbp bounded by repeat sequences
each of 9 Kbp.
They also found an unrelated second pair of
repeat sequences located at
0.67 and 0.88 map units.
A
terminal repeat of the unique long region was also detected,
but no isomerization of the unique long sequence was found
(Johnson, et al., 1991).
DNA has been reported
Infectious laryngotracheitis virus
to have
a
guanine plus
cytosine
percentage of 45% (Plummer, et al., 1969), which is lower than
that for many other animal herpesviruses.
Some workers have
studied the nucleotide sequence of the ILTV gene coding the
205K complex glycoprotein (gb 205) and have found that the
gene is contained within a 3 kb EcoRI restriction fragment
mapping at map coordinates 0.23 to 0.25 in the unique long
region of the ILTV genome and is transcribed from right to
left (Kongsuwan, et al., 1991).
ILTV Proteins and Glycoproteins
Four major glycoproteins of molecular weights 205,
115,
90, and 60 Kd, respectively, have been located both on the
virus and the surface of virus-infected cells (York, et al.,
1987), and are considered to be the major immunogens of ILTV.
Monoclonal antibodies (MCAs) to immunogenic glycoproteins were
produced and characterized by immunoprecipitation and western
9
blotting.
One group of MCA reacted only with the 60 Kd
glycoprotein, by both techniques, while a second group reacted
with
the
205,
115,
and
KD
90
glycoproteins
in
immunoprecipitation and with additional bands of 85 and 160 KD
Glycoproteins
in Western blotting (York,et al.,1987).
with molecular weights less than 60 Kd have been detected in
extracts of virus-infected cells.
In a recent study,
the glycoprotein B
(gB)
gene was
identified using the polymerase chain reaction. Northern blot
analysis using a portion of open reading frame as a probe
identified a 2.7 kb RNA transcript in ILTV-infected chicken
embryo liver cells, and analysis of the amino acid sequence of
the ILTV protein indicated that it shares structural features
with the gB glycoproteins of other herpeseviruses (Poulsen, et
al., 1991).
Pathogenesis
The chicken is the primary natural host affected by ILTV,
but several workers have described a form of ILT in pheasants
and pheasant-chicken crosses
Boycott, 1982).
(Kernohan,
1931;
Crawshaw &
Lesions were experimentally induced in the
upper respiratory tract of young turkeys (Winterfield & So,
1968).
Starlings, crows, doves, ducks, pigeons, and guinea
fowl are not susceptible to ILTV
(Beach,
1931; Brandly &
Bushnell, 1934), although experimental infection of ducks has
been reported (Yamada, et al., 1980).
Natural
infections
by
ILTV
occur
through
upper
10
respiratory and ocular routes
1937).
Ingestion
can
1930; Beaudette,
(Beaudette,
also produce
although
infection,
exposure of nasal epithelium following ingestion is required
Transmission
with this route (Robertson & Egerton, 1981).
occurs more readily from acutely infected birds than through
contact with clinically recovered carrier birds.
Viral replication is
tissues,
limited to upper respiratory tract
with little or no evidence of viremia normally
occurring (Bagust,
et al., 1986;
Hitchner, et al., 1958).
Affected tracheal mucosa becomes
resulting
in
and
erosion
laryngotracheitis virus
is
swollen
and
hemorrhage.
usually present
edematous,
Infectious
in tracheal
tissues and secretions for only 6-8 days postinoculation
(Bagust, et al.,
Egerton, 1981).
1986; Hitchner, et al.,
1977; Robertson &
Mechanical transmission can occur by use of
contaminated equipment and litter (Kingsbury & Jungherr, 1958;
Dobson, 1935; Beaudette, 1937).
Egg transmission of virus has
not been demonstrated, and ILTV infected embryos die before
hatching (Jordan, 1966).
A recent study suggests a possible
chicken
major
histocompatibility
association
complex
(MHC)
of the
with
susceptibility and resistance of chickens to ILTV (Loudovaris,
et al., 1991).
Characteristic signs of an acute infection are nasal
discharge, and moist rales followed by coughing and gasping
(Beach, 1926; Kernohan, 1931).
In severe forms of the disease
11
there is severe dyspnea and expectoration of blood-stained
mucus
(Beach,
1935).
1926;
Hinshaw,et al.,
1931;
Seddon & Hart,
Mild enzootic forms of the disease have been described
in Britain, Australia,
the United States, and New Zealand
(Cover & Benton, 1958; Pulsford & Stokes, 1953; Seddon & Hart,
1935). In mild forms, signs are unthriftiness, reduction in
egg production,
infraorbital
watery eyes,
sinuses,
conjunctivitis,
persistent
nasal
swelling of
discharge,
and
hemorrhagic conjunctivitis (Seddon & Hart, 1935; Pulsford &
Stokes, 1953; Cover & Benton, 1958).
Most chickens recover in
10-14 days, but extremes of 1-4 weeks have also been reported
(Hinshaw, et al., 1931; Beach, 1926). Reactivation of latent
ILTV has not been achieved by using immunosuppressive drugs
(Hughes, et al., 1987., Bagust, 1984).
The incubation period is 6-12 days following natural
exposure (Kernohan, 1931; Seddon & Hart, 1935).
Intratracheal
exposure results in a shorter incubation period of 2-4 days
(Seddon & Hart, 1935; Benton, et al., 1958). Mortality varies
from 5-70% (average 10-20%) in epizootic form of the disease,
which spreads rapidly in susceptible chickens (Beach, 1931;
Hinshaw, et al., 1931; Seddon & Hart, 1935).
forms
of
the disease seen
in recent years
In the benign
in
the USA,
Australia, and the UK, morbidity is low and mortality varies
from 0.1-2% (Curtis & Wallis, 1983; Davidson & Miller, 1988).
Immunity
Chickens
gain
resistance after being
infected;
the
12
duration of
resistance induced from a
appears to be a year or more.
natural
infection
Immunity induced by vaccination
Cell-associated (chicken embryo
is of variable duration.
fibroblast cells) vaccine prepared from an attenuated ILTV
provides adequate protection when given at
1
day of age
intramuscularly or subcutaneously, and the chickens acquire
immunity within 6 days after vaccination.
The protection rate
remains above 60% until 10 weeks post-vaccination (Taneno, et
al.,
1991).
Infectious laryngotracheitis virus infection
causes the production of antibodies that can neutralize virus
(Bagust, et al., 1986).
The humoral response associated with
infection is not a primary mechanism of protection against
ILTV infection.
There is a poor correlation
between serum
antibody titers and the immune status of a flock (Jordan,
1981)
.
Resistance to ILTV due to cell-mediated responses is of
greater importance.
Bursectomized chickens, which can not
produce a humoral response, can develop immunity following ILT
vaccination (Robertson,
1977).
Some workers have reported
transmission of maternal antibodies to ILTV through the egg to
the offspring (Benton, et al., 1960).
interfere with vaccination,
lasting protection.
as compared to
al.,
The antibody does not
and does not provide a long-
Older birds respond well to vaccination
chickens less than two weeks of age (Alls, et
1969; Cover, et al., 1960; Gelenczei & Marty, 1965),
although chickens can be successfully vaccinated as early as
1 day old (Sincovic & Hunt, 1968).
13
Diagnosis
Although
acute
some
signs
of
the
disease
are
characteristic, signs are often similar to other respiratory
diseases, so ILT can not be reliably diagnosed by observation
of signs and lesions. Laboratory confirmation is required for
certain diagnosis.
Virus can be isolated from tracheal and lung tissue by
chicken embryo inoculation, by inoculation of trachea, and
infraorbital
sinuses
susceptible chickens
of
(Hitchner
&
White, 1958), or by cell culture inoculation (Chomiac, et al.,
1960).
Inoculation of ILTV is achieved by inoculation of
suspension from tracheal exudate, tracheal tissue, or lung
tissue onto the CAM of 9-12 day old embryonated egg or into
susceptible cell culture.
Cytopathic effect
(CPE)
observed 24-48 hours later (Hitchner & White, 1958).
can be
Chicken
embryo liver cells and chicken kidney cells are the laboratory
systems of choice for the isolation of ILTV (Hughes & Jones,
1988).
Demonstration
of
intranuclear
inclusion
bodies
in
tracheal and conjunctival tissues stained with Giemsa stain is
also diagnostic of ILTV (Cover & Benton, 1958; Beveridge &
Burnet,
1946).
Intratracheal
inoculation with
tracheal
exudate or tissue suspension from recently affected birds can
provide a reliable diagnostic procedure (Pulsford & Stokes,
1953).
Electron microscopic
(EM)
examination of tracheal
scrapings and recognition of herpes virus particles is also a
14
way of rapid diagnosis (Van Kammen & Spradbrow, 1976).
The
fluorescent antibody test may also be used for identification
of
(Braune
virus
&
Gentry,
Infectious
1965).
laryngotracheitis virus was detected in tracheal tissue from
day 2 to day 14 postinoculation using fluorescein-labelled
anti-LTV globulins (Bagust, et al., 1986; Hitchner, et al.,
1977; Wilks & Kogan,
Recently, an enzyme-linked
1979).
immuno-sorbent assay (ELISA) using monoclonal antibodies was
developed for rapid detection of ILTV antigen in tracheal
exudate
(Van Kammen & Spradbrow,
Enzyme-linked
1976).
immun-osorbent assay systems for detection of ILT antibodies
have also been developed (Ohkubo, et al., 1988; York & Fahey,
1988; York, et al., 1983). Virus neutralization (VN) can be
measured by using the CAM-pock counting technique, and VN
antibodies can also be detected in cell culture
monolayers
(Robertson & Egerton, 1977; Churchill, 1965).
Infectious laryngotracheitis virus DNA was also detected
in tracheal samples in a DNA hybridization assay by using a
non-radioactive probe, and this technique has been found very
effective in detecting field outbreaks and also latent ILTV
infections (Keam, et al., 1991).
Infectious laryngotracheitis virus antigen has also been
detected
in
frozen
tissue
section
by
an
indirect
immunoperoxidase (IP) procedure using monoclonal antibodies
(Guy, et al., 1992).
15
Treatment, Prevention and Control
No drug has yet been found effective in reducing the
severity of ILT.
Vaccination of chickens for ILT has proven
Live attenuated vaccines which
to induce adequate protection.
are given in drinking water and by eye drops are very
effective against ILT.
Mild field isolate SA-2, which has
been used for vaccination in Australia, is able to establish
infections
latent
(Bagust,
responses are thought
resistant
against
a
Cell-mediated
immune
to be protective against infection
Bursectomized
vaccination.
after
1986).
challenge
were
chickens
infection,
while
found
passive
transfer of hyperimmune serum failed to protect against the
infection (Fahey, et al., 1983).
Infectious laryngotracheitis
virus attenuated by passage in cell cultures (Izuchi, et al.,
1984;
Izuchi,
et al.,
1983; Gelenczei & Marty,
1964),
or
embryonated eggs, and selected mild enzootic strains (Pulsford
& Stokes, 1953) are capable of inducing acceptable protection
and are used in vaccines.
Virulent strains of ILT vaccine
should be administered with caution, because vaccination may
result in some carrier birds.
sinuses
(Shibley,
et
al.,
Inoculation of infraorbital
1962),
intranasal
installation
(Benton, et al., 1958), feather follicle inoculation (Molgard
& Cavett, 1947), eye drop (Sinkovic & Hunt, 1968), and oral
administration through drinking water (Hilbink, et al., 1981)
have been used to vaccinate chickens against ILTV.
Most layer
or breeder chickens are now vaccinated by the eye drop method
16
using attenuated ILTV vaccine.
When broiler vaccination is
required, spray or drinking water application is usually used.
Successful vaccination through the drinking water depends upon
ILTV contacting the epithelium of the nasal cavity during
drinking.
Glycoproteins of ILTV, purified from detergent extracts of
virus-infected cells by lectin affinity chromatography, have
also been used for vaccination of chickens, and have been
proven to protect up to 83% of chickens against ILTV.
neutralizing
antibody
responses were
and
delayed-type
Both
hypersensitivity
induced by vaccination with glycoprotein
preparation, but neither correlated with protection (York and
Fahey, 1991).
Proper management procedures help prevent the disease,
including sound sanitation and hygiene measures, control of
movement of staff, service workers, feed, equipment and birds,
as well as rodent and dog control measures (Kingsbury, et al.,
1958).
A resolution pertaining to the proposed eradication
program for ILT was unanimously passed by a United States
Animal
Health Association
eradication program
1990).
committee.
The
consists of four phases
proposed
ILT
(Proc. USAHA;
These four phases cover all aspects of a complete
eradication
program,
including
vaccination, surveillance,
diagnostic
capabilities,
biosecurity, and treatment of the
infected flocks and infected houses.
17
Monoclonal Antibodies
Monoclonal antibodies are a homogenous population of
identical antibodies with a defined specificity secreted by a
Monoclonal
B-cell clone after fusion with tumor cells.
antibodies (MCAs) do not differ structurally from naturally-
occurring polyclonal antibodies.
MCAs
unique
that
is
the
all
The property which makes
molecules
any
in
single
Monoclonal antibodies have same
preparation are identical.
specificity of reaction with any defined antigen every time
when ever reacted.
Since
1975
MCAs
have been
discovery in the field of science.
and
Milstein
published
in
considered
important
an
On August 7, 1975, Kohler
Nature
a
report
describing
"continuous cultures of fused cells secreting antibody of
The first
predefined specificity" (Kohler & Milstein, 1975).
workshop on lymphocyte hybridomas was held on April 3-5, 1978,
in Bethesda, Maryland.
Kohler and Milstein (1975) observed
that somatic cell hybridization could be used to generate a
continuous
antibody.
homogenous
"hybridoma"
cell
line producing
a
monoclonal
Immunologists have since produced large amounts of
monoclonal antibodies against a wide variety of
antigens.
Monoclonal
antibodies
histocompatibility,
have
been
differentiation,
produced
against
tumor or other cell-
surface antigens, as well as viral and bacterial antigens and
certain single antigenic determinants on a wide variety of
18
proteins, nucleic acids, and sugars. Monoclonal antibodies to
certain specific antigens are available commercially.
The primary advantage of MCAs as diagnostic reagents is
their specificity of binding to a single epitope, producing
highly
diagnostic
specific
reagents.
test
Monoclonal
Examples
antibodies are used for many research purposes.
include construction of antigenic maps of the haemagglutin
molecule of influenza virus, in vitro estimation
of mutation
rate or genetic changes of influenza virus by culturing virus
in presence of a single neutralizing monoclonal antibody, use
of
monoclonal
antibodies
in
passive
different antigenic determinants of
ability
of
monoclonal
antibodies
to
protection
against
influenza virus,
separate
and
individual
antigens from a complex mixture (Antczak, 1982; Re, 1987).
For production of MABs, a suitable host is
with an antigen.
immunized
Antibody-secreting B cells are obtained from
the spleen of the immunized host and fused with myeloma cells
in medium containing hypoxanthine, aminopterin, thymidine (HAT
Normal cells have two pathways
medium).
for nucleotide
synthesis, de novo pathway and salvage pathway. Myeloma cells
do not have hypoxanthine-guanine phosphoribosyl transferase
(HGPRT)
which is responsible for the salvage pathway,
so
because of the absence of this enzyme they are sensitive to
aminopterin, which inhibits dihydrofolate reductase and hence
blocks de novo purine and thymidilate synthesis.
are
thrown
in
the
salvage pathway,
Fused cells
being provided with
19
hypoxanthine and thymidine in the medium.
Fused cells have
HGPRT which utilizes the hypoxanthine for purine synthesis and
also have thymidine kinase which utilizes the thymidine for
thymidylate synthesis.
Both of these enzymes are provided by
the spleen cells after fusion.
Unfused B cells die naturally
in the media because they are not immortal like myeloma cells.
Because of the effect of aminopterin, unfused myeloma cells
will be killed too.
Only
fused cells can survive in this HAT
medium because of HGPRT and thymidine kinase provided by the
B cells, which is responsible for the salvage pathway, and
immortalization provided by the myeloma cells.
Positive
hybridomas producing the desired antibody are identified, and
cloning is done to get specific monoclonal antibodies (Goding,
1986; Harlow & Lane,
1988).
Enzyme-linked immuno-sorbent
assay and indirect immunofluorescence tests are most commonly
used for identification of positive hybridomas.
There are
different ways of cloning, but a common way is cloning by
limiting dilution at the rate of <1 cell/well.
Four animal species can be used for MCA production:
mouse, rat, hamster, and rabbit.
Rabbit MCAs have recently
been described (Raybould & Takahashi, 1988).
Mice are the
best choice for immunization and production of MCAs because
mice
are
easier
to
handle
and
there
are
many
anti
immunoglobulin (Ig) reagents available that are specific for
each mouse Ig isotype.
than rat.
Also, mouse MCAs are easier to purify
20
Female BALB/c mice are preferred
if
the
SP2/0-Ag14
myeloma cell line is used as a fusion partner because the
hybridomas resulting from this fusion will be entirely of
BALB/c origin.
Such hybridomas can grow well in BALB/c hosts
for the production of ascites fluid.
The sex of the mice does
not matter except that females are easier to handle and are
less agressive.
21
References
Alexander DJ, Manvell RJ, Collins MS, Brookman SJ, Westbury
of
Characterization
FJ.
I,
Austin
HA,
Morgan
paramyxoviruses isolated from penguins in Antarctica and
109(1­
Arch-Virol
sub-Antarctica during 1976-1979.
2):135 -143.
Alls
1989.
Studies on an ocular
Ipson JR, Vaughan WR.
infectious laryngotracheitis vaccine. Avian Dis 13:36­
AA,
45.
1969.
Andreasen JR, Jr., Glisson JR, Villegas P. Differentiation of
of
isolates
field
Georgia
and
strains
vaccine
infectious laryngotracheitis virus by their restriction
endonuclease fragment patterns. Avian Dis 34:646-656.
1990.
Antczk DF.
use.
Monoclonal antibodies: Technology and potential
J Am Vet Med Assoc 181:1005-1010. 1982.
Atherton JG, Anderson W. Propagation in tissue culture of the
Aust J
virus of infectious laryngotracheitis of fowls.
1957.
35:335-346.
Exp Biol
herpes virus
(Gallid-1)
Laryngotracheitis
Bagust TJ.
Latency establishment by
4..
infection in the chicken.
Avian Pathol
wild and vaccine strains of ILT virus.
1986.
15:581-595.
Gallid-1 herpes virus
Bagust TJ, Calnek BW, Fahey KJ.
Reinvestigation of the
3.
infection in the chicken.
pathogenesis of infectious laryngotracheitis in acute and
early post-acute respiratory disease. Avian Dis 30:179­
190. 1986.
The virus of laryngotracheitis of fowls. Science
Beach JR.
1930.
72:633-634.
Beach JR. A filterable virus, the cause of laryngotrcheitis
1931.
of chickens. J Exp Med 54:809-816.
Beaudette FR.
Annu Rep
Infectious bronchitis.
1930.
51:286.
Beaudette FR.
16:103-105.
New Jersey Aqr Exp Stn
Infectious laryngotracheitis.
Poultry Sci
1937.
Benton WJ, Cover MS, Greene LM. The clinical and serological
laryngotracheitis
certain
to
chickens
response of
Avian Dis 2:383-396. 1958.
viruses.
22
Benton WJ, Cover MS, Krauss WC. Studies on parental immunity
to infectious laryngotracheitis of chickens. Avian Dis
1960.
4:491-499.
Beveridge WIB, Burnet FM.
256.
Med Res Council Spec Rep Ser London
1946.
Some studies on infectious laryngotracheitis- a
J AM Vet Med Assoc 84:588-595.
preliminary report.
Brandly CA.
1934.
Some studies on infectious laryngotracheitis.
Brandly CA.
The continued propagation of the virus upon the CAM of
1935.
J Infect Dis 57:201-206.
the hen's egg.
1.
Studies on certain filterable viruses.
Brandly CA.
Factors concerned with the egg propagation of fowl pox
J Am Vet Med Assoc
and infectious laryngotracheitis.
90:479-487.
1937
Brandly CA, Bushnell JD. A report of some investigations of
13:212-217.
Poult Sci
infectious laryngotracheitis.
1934.
Braune MO, Gentry RF. Standardization of fluorescent antibody
technique for the detection of avian respiratory viruses.
1965.
Avian Dis 9:535-545.
Effect of avian viruses on cultured
Bulow Vv, Klasen A.
Avian Pathol
chicken bone-marrow-derived macrophages.
1983.
12:179-198.
Immunological studies with the virus of infectious
laryngotracheitis of fowls using the developing egg
J Exp Med 63:685-701. 1936.
technique.
Burnet FM.
Chang PW, Jasty V, Fry D, Yates VJ. Replication of a cell­
culture-modified infectious laryngotracheitis virus in
experimentally infected chickens. Avian Dis 17:683-689.
1973.
Chang PW, Sculo F, Yates VJ. An in vivo and in vitro study of
infectious laryngotracheitis virus in chicken leukocytes.
1977.
Avian Dis 21:492-500.
Chomiac TW, Luginbuhl RE, Helmboldt CF.
chicken
embryo
laryngotracheitis.
techniques
Avian Dis
Tissue culture and
infectious
for
4:235-246.
1960.
The use of chicken kidney tissue cultures in
Churchill AE.
the study of the avian viruses of Newcastle disease,
23
infectious laryngotracheitis, and infectious bronchitis.
1965.
Res Vet Sci 6:162-169.
Collins MS, Alexander DJ, Brookman S, Kemp PA, Manvell RJ.
Evaluation of mouse monoclonal antibodies raised against
an isolate of the variant avian paramyxovirus type 1
responsible for current panzootic in pigeons. Arch-Virol
1989.
104(1-2):53-61.
Cover MS, Benton WJ. The biological variation of infectious
1958.
laryngotracheitis virus. Avian Dis 2:375-383.
The effect of parenteral
Cover MS, Benton WJ, Krauss WC.
infectious
response to
on
the
immunity and age
4:467-473.
Avian Dis
laryngotracheitis vaccination.
1960.
The fine structure of
Cruickshank JG, Berry DM, Hay B.
infectious laryngotracheitis virus. Virology 20:376­
378.
1963.
Curtis PE, Wallis AS.
Rec
112:486.
Infectious laryngotracheitis.
Vet
1983.
Proceeding of
Daryl CJ. Transmissible diseases of poultry.
the United States Animal Health Association (94th annual
1990.
6-12,
Oct.
Colorado.
Denver,
meeting).
(HSAHA).
laryngotracheitis subcommittee
Infectious
1990.
337-339.
pp.
Davidson S, Miller K. Recent laryngotracheitis outbreaks in
Pennsylvania. Proc 37th West Poult Conf, pp. 135-136.
1988.
Infectious laryngotracheitis in poultry.
Dobson N.
1935.
15:1467-1471.
Vet Rec
Fahey KJ, Bagust TG, York JJ. Laryngotracheitis herpes virus
The role of humoral antibody
infection in the chicken.
in immunity to a graded challeng infection. Avian Pathol
1983.
12:505-514.
Fitzgerald JE, Hanson LE. A comparison of some properties of
laryngotracheitis and herpes simplex viruses. Am J Vet
1963.
24:1297-1303.
Res
Gelenczei EF, Marty EW. Studies on a tissue-culture modified
Avian Dis
8:105­
infectious laryngotracheitis virus.
122.
1964.
Gelenczei EF, Marty EW.
Strain stability and immunologic
24
characteristics of tissue-culture modified infectious
1965.
laryngotracheitis virus. Avian Dis 9:44-56.
Infectious laryngotracheitis field experiments:
Gibbs CS.
Vaccination. Massachusetts Aar Exp Stn Bull 305:57-58.
1934.
Monoclonal antibodies: Principles and practice.
Goding JW.
1986.
1-5.
Academic press, San Diego. pp.
Goldhaft TM. Viability of pox and laryngotracheitis vaccines.
1961.
Avian Dis 5:196-200.
Subacute or chronic
Graham RF, Throp Jr; F, James WA.
infectious avian laryngotracheitis. J Infect Dis 47 :87­
91.
Guy
1930.
Restriction
Mugner LL, Rose L.
Barnes HJ,
endonuclease analysis of infectious laryngotracheitis
virus:Comparison of modified-live vaccine viruses and
33:316-323.
Avian Dis
North Carolina field isolates.
JS,
1989.
Rapid diagnosis of infectious
Guy JS, Barnes HJ, Smith LG.
laryngotracheitis using a monoclonal antibody-based
21:77-86.
Avian Pathol
immunoperoxidase procedure.
1992.
Cold
Antibodies: A laboratory Manual.
Harlow E, Lane D.
Spring Harbor Laboratory, Cold Spring Harbor, New York.
1988.
139-148.
pp.
Drinking water vaccination
Hilbink F, Smit TH, Yadin H.
1981.
against ILT. Can J Comp Med 45:120-123.
A comparison of embryo and bird
Hitchner SB, White PG.
infectivity using five strains of laryngotracheitis
1958.
37:684-690.
Poult Sci
virus.
Hitchner SB, Fabricant J, Bagust TG. A fluorescent antibody
infectious
of
pathogenesis
of
the
study
1977
21:185-194.
Avian
Dis
laryngotracheitis.
An electron microscope study of the
Holmes IH, Watson DH.
attachment and the penetration of herpes virus in BHK 21
1963.
cells. Virology 21:112-123.
Comparison of cultural methods for
Hughes CS, Jones RC.
primary isolation of infectious laryngotracheitis virus
1988
Avian Pathol 17:295-303.
from field meterial.
Hughes CS, Jones RC, Gaskell RM, Jordan FTW, Bradbury JM.
25
Demonstration in live chickens of the carrier state in
Res Vet Sci 42:407-410.
infectious laryngotracheitis.
1987.
Izuchi T, Hasegawa A, Miyamoto T.
Studies on a live virus
vaccine against infectious laryngotracheitis of chickens.
Biological properties of attenuated strain C7. Avian
I.
1983.
27:918-926.
Dis
Studies on the live virus
Izuchi T, Hasegawa A, Miamoto T.
Evaluation of the
vaccine against ILT of chickens. II.
tissue-culture-modified strain C7 in laboratory and field
1984.
trials. Avian Dis 28:323-330.
Johnson MA, Prideaux CT, Kongsuwan K, Sheppard M, Fahey KJ.
Gallid herpes virus 1 (infectious laryngotracheitis
virus):cloning and physical map of the SA-2 strain. Arch
Virology 119:181-198. 1991.
A review of the literature on infectious
Jordan FTW.
1966.
laryngotracheitis (ILT). Avian Dis 10:1-26.
In
Immunity to infectious laryngotracheitis.
Jordan FTW.
Ross ME, Payne LN, Freeman BM, (eds.). Avian Immunology.
British Poultry Science Ltd., Edinburgh.
245-254.
pp.
1981.
Keam
Detection of
Fahey KJ.
York JJ, Sheppard M,
L,
infectious laryngotracheitis virus in chickens using a
non-radioactive DNA probe. Avian Dis 35:257-262. 1991.
Differences
Keller LH, Benson CE, Davison S, Eckroade RJ.
among restriction endonuclease DNA fingerprints of
and
vaccine strains,
Pennsylvania field isolates,
challenge strains of infectious laryngotracheitis virus.
1992.
Avian Dis 36:575-581.
Kernohan G. Infectious laryngotracheitis of fowls.
1931.
Med Assoc 78:196-202.
J Am Vet
Indirect transmission of
Jungherr EL.
Kingsbury FW,
Avian Dis
infectious laryngotracheitis in chickens.
1958.
2:54-63.
Continuous cultures of fused cells
Kohler G, Milstein C.
specificity.
predefined
of
antibody
secreting
Nature:495.
1975.
Kongsuwan K, Prideaux CI, Johnson MA, Sheppard M, Fahey KJ.
Nucleotide sequence of the gene encoding infectious
Virology
laryngotracheitis virus glycoprotein B.
184:404-410.
1991.
26
Kotiw M, Wilks CR, May JT.
virus
laryngotracheitis
endonucleases. Avian Dis
Differentiation of infectious
restriction
using
strains
26:718-731.
1982.
Differentiation
Kotiw M, Sheppard M, May JT, Wilks CR.
between virulent and avirulent strains of infectious
laryngotracheitis virus by DNA:DNA hybridization using a
cloned DNA marker. Vet Microbiol 11:319-330. 1986.
Leib DA, Bradbury JM, Gaskell RM, Hughes CS, Jones RC.
Restriction endonuclease patterns of some European and
American isolates of avian infectious laryngotrcheitis
1986.
virus. Avian Dis 30: 835-837.
Leib DA, Bradbury JM, Hart CA, McCarthy K. Genome isomerism
in two alphaherpesviruses: Herpes saimiri-1 (herpesvirus
tamaerinus) and avian infectious laryngotracheitis virus.
Arch Virol 93:287-294. 1987.
Fahey KJ.
Genetic
Calnek BW, Yuu BH,
Loudovaris I,
susceptibility of chicken macrophages to in vitro
infection with infectious laryngotracheitis virus. Avian
1991.
Pathol 20:291-302.
Genetic resistance to
Yuu BH, Fahey KJ.
I,
infectious laryngotracheitis in inbred lines of white
1991.
leghorn chickens. Avian Pathol 20:357-362.
Loudovaris
May HG, Tittsler RP. Tracheo-laryngitis in poultry. JAM Vet
1925.
Med Assoc 67:229-231.
The feather follicle method of
Molgard PC, Cavett JW.
Poult
vaccinating with fowl laryngotracheitis vaccine.
Sci
26:263-267.
1947.
Electron microscopy of herpes
Nii S, Morgan C, Rose HM.
J Virol
Sequence of development.
II.
simplex virus.
1968.
(Kyoto)
2:517-536.
Ohkubo Y, Shibata K, Mimura T, Taskashima I. Labeled avidin­
biotin enzyme-linked immunosorbent assay for detecting
in
antibody to infectious laryngotracheitis virus
1988.
chickens. Avian Dis 32:24-31.
Pirozok RP, Helmbolt CF, Jungherr EL. A rapid histological
avian
infectious
of
for
diagnosis
the
technique
130:406-407.
J Am Vet Med Assoc
laryngotracheitis.
1957.
Plummer G, Goodheart CR, Henson D, Bowling CP. A comparative
study of the DNA density and behavior in tissue culture
27
of fourteen different herpesviruses.
137.
Virology
39:134­
1969.
Poulsen DJ, Burton CRA, O'Brian JJ, Rabin SJ, Keeler CL Jr.
Identification of the infectious laryngotracheitis virus
glycoprotein gB gene by the polymerase chain reaction.
Virus Genes 4:335-348. 1991
Pulsford MF, Stokes J. Infectious laryngotracheitis in South
1953.
Australia. Aust Vet J 29:8-12.
Effect of infectious
Raggi LG, Brownell JR, Stewart GF.
laryngotracheitis on egg production and quality. Poultry
1961.
Sci 40:134-140.
Raybould TJG, Takahashi M. Production of stable rabbit-mouse
of
defined
MAB
rabbit
secrete
that
hybridomas
1988.
specificity. Science 233:566-568.
Re R.
The diagnostic and therapeutic promise of monoclonal
1987.
antbodies. Modern Med 55:21-24.
Development of nuclear
Reynolds HA, Watrach AW, Henson LE.
inclusion bodies of infectious laryngotracheitis. Avian
Dis
12:332-347.
1968.
The role of bursa-dependent responses in
Robertson GM.
Res Vet Sci
immunity to infectious laryngotracheitis.
22:281-284.
1977.
Robertson GM, Egerton JR. Micro-assay systems for infectious
1977.
laryngotracheitis virus. Avian Dis 21:133-135.
Replication of infectious
Egerton JR.
Robertson GM,
following
chickens
in
virus
laryngotracheitis
1981.
57:119-123.
vaccination. Aust Vet J
The family He pesviridae:General description,
Roizman B.
Roizman (ed.). The
In B.
taxonomy and classification.
Plenum Press, New
1.
pp. 1-23.
Vol.
Herpesviruses.
York.
1982.
Characterization of infectious
Russell RG, Turner AJ.
Antigenic comparison by
1.
laryngotracheitis viruses.
kinetics of neutralization and immunization studies. Can
J Comp Med 47:163-171. 1983.
Seddon HR, Hart L. The occurrence of infectious
laryngotracheitis in New South Wales. Aust Vet J 11:212­
222.
1935.
28
A quick method for the diagnosis of avian pox and
4:474-477.
Avian Dis
infectious laryngotracheitis.
Sevoian M.
1960.
Shibley GP, Luginbuhl RE, Hemboldt CF. A study of infectious
Comparison of serological and
laryngotracheitis. I.
1962.
immunogenic properties. Avian Dis 6:59-71.
Sinkovic B, Hunt S. Vaccination of day-old chickens against
by
conjunctival
laryngotracheitis
infectious
instillation. Aust Vet J 44:55-57. 1968.
Kawai T, Tukuyama Y, Eto M.
the cell-associated vaccine
prepared by an attenuated infectious laryngotracheitis
1991.
virus. J Vet Med Sci 53:671-676.
Taneno, A, Honda T, Sakai E,
Safety and efficacy of
Rapid diagnosis of some avian
Van Kammen A, Spradbrow PB.
1976.
20: 748-751.
Avian
Dis
virus diseases.
Watrach AM, Vatter AE, Hanson LE, watrook MA, Rhades HE.
Electron microscopic studies of the virus of infectious
laryngotracheitis. Am J Vet Res 20:535-544. 1959.
Watrach AM, Hanson LE, Watrook MA.
infectious laryngotracheitis virus.
608.
structure of
Virology 21:601­
The
1963.
Immunofluorescence diagnostic test for
Wilks CR, Kogan VG.
avian infectious laryngotracheitis. Aust Vet J 55:385­
388.
1979.
So IG.
Susceptibility of
Winterfield RW,
Avian Dis
infectious laryngotracheitis.
turkeys to
12:191-202.
1968
Susceptibility of
Yamada S, Matsuo K, Fukuda T, Uchinuno Y.
ducks to the virus of infectious laryngotracheitis.
1980.
Avian Dis 24:930-938.
York JJ, Fahey KJ. Diagnosis of infectious laryngotracheitis
using a monoclonal antibody ELISA. Avian Pathol 17:173­
182.
1988.
York JJ, Fahey KJ, Bagust TG.
Development and evaluation of
an ELISA for the detection of antibody to infectious
laryngotracheitis virus in chickens.
421.
Avian Dis
27:409­
1983.
Immunogenic glycoproteins of
York JJ, Sonza S, Fahey KJ.
Virology
infectious laryngotracheitis herpesvirus.
1987.
161:340-347.
29
York
Vaccination with affinity purified
Fahey KJ.
JJ,
infectious
against
protects chickens
glycoproteins
20:693­
Avian Pathol
laryngotracheitis herpes virus.
704.
1991.
30
CHAPTER II
PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST THE USDA
CHALLENGE STRAIN OF INFECTIOUS LARYNGOTRACHEITIS VIRUS
31
Introduction
Infectious laryngotracheitis virus (ILTV) is assigned to
the subfamily Alphaherpesvirinae, of the Herpesviridae family
on the basis of its rapid replication and latency in neurons.
It is an enveloped virus, icosahedral, with double stranded
DNA which has a density of 1.704 g/ml, similar to the DNA of
other herpes viruses
(Plummer,
et al.,
1969).
Four major
glycoproteins with molecular weights 205, 115, 90, and 60 kD
have been described in the SA-2 strain of ILTV (York, et al.,
1987).
These glycoproteins are considered to be the
major
immunogens of ILTV and are present on the virus envelope and
on
the
surface
of
virus-infected
cells.
Infectious
laryngotracheitis virus DNA has a molecular weight of about
100 x 106, with the genome having two isomeric forms (Kotiw,
et al., 1982; Leib, et al., 1987).
Some workers have reported
that ILTV has a guanine plus cytosine percentage of 45% which
is lower than other animal herpesviruses (Plummer,
et al.,
1969).
The envelope
glycoproteins are important immunogens
capable of inducing humoral and probably cellular immune
responses.
The involvement of other virion components in
inducing immune responses has not been investigated
(Watari,
et al., 1987).
Chicken antisera and rabbit antisera have been used to
immunoprecipitate four major glycoproteins of 205, 115,
and 60kD molecular weight.
90,
Some additional glycoproteins also
32
have been recognized by immune chicken and rabbit antisera in
western blotting using a glycoprotein fraction which was
purified from virus-infected cells (York, et al., 1987).
The
same workers have also produced monoclonal antibodies (MCAs)
and have
characterized them by immunoprecipitation and
western blotting. They have defined 5 groups of MCAs. Group 1
reacted with the 60kD by both techniques, and group 2 MCAs
reacted with
the
205,
115,
and
glycoproteins
90kD
in
immunoprecipitation. The reactivity of group 1 and group 2
MCAs in immunofluorescence suggests that the antigens are
present on the surface
of virus infected cells. Group 2
antibodies recognized at
least three epitopes,
confirmed by ELISA additivity assays.
Group 3,
which was
4,
and 5
antibodies recognized several proteins with lower molecular
weights from 45 to 24kD.
immunofluorescence studies,
Furthermore,
on the basis of
it was found that the antigens
recognized by groups 3, 4, and 5
were nuclear and cytoplasmic
antigens
that were not present on the surface of virus
infected
cells (York, et al., 1990).
All strains of ILT virus isolated throughout the world so
far are antigenically homogenous on the basis of
their
reactions in virus neutralization tests. However, restriction
endonucleases have been used to study the DNA patterns and
some workers have found differences among some strains of ILTV
(Kotiw, et al., 1982; Leib, et al., 1986).
Once monoclonal antibodies are produced, they can be used
33
in
various
diagnostic
tests
such
as
the
indirect
immunofluorescent antibody test (IFAT), virus neutralization
(VN), enzyme
linked immunosorbent assay (ELISA), etc.
Virus
neutralization activity has been assayed by some workers using
the plaque reduction method
(York, et al., 1989).
There are
various techniques to characterize these antibodies such as
western blotting, immunoprecipitation test, IFAT, ELISA.
The objective
of the present research was to produce
monoclonal antibodies against the USDA challenge strain of
infectious laryngotracheitis virus and to perform an initial
investigation of their use in an indirect immunofluorescence
diagnostic test.
Materials and Methods
Source of Virus:
Infectious laryngotrachetis virus
strains include the United States Department of Agriculture
(USDA) challenge strain, provided by the National Veterinary
Services Laboratory in Ames, Iowa; 86-1169, a Georgia field
isolate; and S 88 00224, a California field isolate.
viruses including a parrot herpesvirus
Other
(Pacheco's disease
virus) and avian adenovirus 301, were obtained from The Avian
Serology Laboratory, Veterinary Diagnostic Laboratory, Oregon
State University.
The parrot herpesvirus was isolated in
chicken embryo liver cells from the liver of a parrot that
died acutely.
Virus Growth and Purification:
Chicken embryo liver
cells were made from 14-day old chicken embryos.
The livers
34
were removed aseptically and placed
saline (PBS)
in phosphate-buffered
(0.16% sodium phosphate dibasic, 0.05% potassium
phosphate monobasic, and 0.73% sodium chloride; pH 7.2) in a
beaker.
Calcium and magnesium free PBS was used. After
chopping
with
scissors, livers were washed 5-6 times with
PBS to eliminate the red blood cells (RBCs) and other fibrous
tissue.
Versene-trypsin (Sigma, St. Louis, MO 63178 USA;
Trypsin 20 mg/ml, Versene 2 mg/ml)
cells.
Trypsinization was
chilled
fetal
bovine
was used to separate the
stopped by adding sufficient
serum
(FBS)
to
produce
an
8%
concentration, and cells were centrifuged at 750 X g for 5
minutes.
Packed
cells
were diluted at the rate of 1:150 in
minimal essential medium (MEM)
(Sigma, M0643) containing 10%
FBS, gentamicin (50 ug/ml), and Amphotericin-B (2.5ug /ml) (pH
7.0-7.1).
Five ml of diluted cells were added to 60-mm tissue
culture plates and were kept in a 5% CO2 incubator at 37C
overnight until a liver cell monolayer formed.
Each tissue
culture plate with a grown monolayer was infected with 0.1 ml
of 1:10 dilution of ILT cell culture supernatant.
After
infection, cells were incubated 1 hour at 37C in a
5% CO2
atmosphere to allow
virus adsorption.
Infected plates were
fed with growth medium containing 4% FBS and incubated at 37C
and 5% CO2 for 24 hours.
Only the cells which were infected
with the USDA challenge strain of ILTV were used for the
purification of virus for monoclonal antibody production.
35
Other viruses including Parrot herpesvirus, and adenovirus 301
were also used to infect the cells for certain comparative
studies.
Cells having a good growth of virus,
extensive cytopathic effect
were washed with TEN buffer
(CPE)
as determind by
characteristic of ILTV,
(0.01 M tris, 0.001 M EDTA, 0.1
M sodium chloride in distilled water)
Cells were
(pH 7.2).
scraped from the tissue culture plates and collected in a 50
ml tube.
Virus particles were released by sonification and
the solution was
centrifuged at 5930 X g for 25 minutes to
The supernatant
separate the cellular debris.
(32 ml) was
and 16 ml of the supernatant was layered onto
retained,
discontinuous sucrose gradients in centrifuge tubes containing
8
ml
65%
(w/w)
sucrose under
10
ml
30%
sucrose.
(w/w)
Supernatant was centrifuged through this discontinuous sucrose
100,000 X g for 1 hour.
gradient at
Visible virus bands were
collected (15 ml) from the 30%-65% sucrose interface.
of this fluid was added to the top of each tube
containing continuous
sucrose gradients.
Two ml
(6 tubes)
The continuous
sucrose gradient was made from four different concentrations
of sucrose in the same tube: 60% sucrose (w/w) was at the
bottom of the tube under 50%,
respectively.
100,000 X g
(15 ml).
Virus was
for 19 hours.
40%,
and 30% sucrose
(w/w)
centrifuged through the gradient at
Visible virus bands were collected
The refractory index of the virus-sucrose solution
was 1.410 and the density was
1.20 g/cm3­
36
After diluting the virus and sucrose in 15 ml TEN buffer,
the solution was placed in a concentrator
(100,000 MW
(Amicon, Beverly MA 01915 USA) and centrifuged
)
1250 X g
at
This centrifugation process was repeated 3
for 45 minutes.
times to eliminate
Dialysis was performed by
sucrose.
keeping dialysis tubing containing virus-sucrose solution in
The
TEN buffer for 48 hours to completely eliminate sucrose.
dialysis tube has a semi-permeable membrane which allows only
Dialysis
sucrose and TEN buffer to pass through the membrane.
was
followed
another
by
in
a
Five ml of purified ILTV was obtained.
concentrator.
Virus particles
microscopy.
centrifugations
three
were seen in the solution by electron
Spectrophotometry was used to measure protein
concentration as 68.117 ug/ml and nucleic acid concentration
as 9.8948 ug/ml.
Purified virus was stored at -70C.
Mouse Immunization:
Purified
virus was thawed in a
water bath at 37 C, and 0.8 ml of virus was mixed with 0.8 ml
of Freund's complete adjuvant (Sigma) by sonification.
10-week-old
female
intraperitoneally
(IP)
BALB/c
mice
were
Five
immunized
with 0.2 ml of emulsion containing
virus and Freund's complete adjuvant.
The mice were marked by
ear punch for individual identification.
Three weeks later a
second injection (0.2 ml, IP) of ILTV antigen, consisting of
incomplete Freund's adjuvant mixed in equal amount with the
purified
virus was given to all 5 mice.
Three weeks after the first injection of antigen, blood
37
was obtained from the conjunctival venous sinus of each mouse
using 100 ul heparinized capillary tubes.
Serum 1:100 dilution was checked for
by centrifugation.
by the indirect immunofluorescence
antibody against ILTV
test.
Serum was separated
Subsequently serum was checked every week for antibody
against ILTV until fusion.
respond best.
Mice 1,
3,
continued to
and 5
Four weeks after the first injection a third
injection (0.2 ml, IP) of ILTV in incomplete Freund's adjuvant
was given to all 5 mice.
An intravenous booster injection of
the purified virus without any adjuvant was given to mice 1,
3, and 5 four days before fusion, that is 8 weeks after the
first injection.
Five female BALB/c mice of the same age and
sex were kept as unimmunized control mice for a source of
negative control sera.
Serum from mice responding to the
viral antigen was also collected for use as positive control
serum before mice were sacrificed.
Fusion and Cloning:
with spleen
challenge
cells
strain
antibodies
of
of
SP2/0-Ag14 myeloma cells were fused
ILTV
for
(Kohler & Milstein,
Immunology, 1991).
immunized with the USDA
the mice
production
1975;
of
monoclonal
Current Protocol in
One day before fusion, feeder plates were
made from spleen cells of a female BALE /c mouse that had not
experienced any antigen exposure.
Mice were killed by
dislocating the cervical vertebrae and disinfected with 70%
alcohol before placing them in a sterile cabinet.
Spleens
from
three
immunized
mice
were
removed
38
aseptically, minced, and washed twice in complete Dulbecco's
Modified
medium
Eagle's
without
(Sigma)
(DMEM)
serum,
containing 1 ml penicillin/streptomycin (100X) in 100 ml of
in 100 ml of DMEM.
1 ml of L- glutamine (100X)
DMEM,
was prepared according to supplier's directions
medium (DMEM)
pH
(Sigma,
The
The
7.2).
medium
filtered
was
and
kept
Spleen cells were mixed by repeatedly and
refrigerated.
gently pipetting them with the media.
centrifuged at 750 X g for 5 minutes.
The spleen cells were
Cells were counted with
The spleen cells were mixed with SP2/0-AG 14
a hemocytometer.
mouse myeloma cells at the ratio of 1:1 (Schulman, et al.,
The combined cells were centrifuged at 750 X g for 5
1978).
minutes,
the supernatant was discarded,
1
ml
of
50%
(Sigma) was added to the cells as a
polyethylene glycol (PEG)
fusion reagent
and
(Poste & Allison,
1973).
Cells were mixed
very slowly in 2 ml of serum free DMEM and then in 7 ml of
serum free DMEM by stirring the cells very gently
pipet.
Cells
hypoxanthine
were
centrifuged
aminopterin
thymidine
with the
resuspended
and
(HAT)
(Sigma)
in
media
containing 20% FBS, HAT (5 x 10-3 M hypoxanthine, 2 x 10-5 M
aminopterin,
8
x
10-4
M thymidine),
pen/strep
(10,000
u
penicillin/ml, 10 mg streptomycin/ml), L-glutamine (200mM),
buffer
hepes
(Sigma)
(1
M),
and
OPI
(Sigma)
(0.15
g
oxaloacetate, 0.5 g pyruvate, and 0.0082 g bovine insulin per
10 ml).
One hundred ul of cell suspension was added to each well
39
of 96 well tissue culture plates (feeder plates) and incubated
at 37C in a
5% CO2 atmosphere.
For the first feeding,
containing HAT,
Cells were fed
every 2 days.
the wells were fed with the media
while for the second feeding hypoxanthine
thymidine media (HT media) was used.
After that, wells were
Screening
fed with complete DMEM containing 10% serum.
normally begins 10-14 days after fusion.
Supernatant fluids
from wells were screened by IFAT for specific ILTV antibodies
when the culture supernatant became acidic (yellowish) and the
hybridomas were grown enough to produce specific antibodies.
The hybridomas grow in the form of a clump of cells which is
visible using a microscope.
The hybridomas that were found to
be producing antibodies were cloned by limiting dilution
calculated to give <1 cell/well.
Ten cells per ml were
calculated by serial dilution and were plated onto feeder
plates at the rate of 100 ul/well to give <1 cell/well.
Cloning was done twice.
Fusion was done according the procedure given in Current
Protocols in Immunology, Green Publishing Associates and Jhon
Wiley and Sons, 1991., with slight modeficaton.
Production of Cloned Antibodies:
Monoclonal antibodies
were produced by growing cloned hybridomas in 80
cm'
culture flasks in complete DMEM containing 10% FCS.
tissue
Extinct
cultures of the cells were made by incubating the cells at 37C
in a 5% CO2atmosphere for 20 days without providing any media
after the cells were sufficiently grown.
This gave a very
40
After 20 days
high titre of antibody in the cell supernatent.
the cells were centrifuged and the supernatant was collected
in small aliquots and stored at -20C.
Screening
(IFAT):
By Using
Indirect
Immunofluorescence
Test
Infectious laryngotrachetis virus infected cells and
uninfected cells were collected from tissue culture flasks.
The cells were washed with PBS, and were centrifuged at 450 X
g.
The supernatant was discarded and the cell pellet was
suspended in 0.01M FA buffer (containing Na2HPO4, NaH2PO4.H20,
and NaCl) with 2% bovine serum albumin (BSA) and centrifuged
as before.
was
The supernatant was discarded and the cell pellet
resuspended
microliter
in
FA buffer
containing
One
BSA.
of the ILTV-infected cells was smeared on each
top-row well of teflon-coated slides
(Cel-line Associates,
P.O. Box 35. Newfield, NJ 08344).
Inc.
2%
Slides for other
virus-infected cells such as ILTV S 88 00224, ILTV 86-1169,
adenovirus 301, and parrot herpes virus were made in the same
way.
Uninfected cells were smeared on the wells on the bottom
row of the slide to serve as negative cell controls.
slides
were
fixed
in
acetone
for
10
minutes.
All
Twenty
microliters of undiluted hybridoma supernatant was added to
infected and uninfected wells. Negative control sera
(1:5
dilution) and positive control sera (1:100 dilution) were also
used at the time of screening of hybridomas for comparison.
After putting hybridoma
supernatant
or
control
sera
on
infected and uninfected cells, the slides were incubated for
41
20 minutes at 37C in a 5% CO2
atmosphere.
Slides were washed
with FA buffer and were soaked in FA buffer for 20 minutes.
Slides were washed with double distilled water, rinsed, and
Twenty microliters of rabbit anti-mouse
air dried completely.
immunoglobulin (Sigma; Jackson Immunoresearch Laboratories,
Inc,
872 Baltimore Pike,
(1:200
dilution)
PO Box 9,
West Grove,
PA 19390)
was added to each well of infected and
uninfected cells and incubated for 20 minutes.
Slides were
washed with FA buffer and soaked in FA buffer for 20 minutes.
Slides were washed with double distilled water, rinsed, and
counterstained for 5 minutes with 0.0001% Evan's blue.
were
air
dried,
mounted
with
mounting
fluid
Slides
(Difco
Laboratories, Detroit, Michigan USA) having a pH of 9.0, and
were examined under a fluorescent microscope.
Immunoqlobulin Subclass:
The isotypes of the monoclonal
antibodies were determined by a mouse monoclonal antibody
isotyping kit
IgG2a,
IgG2b,
procedures.
(Sigma)
IgG3,
containing rabbit anti-mouse
IgM,
IgG1,
and IgA following manufacturer's
The immunotype kit consists of nitrocellulose
strips on which the entire assay is completed.
The precoated
strip captures the relevant mouse immunoglobulin and reveals
the
isotype by self-description.
42
RESULTS
Monoclonal Antibodies.
Of 234 growing hybridomas,
15
showed positive reactions with cells infected with ILTV USDA
challenge strain and no reaction with uninfected control
cells.
Five of the 15 died during the process of expansion
Four of the remaining 10 proved to give negative
and cloning.
reactions, and 3 reacted both with infected and non-infected
cells on the second screening after cloning by limiting
dilution.
Three hybridomas were found positive against the
USDA challenge strain of ILTV and were designated as 2D1D8,
2E11G2, 2C6C7.
Isotypic
Characterization.
Monoclonal
antibodies
secreted by the three hybridomas were isotyped using the
isotyping kit, and all three hybridomas 2D1D8, 2E11G2, and
2C6C7 were found to secrete IgM (Table 1).
Indirect Immunofluorescence Assay.
antibodies
2D1D8,
2E11G2,
2C6C7
All three monoclonal
reacted in
an
identical
manner, fluorescence being found mainly along the peripheries
of
the infected cells.
Monoclonal antibodies were also
examined against other viruses including S 88 00224 isolate of
ILTV, 86-1169 isolate of ILTV, a parrot herpesvirus, and avian
adenovirus 301
(Table 2).
The other two strains of ILTV
reacted in the same manner as the
USDA challenge strain of
ILTV against all three monoclonal antibodies.
Fluorescence
was observed mostly at the peripheries of the infected cells
(Fig.1).
Cells infected with parrot herpesevirus and avian
43
adenovirus 301 did not produce any fluorescence with any of
the monoclonal antibodies.
The reaction of all three monoclonal antibodies with the
different isolates of ILTV and production of fluorescence was
almost identical. This suggests that the other isolates of
ILTV share the same antigen and the same epitope as the USDA
challenge strain of ILTV.
44
Table 1.
Isotypes of monoclonal antibodies secreted by the
three cloned hybridomas producing ILTV-specific antibodies.
Isotypes
Monoclonal antibody supernatants
2D1D8
IgM
IgG1
IgG2a
IgG2b
IgG3
IgA
+
2E11G2
2C6C7
45
Table 2.
Reaction of monoclonal antibodies with different
strains of ILTV and some other viruses as determined by
indirect immunofluorescence.
Virus
Strain
ILTV USDA
Monoclonal antibody supernatant
2C6C7
2D1D8
2E11G2
+
+
+
ILTV
S 88 00224
+
+
+
ILTV
86-1169
+
+
+
Avian Adenovirus 301
Parrot herpes virus
46
Figure
1.
infected
Indirect immunofluorescence staining of cells
with
USDA
challenge
strain
of
infectious
laryngotracheitis virus with 2D1D8 monoclonal antibody.
47
Figure
2.
Indirect immunofluorescence staining of cells
infected with avian adenovirus 301 with 2D1D8 monoclonal
antibody.
48
Discussion
Three monoclonal antibodies designated 2D1D8, 2E11G2, and
2C6C7 were obtained.
Although not yet characterized, these
monoclonal antibodies probably are directed against proteins
or
against
glycoproteins which
major
as
MCAs which are directed against viral
characterize
All three MCAs produced
proteins or against glycoproteins.
nearly
considered
Western blotting can be used
immunogens (York, et al., 1987).
to
are
reactions
identical
with
ILTV-infected
cells.
Fluorescence was observed mainly at the surface of the virus-
infected cells and fluorescence was also observed within the
These patterns of fluorescence by the monoclonal
nucleus.
suggest
antibodies
the reaction of monoclonal antibodies
against glycoproteins and against viral proteins because
cytoplasmic
antigens
whereas
fluorescence
suggests
the
presence
of
viral
expressed on the surface of virus-infected cells,
nuclear
fluorescence
suggests
the
reaction
of
monoclonal antibodies against viral structural proteins (York,
et,
al.,
1987).
Monoclonal
antibodies
specific
for
glycoproteins have been used to investigate the role of
glycoproteins in adsorption and penetration of cells, and cell
fusion and neutralization (Spear, 1985).
Each of the three monoclonal antibodies cross-reacted
with the other strains of ILTV,
and the fluorescence was
nearly identical to that observed against the USDA challenge
strain.
The fluorescence patterns were similar to those seen
49
with
the
USDA
challenge
All
strain.
three monoclonal
antibodies were found to not react with avian adenovirus 301
or parrot herpes virus.
While screening, some of the hybridoma supernatants were
found to react both with infected and non-infected cells,
which indicates that
some spleen cells were also producing
chicken
embryo
liver
cells
or
cell
antibodies
against
proteins.
This suggests that some cell antigens remained
after the virus purification steps and were injected with the
virus at the time of immunization.
This non-specific reaction
was also observed when the immunized mouse serum was checked
using the indirect immunofluorescent test before the mice were
sacrificed.
All 5 mice showed this serum antibody reaction
before being sacrificed.
Monoclonal antibodies 2D1D8, 2E11G2, and 2C6C7 were each
found to be of isotype IgM.
It is possible that the cells
that were responsible for producing antibodies other than IgM
could not fuse or could not survive during the process of
fusion, expansion, and cloning.
Monoclonal antibodies are an important tool for certain
other assays.
Glycoproteins are considered to be the major
immunogens for ILTV.
Identification of ILTV glycoproteins has
been done in Australia using different ILTV strains, and minor
differences have been found.
The largest glycoprotein that
has been detected measures 205Kd
(York,
et,
al.,
1987).
Identification of similar glycoproteins in ILTV strains found
50
in
the United States may be possible
if
the monoclonal
antibodies produced by this project are directed against those
glycoproteins.
The further characterization of these MCAs
remains to be done.
Different isolates of ILTV can not be differentiated by
conventional serological assays (Cover and Benton, 1958).
It
is possible that specific monoclonal antibodies could be
produced which could differentiate among virus isolates.
51
References
herpes virus
(Gallid-1)
Laryngotracheitis
TJ.
Bagust
infection in the chicken. 4. Latency establishment by
Avian Pathol
wild and vaccine strains of ILT virus.
15:581-595.
1986.
Cover MS, Benton WJ. The biological variation of infectious
1958.
laryngotracheitis virus. Avian Dis 2:375-383.
Current Protocols in Immunology. Green Publishing Associate
1991.
and John Wiley and Sons. pp. 2.5.1-2.5.17.
Continuous cultures of fused cells
Kohler G, Milstein C.
specificity.
predefined
of
antibody
secreting
Nature:495.
1975.
Kotiw M, Wilks CR, May JT.
virus
laryngotracheitis
Avian Dis
endonucleases.
Differentiation of infectious
restriction
using
strains
26:718-731.
1982.
Leib DA, Bradbury JM, Gaskell RM, Hughes CS, Jones RC.
Restriction endonuclease patterns of some European and
American isolates of avian infectious laryngotrcheitis
Avian Dis 30: 835-837. 1986.
virus.
Leib DA, Bradbury JM, Hart CA, McCarthy K. Genome isomerism
in two alphaherpesviruses: Herpes saimiri-1 (herpesvirus
tamaerinus) and avian infectious laryngotracheitis virus.
Arch Virol 93:287-294. 1987.
Plummer G, Goodheart CR, Henson D, Bowling CP. A comparative
study of the DNA density and behavior in tissue culture
of fourteen differenr herpesviruses. Virology 39:134­
137.
1969.
A better cell for making
Schulman M, Wide CD, Kohler G.
Nature
secreting specific antibodies.
hybridomas
276:269-270.
1978.
Glycoproteins specified by herpes simplex virus.
Spear PG.
3:315-356.
The herpes viruses.
(ed).
In: Roizman B.
1985.
Watari E, Dietzshold B, Szokan G, Heber-Kalz E. A synthetic
from
lethal
long-term protection
induces
peptide
infection with herpes simplex virus
165:459-470.
2.
J Exp Med
1987.
Immunogenic glycoproteins of
York JJ, Sonza S, Fahey KJ.
infectious laryngotracheitis herpesvirus. Virology
1987.
161:340-347.
52
York JJ, Young JG, Fahey KJ.
The appearence of viral antigen
and antibody in the trachea of naive and vaccinated
chickens infected with infectious laryngotracheitis
virus.
York
Avian Pathol
18: 643-658.
1989.
Antigens of
Sonza S, Brandon MR, Fahey KJ.
JJ,
infectious laryngotracheitis herpes virus defined by
monoclonal antibodies.
Arch Virol
115:147-162.
1990.
53
Bibliography
Alexander DJ, Manvell RJ, Collins MS, Brookman SJ, Westbury
of
Characterization
FJ.
I,
Austin
HA,
Morgan
paramyxoviruses isolated from penguins in Antarctica and
109(1­
Arch-Virol
sub-Antarctica during 1976-1979.
2):135 -143.
Alls
1989.
Studies on an
AA,
Ipson JR, Vaughan WR.
infectious laryngotracheitis vaccine. Avian Dis
45.
ocular
13:36­
1969.
Andreasen JR, Jr., Glisson JR, Villegas P. Differentiation of
of
isolates
field
Georgia
and
strains
vaccine
infectious laryngotracheitis virus by their restriction
endonuclease fragment patterns. Avian Dis 34:646-656.
1990.
Antczk DF.
use.
Monoclonal antibodies: Technology and potential
1982.
J Am Vet Med Assoc 181:1005-1010.
Atherton JG, Anderson W. Propagation in tissue culture of the
Aust J
virus of infectious laryngotracheitis of fowls.
1957.
Exp Biol 35:335-346.
herpes virus
(Gallid-1)
Laryngotracheitis
TJ.
Bagust
Latency establishment by
4.
infection in the chicken.
wild and vaccine strains of ILT virus.
15:581-595.
Avian Pathol
1986.
Gallid-1 herpes virus
Bagust TJ, Calnek BW, Fahey KJ.
Reinvestigation
of the
3.
infection in the chicken.
pathogenesis of infectious laryngotracheitis in acute and
early post-acute respiratory disease. Avian Dis 30:179­
190. 1986.
The virus of laryngotracheitis of fowls. Science
Beach JR.
1930.
72:633-634.
Beach JR. A filterable virus, the cause of laryngotrcheitis
1931.
of chickens. J Exp Med 54:809-816.
Beaudette FR.
Annu Rep
Infectious bronchitis.
1930.
51:286.
Beaudette FR.
16:103-105.
New Jersey Agr Exp Stn
Infectious laryngotracheitis.
Poultry Sci
1937.
Benton WJ, Cover MS, Greene LM. The clinical and serological
to
certain laryngotracheitis
chickens
response of
1958.
viruses. Avian Dis 2:383-396.
54
Benton WJ, Cover MS, Krauss WC. Studies on parental immunity
to infectious laryngotracheitis of chickens. Avian Dis
1960.
4:491-499.
Beveridge WIB, Burnet FM.
256.
Med Res Council Spec Rep Ser London
1946.
Some studies on infectious laryngotracheitis- a
Brandly CA.
J AM Vet Med Assoc 84:588-595.
preliminary report.
1934.
Some studies on infectious laryngotracheitis.
Brandly CA.
The continued propagation of the virus upon the CAM of
1935.
the hen's egg. J Infect Dis 57:201-206.
1.
Studies on certain filterable viruses.
Brandly CA.
Factors concerned with the egg propagation of fowl pox
J Am Vet Med Assoc
and infectious laryngotracheitis.
90:479-487.
1937
Brandly CA, Bushnell JD. A report of some investigations of
13:212-217.
Poult Sci
infectious laryngotracheitis.
1934.
Braune MO, Gentry RF. Standardization of fluorescent antibody
technique for the detection of avian respiratory viruses.
1965.
Avian Dis 9:535-545.
Effect of avian viruses on cultured
Bulow Vv, Klasen A.
Avian Pathol
chicken bone-marrow-derived macrophages.
12:179-198.
1983.
Immunological studies with the virus of infectious
laryngotracheitis of fowls using the developing egg
J Exp Med 63:685-701. 1936.
technique.
Burnet FM.
Replication of a cell­
Chang PW, Jasty V, Fry D, Yates VJ.
culture-modified infectious laryngotracheitis virus in
experimentally infected chickens. Avian Dis 17:683-689.
1973.
Chang PW, Sculo F, Yates VJ. An in vivo and in vitro study of
infectious laryngotracheitis virus in chicken leukocytes.
1977.
Avian Dis 21:492-500.
Tissue culture and
Chomiac TW, Luginbuhl RE, Helmboldt CF.
for
infectious
techniques
embryo
chicken
1960.
Avian Dis 4:235-246.
laryngotracheitis.
The use of chicken kidney tissue cultures in
Churchill AE.
the study of the avian viruses of Newcastle disease,
55
infectious laryngotracheitis, and infectious bronchitis.
1965.
Res Vet Sci 6:162-169.
Collins MS, Alexander DJ, Brookman S, Kemp PA, Manvell RJ.
Evaluation of mouse monoclonal antibodies raised against
an isolate of the variant avian paramyxovirus type 1
responsible for current panzootic in pigeons. Arch-Virol
1989.
104(1-2):53-61.
Cover MS, Benton WJ. The biological variation of infectious
1958.
laryngotracheitis virus. Avian Dis 2:375-383.
The effect of parenteral
Cover MS, Benton WJ, Krauss WC.
to
infectious
response
the
immunity and age on
4:467-473.
Avian Dis
laryngotracheitis vaccination.
1960.
The fine structure of
Cruickshank JG, Berry DM, Hay B.
20:376­
Virology
infectious laryngotracheitis virus.
378.
1963.
Curtis PE, Wallis AS.
Rec
112:486.
Infectious laryngotracheitis.
Vet
1983.
Current Protocols in Immunology. Green Publishing Associate
and John Wiley and Sons. pp. 2.5.1-2.5.17. 1991.
Transmissible diseases of poultry. Proceeding of
Daryl CJ.
the United States Animal Health Association (94th annual
Denver, Colorado. Oct. 6-12, 1990.
meeting).
(HSAHA).
subcommittee
laryngotracheitis
Infectious
1990.
pp.
337-339.
Davidson S, Miller K. Recent laryngotracheitis outbreaks in
Pennsylvania. Proc 37th West Poult Conf, pp. 135-136.
1988.
Infectious laryngotracheitis in poultry.
Dobson N.
1935.
15:1467-1471.
Vet Rec
Fahey KJ, Bagust TG, York JJ. Laryngotracheitis herpes virus
The role of humoral antibody
infection in the chiCken.
in immunity to a graded challeng infection. Avian Pathol
1983.
12:505-514.
Fitzgerald JE, Hanson LE. A comparison of some properties of
laryngotracheitis and herpes simplex viruses. Am J Vet
1963.
Res 24:1297-1303.
Gelenczei EF, Marty EW. Studies on a tissue-culture modified
Avian Dis
8:105­
infectious laryngotracheitis virus.
122.
1964.
56
Strain stability and immunologic
Gelenczei EF, Marty EW.
charactersitics of tissue-culture modified infectious
1965.
laryngotracheitis virus. Avian Dis 9:44-56.
Infectious laryngotracheitis field experiments:
Gibbs CS.
Vaccination. Massachusetts Aar Exp Stn Bull 305:57-58.
1934.
Monoclonal antibodies: Principles and practice.
Goding JW.
1-5.
1986.
Academic press, San Diego. pp.
Goldhaft TM. Viability of pox and laryngotracheitis vaccines.
Avian Dis 5:196-200. 1961.
Subacute or chronic
Graham RF, Throp Jr; F, James WA.
infectious avian laryngotracheitis. J Infect Dis 47:87­
91.
Guy
1930.
Restriction
Rose L.
Mugner LL,
endonuclease analysis of infectious laryngotracheitis
virus:Comparison of modified-live vaccine viruses and
33:316-323.
Avian Dis
North Carolina field isolates.
JS,
Barnes
HJ,
1989.
Rapid diagnosis of infectious
Guy JS, Barnes HJ, Smith LG.
laryngotracheitis using a monoclonal antibody-based
21:77-86.
Avian Pathol
immunoperoxidase procedure.
1992.
Cold
Antibodies: A laboratory Manual.
Harlow E, Lane D.
Spring Harbor Laboratory, Cold Spring Harbor, New York.
pp.
1988.
139-148.
Drinking water vaccination
Hilbink F, Smit TH, Yadin H.
1981.
45:120-123.
Can
J
Comp
Med
against ILT.
A comparison of embryo and bird
Hitchner SB, White PG.
infectivity using five strains of laryngotracheitis
Poult Sci 37:684-690. 1958.
virus.
A fluorescent antibody
Hitchner SB, Fabricant J, Bagust TG.
of
infectious
pathogenesis
of
the
study
1977
Avian Dis 21:185-194.
laryngotracheitis.
An electron microscope study of the
Holmes IH, Watson DH.
attachment and the penetration of herpes virus in BHK 21
1963.
Virology 21:112-123.
cells.
Comparison of cultural methods for
Hughes CS, Jones RC.
primary isolation of infectious laryngotracheitis virus
1988
from field meterial. Avian Pathol 17:295-303.
57
Hughes CS, Jones RC, Gaskell RM, Jordan FTW, Bradbury JM.
Demonstration in live chickens of the carrier state in
Res Vet Sci 42:407-410.
infectious laryngotracheitis.
1987.
Izuchi T, Hasegawa A, Miyamoto T.
Studies on a live virus
vaccine against infectious laryngotracheitis of chickens.
Biological properties of attenuated strain C7. Avian
I.
1983.
Dis 27:918-926.
Studies on the live virus
Izuchi T, Hasegawa A, Miamoto T.
vaccine against ILT of chickens. II. Evaluation of the
tissue-culture-modified strain C7 in laboratory and field
1984.
Avian Dis 28:323-330.
trials.
Johnson MA, Prideaux CT, Kongsuwan K, Sheppard M, Fahey KJ.
Gallid herpes virus 1 (infectious laryngotracheitis
virus):cloning and physical map of the SA-2 strain. Arch
1991.
Virology 119:181-198.
A review of the literature on infectious
Jordan FTW.
1966.
laryngotracheitis (ILT). Avian Dis 10:1-26.
In
Immunity to infectious laryngotracheitis.
Jordan FTW.
Ross ME, Payne LN, Freeman BM, (eds.). Avian Immunology.
British Poultry Science Ltd., Edinburgh.
245-254.
pp.
1981.
Keam
Detection of
L,
York JJ, Sheppard M, Fahey KJ.
infectious laryngotracheitis virus in chickens using a
non-radioactive DNA probe. Avian Dis 35:257-262. 1991.
Differences
Keller LH, Benson CE, Davison S, Eckroade RJ.
of
DNA
fingerprints
endonuclease
restriction
among
and
vaccine strains,
Pennsylvania field isolates,
challenge strains of infectious laryngotracheitis virus.
1992.
Avian Dis 36:575-581.
Kernohan G. Infectious laryngotracheitis of fowls.
1931.
Med Assoc 78:196-202.
J Am Vet
transmission of
Indirect
Jungherr EL.
Kingsbury FW,
Avian Dis
infectious laryngotracheitis in chickens.
1958.
2:54-63.
Continuous cultures of fused cells
Kohler G, Milstein C.
predefined
specificity.
of
antibody
secreting
Nature:495.
1975.
Kongsuwan K, Prideaux CI, Johnson MA, Sheppard M, Fahey KJ.
Nucleotide sequence of the gene encoding infectious
58
laryngotracheitis virus
184:404-410. 1991.
Kotiw M, Wilks CR, May JT.
virus
laryngotracheitis
Avian Dis
endonucleases.
glycoprotein
B.
Virology
Differentiation of infectious
restriction
using
strains
26:718-731.
1982.
Differentiation
Kotiw M, Sheppard M, May JT, Wilks CR.
between virulent and avirulent strains of infectious
laryngotracheitis virus by DNA:DNA hybridization using a
1986.
cloned DNA marker. Vet Microbiol 11:319-330.
Leib DA, Bradbury JM, Gaskell RM, Hughes CS, Jones RC.
Restriction endonuclease patterns of some European and
American isolates of avian infectious laryngotrcheitis
1986.
Avian Dis 30: 835-837.
virus.
Genome isomerism
Leib DA, Bradbury JM, Hart CA, McCarthy K.
in two alphaherpesviruses: Herpes saimiri-1 (herpesvirus
tamaerinus) and avian infectious laryngotracheitis virus.
1987.
Arch Virol 93:287-294.
Fahey KJ.
Genetic
Yuu BH,
Loudovaris I, Calnek BW,
susceptibility of chicken macrophages to in vitro
infection with infectious laryngotracheitis virus. Avian
1991.
Pathol 20:291-302.
I,
Yuu BH, Fahey KJ.
Genetic resistance to
infectious laryngotracheitis in inbred lines of white
1991.
leghorn chickens. Avian Pathol 20:357-362.
Loudovaris
May HG, Tittsler RP. Tracheo-laryngitis in poultry. JAM Vet
Med Assoc 67:229-231. 1925.
The feather follicle method of
Molgard PC, Cavett JW.
vaccinating with fowl laryngotracheitis vaccine. Poult
1947.
Sci 26:263-267.
Electron microscopy of herpes
Nii S, Morgan C, Rose HM.
J Virol
simplex virus.
II.
Sequence of development.
2:517-536.
1968.
(Kyoto)
Ohkubo Y, Shibata K, Mimura T, Taskashima I. Labeled avidin­
biotin enzyme-linked immunosorbent assay for detecting
in
laryngotracheitis virus
antibody to infectious
1988.
chickens. Avian Dis 32:24-31.
A rapid histological
Pirozok RP, Helmbolt CF, Jungherr EL.
of
infectious
avian
technique for
the
diagnosis
130:406-407.
J Am Vet Med Assoc
laryngotracheitis.
1957.
59
Plummer G, Goodheart CR, Henson D, Bowling CP. A comparative
study of the DNA density and behavior in tissue culture
of fourteen different herpesviruses. Virology 39:134­
137.
1969.
Poulsen DJ, Burton CRA, O'Brian JJ, Rabin SJ, Keeler CL Jr.
Identification of the infectious laryngotracheitis virus
glycoprotein gB gene by the polymerase chain reaction.
1991
Virus Genes 4:335-348.
Pulsford MF, Stokes J. Infectious laryngotracheitis in South
1953.
Australia. Aust Vet J 29:8-12.
Effect of infectious
Raggi LG, Brownell JR, Stewart GF.
laryngotracheitis on egg production and quality. Poultry
Sci
40:134-140.
1961.
Raybould TJG, Takahashi M. Production of stable rabbit-mouse
of
defined
rabbit
MAB
secrete
that
hybridomas
1988.
Science
233:566-568.
specificity.
Re R.
The diagnostic and therapeutic promise of monoclonal
Modern Med 55:21-24. 1987.
antbodies.
Development of nuclear
Reynolds HA, Watrach AW, Henson LE.
inclusion bodies of infectious laryngotracheitis. Avian
Dis
12:332-347.
1968.
The role of bursa-dependent responses in
Robertson GM.
Res Vet Sci
immunity to infectious laryngotracheitis.
22:281-284.
1977.
Robertson GM, Egerton JR. Micro-assay systems for infectious
1977.
laryngotracheitis virus. Avian Dis 21:133-135.
Replication of infectious
Egerton JR.
Robertson GM,
following
in
chickens
virus
laryngotracheitis
vaccination. Aust Vet J 57:119-123. 1981.
The family Herpesviridae:General description,
Roizman B.
taxonomy and classification. In B. Roizman (ed.). The
Plenum Press, New
1.
pp. 1-23.
Herpesviruses.
Vol.
York.
1982.
Characterization of infectious
Turner AJ.
Russell RG,
Antigenic comparison by
1.
laryngotracheitis viruses.
kinetics of neutralization and immunization studies. Can
1983.
J Comp Med 47:163-171.
The
occurrence
of
infectious
Hart
L.
HR,
Seddon
laryngotracheitis in New South Wales. Aust Vet J 11:212­
222.
1935.
60
A quick method for the diagnosis of avian pox and
Avian Dis
4:474-477.
infectious laryngotracheitis.
Sevoian M.
1960.
Schulman M, Wide CD, Kohler G.
hybridomas secreting
276:269-270.
1978.
A better cell for making
specific
antibodies.
Nature
Shibley GP, Luginbuhl RE, Hemboldt CF. A study of infectious
Comparison of serological and
laryngotracheitis. I.
immunogenic properties. Avian Dis 6:59-71. 1962.
Sinkovic B, Hunt S. Vaccination of day-old chickens against
infectious
laryngotracheitis
by
conjunctival
Aust Vet J 44:55-57.
1968.
instillation.
Glycoproteins specified by herpes simplex virus.
Spear PG.
(ed).
The herpes viruses.
3:315-356.
In: Roizman B.
1985.
Kawai T, Tukuyama Y, Eto M.
the cell-associated vaccine
prepared by an attenuated infectious laryngotracheitis
1991.
virus. J Vet Med Sci 53:671-676.
Taneno, A, Honda T, Sakai E,
Safety and efficacy of
Rapid diagnosis of some avian
Van Kammen A, Spradbrow PB.
1976.
virus diseases. Avian Dis 20: 748-751.
Watari E, Dietzshold B, Szokan G, Heber-Kalz E. A synthetic
from
lethal
peptide
induces
long-term protection
infection with herpes simplex virus
165:459-470.
2.
J Exp Med
1987.
Watrach AM, Vatter AE, Hanson LE, watrook MA, Rhades HE.
Electron microscopic studies of the virus of infectious
laryngotracheitis.
Am J Vet Res 20:535-544.
1959.
Watrach AM, Hanson LE, Watrook MA.
infectious laryngotracheitis virus.
608.
The structure of
21:601­
Virology
1963.
Wilks CR, Kogan VG.
Immunofluorescence diagnostic test for
avian infectious laryngotracheitis. Aust Vet J 55:385­
388.
1979.
Winterfield RW,
So IG.
Susceptibility of
infectious laryngotracheitis.
Avian Dis
turkeys to
12:191-202.
1968.
Yamada S, Matsuo K, Fukuda T, Uchinuno Y.
Susceptibility of
61
ducks to the virus of infectious
Avian Dis 24:930-938. 1980.
York JJ, Young JG, Fahey KJ.
laryngotracheitis.
The appearence of viral antigen
and antibody in the trachea of naive and vaccinated
chickens infected with infectious laryngotracheitis
virus.
Avian Pathol
18: 643-658.
1989.
York JJ, Fahey KJ. Diagnosis of infectious laryngotracheitis
using a monoclonal antibody ELISA. Avian Pathol 17:173­
182.
1988.
York JJ, Fahey KJ, Bagust TG.
Development and evaluation of
an ELISA for the detection of antibody to infectious
laryngotracheitis virus in chickens.
421.
Avian Dis
27:409­
1983.
Immunogenic glycoproteins of
York JJ, Sonza S, Fahey KJ.
Virology
infectious laryngotracheitis herpesvirus.
1987.
161:340-347.
York
Antigens of
JJ,
Sonza S, Brandon MR, Fahey KJ.
infectious laryngotracheitis herpes virus defined by
monoclonal antibodies.
York
Arch Virol
115:147-162.
1990.
Vaccination with affinity purified
JJ,
Fahey KJ.
glycoproteins protects
chickens
against
infectious
20:693­
laryngotracheitis herpes virus.
Avian Pathol
704.
1991.