AN ABSTRACT OF THE THESIS OF
Ferhat Abbas for the degree of Doctor of Philosophy in
Comparative Veterinary Medicine presented on November 22,
1994.
Title:
Studies of Immunological and Molecular
Biological Techniques with Infectious Laryngotracheitis
Virus of Chickens.
Abstract approved:
Redacted for Privacy
Monoclonal antibodies (MCA) produced against infectious
laryngotracheitis virus (ILTV) of chickens reacted in
western blotting experiments with several different ILTV
protein bands in the absence of tunicamycin which inhibits
carbohydrate synthesis.
Most of the MCA lost their
reactivity in western blotting experiments when extracts of
tunicamycin-treated ILTV CELC were used, suggesting their
specificity for carbohydrate-based epitopes.
In an indirect
immunofluorescence test most of the MCA bound primarily to
cytoplasmic antigens except some MCA which bound primarily
to nuclear antigens.
Additivity ELISA was also performed to
study whether MCA are against the same epitope or different
epitopes.
The polymerase chain reaction (PCR) was developed as a
diagnostic technique for detection of ILTV using primers
made from a portion of the ILTV thymidine kinase gene.
The
647-basepair amplified ILTV PCR product was labeled to
create a non-radioactive, biotinylated DNA probe.
Hybridization was performed using the probe to detect ILTV.
Both PCR and hybridization detected ILTV, and neither
hybridization nor PCR gave positive results with any other
pathogen.
Hybridization was specific for ILTV, However,
slight hybridization occurred with CELC DNA when relatively
relaxed conditions were used.
In another experiment, diagnostic tests to detect ILTV
in tracheas of experimentally-infected chickens, including
the indirect fluorescent antibody test (IFAT),
immunoperoxidase (IP), virus isolation (VI), histopathology,
PCR, and hybridization, were performed and compared.
Using
virus isolation as a reference, the sensitivity and
specificity of the tests were calculated.
The IP test and
IFAT performed better than any other test used in this
study.
Studies of Immunological and Molecular Biological Techniques
With Infectious Laryngotracheitis Virus of Chickens
by
Ferhat Abbas
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed November 22, 1994
Commencement June 1995
Doctor of Philosophy thesis of Ferhat Abbas presented on
November 22, 1994
APPROVED:
Redacted for Privacy
.
Majo
ofessor, representing Veterinary Medicine
Redacted for Privacy
can
'ollege of Veterinary Medicine
Redacted for Privacy
Dean of Graduate schoo
I understand that my thesis will become part of the
permanent collection of Oregon State University libraries.
My signature below authorizes release of my thesis to any
reader upon request.
Redacted for Privacy
Ferhat Abbas, Author
1
ACKNOWLEDGEMENT
I would like to thank Dr. James R. Andreasen Jr. for
serving as my major professor for his time and assistance.
I also want to thank him for his guidance and very
intelligent suggestions which provided direction at the
critical time and made every thing possible to finish this
study.
I want to thank Dr. Christopher J. Bayne, Dr. Donald E.
Mattson, Dr. William G. Dougherty, and Dr. Alfred R. Menino
Jr. for their time, suggestions, and constant help.
I extend special thanks to Rocky J. Baker and Anita
Sonn for their understanding, technical guidance, and great
help.
I am also thankful to my fellow graduate students who
created an environment where ideas and experience are freely
shared.
I would also like to express my special thanks to my
father Haji Najaf Ali Khan, my mother, and my brothers and
sisters, especially my sister Zarifa Abdul Salam who is like
mother to me, for their love, patience, understanding, and
emotional support.
Finally, all thanks to "Allah" who helped me to
complete my education in this country.
11
TABLE OF CONTENTS
page
CHAPTER I
LITERATURE REVIEW
Infectious laryngotracheitis (ILTV)
Introduction
Morphology and classification
Virus replication
Growth of ILTV
Pathogenesis
Immunity
Prevention and control
Strain classification
ILTV genome
ILTV proteins and glycoproteins
ILTV glycoproteins detected in western
blotting and immunoprecipitation
studies
ILTV glycoproteins: size comparison with
other herpesviruses
Antigenically distinct families of ILTV
glycoproteins
Immunogenic ILTV glycoproteins
ILTV polypeptides: comparison with other
herpesviruses
Monoclonal antibodies
Introduction
How MCA are produced
Use of MCA to study ILTV
Detection and diagnosis of ILTV
VI procedure
Histopathology
Fluorescent antibody test (FAT)
IP test
PCR
Hybridization
ELISA or serum neutralization
References
1
1
1
3
4
9
9
12
14
16
17
20
20
22
23
24
25
28
28
30
37
41
42
43
43
44
44
45
47
48
iii
CHAPTER II
CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST
INFECTIOUS LARYNGOTRACHEITIS VIRUS
63
Summary
64
Introduction
65
Materials and methods
67
Viruses
MCA
Indirect fluorescent antibody test
67
67
Isotyping
Virus neutralization (VN)
Western blotting
Additivity ELISA
68
68
68
69
72
(IFAT)
Results
75
Isotypic characterization
75
IFAT
75
VN test results
81
Coomassie staining of PAGE gels
81
Western blotting results
81
Effect of tunicamycin on western blotting
results
82
Additivity ELISA test
86
Discussion
88
References
96
CHAPTER III
USE OF THE POLYMERASE CHAIN REACTION AND A NONRADIOACTIVE DNA PROBE TO DETECT INFECTIOUS
LARYNGOTRACHEITIS VIRUS
100
Summary
101
Introduction
102
Materials and methods
103
Template DNA
Polymerase chain reaction (PCR)
Sequencing of PCR product
DNA probe
Hybridization
103
104
105
106
107
iv
Results
PCR
Hybridization
108
108
112
Discussion
117
References
120
CHAPTER IV
COMPARISON OF DIAGNOSTIC TESTS FOR INFECTIOUS
LARYNGOTRACHEITIS
122
Summary
123
Introduction
123
Materials and methods
125
Chickens
Challenge and sample collection
Monoclonal antibodies (MCA) to ILTV
IFAT
IP test
PCR
Hybridization
Histopathology
VI procedure
Statistics
Results
Response to ILTV challenge
Diagnostic test results
125
126
127
127
128
129
130
131
131
132
132
132
133
Discussion
137
References
141
BIBLIOGRAPHY
144
LIST OF FIGURES
page
Figures
2.1.
2.2.
2.3.
2.4.
3.1.
3.2.
3.3.
3.4.
3.5.
Indirect immunofluorescence staining of CELC
infected with USDA Challenge strain of ILTV
illustrating nuclear staining produced with
MCA D
77
Indirect immunofluorescence staining of CELC
infected with USDA Challenge strain of ILTV
illustrating cytoplasmic staining produced
with MCA C
78
Western blot produced by MCA with a detergent
extract of CELC infected with the USDA
challenge strain of ILTV in the absence of
tunicamycin
83
Western blot produced by MCA with a detergent
extract of CELC infected with the USDA
challenge strain of ILTV in the presence of
tunicamycin
84
PCR products electrophoresed on a 4% agarose
gel, stained with ethidium bromide, and
viewed under ultraviolet radiation
(ILTV strains)
110
PCR products electrophoresed on a 4% agarose
gel, stained with ethidium bromide, and
viewed under ultraviolet radiation,
(Pathogens other than ILTV)
111
Hybridization of a biotinylated DNA probe
with ILTV DNA
114
Hybridization of biotinylated ILTV DNA probe
with DNA extracted from other viruses,
bacteria, and mycoplasmas
115
Hybridization of a biotinylated ILTV DNA
probe with ILTV DNA at 2 different
stringencies
116
vi
LIST OF TABLES
page
Tables
2.1. Reaction of monoclonal antibodies by indirect
2.2.
immunofluorescence with strains of infectious
laryngotracheitis virus (ILTV) and 2 other
viruses
79
Titration by indirect immunofluorescence of
monoclonal antibodies (MCA) produced against
infectious laryngotracheitis virus
80
2.3.
Characterization of monoclonal antibodies (MCA)
raised against infectious laryngotracheitis
85
virus (ILTV) by western blotting
2.4.
Additivity ELISA additvity indices (AI) (%)
calculated for pairs of monoclonal antibodies
(MCA) produced against infectious
laryngotracheitis virus
87
4.1. Results of the virus isolation (VI),
histopathology, indirect fluorescent antibody
(IFAT) immunoperoxidase (IP), polymerase chain
reaction (PCR), and hybridization diagnostic
tests for detection of ILTV in tracheas of
135
experimentally-infected chickens
4.2.
Sensitivity and specificity of the
histopathology, indirect fluorescent antibody
(IFAT) immunoperoxidase (IP), polymerase chain
reaction (PCR), and hybridization diagnostic
tests for detection of ILTV in tracheas of
experimentally-infected chickens, based on
virus isolation (VI) results as a standard... 136
Studies of Immunological and Molecular Biological Techniques
With Infectious Laryngotracheitis Virus of Chickens
CHAPTER 1
Literature Review
INFECTIOUS LARYNGOTRACHEITIS
Introduction.
Infectious laryngotracheitis (ILT) is an
acute disease of chickens caused by an alphaherpesvirus,
infectious laryngotracheitis virus (ILTV).
ILTV is
cuboidal, enveloped, ether-sensitive, and contains a core
composed of DNA.
ILTV has all the characteristics of the
family Herpesviridae and is included as a member of the
subfamily Alphaherpesvirinae.
The members of this subfamily
are classified on the basis of a variable host range, short
reproductive cycle and rapid spread in cell culture, and
capacity to establish latent infections in ganglia (Roizman,
1982).
More than 80 distinct herpesviruses have been
isolated from a wide variety of animal species and have been
partially characterized.
These herpesviruses share
structural features (Roizman, 1982).
word which means "creep".
"Herpes" is a Greek
This term was used in medicine to
describe a variety of skin conditions and diseases (Roizman,
1982).
A typical herpesvirus has the 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 envelope, and (c)
intranuclear assembly of the capsid around the genome.
The
2
subfamily Alphaherpesvirinae contains the genera Simplex
virus including herpes simplex virus (HSV)
1 and
2,
circopithecine herpesvirus 1, and bovine mammillitis virus;
and Varicellovirus, including varicella-zoster virus (VZV),
equine herpesvirus type 1 (EH1), and pseudorabies virus
(PRV).
ILTV is not yet assigned to a genus.
ILT has been known by various names such as infectious
bronchitis, infectious tracheitis, tracheo-laryngitis,
chicken "flu" and Canadian "flu" (Beach, 1926).
The term
laryngotracheitis was used in 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.
Disease due to ILTV affects chickens of all ages but
the most characteristic signs are observed in adult birds,
including respiratory depression, gasping, and expectoration
of bloody exudate.
ILT also is responsible for egg
production losses and mortality.
Laryngotracheitis was first described in 1925 based on
an outbreak that occurred in October and November of 1923
(May & Tittsler, 1925).
Some reports indicate that ILT was
introduced into the United States in 1921 by show birds from
Canada (Beach, 1926).
The disease may have existed earlier,
as high mortality among live poultry was observed but no
specific pathogen was isolated.
After further study it was
3
concluded that this particular disease, if not new, had not
been previously extensively studied (Beach, 1926).
The first report of ILT in the United States involved a
Rhode Island flock in November, 1923 (May & Tittsler, 1925).
The cause of the disease was then unknown, and it was
suggested that losses might be due to exposure to cold (May
& Tittsler, 1925).
To further investigate the cause of the
disease, it was experimentally transmitted to healthy birds
by means of a throat swab from dead birds (Graham, et al.,
1930; May & Tittsler, 1925).
A mild form of ILT also was
reported and designated as the subacute or chronic form of
the disease (Graham, et al., 1930).
The cause of ILT was
first reported to be a filterable virus in 1930 (Beaudette,
1930).
Morphology and classification.
ILTV appears similar in
structure to HSV by electron microscopy.
The ILTV
nucleocapsid is 80 to 100 nm in diameter which is
characteristic of all herpesviruses.
Herpesviruses have
four main structural components: the core, an icosahedral
capsid that encloses the core, a material asymmetrically
distributed around the capsid and known as tegument, and an
outer membrane or envelope which surrounds the capsid and
tegument.
The envelope is 195 to 250 nm in diameter with
fine projections on its surface (Cruickshank, et al., 1963;
Watrach, et al., 1963).
Due to variability in the thickness
of the tegument, the size of herpes virions may vary from
4
120 to 300 nm.
Size also depends on the envelope's
integrity, since damaged envelopes become permeable, causing
the virion diameter to increase (Roizman, 1982).
Electron
microscopic studies show the envelope to have a trilaminar
appearance, suggesting that it is derived from patches of
altered cellular membranes (Morgan, et al., 1959).
The
presence of lipids in the membrane was demonstrated using
lipid solvents and detergents (Spear & Roizman, 1972).
The
herpesvirus envelope contains spikes or projections which
are shorter than those of many other enveloped viruses; the
spikes on HSV virions are approximately 8 nm long (Wildy &
Watson, 1963).
The capsid has icosahedral symmetry and is
composed of 162 elongated hollow capsomeres.
Virus replication.
The replication process ranges in
duration from 12 hours for PRV to over 70 hours for human
cytomegalovirus (HCMV).
HSV replication requires about 18
hours (Roizman & Betterson, 1986).
HSV replication has been
studied in more detail than any other alphaherpesvirus.
Relatively little is known specifically about ILTV
replication, but it is considered to be similar to PRV and
HSV.
After being adsorbed to the cell surface by attaching
to cell receptors on the plasma membrane, ILTV enters the
cell by pinocytosis.
The rate of adsorption depends on the
volume of inoculum and the cell system.
Enveloped particles
adsorb more readily than naked particles (Holmes & Watson,
1963).
Host-cell enzymes disrupt the ILTV envelope and
5
capsid after viral entry in the cell, releasing viral DNA.
ILTV DNA migrates to the cell nucleus and its transcription
occurs in the nucleus.
After 10 to 12 hours, ILTV
Alpha genes, the
components can be observed in the nucleus.
first to be expressed, code for alpha proteins, which are
responsible for the regulation and expression of beta genes
(Prideaux, et al., 1992).
Beta proteins, in turn, turn on
the genes responsible for gamma proteins.
Two gamma
proteins, structural proteins known as gamma 1 and gamma 2,
are synthesized (Prideaux, 1992).
In HSV, gamma proteins 1,
5, and 2 are capsid protein, glycoprotein gB, and
glycoprotein gC (Roizaman & Betterson 1986).
HSV mRNA is
capped, methylated, and polyadenylated (Stringer, et al.,
1977).
The organization of the ILTV genome is not known.
The
properties and functions of HSV proteins have been
extensively studied, but efforts to study ILTV proteins have
just begun.
ILTV has an alpha polypeptide of 200 kd which
is produced in large quantities, as in PRV (Ihara, et al.,
1983) and EH1 (Caughman, et al., 1985), for a short period
of time, since the alpha genes are expressed only briefly.
ILTV also has two beta polypeptides of 115 and 82 kd that
are synthesized between 4 and 16 hours postinfection
(Prideaux, et al., 1992).
ILTV DNA replication between 8
and 12 hour postinfection results in a rapid increase in
ILTV DNA (Prideaux, et al., 1992).
Two gamma 1 ILTV
6
polypeptides of 155 and 130 kd are synthesized at 4 hours
postinfection and also in the absence of DNA replication,
while 11 gamma 2 ILTV polypeptides of 170, 96, 90, 88, 68,
66, 47, 34, 26, 21, and 18 kd are synthesized only after DNA
replication (Prideaux, et al., 1992).
A recent study described three assembly intermediates
of ILTV, designated A, B, and C, discovered in ILTV-infected
cells by analyzing [3H]thymidine- and [35S]methionine- labeled
virus particles.
Electron microscopic studies showed that
intermediate A was an empty capsid, B was a procapsid
containing nonhomogeneous scaffolding protein, and C was a
capsid filled with DNA (Guo, et al., 1993).
Procapsids and
capsids also have been observed with other herpesviruses
(Lee, et al., 1988).
The ILTV procapsid was found only in
the nucleus, while the capsid was found in both nucleus and
cytoplasm, suggesting that packaging of DNA occurs before
translocation through the nuclear membrane into the
cytoplasm and envelopment by nuclear membrane (Guo, et al.,
1993).
All herpesviruses are surrounded by an envelope
containing glycoproteins.
The ILTV viral envelope is formed
as the nucleocapsid migrates through the cell's nuclear
membrane.
Enveloped virus particles accumulate in a
vacuolar membrane in the cytoplasm.
The vacuoles containing
virus particles migrate to the plasma membrane and are
released upon disruption of the cell membrane (Nii, et al.,
7
1968).
A recent study reported, however, that the enveloped
particles migrate through the lumen of endoplasmic reticulum
into vacuoles in the cytoplasm, where the envelope is
removed.
The viral capsid then is released into the
cytoplasm, migrates to the Golgi, and obtains a new envelope
from the membrane of the trans Golgi network which has fully
processed ILTV glycoprotein (Guo, et al., 1993).
A similar
multistep pathway including envelopment and de-envelopment
also has been reported for PRV (Whealy, et al., 1991).
The
enveloped ILTV particles in the perinuclear membrane were
smaller than those in vacuoles, suggesting two different
envelopes (Guo, et al., 1993).
A similar difference between
envelopes also has been reported for human herpesvirus-6
(Roffman, et al., 1990).
In other herpesviruses such as
VZV, naked nucleocapsids are released from the perinuclear
space, envelopment and modification of glycoprotein occur
into the glycoprotein-loaded cytoplasmic vacuoles, and virus
is released by reverse phagocytosis (Jones & Grose, 1988).
Observed by electron microscopy in intracellular
vacuoles, ILTV contains an electron-dense material, possibly
the tegument, that is similar to the dense bodies seen in
HSV 1 (Nii & Yasuda, 1976), HSV 2 (Atkinson, et al., 1978),
and HCMV (Fiala, et al., 1976).
Unlike other herpesviruses,
the ILTV dense bodies have a core that resembles the core
seen in DNA-filled capsids and are infective.
This suggests
that budding through the cytoplasmic membrane is not
8
required for ILTV maturation, because infectious virions can
be isolated from within infected cells (Guo, et al., 1993).
The cytoplasm of ILTV-infected cells also contains tubules
45 to 50 nm wide (Guo, et al., 1993).
Similar tubules are
reported with HSV-2 (Oda & Mori, 1976).
To study the similarity between the assembly
intermediates and DNA packaging pathways of ILTV and
bacteriophage 029, genes coding bacteriophage 029 prohead
proteins, that is gp7 (scaffolding protein), gp8 (capsid
protein), and gp10 (portal protein), were cloned into
Escherichia coli using a plasmid.
Proheads were purified,
and a white body located within the prohead, which is
scaffolding protein, appeared similar to core bodies in ILTV
procapsids (Guo, et al., 1993).
This scaffolding protein is
seen as a single spot in bacteriophage 029 while multiple
spots are seen in ILTV procapsids (Guo, et al., 1993).
The amount of intracellular virus increases rapidly 12
hours after infection, and a maximum virus titer occurs 30
to 36 hours postinfection.
The virus titer gradually drops
thereafter (Reynolds, et al., 1968).
A maximum ILTV titer
of 106 plaque forming units/ml of tracheal exudate occurs on
day 3 postinoculation in 8-week-old white leghorn chickens.
Virus also has been isolated from the lungs and livers of
infected chickens, but virus titers were low in those organs
(Chang, et al., 1973).
9
Growth of ILTV.
Chicken embryo liver (CEL), chicken
embryo kidney (CEK), and chicken kidney (CK) primary cell
cultures have been used to grow ILTV (Atherton & Anderson,
1957).
CEL and CK are the preferred culture systems (Hughes
& Jones, 1988).
ILTV also may be grown in embryonating
chicken eggs, although ILTV growth causes embryo death
(Brandly, 1935; Brandly, 1937).
Proliferative and necrotic
opaque plaques occur on the chorio-allantoic membrane (CAM)
due to virus growth.
Recently, ILTV was grown in an avian hepatoma cell
The hepatoma cell is the only cell line known to
line.
support the growth of ILTV (Scholz, et al., 1993).
Pathogenesis.
ILTV affects pheasants and pheasant-
chicken crosses (Crawshaw & Boycott, 1982; Kernohan, 1931)
but chickens are the primary and most-commonly affected
hosts.
Turkeys may be experimentally infected (Winterfield
& So, 1968).
ILTV does not cause disease in starlings,
crows, doves, ducks, pigeons, and guinea fowl (Beach, 1931;
Brandly & Bushnell, 1934), although experimental infection
of ducks has been reported (Yamada, et al., 1980).
The
chicken major histocompatibility complex (MHC) may be
associated with susceptibility and resistance of chickens to
ILTV (Loudovaris, et al., 1991).
Infection with ILTV occurs mainly through upper
respiratory and ocular routes (Beaudette, 1930; Beaudette,
1937), but ingestion has been implicated as another route of
10
infection.
Ingestion may produce disease by exposing nasal
epithelium to the virus (Robertson & Egerton, 1981).
Acutely infected birds are the main source of spreading the
disease, but transmission occurs at lower rates through
contact with recovered carrier birds.
Mechanical
transmission can occur due to contaminated equipment and
litter (Beaudette, 1937; Dobson, 1935; Kingsbury & Jungherr,
1958).
Egg transmission of virus has not been reported, and
ILTV-infected embryos die before hatching (Jordan, 1966).
Infectious laryngotracheitis virus is present in the
trachea of infected chickens for only 6 to 8 days after
infection (Bagust, et al., 1986; Hitchner, et al., 1977;
Robertson & Egerton, 1981).
In the acute phase of disease,
virus replication occurs mainly in the upper respiratory
tract, with no evidence of viremia (Bagust, et al., 1986;
Hitchner, et al., 1958).
After infection the tracheal
mucosa becomes edematous, inflamed, and hemorrhagic.
There are two distinct forms of the disease caused by
ILTV, the mild form and the severe form.
Mild forms of the
disease have been reported in Britain, Australia, the United
States, and New Zealand (Cover & Benton, 1958; Pulsford &
Stokes, 1953; Seddon & Hart, 1935).
The signs commonly seen
in severe acute ILTV infection are nasal discharge, moist
rales, coughing and gasping (Beach, 1926; Kernohan, 1931),
dyspnea, and expectoration of blood-stained mucus (Beach,
1926; Hinshaw, et al., 1931; Seddon & Hart, 1935).
In mild
11
disease, the signs include unthriftiness, reduction in egg
production, watery eyes, conjunctivitis, swelling of
infraorbital sinuses, and persistent nasal discharge (Cover
& Benton, 1958; Pulsford & Stokes, 1953; Seddon & Hart,
1935;).
Chickens recover from the disease in 10-14 days,
but latent infections (Bagust, 1986) and cases lasting for
weeks also have been reported (Beach, 1926; Hinshaw, et al.,
1931).
Recovered chickens may be latently-infected carriers
of the disease.
Reactivation of latent ILTV occurs due to
stress, but has not been achieved experimentally with
immunosuppressive drugs (Bagust, 1984; Hughes, et al.,
1987).
ILTV has an incubation period of 6 to 12 days in
naturally infected chickens (Kernohan, 1931; Seddon & Hart,
1935) but shorter incubation periods of 2 to 4 days can
occur with intratracheal exposures (Benton, et al., 1958;
Seddon & Hart, 1935).
Mortality varies from 5 to 70%, but
averages 10 to 20% of susceptible chickens in the severe
form of the disease (Beach, 1931; Hinshaw, et al., 1931;
Seddon & Hart, 1935).
In the mild form of the disease
morbidity is low and mortality varies from 0.1 to 2% (Curtis
& Wallis, 1983; Davidson & Miller, 1988).
ILTV has occurred in association with secondary
bacteria such as Pasteurella haemolytica (Shaw, et al.,
1990).
In association with P. haemolytica, an increased
mortality and tracheal inflammation were observed in laying
12
hens and pullets, and both ILTV and P. haemolytica were
isolated from their internal organs (Shaw, et al., 1990).
Immunity.
Specific neutralizing antibodies are
produced against ILTV after infection (Bagust, et al.,
1986).
The humoral response, however, is not a primary
mechanism of protection against ILTV.
Serum antibody titers
do not assure protective immunity in a flock (Jordan, 1981).
Immunity to ILTV develops in chickens after infection
and protects against reinfection.
Cell-mediated immune
responses provide major protection against ILTV in
vaccinated chickens.
Humoral immune responses are not as
effective as cell-mediated immune responses in protecting
against ILTV, since bursectomized chickens resisted
challenge, and passive transfer of hyperimmune serum failed
to protect against infection (Fahey, et al., 1983;
Robertson, 1977).
In one study, chickens were infected at 1 and 14 days
of age with chicken anemia agent (CAA) and infectious bursal
disease virus (IBDV).
Two weeks postinfection, birds were
vaccinated with ILTV and later challenged to evaluate the
protection rate.
Chickens infected with IBDV, at both day 1
and 14 did not have decreased protection against ILTV
challenge, but chickens infected with CAA at day 1 had a
protection rate of 7.1%.
As IBDV infection damages humoral
immunity and CAA infection damages cell-mediated immunity,
the results suggest the importance of cell-mediated immune
13
responses against ILTV.
Chickens infected at day 14 with
CAA and vaccinated 2 weeks later with ILTV had increased
protection compared to control chickens.
This suggests
that, at 2 weeks of age, chickens had a developed immune
system, and that the higher protection rate than control
chickens in response to ILTV vaccination may be because of
stimulation of immune responses due to CAA infection (Cloud,
et al., 1992).
The duration of resistance varies.
A natural infection
results in resistance for one year or more, while immunity
induced by vaccination is of variable duration.
Although
not used in the United States, a cell-associated (chicken
embryo fibroblast cell) vaccine has been developed from an
attenuated ILTV which protects when given at 1 day of age
intramuscularly or subcutaneously, and the chickens become
immune to ILTV within 6 days after vaccination.
The vaccine
provides more than 60% protection until 10 weeks postvaccination (Taneno, et al., 1991).
Transmission of
maternal antibodies against ILTV through the egg to the
offspring also occurs (Benton, et al., 1960).
Birds more than two weeks of age respond well to
vaccination (Alls, et al., 1969; Cover, et al., 1960;
Gelenczei & Marty, 1965), and there are reports of
successful vaccination as early as 1 day of age (Sincovic &
Hunt, 1968).
14
Prevention and control.
treat ILT.
No effective drug exists to
Live attenuated vaccines given in drinking water
and by eye drop provide effective protection against ILTV.
ILTV attenuated by passage in cell cultures (Gelenczei &
Marty, 1964; Izuchi, et al., 1984; Izuchi
et al., 1983) or
embryonated eggs, and selected mild enzootic strains
(Pulsford & Stokes, 1953) used as vaccines can induce
protection against ILTV.
Vaccination with virulent strains
of ILTV is not as safe as using attenuated virus since
virulent virus may cause disease.
still may cause latent infections.
Attenuated virus vaccines
Inoculation of
infraorbital sinuses (Shibley, et al., 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.
Layers are mostly vaccinated by eye drop
using attenuated virus vaccines, while broilers are commonly
vaccinated by spray or drinking water.
ILTV contacting the
epithelium of the nasal cavity during drinking results in
successful vaccination.
Field isolate SA-2, used for vaccination in Australia,
causes latent infections (Bagust, 1986).
Current vaccines
against ILTV may be flawed because they cause latent
infections.
A recombinant vaccine for ILTV potentially
could be constructed.
A 4 kilobase pairs (Kbp) ILTV DNA
15
fragment was cloned in plasmid pUC13, sequenced, and the
beta-galactosidase gene of Escherichia coli was integrated
by homologous recombination into this ILTV DNA fragment and
expressed under the control of a CMV promoter (Guo, et al.,
A recombinant ILTV vaccine might be used not only
1994).
against
ILTV, but also against other avian pathogens since
ILTV has the capacity to incorporate foreign DNA in its
genome (Guo, et al., 1994).
Purified ILTV glycoproteins also have been used to
vaccinate chickens, and have protected up to 83% of
vaccinates against ILTV.
Vaccination with a glycoprotein
preparation resulted in both neutralizing antibody and
delayed-type hypersensitivity responses, but neither
correlated with protection (York & Fahey, 1991).
Management is a key factor in preventing the disease.
Management considerations include sound sanitation and
hygiene measures, control of movement of staff, service
workers, feed, equipment and birds, and rodent and dog
control measures (Kingsbury, et al., 1958).
The United States Animal Health Association in 1990
passed a resolution calling for an ILTV eradication program.
The proposal includes most of the aspects of a complete
eradication program, including diagnostic capabilities,
vaccination, surveillance, biosecurity, and elimination of
infected flocks and infected houses (Daryl, 1990).
16
Strain classification.
Based on virus neutralization
or immunofluorescence tests using reference-specific
antiserum, most ILTV strains are antigenically similar,
although a few strains are relatively poorly neutralized by
such antisera (Burnet, 1936; Pulsford & Stokes, 1953).
Minor antigenic variation among strains of ILTV has been
reported (Russell & Turner, 1983; Shibley, et al., 1962).
Restriction endonucleases have been used to study ILTV
DNA and to differentiate certain antigenically-similar
strains.
Differences in DNA fragment patterns have been
found between an Australian vaccine strain and a field
strain by this method (Kotiw, et al., 1982).
Others found
differences between American and European ILTV strains and
between an American field strain and a vaccine strain (Leib,
et al., 1986).
Further studies found differences between
live ILTV vaccines and reference strains of ILTV, but found
ILTV field isolates from North Carolina to be
indistinguishable from vaccine strains (Guy, et al., 1989).
However, differences between DNA patterns of vaccine strains
and Georgia field isolates of ILTV were reported (Andreasen,
et al., 1990).
Differences among restriction endonuclease
DNA fingerprints of Pennsylvania field isolates, vaccine
strains, and challenge strains of ILTV also have been
reported (Keller, et al., 1992).
Cloned DNA fragment,
reciprocal DNA:DNA hybridization also has been used for
differentiating ILTV strains (Kotiw, et al., 1986).
17
Different antigenically-similar paramyxovirus strains
have been classified by using monoclonal antibodies (MCA) in
reactions such as the indirect immunoperoxidase test,
hemagglutination inhibition test, and the binding pattern of
MCA with the infected cells (Alexander, et al., 1989;
Collins, et al., 1989).
However, no ILTV strain-specific
MCA has yet been reported.
ILTV genome.
Compared to other herpesvirus
subfamilies, alphaherpesviruses have shorter DNA distances
based on statistical analysis of relative abundance of
dinucleotides in pairs of genomes, greater similarity of
amino acid sequences, and are less diverse.
Among
alphaherpesviruses, the protein sequences of EH1 and PRV are
the most similar (Karlin, et al., 1994).
In common with other herpesviruses, ILTV has a linear,
double-stranded DNA genome, with a DNA density of 1.704 g/m1
(Plummer, et al., 1969).
Hybridization experiments using
ILTV clones determined the ILTV genome to consist of 155
Kbp.
The genome has a long unique sequence of 120 Kbp and a
short unique sequence of 17 Kbp that is bounded by repeat
sequences each of 9 Kbp.
An unrelated second pair of repeat
sequences also was located at 0.67 and 0.88 map units.
A
terminal repeat of the unique long region also was detected,
but no isomerization of the unique long sequence was found
(Johnson, et al., 1991).
ILTV DNA is more homologous to
other alphaherpesviruses than to gammaherpesviruses, as
18
shown by a study in which ILTV DNA was sheared randomly and
cloned in the M13 bacteriophage.
The clones containing ILTV
were sequenced and compared by computer with known sequences
of other herpesviruses.
Twenty genes were similar to VZV
and 19 genes were similar to HSV type 1, but only 12 genes
showed slight homology with Epstein-Barr virus (EBV)
(Griffin, 1989).
Using restriction enzyme maps, the ILTV genome coding
capacity, approximately 70 distinct genes, is similar to HSV
1 (Johnson, 1991).
Herpesviruses from the same genera,
compared by hybridization, show more homology than
herpesviruses from unrelated genera; but under very relaxed
conditions of hybridization, there is slight homology (BenPorat, et al., 1983).
Homologous regions between
herpesviruses and host DNA sequences have been reported for
EBV (Hayward, 1980) and HSV 1 (Puga, 1982).
Most herpesviruses have a molecular weight between 80
and 150 X 106 daltons, the variation being due to terminal
and internal reiterated sequences (Roizman & Batterson,
1986).
The molecular weight of ILTV DNA was estimated by
summation of restriction endonuclease fragments using Bam HI
to be 102.1 X 106 and using Hind III to be 97.35 X 106.
The
difference between the estimates may be due to the presence
of submolar fragments.
Genomic isomerization results from the inversion of
long and or short sequences that are bounded by repeat
19
sequences.
Genomic isomerism among alphaherpesviruses ILTV,
VZV, EH1, and PRV involves two isomers of DNA with molecular
weights of 100 X 106, 100 X 106, 94 X 106, and 91 X 106
respectively (Leib, et al., 1986; Roizman & Betterson,
1986).
Other alphaherpesviruses such as HSV 1, HSV 2, and
BH2 have four genomic isomers with molecular weights of 96 X
106, 96 X 106, and 88 X 106 respectively (Roizman, 1982,
Roizman & Betterson, 1986).
Most gammaherpesviruses have
one isomer, except Marek's disease herpesvirus and turkey
herpesvirus which have 4 isomers.
All betaherpesviruses
have 4 isomers (Roizman & Betterson, 1986).
The ILTV
genomic data about molecular weight and isomerism also are
supported by other workers (Kotiw, et al., 1982).
The
percentage of guanine plus cytosine in ILTV DNA is 45%
(Plummer, et al., 1969), which is lower than many other
animal herpesviruses, such as HSV 1, 67%; HSV 2, 69%; BH1,
72%; EH1, 57%; and PRV, 74% (Roizman & Betterson, 1986).
The gene encoding the ILTV 205 kd complex glycoprotein
(gB 205) has been sequenced; the gene lies 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).
Based on a study comparing chicken, human, and
herpesvirus genomic sequences, chicken sequences are more
similar to herpesvirus sequences, which may suggest that
20
chickens or other avian species are natural hosts of
herpesviruses (Karlin, et al., 1994).
ILTV PROTEINS AND GLYCOPROTEINS
Over 33 viral proteins have been reported for HSV 1
(Spear & Roizman, 1972).
Six antigenically distinct
glycoprotein of HSV 1, gB, gC, gD, gE, gG, gH have been
described (Ackerman, et al., 1986; Baucke & Spear, 1979;
Buckmaster, et al., 1984; Cassai, 1975; Richman, et al.,
1986; Roizman, et al., 1984; Showalter, et al., 1981; Spear,
1976; Spear, 1972).
Glycoproteins gB, gC, gD, and gE are
sulphated (Hope & Marsden, 1983) and glycoprotein gE carries
fatty acids (Johnson & Spear, 1982).
Glycoprotein gB is
required for the fusion of the viral envelope to the
cellular plasma membrane (Little, 1981).
contains 6 polypeptides.
The capsid
Empty capsids lack one protein
located on the surface of DNA-containing capsids and are
smaller (Braun, et al., 1984; Gibson & Roizman, 1972).
ILTV glycoproteins detected in western blotting and
immunoprecipitation studies.
Four major glycoproteins
detected in western blotting and immunoprecipitation by MCA
and chicken and rabbit immune sera, 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-
21
infected cells.
Besides those 4 major glycoproteins, ILTV
glycoproteins of 160, 125, 105, 85, and 70 kd are detected
by chicken and rabbit immune sera in western blotting.
However MCA only detected glycoproteins of 160 and 85 kd in
western blotting in addition to the four major
glycoproteins.
In immunoprecipitation studies using MCA and
chicken or rabbit immune sera, no additional ILTV
glycoproteins were detected other than the four major
glycoproteins (York, et al., 1987).
Glycoproteins 125 and
105 were not detected in western blotting using MCA.
The
envelope glycoproteins are important immunogens capable of
inducing humoral and cellular immune responses (York &
Fahey, 1990).
In characterizing MCA by immunoprecipitation and
western blotting, York, et al. obtained information about
the ILTV glycoproteins (York, et al., 1987).
groups of MCA.
They defined 5
Group I reacted with the 60 kd glycoprotein
by both techniques, and group II MCA reacted with the 205,
115, and 90 kd glycoprotein in immunoprecipitation.
The
reactivity of group I and group II MCA in immunofluorescence
suggests that the antigens are present on the surface of
virus-infected cells.
Group II antibodies recognized at
least three epitopes, which was confirmed by additivity
ELISA assays.
Group III, IV, and V antibodies recognized
several proteins with lower molecular weights between 45 and
24 kd.
Low molecular weight nucleocapsid proteins have been
22
described for other herpesviruses too (Friedrichs & Grose,
1986).
On the basis of immunofluorescence studies, the
antigens recognized by groups III, IV, and V appeared to be
nucleocapsid proteins since they were not present on the
surface of virus-infected cells (York, et al., 1990).
ILTV glycoproteins: size comparison with other
herpesviruses.
Glycoproteins are considered to be the major
immunogens of ILTV.
Characterization of vaccine-strain ILTV
glycoproteins has been done in Australia using in vitro
labelling of the carbohydrate portion.
Minor differences
were found in the molecular weights of glycoproteins from
vaccine and virulent strains of ILTV.
The largest
glycoprotein detected, measuring 205 kd, was strongly
labelled by glucosamine and mannose, indicating that it
contains a high proportion of these sugars or that it
represents the largest proportion of glycoprotein in ILTVinfected cells (York, et al., 1987).
HSV 1 glycoproteins
ranging from 59 to 129 kd (Spear, 1976), HCMV glycoproteins
ranging from 52 to 130 kd (Farrar & Oram, 1984), and Marek's
disease virus glycoproteins ranging from 50 to 115 kd
(Ikuta, et al., 1984) also have been reported.
Glycoproteins of 205, 160, 115, 90, 85, 60, 55, 35 to 40,
18, and 13 kd have been found with ILTV (York, et al.,
1987).
Glycoproteins 205, 115, 90, and 60 kd were detected
by chicken and rabbit antisera prepared against both
virulent and vaccine strains of ILTV, suggesting the
23
similarity of glycoproteins of different strains (York, et
al., 1987).
Antigenically distinct families of ILTV glycoproteins.
On the basis of reactivity in western blotting and detection
by MCA, two antigenically distinct families of ILTV antigens
have been recognized.
One family contains glycoproteins of
molecular weights 205, 160, 115, 90, and 85 kd, and the
other family contains a single glycoprotein of 60 kd
molecular weight (York, et al., 1990).
MCA have defined
several groups of ILTV antigens against which an immune
response is stimulated, and most of these antigens consist
of more than one antigenically-related protein band in
western blotting experiments.
MCA against ILTV detected
multiple glycoprotein bands in western blotting (York, et
al., 1990), perhaps because ILTV glycoproteins share
antigenic determinants or because one protein is the posttranslational product of other larger protein, as is the
case with BH1 and PRV (Hampl, et al., 1984; van Drunen
Little-van den Hurk & Babiuk, 1986; van Drunen Little-van
den Hurk, et al., 1984).
Denaturation by reducing
conditions some times used in western blotting may be
responsible for reports that some ILTV proteins not detected
in immunoprecipitation are detected in western blotting
using MCA (York, et al., 1987).
Detection of multiple
glycoprotein bands by MCA also has been described for other
herpesviruses (Hampl, et al., 1984; van Drunen Little-van
24
den Hurk & Babiuk, 1986; van Drunen Little-van den Hurk, et
al., 1984).
The largest glycoprotein detected for ILTV is 205 kd
(York, et al., 1987).
Larger glycoproteins occur on other
herpesviruses, including a glycoprotein of 129 kd on HSV 1
(Spear, 1976), a 350/300 kd and a 250/200 kd glycoprotein on
EBV (Edson & Thorley-Lawson, 1981), and a 250 kd
glycoprotein on HCMV (Farrar & Dram, 1984).
Immune chicken
serum or serum from rabbits immunized either with purified
glycoprotein or whole virus give similar profiles in western
blotting, which indicates that ILTV glycoproteins are both
antigenic and immunogenic (York, et al., 1987).
Important ILTV proteins could be sequenced and cloned
to produce recombinant vaccines.
However, for such a
vaccine to be effective it should stimulate cell-mediated as
well as antibody-mediated immune responses.
Such vaccines
have been produced against other herpesviruses, and
immunization of mice and guinea pigs with recombinant
vaccines expressing HSV 1 glycoprotein was effective
(Cantine, et al., 1987; Cremer, et al., 1985; McDermott, et
al., 1989; Paoletti, et al., 1984; Wachsman, et al., 1987;
Weir, et al., 1989).
Immunogenic ILTV glycoproteins.
ILTV glycoprotein
immunoprecipitated with MCA can cause delayed-type
hypersensitivity in chickens by stimulating the cellmediated immune response (York & Fahey, 1990).
Purified
25
ILTV glycoproteins were used to immunize chickens and to
Such injections of
study their immune responses.
glycoproteins detected by group I and group II MCA caused
thickening of the wattles in cockerels (York & Fahey, 1990).
Immune responses also may be directed against nonglycosylated antigens (Arvin, et al., 1987, Martin, et al.,
1988), and some such responses may be protective (Reddehase,
et al., 1987).
Both glycosylated and nonglycosylated ILTV
antigens have been identified.
Purified HSV 1 glycoproteins
gB, gC, gD, and gE induced a neutralizing antibody response.
In other studies, MCA produced against these glycoproteins
passively protected against challenge (Marsden, 1987).
However, nonneutralizing antibodies also may confer
protection (Marsden, 1987).
Another study found that gC and
gB are target antigens for virus-specific cytotoxic T cells
(Blacklaws, et al., 1987; Glorioso, et al., 1985).
Synthetic peptides corresponding to the N-terminus of gD
were used to immunize mice resulting in protection, but no
neutralizing antibody could be demonstrated in the immunized
mice (Watari, et al., 1987).
ILTV polypeptides: comparison with other herpesviruses.
Of 16 identified ILTV polypeptides (alpha polypeptide 200
kd; beta polypeptides 115 and 82 kd; gamma polypeptides 170,
155, 130, 96, 90, 88, 68, 66, 47, 34, 26, 21, and 18 kd),
representing about 20% of the ILTV genome coding capacity,
chicken and rabbit ILTV antisera identified only 9
26
polypeptides (205, 160,125, 115, 105, 90, 85, 70, and 60 kd)
in western blotting and immunoprecipitation.
Of 10
glycoproteins described by York, et al., (1987), 8 were
identified using metabolic labelling with (mC)glucosamine to
have similar molecular weights to gamma 2 polypeptides
(Prideaux, et al., 1992).
ILTV gamma polypeptides are
synthesized in the absence as well as the presence of viral
DNA replication (Prideaux, et al., 1992) and account for
most of the ILTV glycoprotein, as with HSV 1 (Prideaux, et
al., 1992; Roizman & Sears, 1990).
In a recent study, the ILTV glycoprotein B (gB) gene
was identified using the polymerase chain reaction.
Northern blot analysis using a portion of the open reading
frame as a probe identified a 2.7 kb RNA transcript in ILTVinfected CEL cells.
Analysis of the amino acid sequence of
the ILTV protein indicated that it shares structural
features with the gB glycoprotein of other herpesviruses
(Poulsen, et al., 1991).
In another study, computer
sequencing analysis of random fragments of ILTV DNA
identified a 3698 base pair portion of the genome
containing the entire gene encoding glycoprotein gB, a 205
kd 873 amino acid transmembrane glycoprotein.
The C-
terminus of the gene was homologous to the gB gene of HSV 1
(Griffin, 1991; Kongsuwan, et al., 1991).
Comparing the
ILTV gB gene with the gB gene of other herpesviruses,
extensive homology occurs throughout the molecule except at
27
the amino and carboxyl termini (Kongsuwan, et al., 1991).
Comparison of ILTV gB with the homologous sequences of other
herpesviruses also shows 10 conserved cysteine residues on
the surface of the molecule.
Most of the N-linked
glycosylation sites are conserved (Griffin, 1991).
The gB
gene has hydrophobic sequences at the amino and carboxyl
terminals, is located at map coordinates 0.23-0.25 in the
unique long (UL) region of the ILTV genome, and is
transcribed from right to left (Kongsuwan, et al., 1991).
Recombinant bacteriophages expressing ILTV glycoprotein
60 (gp60) obtained from an ILTV genome library were screened
by MCA specific for the 60 kd glycoprotein to locate the DNA
fragment which contains the gp60 gene (Kongsuwan, et al.,
1993a).
Using the fragment as a hybridization probe, the
gp60 gene was located at map unit 0.72-0.77 in the UL region
of the ILTV genome.
The 1.2 kd gp60 gene also was sequenced
(Kongsuwan, et al., 1993a).
An ILTV gene designated p32, encoding a polypeptide of
298 amino acids (30 kd) with 4 N-glycosylation sites and a
signal sequence at the N-terminal region, was identified,
sequenced, and found to be located upstream of the
glycoprotein gp60 gene (Kongsuwan, et al., 1993b).
The
amino terminal of p32 is homologous to glycoprotein X (gX)
of PRV and to its homolog in EH1 and to glycoprotein G (gG)
of HSV 2 and EH4.
Within this conserved amino region was
found one conserved N-linked glycosylation site and 4
28
conserved cysteine residues, 3 of which have the
characteristic spacing of amino acids between the 3 cysteine
molecules that other alphaherpesviruses glycoproteins have
(Kongsuwan, et al., 1993b).
PRV gX is immunogenic but non-
essential, and can be deleted without reducing the
infectivity of PRV (Marchioli, et al., 1987).
A vaccine
consisting of a wild-type strain with a gX deletion has been
constructed for PRV (Marchioli, et al., 1987).
Based on the
similarities between ILTV p32 and gX of PRV, a similar
vaccine for ILTV might be constructed.
ILTV p32 is detected
by chicken anti-p32 serum as a 65 kd glycoprotein in sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE), suggesting its increased molecular weight is due to
glycosylation of N-linked glycosylation sites (Kongsuwan, et
al., 1993b).
ILTV p32, like gX of PRV, is immunogenic but
failed to protect against challenge when chickens were
vaccinated intraperitoneally (Kongsuwan, et al., 1993b).
MONOCLONAL ANTIBODIES
Introduction.
Monoclonal antibodies (MCA) do not
differ structurally from naturally-occurring polyclonal
antibodies.
The property which makes MCA unique is that all
the molecules in any single preparation are identical.
MCA
are a homogeneous population of identical antibodies with a
defined specificity, secreted by a B-cell clone after fusion
29
with tumor cells.
MCA have the same specificity of reaction
with any defined antigen every time whenever reacted.
On August 7, 1975, Kohler and Milstein published in
Nature a report describing
"continuous cultures of fused
cells secreting antibody of predefined specificity" (Kohler
& Milstein, 1975).
They observed that somatic cell
hybridization could be used to generate a continuous
"hybridoma" cell line producing a monoclonal antibody.
Immunologists have since produced large amounts of
homogeneous monoclonal antibodies against a wide variety of
antigens.
MCA have been produced against histocompatibility,
differentiation, tumor, or other cell-surface antigens, as
well as viral and bacterial antigens and certain single
antigenic determinants on a wide variety of proteins,
nucleic acids, and sugars.
MCA to certain specific antigens
are available commercially.
The primary advantage of MCA as diagnostic reagents is
their specificity of binding to a single epitope, producing
highly specific diagnostic test reagents.
many research purposes.
MCA are used for
Examples include construction of
antigenic maps of the hemagglutinin molecule of influenza
virus, in vitro estimation of mutation rate or genetic
changes of influenza virus by culturing virus in the
presence of a single neutralizing monoclonal antibody, use
of MCA in passive protection against different antigenic
30
determinants of influenza virus, and ability of MCA to
separate individual antigens from a complex mixture
(Antczak, 1982; Re, 1987).
Like natural immunoglobulin (Ig) antibodies, MCA may be
of any of the five different classes such as IgG, IgM, IgA,
IgE, and IgD.
These classes are based on differences in the
immunoglobulin heavy chains.
Within the IgG and IgA
immunoglobulin classes, there is predictable variation
insufficient to designate a distinct class.
However, based
on this variation subisotypes of those immunoglobulins,
including IgGl, IgG2a, IgG2b, IgG3, IgAl, and IgA2, have
been described.
These subisotypes may have distinctly
different biological properties including catabolic rate,
complement fixation, and placental transfer (Roit, 1984).
How MCA are produced.
For production of MCA, a
suitable host is immunized with an antigen.
Although mice
are usually used for this purpose, four animal species can
be used for MCA production: mouse, rat, hamster, and rabbit.
Rabbit MCA have recently been described (Raybould &
Takahashi, 1988).
Mice are the most common choice for
immunization and production of MCA because mice are easier
to handle and there are many anti-immunoglobulin reagents
available that are specific for each mouse Ig isotype.
Also, mouse MCA are easier to purify than rat MCA.
Female BALB/c mice are preferred if the SP2/0-Ag14
myeloma cell line is used as a fusion partner because the
31
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 aggressive.
In one popular protocol, purified antigen is thawed and
mixed with an equal amount of Freund's complete adjuvant by
sonification.
Female BALB/c mice are immunized with the
antigen intraperitoneally (IP).
Three to four weeks later a
second injection of antigen, consisting of incomplete
Freund's adjuvant mixed in equal amount with the purified
antigen is given to the mice (Coligan, et al., 1991).
Three weeks after the first injection of antigen, blood
is obtained from the conjunctival venous sinus of each mouse
using 100 gl heparinized capillary tubes.
Serum is
separated by centrifugation and is checked for antibody
against the antigen using an indirect immunofluorescence
test (IFAT).
Serum is subsequently checked weekly for
antibody until fusion is performed.
Four weeks after the
first injection a third injection of antigen in incomplete
Freund's adjuvant is given to the mice.
An intravenous
booster injection of the purified antigen without any
adjuvant is given to mice 24-48 hours before fusion
(Coligan, al., 1991).
One day before fusion, feeder plates are made from
spleen cells of a female BALB/c mouse that has not
32
experienced any antigen exposure.
Mice are killed by
dislocating the cervical vertebrae and disinfected with 70%
alcohol before placing them in a sterile cabinet.
Spleens from immunized mice are removed aseptically,
minced, and washed twice in complete Dulbecco's modified
Eagle's medium (DMEM) without serum, containing 1 ml
penicillin/streptomycin (100X) in 100 ml of DMEM, 1 ml of Lglutamine (100X) in 100 ml of DMEM.
SP2/0-Ag14 myeloma
cells are fused with spleen cells of the immunized mice for
production of monoclonal antibodies (Coligan, et al., 1991;
Kohler & Milstein, 1975).
The spleen cells are mixed with
SP2/0-AG 14 mouse myeloma cells at the ratio of 1:1 and
centrifuged.
The supernatant is discarded and 1 ml of 50%
polyethylene glycol (PEG) in DMEM, is added to the cells as
a fusion reagent.
Cells are 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 and very slowly with the
pipet.
Cells are centrifuged and resuspended in
hypoxanthine aminopterin thymidine (HAT) medium containing
20% fetal bovine serum (FBS), HAT, penicillin, streptomycin,
L-glutamine, Hepes buffer, and OPI (oxaloacetate,pyruvate,
bovine insulin) (Coligan, et al., 1991).
One hundred gl of cell suspension is added to each well
of 96-well tissue culture plates (feeder plates) and
incubated at 37C in a 5% CO2 atmosphere.
every 2 days.
Cells are fed
For the first feeding, the wells are fed with
33
HAT medium, while for the second feeding hypoxanthine
thymidine (HT) medium is used.
Subsequently, wells are fed
with complete DMEM containing 10% FBS, to allow hybridomas
to grow from fused cells.
Normal cells have two pathways for nucleotide
synthesis, the de novo pathway and the salvage pathway.
Myeloma cells lack hypoxanthine-guanine phosphoribosyl
transferase (HGPRT) which is essential to the salvage
pathway, so they must use the de novo pathway.
They are
thus sensitive to the aminopterin in HAT medium, which
inhibits dihydrofolate reductase and hence blocks de novo
purine and thymidilate synthesis.
Because of the effect of
aminopterin, unfused myeloma cells are killed in HAT medium.
Unfused splenic B cells die naturally in the medium because
they are not immortal like myeloma cells.
Fused cells have
both salvage pathway enzymes, HGPRT and thymidine kinase,
from their spleen cell component.
Fused cells must utilize
the salvage pathway, being provided with hypoxanthine and
thymidine in the HAT medium.
A fused cell's HGPRT utilizes
the hypoxanthine for purine synthesis and its thymidine
kinase utilizes the thymidine for thymidylate synthesis.
Thus, only fused cells can survive in HAT medium because 1)
they have the enzymes HGPRT and thymidine kinase which are
necessary to utilize the salvage pathway, provided by the
splenic B cells, and 2) because of immortalization provided
by the myeloma cells.
34
Screening of hybridomas for production of specific MCA
normally begins 10-14 days after fusion.
Supernatant fluids
from wells are screened for specific antibodies when the
culture supernatant becomes acidic (yellowish) and the
hybridomas are sufficiently mature to produce specific
antibodies.
The hybridomas grow in the form of a clump of
cells which is visible using a microscope.
Enzyme-linked
immuno-sorbent assay (ELISA) and IFAT are most commonly used
for identification of positive hybridomas.
In the IFAT
procedure, virus-infected cells against which MCA are
produced and uninfected cells are collected, washed with
PBS, and centrifuged at 450 X g.
The supernatant is
discarded and the cell pellet is suspended in 0.01M FA
buffer (pH 7.6 containing Na2HPO4, NaH2PO4.H20, and NaC1) with
2% bovine serum albumin (BSA) and centrifuged as before.
The supernatant is discarded and the cell pellet is
resuspended in FA buffer containing 2% BSA.
One microliter
of the virus-infected cells is smeared on wells of tefloncoated slides.
Uninfected cells also are smeared on other
wells on the slides to serve as negative cell controls.
Slides are air dried then fixed in acetone for 10 minutes.
Twenty microliters of undiluted hybridoma supernatant is
added to infected and uninfected wells, and the slides are
incubated for 20 minutes at 37C in a 5% CO2 atmosphere.
Slides are washed with FA buffer, soaked in FA buffer for 20
minutes, washed with double distilled water, rinsed, and air
35
dried completely.
Twenty microliters of anti-mouse
immunoglobulin (1:200 dilution) is added to each well
containing infected and uninfected cells and incubated for
20 minutes.
Slides are washed with FA buffer, soaked in FA
buffer for 20 minutes, washed with double distilled water,
rinsed, counterstained for 5 minutes with 0.001% Evan's
blue, air dried, mounted with mounting fluid, and examined
under a fluorescent microscope.
The hybridomas that are found to be producing
antibodies are cloned by limiting dilution calculated to
give less than 1 cell per well.
A concentration of ten
cells per ml is produced by serial dilution, and the cell
suspension is plated onto feeder plates at the rate of 100
gl per well to give less than 1 cell per well (Coligan, et
al., 1991).
Screened, cloned hybridomas producing specific
MCA are grown in 80 cm2 tissue culture flasks, centrifuged,
and the cell pellet is suspended in freezing medium (DME
70%, FBS 20%, DMSO 10%) and preserved in liquid nitrogen.
To produce MCA ascites fluid, 8-week-old female BALB/c
mice are injected IP with 0.5 ml of incomplete Freund's
adjuvant 1 week before inoculation with the hybridoma cells.
Hybridoma cells are thawed in a water bath and suspended in
a 24-well plate at the rate of 1 x 106 cells per ml in DMEM
containing 20% FBS, 1 ml penicillin/streptomycin (100X) in
100 ml of DMEM, and .1 ml of L-glutamine (200mM) in 100 ml of
DMEM.
Hybridoma cells are transferred to 25 cm2 flasks
36
after 24 hours of incubation at 37 C in a 5% CO2 atmosphere.
Whenever the medium becomes yellow, hybridoma cells are
transferred into new tissue-culture flasks and are fed with
DMEM containing antibiotics, L-glutamine, and 10% FBS.
Hybridoma supernatant is used to perform IFAT to assure that
the hybridoma cells are still producing specific MCA.
Hybridoma cells are transferred to 50 ml conical centrifuge
tubes.
Antibody-secreting cells are centrifuged at 500 X g
at room temperature for 5 minutes.
The supernatant is
discarded and hybridoma cells are washed three times by
resuspending in 50 ml of PBS and centrifuging for 5 minutes
at 500 X g at room temperature.
The supernatant is
discarded and cells are counted with a hemocytometer.
Trypan blue (0.1% in ethyl alcohol) is mixed with the
hybridoma cells at a 1:10 ratio to identify dead cells.
Hybridoma cells are resuspended at the rate of 2.5 X 106
living cells per ml in PBS.
Using a 22 gauge needle, 1 ml
of the cell suspension is injected IP into each mouse.
Seven to ten days after the injection mice develop ascites.
Ascites fluid is collected by inserting an 18 gauge needle
into the abdominal cavity and allowing ascites fluid to drip
into a sterile polypropylene centrifuge tube.
Ascites fluid
is centrifuged at 1500 X g for 10 minutes at room
temperature.
collected.
The pellet is discarded and the supernatant is
Ascites fluid is heat-inactivated for 45 minutes
in a 56 C water bath, filter sterilized through a 0.45 gm
37
filter, aliquoted, and stored undiluted at -70 C.
Ascites
fluid from each mouse sample is diluted 1:10 in FA buffer,
and IFAT is performed to ensure the ascites fluid contains
specific MCA.
MCA also may be collected from cloned hybridomas grown
in 80 cm2 tissue culture flasks in complete DMEM containing
10% FBS.
Extinct cultures cf the cells are made by
incubating the cells at 37C in a 5% CO2atmosphere for 15 to
20 days without providing fresh medium after the cells are
sufficiently grown.
This produces a high titer of antibody
in the cell supernatant.
After 15 to 20 days the cultures
are centrifuged, the supernatant is collected in small
aliquots, and is stored at -20C.
Use of MCA to study ILTV.
MCA against ILTV have been
produced and characterized by immunoprecipitation and
western blotting.
MCA have been produced against ILTV and
have been described (York, et al., 1987).
One other group
also has produced MCA against ILTV and has used them to
compare diagnostic tests such as IFAT and the IP test (Guy,
et al., 1992).
IFAT reactions using MCA specific for ILTV have been
performed to study the pattern of reactivity and other
properties of the MCA.
In such IFAT studies infected cells
are examined for the location of specific fluorescence in
ILTV-infected cells.
MCA are used as primary antibodies to
38
bind viral antigens.
Secondary antibodies, conjugated with
a fluorochrome then bind MCA and make it possible to see the
fluorescence reaction with a fluorescent microscope.
This
test helps in diagnosing the presence of ILTV antigen as
well as in studying the reactivity pattern of MCA.
Five
groups of MCA have been described against ILTV and their
reactivity pattern has been studied in immunofluorescence
studies (York, et al., 1990).
The details of reactivity
patterns of the 5 groups of MCA were discussed earlier in
conjunction with ITLV proteins and glycoproteins.
Those
results using MCA in IFAT studies reveal information about
the MCA as well as about viral antigens.
IFAT studies using
MCA help determine the cellular location of viral antigens
against which immune responses are stimulated.
MCA also can be used to detect ILTV in experimentallyinfected chicken tracheas by the IP test.
ILTV-infected
cells are smeared on slides, fixed in acetone, and soaked in
PBS.
ILTV-infected cells are incubated in blocking serum,
then in diluted ILTV-specific MCA, and then incubated with
biotinylated diluted secondary antibody.
Slides are
incubated in Vectastain ABC reagent (contains avidin and
biotinylated horseradish peroxidase), incubated in substrate
solution for 5 min, counterstained, mounted and read.
positive ILTV-infected cell stains reddish-brown.
Some
workers have used MCA to detect ILTV in experimentally-
A
39
infected chicken tracheas by the IP test (Guy, et al.,
1992).
MCA also have been used to study ILTV proteins and
glycoproteins in western blotting and immunoprecipitation
experiments (York, et al., 1990).
In western blotting
experiments, ILTV proteins are extracted from ILTV-infected
cells, separated on a gel by electrophoresis, transferred
onto a nitrocellulose membrane, and then detected by MCA.
Major immunogens of ILTV can be identified with this
technique.
Five groups of MCA described by York, et al.
(1990) were described based on such studies.
The details of
the 5 groups of MCA are discussed earlier in conjunction
with ITLV proteins and glycoproteins.
These results also
indicate the ILTV antigens against which immune responses
are stimulated.
Such studies also may provide information
about the conformation of ILTV proteins and whether the
proteins are glycosylated.
Using drugs like tunicamycin,
which inhibits carbohydrate synthesis, studies with ILTV
have been done (York, et al., 1990).
Of group II MCA, 131-6
and 23-1 were unable to detect any protein in extracts of
tunicamycin-treated cells while MCA 22-37 recognized three
protein bands of 135, 73, and 68 kd and MCA 12-1 recognized
two protein bands of 135 and 102 kd.
Groups III, IV, and V
MCA recognized the same protein bands in extracts prepared
in the presence or the absence of tunicamycin, suggesting
40
that the proteins they bind do not contain any carbohydrate
residue (York, et al., 1990).
Additivity ELISA studies also are useful to study viral
proteins.
In this technique a combination of two MCA in an
antigen-saturating concentration are used in series in an
ELISA test.
If MCA are specific for the same epitope,
binding of one MCA blocks subsequent binding of the other
If the 2 MCA are specific for different epitopes, they
MCA.
bind separately and the ELISA reading increases.
Using this
technique, group II MCA that detected the same proteins in
western blotting were found to be specific for different
epitopes, suggesting the presence of more than one epitope
on the same viral protein.
Group I MCA all bound the same
epitope (York, et al., 1990).
MCA may serve as reagents to distinguish different
viral isolates.
Work on antigenic relationships in
paramyxoviruses has classified different strains using MCA
in such reactions as the IP test, hemagglutination
inhibition test, and the binding pattern of MCA with the
infected cells (Alexander, et al., 1989; Collins, et al.,
1989).
However, no ILTV strain-specific MCA has yet been
reported.
Using in vitro labeling, minor differences in
molecular weights have been found in vaccine and virulent
isolates of ILTV in western blotting experiments using MCA
(York, et al., 1987).
41
In other experiments, chicken and rabbit immune sera
against ILTV were produced and compared with group I and
group II MCA in western blotting and immunoprecipitation
studies.
In immunoprecipitation studies chicken and rabbit
immune sera detected the same bands detected by group I and
group II MCA.
Additional protein bands were detected in
western blotting by chicken and rabbit immune sera (York, et
al., 1990) as discussed earlier in conjunction with ITLV
proteins and glycoproteins.
ILTV glycoproteins detected by MCA were used to study
humoral and cell-mediated responses to those glycoproteins
as mentioned earlier.
Glycoproteins that induce both
humoral and cell-mediated immune responses may prove to be
important protective immunogens and could be useful in a
subunit vaccine (York, et al., 1990).
Group I MCA specific for the ILTV 60 kd glycoprotein
(gp60) were used in immunoscreening to detect recombinant
bacteriophages expressing the gp60 glycoprotein as discussed
earlier in conjunction with ILTV proteins and glycoproteins.
That allowed subsequent gp60 gene sequencing to be done, and
the DNA sequences coding for the gp60 epitope were
identified (Kongsuwan, et al., 1993a).
DETECTION AND DIAGNOSIS OF ILTV
Although some signs of severe acute disease are
characteristic, signs are often similar to other respiratory
diseases, so ILT can not be reliably diagnosed solely by
42
observation of "signs" and lesions.
is required for certain diagnosis.
Laboratory confirmation
Such laboratory
confirmation is commonly accomplished by means of virus
isolation (VI) and identification, by histopathology, or by
IFAT.
Other more experimental techniques include the IP
test, the polymerase chain reaction (PCR), and DNA probe
hybridization.
VI procedure.
Virus can be isolated from tracheal and
lung tissue by chicken embryo inoculation or by cell culture
inoculation (Chomiac, et al., 1960).
VI by inoculation of
the trachea or infraorbital sinuses of susceptible chickens,
although possible, is rarely used (Hitchner & White, 1958).
ILTV VI is achieved by inoculation of suspensions from
tracheal exudate, tracheal tissue, or lung tissue onto the
CAM of 9 to 12 day old embryonated chicken eggs or into
susceptible cell cultures.
The virus produces pocks on the
CAM of embryonated eggs after about 5 days.
In cell
culture, cytopathic effect (CPE) can be observed 24 to 48
hours after inoculation (Hitchner & White, 1958).
Chicken
embryo liver cells and chicken kidney cells are the
laboratory systems of choice for the isolation of ILTV
(Hughes & Jones, 1988).
VI and identification are widely
used and provide satisfactory results, but are timeconsuming and expensive.
Electron microscopic (EM)
examination of tracheal scrapings and recognition of
43
herpesvirus particles is also a means of rapid diagnosis or
of confirming VI results (Van Kammen & Spradbrow, 1976).
Histopathology.
Histopathology for the demonstration
of intranuclear inclusion bodies and typical syncytial cells
in tracheal and conjunctival tissues also is diagnostic of
ILTV (Beveridge & Burnet, 1946; Cover & Benton, 1958).
Histopathology is very useful, but requires skilled labor
for fixation, processing, and evaluation of tissue.
Fluorescent antibody test (FAT).
Direct or indirect
FAT also may be used for identification of ILTV (Braune &
Gentry, 1965).
ILTV was detected in chicken tracheal tissue
from day 2 to day 14 postinoculation using fluoresceinlabelled anti-ILTV globulins in DFAT (Bagust, et al., 1986;
Hitchner, et al., 1977; Wilks & Kogan, 1979).
However, the
DFAT for ILTV suffers from non-specific reactions.
IFAT
reactions using MCA specific for ILTV have been performed
for diagnostic and research purposes.
MCA are used as
primary antibodies to bind viral antigens.
Secondary
antibodies, conjugated with a fluorochrome then bind MCA and
make it possible to see the fluorescence reaction with a
fluorescent microscope.
The IFAT is rapid, and suffers from fewer non-specific
reactions than DFAT, but still requires a fluorescence
microscope.
It has not been widely used for diagnostic
purposes due to lack of specific MCA against ILTV.
44
IP test.
Slides containing ILTV-infected cells are
incubated in blocking serum (provided with the kits), then
in (diluted) ILTV-specific MCA, incubated with a
biotinylated (diluted) secondary antibody, then incubated in
a reagent containing avidin conjugated to biotinylated
horseradish peroxidase, incubated in substrate solution,
counterstained, mounted and examined under a microscope.
A
reddish-brown staining of the ILTV-infected cells represents
a positive reaction.
The IP technique is simple, rapid,
provides a relatively permanent record, and requires only a
conventional light microscope.
ILTV antigen has been
detected in frozen tissue sections by an indirect IP
procedure using MCA (Guy, et al., 1992).
The procedure has
not been widely employed, however, due to lack of strain
specific MCA against ILTV.
PCR.
The PCR requires a pair of primers that are made
from a known sequence of the ILTV genome.
Such primers have
been used for amplification of ILTV DNA (Williams, et al.,
1992).
In a PCR reaction these primers hybridize to the
specific corresponding sites on the template DNA.
Then Taq
polymerase binds to these primers and extends the DNA
template using the nucleotides provided in the PCR mixture.
PCR is run in a series of cycles, using different
temperatures to cause serial denaturation of the DNA
template, annealing of primers, and polymerization.
A total
of 35 to 40 cycles are run to obtain detectable quantities
45
of amplified product.
Strict precautions must be taken to
avoid contamination by extraneous DNA.
PCR products are
examined by agarose gel electrophoresis and the size of the
PCR product is determined by comparison with molecular
weight standards.
Experimentally, ILTV has been detected in tracheas,
turbinate tissue, and trigeminal ganglia using PCR
(Williams, et al., 1992).
Latent virus was detected in the
trigeminal ganglia of recovered chickens 61 days
postinfection using PCR, after VI failed to detect virus
(Williams, et al., 1992).
ILTV also has been detected in
joint and bone samples by PCR, possibly reflecting infection
of bone marrow macrophages (Jones, et al., 1993; Von Bulow &
Klasen, 1983).
PCR is a very specific and sensitive technique for
detection of ILTV, but extraction or purification of DNA may
be required and the technique is labor intensive.
It has
not been widely employed as a diagnostic technique for
ILTV.
Hybridization.
For DNA hybridization studies a labeled
or a non-labeled DNA probe is used.
A DNA probe is a DNA
fragment that binds to complementary target sequences in
hybridization studies.
A specific amplified PCR product may
be gel purified, extracted from the gel, and labeled for use
as a DNA probe for hybridization.
46
To perform hybridization, a DNA sample is transferred
and fixed onto a nylon membrane by treating with ultraviolet
light.
Following prehybridization, hybridization is
performed by incubating the nylon membrane in hybridization
solution containing the biotinylated DNA probe.
Hybridization provides the necessary conditions for binding
of the target DNA on the nylon membrane by a specific,
labeled DNA probe.
After hybridization, washings with
stringent salt solutions, blocking of the nylon membrane,
incubation with streptavidin-alkaline phosphatase conjugate,
washings with Tween-tris buffered saline (TTBS) and final
wash buffer, incubation with detection reagent, and exposure
to X-ray films are performed.
A minimum of 500 ng ILTV DNA was detected in tracheal
samples in a DNA hybridization assay by using a 1.4 kb nonradioactive probe, and this technique has been found to be
effective in diagnosing field outbreaks and also detecting
latent ILTV infections (Keam, et al., 1991).
Others have
described a much more sensitive 1.1 kb non-isotopically
labeled DNA probe and have detected 40 pg of ILTV DNA
extracted from purified ILTV (Key, et al., 1994).
Like PCR, hybridization is a very specific technique
for detection of ILTV, but involves extraction or
purification of DNA and the technique is labor intensive.
It has not yet been widely used in the diagnosis of ILTV.
47
ELISA or serum neutralization (SN).
An antigen-capture
ELISA using monoclonal antibodies was developed for the
rapid detection of ILTV antigen in tracheal exudate (Van
Kammen & Spradbrow, 1976).
ELISA systems for detection of
chicken antibodies to ILTV antibodies also have been
developed (Ohkubo, et al., 1988; York & Fahey, 1988; York,
et al., 1983).
infection.
Neither is widely used to diagnose ILTV
Virus neutralization (VN) titers of chicken
serum can be measured by using the CAM-pock counting
technique, or by plaque production in cell culture
monolayers (Churchill, 1965; Robertson & Egerton, 1977).
Detection of antibodies by SN or ELISA may occasionally be
used diagnostically but serological tests do not provide a
timely diagnosis.
48
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63
CHAPTER II
Characterization of Monoclonal Antibodies Against
Infectious Laryngotracheitis Virus
Ferhat Abbas ,A James R. Andreasen, Jr.,A Rocky J. Baker,A
Donald E. Mattson,A James S. GuyB
ACollege of Veterinary Medicine, Oregon State University,
Corvallis, Oregon 97331
BDepartment of Microbiology, Pathology, and Parasitology,
College of Veterinary Medicine,
North Carolina State University,
Raleigh, North Carolina 27606
64
SUMMARY
Ten monoclonal antibodies (MCA) produced against
infectious laryngotracheitis virus (ILTV) of chickens
reacted with several different ILTV protein bands in western
blotting experiments.
MCA C, E, 11, and 131-6 detected
bands of 205, 160, 115, and 90 kd, and several bands less
than 49 kd.
MCA D detected a band of 90 kd along with
several bands less than 49 kd.
MCA 4 and 5 each detected
bands of 205, 160, 100, 90, and 70 kd.
MCA 9 detected the
same bands detected by MCA 4 and 5 except the 160 kd band.
MCA 10 detected bands of 100, 90, and 70 kd and several
bands less than 49 kd.
MCA C, D, E, and 131-6 were unable
to react with any band when the ILTV was grown in the
presence of tunicamycin, which inhibits glycosylation,
suggesting that those MCA are specific for carbohydratebased epitopes.
MCA 6 reacted with only one band of 100 kd
in the absence or presence of tunicamycin.
The remaining
MCA detected only a 70 kd band in the presence of
tunicamycin except MCA 5, which reacted with bands of 70 and
90 kd.
In an indirect immunofluorescence test of 9 MCA,
each bound primarily to cytoplasmic antigens except MCA D,
9, and 10, which bound primarily to nuclear antigens.
MCA 9
and 10 also bound very weakly to cytoplasmic antigens, and
MCA E, 4, and 6 also bound weakly to nuclear antigens.
MCA 4 and 6 neutralized ILTV infectivity.
Only
65
INTRODUCTION
Infectious laryngotracheitis virus (ILTV) causes an
acute disease of chickens characterized by respiratory
depression, gasping, and expectoration of bloody exudate.
ILTV is assigned to the subfamily Alphaherpesvirinae, of the
Herpesviridae family.
Monoclonal antibodies (MCA) produced against the SA-2
strain of ILTV were characterized by immunoprecipitation and
by western blotting, defining 5 groups of MCA (York, et al,
1990; York, et al., 1987).
Group I reacted with a 60 kd
ILTV glycoprotein by both techniques, and the group II MCA
reacted with 205, 160, 115, 90, and 85 kd glycoproteins in
immunoprecipitation.
The reactivity of group I and group II
MCA in immunofluorescence suggested that they detect
antigens on the membrane surface of virus-infected cells.
Groups III, IV, and V MCA recognized several smaller
proteins between 45 and 24 kd.
Immunofluorescence studies
demonstrated that the antigens recognized by groups III, IV,
and V were nucleocapsid proteins and were not present on the
surface of virus-infected cells (York & Fahey, 1990; York,
et al., 1990).
In other herpesviruses, glycoproteins also
are major immunogens capable of inducing humoral and
cellular responses, but the involvement of other virion
components in inducing immune responses has not been
investigated (Watari, et al., 1987).
66
York, et al. further characterized their MCA as to
their specificity against glycosylated proteins or nonglycosylated proteins (York, et al., 1990).
To do that they
used detergent extracts of ILTV-infected chicken-embryo
liver cells (CELC) treated with tunicamycin, a drug that
inhibits glycosylation (Takatsuki, et al., 1985), in western
blotting experiments.
Comparing those results with the
results obtained with extracts from ILTV-infected CELC not
treated with tunicamycin, they found that Group I MCA failed
to detect any bands in extracts from tunicamycin-treated
cells (York, et al., 1990).
Of the Group II MCA, 131-6, and
23-1 did not detect any bands, but MCA 22-37 recognized
three bands of 135, 73, and 68 kd, and MCA 12-1 recognized
two bands of 135 and 102 kd from the tunicamycin-treated
extracts (York, et al., 1990).
The MCA in groups III, IV,
and V recognized the same bands, whether or not tunicamycin
was used (York, et al., 1990).
The specificity of various MCA for the same or
different epitopes was studied by additivity ELISA.
York,
et al. found all Group II MCA to have specificity for
different epitopes, although those MCA detected the same
bands in western blotting.
Group II antibodies recognized
at least three epitopes was confirmed by additivity ELISA
assays (York, et al., 1990).
By comparison, all Group I MCA
were specific for the same epitope (York, et al., 1990).
67
The objective of the present research was to perform an
initial characterization of MCA produced against the USDA
challenge strain of ILTV.
We also compared our MCA to MCA
produced by other researchers.
MATERIALS AND METHODS
Viruses.
ILTV strains utilized 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; 58800224, a
California field isolate; and 94-11221, an Oregon field
isolate.
Other viruses, including a parrot herpesvirus
isolated in CELC from the liver of a parrot that died
acutely, and avian adenovirus isolate 301, served as
negative controls.
ILTV was grown in CELC using described
techniques (Villegas, 1989).
MCA.
Mouse MCA, in the form of ascites fluid and
extinct culture supernatant fluid, designated C, D, and E
were produced against the USDA challenge strain of ILTV at
Oregon State University using described techniques (Abbas,
1992; Coligan et al., 1991).
Six other mouse monoclonal
antibodies against ILTV designated 4, 5, 6, 9, 10, and 11,
produced at North Carolina State University, were obtained
from Dr. James S. Guy as ascites fluid.
Additionally, a
mouse MCA (ascites fluid) against ILTV designated 131-6,
originally produced by Dr. Jennifer York (York, et al.,
68
1990; York, et al., 1987), was purchased for use as a
control in western blotting studies (JCU Tropical
Biotechnology Pty. Ltd., James Cook University, Townsville,
Queensland, Australia).
Indirect immunofluorescence test (IFAT).
IFAT was
performed as described (Abbas, 1992; Adair, et al., 1985).
Ten-fold dilutions of MCA ascites fluid and supernatant from
extinct cultures were evaluated by IFAT to determine the
best readable dilution for each MCA.
Titration of ascites
fluid (all MCA) and supernatant from extinct cultures (MCA
C, D, and E) also was performed by IFAT.
Isotyping.
The isotypes of the MCA were determined
with a mouse MCA isotyping kit ((Sigma Immunochemicals ISO].) Sigma Chemical Company, St. Louis, Mo.)
containing
rabbit antibodies against mouse isotypes IgGl, IgG2a, IgG2b,
IgG3, IgM, and IgA following the manufacturer's
instructions.
The kit contains precoated nitrocellulose
strips on which the entire assay is completed.
The strips
capture the relevant mouse immunoglobulin in such a way
that, on binding of an immunoglobulin, a designated letter
appears which clearly indicates the isotype.
Virus neutralization test (VN).
The VN test was
performed in 96-well microtiter plates using the constantvirus diluting serum (beta) method (Andreasen, et al.,
69
1990).
Five hundred TCIDm of virus was added to each well
of the plate, except the last two wells of each row, that
were used as control wells.
Fifty Al undiluted MCA (ascites
fluid) was added to the first well of each row.
Serum and
virus were mixed with a pipet, and 50 Al was transferred to
the next well of the same row and so on to make doubling
dilutions of the MCA ranging from 1:2 to 1:512.
Fifty Al
undiluted MCA was added to a well in each row as a
cytotoxicity control, and the last well in each row was used
as a CELC control.
CELC were grown according to a described
procedure (Villegas, 1989).
Back titration of virus also
was done to ensure its viability and titer.
was performed at room temperature for 60 min.
Neutralization
Following
neutralization, 100 Al of CELC was added to each well of the
microtiter plate.
The microtiter plates were incubated for
5 days in a CO2 atmosphere at 37 C and were examined daily
for cytopathic effect (CPE) using an inverted microscope.
The wells containing CPE were identified and marked on the
plate.
Total absence of viral CPE was considered to be
neutralization.
In other experiments, various combinations
of those MCA that failed to neutralize ILTV were evaluated
in VN tests performed as above.
Western blotting.
ILTV was grown as previously
described (Villegas, 1989), but with slight modification in
some experiments.
Briefly, after adsorption of the ILTV
70
onto CELC, unadsorbed virus was removed by washing the cells
with phosphate-buffered saline (PBS) and the cells were fed
with medium containing 1 gg/ml tunicamycin (Calbiochem
Novabiochem Corporation, La Jolla, Cal.).
A detergent extract of ILTV-infected CELC was prepared
according to a described procedure (York, et al., 1987).
Briefly, CELC were infected, and 24 hours postinfection
cells were scraped from tissue-culture plates and washed
three times with cold PBS.
The cell pellet was suspended at
a density of no greater than 30 x 106 cells/ml in cold lysis
buffer (0.05 M Tris-HC1, pH 8.0; 0.15 M NaCl; 1 mM
phenylmethylsulfonyl fluoride (PMSF); 0.01% NaN3; 1% nonidet
P-40; 1% sodium deoxycholate).
The cell suspension was
placed on ice for 15 min, sonicated, and clarified in a
microcentrifuge at 12,000 rpm for 10 min.
was collected and saved at -20 C.
The supernatant
Extracts were prepared
similarly from ILTV-infected cells treated with tunicamycin
and from uninfected CELC.
Western blotting was performed according to a
described procedure (Wiens, et al., 1990).
Briefly,
detergent extracts of ILTV-infected CELC were separated
under reducing conditions by discontinuous polyacrylamide
gel electrophoresis (PAGE).
After transferring the extracts
to a nitrocellulose membrane by electrophoresis in transblot
buffer, the membrane was incubated in Tween tris-buffered
saline (TTBS) (Tris-base 6.07 g, disodium EDTA 0.41 g, NaC1
71
8.7 g, Tween-20 1.0 ml, distilled water 1000 ml) containing
The
1% bovine serum albumin (BSA) at 37 C for 1 hour.
nitrocellulose was washed in TTBS for 5 min on a shaker.
The washing was repeated three times, and the nitrocellulose
paper was cut in 5 mm strips, each representing the contents
(Detergent extract of ILTV-infected CELC) of one well
transferred from the gel and the strips were incubated
separately for 1 hour at room temperature with gentle
shaking in various primary antibodies (MCA diluted 1:200 in
TTBS).
After washing again, the nitrocellulose strips were
incubated individually in secondary goat antimouse
peroxidase-labeled antibody (Hyclone Laboratories Inc.,
Logan, Utah), diluted 1:500, for 1 hour, then washed as
before.
Substrate solution was made by mixing 2 ml stock
substrate (1,4-chloro-naphthol 0.15 g, methanol 50 ml) in 10
ml 10 mM PBS containing 10 Al hydrogen peroxide.
The
nitrocellulose strips were incubated in substrate solution
for 15 min at 37 C.
The nitrocellulose paper was rinsed in
distilled water for 5 min to stop blot development.
The
strips were dried, photographed, and saved in the dark.
Prestained SDS-PAGE standards (Bio-Rad Laboratories,
Hercules, Cal.) and western blots prepared using MCA 131-6
as primary antibody were used as standards.
MCA 131-6
detects ILTV glycoproteins of 205, 160, 115, and 90 kd
molecular weight in extracts not treated with tunicamycin
72
and does not detect any protein in extracts treated with
tunicamycin (York, et al., 1987).
In another experiment, after electrophoresis the PAGE
gel was incubated in Coomassie blue solution (50% methanol
(v/v), 0.05% Coomassie R-250 (Bio-Rad), 10% acetic acid, 40%
distilled water) at room temperature for 15 min on a shaker.
The gel was rinsed with a solution containing 45% methanol
and 10% acetic acid in distilled water and then incubated in
the same solution for 10 min at room temperature on a
shaker.
The gel was rinsed with 10% acetic acid, incubated
in 10% acetic acid for at least 1 hour, and the gels made
from uninfected CELC were compared to gels made from ILTVinfected CELC.
Additivity ELISA.
described (Abbas, 1992).
ILTV was grown and purified as
A protein assay of the purified
ILTV was performed in 96-well microtiter plates using a
commercial protein assay reagent kit according to the
protocol provided by the manufacturer (BCA Protein Assay
Reagent, Pierce Chemical Company, Rockford, Ill.).
A set of
protein standards was made by diluting the stock BSA
provided with the kit.
Ten gl of protein standards and of
virus sample were added to separate wells and incubated with
working reagent for 1 hour at 60 C.
Absorbance was read at
562 nm with a microtiter plate reader.
A standard curve was
made by plotting absorbance against protein concentration
73
for the standards.
The protein concentration of the ILTV
sample was determined using the standard curve.
To ensure that the working MCA concentration saturated
the antigen, a saturation curve was made for each MCA to
determine the proper MCA dilution..
To ensure that the
plateau was reached because of saturation and not because
the conjugate was exhausted, the experiment was done with 2
concentrations of conjugate (1 gg/well, and 2 pg /well).
The additivity ELISA was performed as described
(Voller, et al., 1979), using pairs of MCA, each at an
antigen-saturating concentration, as calculated from its
saturation curve.
Each of 9 MCA was tested in combination
with the other 8.
A total of 200 gl/well of purified ILTV antigen (5
ng/well) dissolved in coating buffer (Na2CO3 1.59 g, NaHCO3
2.93 g, distilled water 1000 ml, pH 9.5) was added to each
well, and the ELISA plate (Costar Corporation, Kennebunk,
Me.) was incubated overnight at 4 C.
Each well was washed 3
times with washing buffer (PBS, pH 7.4, containing 0.05%
Tween-20 and 1% BSA).
After washing the plate, 200 gl of
ascites fluid diluted in washing buffer was added to each
well, wells were sealed with adhesive tape, and were
incubated for 2 hours at room temperature.
The plate was
washed 3 times with washing buffer and 200 gl of conjugate
(goat antimouse immunoglobulins, peroxidase-labeled)
(Hyclone) was added to each well, sealed with adhesive tape
74
and incubated overnight at 4 C.
After 3 washings with
washing buffer, 200 gl of substrate was added to each well
and the wells were sealed with adhesive tape and incubated
30 min at room temperature.
Substrate solution was made by
mixing 75 gl of 40mM stock ABTS (2,2-azino-bis 3ethylbenzthiazoline-6-sulfonic acid) in 10 ml of citrate
buffer (citric acid monohydrate 0.21 g, distilled water 100
ml, pH 4.00) in a separate beaker.
Twenty-five gl of
hydrogen peroxide (30%), diluted 1:10 in citrate buffer, was
mixed in 10 ml diluted ABTS.
After incubating in substrate
solution for 30 min, 100 gl of stopping buffer (5 X sodium
dodecyl sulfate (SDS) diluted 1:4 in distilled water) was
added to each well to block the enzymatic reaction.
Absorption was read at 410 nm by an automatic ELISA plate
analyzer.
The additivity indices (AI) for a pair of MCA was
calculated according to the formula
AI =
2A1+2
- 1)
(
Al + A2
x 100
where Al and A2 are the absorbances of two antibodies alone
and
A1+2
is the absorbance of two mixed antibodies.
An AI is
close to zero for two antibodies binding at the same site or
close to 100% for two antibodies binding at different sites
(Friguet, et al., 1983).
75
RESULTS
Isotypic characterization.
found to be isotype IgM.
MCA C, D, and E were each
MCA 4, 5, 6, 9, 10, and 11 were
each determined to be isotype IgG2b.
IFAT.
MCA D, 9, and 10 reacted in a similar manner,
producing fluorescence primarily in the nucleus of ILTVinfected CELC.
MCA D, however, produced more intense,
uniform nuclear fluorescence than MCA 9 and 10 and produced
no cytoplasmic fluorescence (Fig. 2.1).
MCA 9 and 10
produced mainly pinpoint, much less intense, nuclear
fluorescence and also weak cytoplasmic staining.
MCA C, 5, and 11 produced generalized cytoplasmic
staining, with accumulation of antigen around the multiple
nuclei of syncytia (Fig. 2.2), but nuclei did not fluoresce,
appearing brownish black.
MCA E, 4, and 6 produced diffuse,
generalized cytoplasmic fluorescence and spotty nuclear
fluorescence.
Nuclear fluorescence due to MCA 4 was more
prominent than with MCA 6, or E.
Results of IFAT with CELC infected with ILTV isolates
S8800224, 86-1169, and 94-11221, and with parrot herpesvirus
and avian adenovirus 301 are presented in Table 2.1.
CELC
infected by those ILTV isolates fluoresced similarly to CELC
infected with the USDA challenge strain of ILTV.
With all
MCA, cytoplasmic fluorescence was observed mostly at the
periphery of infected cells but MCA D, 9, and 10, produced
76
nuclear fluorescence as described above.
No MCA reacted
with uninfected CELC (data not shown) or with CELC infected
by the parrot herpesvirus or avian adenovirus 301 (Table
2.1) .
The results of IFAT titration of ascites fluid (all
MCA) and supernatants from extinct cultures (MCA C, D, and
E) are given in Table 2.2.
The IFAT working dilution for
MCA ascites fluids was 1:1000, except for MCA 9 and 10 for
it was 1:100.
The IFAT working dilution for supernatant
from extinct cultures for MCA C, D, and E was 1:10.
Dilutions 1:10 with supernatant and 1:1000 with ascites
fluid for MCA C, D, and E produced similar fluorescence.
Figure 2.1.
Indirect immunofluorescence staining of
CELC infected with the USDA challenge strain of ILTV
illustrating nuclear fluorescence (open arrowhead) produced
with MCA D.
Bar = 25 Am.
Figure 2.2.
Indirect immunofluorescence staining of
CELC infected with the USDA challenge strain of ILTV
illustrating cytoplasmic fluorescence (open arrowhead)
produced with MCA C.
Bar = 25 gm.
79
Table 2.1. Reaction' of monoclonal antibodies by
indirect immunofluorescence with strains of infectious
laryngotracheitis virus (ILTV) and 2 other viruses.
Virus
Monoclonal antibody
C
D
E
4
5
6
9
10
11
+
+
+
+
+
+
+
+
+
ILTV S8800224
+
+
+
+
+
+
+
+
+
ILTV 94-11221
+
+
+
+
+
+
+
+
+
ILTV 86-1169
+
+
+
+
+
+
+
+
+
Avian
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
ILTV
USDA
adenovirus
Parrot
herpesvirus
= specific immunofluorescence; - = no fluorescence.
80
Table 2.2. Titration' by indirect immunofluorescence
of monoclonal antibodies (MCA) produced against infectious
laryngotracheitis virus.
Ten-fold dilutions of MCA
MCA
10-1
10-2
10-3
1o4
10-5
Cb
+++++
++++
+++d
++
+
Db
+++++
++++
+++d
++
+
El'
+ + + ++
++++
+ + +d
++
+
4b
+ + + ++
+++
4I1-4
++
+
5b
+++++
++++
-1I0
++
++
6b
++++
+++
AiFd
++
9b
++
++d
10b
++
++d
116
++++
-II0
++
++++
cc
+++d
++
Dc
+ + +d
++
Ec
+ + +d
++
10-6
++
'+++++ = Highest intensity of specific fluorescence; + =
lowest intensity of specific fluorescence; - = no
fluorescence.
bTitration of MCA as ascites fluid.
qitration of MCA as extinct hybridoma culture supernatant.
dDilution of MCA selected for use in IFAT.
81
VN test results.
Only MCA 4 and 6 caused
neutralization in the VN test.
1:256 and of MCA 6 was 1:128.
The VN titer of MCA 4 was
Viral CPE occurred after 24
hours in the presence of non-neutralizing MCA.
No
combination of any 2, 3, or 4 non-neutralizing MCA caused
neutralization.
Coomassie staining of PAGE gels.
Two viral bands, 205
kd and 90 kd, were present in gels made from extracts of
ILTV-infected CELC that were not present in gels made from
extracts of uninfected CELC (data not shown).
Only the 90
kd band was seen in gels made from ILTV-infected CELC
extracts treated with tunicamycin.
These 205 kd and 90 kd
viral protein bands were faint.
Western blotting results.
Table 2.3 and Fig. 2.3.
Results are presented in
MCA C, E, and 11 detected the same
205, 160, 115, and 90 kd protein bands and several bands
less than 49 kd as did MCA 131-6.
MCA C reacted weakly with
all bands while MCA E reacted weakly with the 160 kd band.
MCA D reacted strongly with a band of 90 kd and weakly with
several bands less than 49 kd.
single band of 100 kd.
MCA 6 reacted only with a
MCA 4 and 5 reacted with varying
intensity with bands of 205, 160, 100, 90, and 70 kd.
MCA 9
reacted with the same bands detected by MCA 4 and 5 except
the 160 kd band.
MCA 5 reacted weakly with the 205, 160,
and 100 kd bands, while MCA 4 and 9 reacted weakly with all
82
bands.
MCA 10 reacted weakly with the same bands detected
by MCA 4 and 5 except the 205 and 160 kd bands, but it also
reacted with several bands less than 49 kd.
No MCA reacted
with any proteins from extracts of uninfected CELC.
Effect of tunicamycin on western blotting results.
Results are presented in Table 2.3 and Fig. 2.4.
MCA 131-6,
C, D, and E did not react with any band in extracts of
tunicamycin-treated ILTV-infected CELC.
11 reacted only with the 70 kd band.
bands of 90 and 70 kd.
MCA 4, 9, 10, and
MCA 5 reacted with two
MCA 4, 5, 9, and 10, reacted with a
70 kd band in extracts both treated and not treated with
tunicamycin, but MCA 11 reacted with a 70 kd band only in
tunicamycin-treated extracts.
MCA 6 reacted with the 100 kd
band, which was also the only band MCA 6 detected in the
absence of tunicamycin.
All MCA except MCA 6 lost some
reactivity with detergent extracts of cells infected in the
presence of tunicamycin.
83
KD
2
3
4
5
6
7
8
9 10
11 S
KD
205
205
116
116
80
Iv.
49
". 49
Figure 2.3.
80
Western blot produced by MCA with a
detergent extract of CELC infected with the USDA challenge
strain of ILTV in the absence of tunicamycin and
electrophoresed through a 4-20 % gel.
Lane 1: MCA C; Lane
2: MCA D; Lane 3: MCA E; Lane 4: MCA 131-6; Lane 5: MCA D
against detergent extract from uninfected CELC; Lane 6: MCA
9; Lane 7: MCA 10; Lane 8: MCA
MCA 5; Lane 11: MCA 6.
11; Lane 9: MCA 4; Lane 10:
Prestained molecular weight
standards are indicated on both sides of the figure as lane
S.
84
KD
S
1
2
3
4
5
6
7
8
9
10
11
S
205
205
116 r
116
80 r
80
49 I,
Figure 2.4.
04
49
Western blot produced by MCA with a
detergent extract of CELC infected with the USDA challenge
strain of ILTV in the presence of tunicamycin and
electrophoresed through a 4-20 % gel.
Lane 1: MCA 11; Lane
2: MCA 9; Lane 3: MCA 10; Lane 4: MCA 131-6; Lane 5: MCA D
against detergent extract from uninfected CELC; Lane 6: MCA
C; Lane 7: MCA D; Lane 8: MCA E; Lane 9: MCA 4; Lane 10: MCA
5; Lane 11: MCA 6.
Prestained molecular weight standards
are indicated on both sides of the figure as lane S.
85
Table 2.3. Characterization of monoclonal antibodies
(MCA) raised against infectious laryngotracheitis virus
(ILTV) by western blotting.
Protein bands (kd)
MCA
205
131-6
+ +b
160
115
++
++
100
<49'
++
++
++
++
+C
4
++C
5
++
++C
6
+C
9
+c
10
11
70
++
D
E
90
++
++
++
++
a = Several bands less than 49 kd.
b = In the absence of tunicamycin, ++ = strong staining; + =
weak staining
= Bands stained by MCA in blots prepared from extracts of
both tunicamycin-treated and untreated ILTV-infected CELC.
d = Band stained by MCA in blot prepared from extracts of
tunicamycin-treated ILTV-infected CELC, but not detected in
untreated ILTV-infected CELC.
86
Additivity ELISA test.
A saturation curve plateau was
reached at a dilution of 1:100 with MCA 4, 5, 6, 9, 10, and
E, at 1:200 with MCA 11 and C, and at 1:400 with MCA D.
Similar results were obtained using 2 conjugate
concentrations.
The AI results are shown in Table 2.4.
MCA
C and E are specific for the same epitope or very closely
related epitopes.
their specificity.
epitopes.
MCA 9 and 10 also have some similarity in
All other MCA are specific for unrelated
87
Table 2.4. Additivity ELISA additivity indices*
(AI)(%) calculated for pairs of monoclonal antibodies (MCA)
produced against infectious laryngotracheitis virus.
MCA
MCA
4
5
6
9
10
11
C
4
5
87
6
81
93
9
104
79
101
10
91
94
88
51
11
78
83
97
81
86
-
C
107
102
101
89
97
91
-
D
80
95
89
94
89
97
92
-
E
97
106
96
92
81
98
24
89
'See text for method of calculation of AI.
88
DISCUSSION
In the IFAT, each of nine MCA reacted with each ILTV
isolate, producing identical patterns of fluorescence, and
suggesting that different ILTV strains share antigenic
determinants and that MCA produced against one strain may
react with other ILTV strains.
Comparisons with additional
ILTV strains still are required, however.
No MCA reacted
with avian adenovirus 301 or parrot herpesvirus, suggesting
the specificity of the MCA-ILTV reaction.
MCA C, D, and E, are of isotype IgM.
It is possible
that spleen cells producing antibodies other than IgM could
not fuse or could not survive the process of fusion,
expansion, and cloning.
Or, perhaps the immune response was
still early at the time spleen cells were harvested.
Five groups of ILTV MCA have been described (York, et
al., 1990; York, et al., 1987).
In western blotting, MCA C,
E, and 11 reacted like MCA 131-6 which belongs to group II
MCA, and detected the same 205, 160, 115, and 90 kd bands as
the group II MCA.
MCA 131-6 is reported also to detect an
85 kd band but neither MCA 131-6 nor MCA C, E, or 11
detected an 85 kd band in this study.
MCA D, 4, 5, 6, 9,
and 10 do not match any of the 5 MCA groups (York, et al.,
1990; York, et al., 1987).
No MCA in this study detected
the 60 kd band that defines group I MCA (York, et al., 1990;
York, et al., 1987).
89
The additivity ELISA AI results show that all MCA
except MCA C and E are directed against unrelated epitopes.
These AI results support the results obtained with western
blotting and the VN test.
MCA 9 and 10 had an AI of 51%
which may indicate that they bind somewhat related epitopes
or overlap when binding physically-close epitopes.
The cytoplasmic fluorescence seen at the surface of
virus-infected cells suggests the MCA are directed against
membrane glycoproteins, while nuclear fluorescence suggests
the MCA are directed against viral structural proteins
(York, et al, 1987).
Similar patterns of fluorescence have
been observed in other herpesviruses (Spear, 1985).
A MCA
against varicella-zoster virus p32/p36 complex which
produces nuclear staining has been reported to be specific
for an antigen involved in nucleocapsid assembly and the
encapsidation of viral DNA (Friedrichs & Grose, 1986).
The data for MCA 6, however, suggest that it detects a
nonglycosylated protein on the surface of ILTV-infected
CELC, as MCA 6 produces cytoplasmic fluorescence and also
detects a single protein of 100 kd from both tunicamycintreated and untreated ILTV-infected CELC extracts in western
blotting.
MCA 4, 5, and 11 similarly produce primarily
cytoplasmic fluorescence and also detect proteins in
tunicamycin-treated extracts.
These results suggest that
there may be nonglycosylated proteins on the surface of
90
ILTV-infected cells or that antigenic epitopes occur on nonglycosylated portion of glycoproteins.
By contrast MCA D, which produces only nuclear
staining, detects a protein in western blotting that it does
not detect in tunicamycin-treated extracts, suggesting it
detects a glycosylated protein.
This nuclear protein may be
similar to an epitope on a glycosylated protein, or the
protein may be nonglycosylated but reducing conditions or
denaturation may have altered the epitope structure, making
it undetectable in tunicamycin-treated extracts.
MCA specific for glycoproteins are useful for
investigating the role of glycoproteins in adsorption and
penetration of cells, cell fusion, and neutralization
(Spear, 1985).
Glycoproteins are the major components
involved in binding, penetration, and production of CPE.
ILTV-infected CELC fed with a medium containing tunicamycin
produced virus that failed to cause CPE (York, 1990).
Only MCA 4 and 6 neutralize ILTV.
These two MCA may
bind epitopes involved with virus adsorption or penetration.
Based on the demonstrated involvement of glycosylated
proteins in viral infectivity, such epitopes could be
located on a surface glycoprotein.
MCA 4 detected proteins
of 205, 160, 100, 90, and 70 kd in western blots, but in
tunicamycin-treated extracts it detected only the 70 kd
protein.
This suggests that MCA 4 binds a glycosylated
protein antigen in which carbohydrate residues are important
91
to the epitope conformation, although the 70 kd protein
results make that conclusion uncertain.
MCA 6 detected a
nonglycosylated protein of 100 kd and neutralized viral
These results suggest both nonglycosylated and
infectivity.
glycosylated proteins may be involved in viral infectivity.
As no other MCA neutralized the virus and other MCA bind
different epitopes than MCA 4 and 6, not all antigenic
epitopes are essential to viral infectivity.
Combinations
of non-neutralizing MCA also failed to neutralize the virus,
so binding more than one epitope did not improve
neutralization.
Proteins detected by the neutralizing antibodies might
prove useful in a recombinant ILTV vaccine.
Such vaccines
are protective against other herpesviruses (Cantin, et al.,
1987; Cremer, et al., 1985; McDermott, et al., 1989;
Paoletti, et al., 1984; Wachsman, et al., 1987; Weir, et
al., 1989).
Immune responses may also be directed against
non-glycosylated antigens (Arvin, et al., 1987; Martin, et
al., 1988) and such responses may be protective (Reddehase,
et al., 1987).
The failure of some MCA to react with extracts of
tunicamycin-treated CELC suggests that those MCA bind
carbohydrate epitopes or that the absence of carbohydrate
alters the antigenic conformation of the epitopes.
Tunicamycin is an antibiotic extracted from Streptomyces
lysosuperficus which inhibits membrane glycosylation
92
(Takatsuki, et al., 1985).
The antibiotic is composed of 4
major elements which are uracil, aminodialdose (tunicamine),
N-acetylglucosamine, and a fatty acid (Elbein, 1971).
The
N-acetylglucosamine is responsible for the antiviral
activity of tunicamycin (Takatsuki & Tamura, 1971).
MCA 11 detected the 70 kd protein band only in the
presence of tunicamycin, suggesting that this 70 kd protein
may be one of the glycosylated proteins detected by MCA 11
in the absence of tunicamycin and that inhibition of
glycosylation may have reduced the size of the protein but
left the epitope still detectable by MCA 11.
Some of the
group II MCA described by York, et al. (York, et al., 1990)
also reacted in a similar way in western blotting.
Of the
group II MCA, MCA 22-37 detected three protein bands of 135,
73, and 68 kd and 12-1 detected two bands of 135 and 102 kd
only in the presence of tunicamycin.
None of these protein
bands were detected by group II MCA in the absence of
tunicamycin (York, et al., 1990).
It is not known whether
the 70 kd protein detected by MCA 11 is the same as the 70
kd protein detected by MCA 4, 5, 9, and 10.
The 70 kd
protein detected by MCA 4, 5, 9, and 10 may lack
glycosylation because MCA that bind that protein also bind
it in the presence of tunicamycin.
Alternatively, the
inhibition of its glycosylation by tunicamycin may not
affect the conformation of its epitopes.
93
The 90 kd protein was detected by all MCA except MCA 6,
but only MCA 5 detected it in extracts treated with
tunicamycin, suggesting the presence of more than one
epitope on the 90 kd protein.
Occurrence of multiple epitopes also could explain why
MCA 4 neutralized the virus when other MCA that bind the
same proteins as MCA 4 did not.
MCA 6 detected only the 100
kd protein and neutralized the virus, so the 100 kd protein
contains an epitope that, if bound, results in inactivation
of the virus.
MCA 4 also neutralized the virus and detected
the 100 kd protein, but additivity ELISA results indicate
MCA 4 and 6 bind different epitopes.
Some MCA reacted with similar bands but with different
intensities, which may be due to variation in MCA
concentration or due to differing levels of protein
production.
Most MCA detect multiple ILTV protein or
glycoprotein bands in western blotting, as others also have
reported (York, et al., 1990).
This can occur if ILTV
glycoproteins contain similar antigenic determinants, or if
one protein band represents the post-translational product
of another protein, as occurs with bovine herpesvirus type 1
and pseudorabies virus (Hampl, et al., 1984; van Drunen
Littel-van den Hurk & Babiuk, 1986; van Drunen Littel-van
den Hurk, et al., 1984).
Denaturation under the reducing
conditions used in western blotting also might produce such
results, as certain ILTV proteins not detected by
94
immunoprecipitation were detected by MCA in western blotting
(York, et al., 1987).
Assembly of a protein unit from
subunits also would explain different-sized proteins with
similar antigenic determinants.
Glycoproteins 205, 160, and 90 kd probably are primary
ILTV immunogens because they are detected by most MCA.
The
60 kd glycoprotein, not detected by any MCA in this study,
also is a prominent ILTV immunogen (York, et al., 1990;
York, et al., 1987).
Antibodies directed against major
immunogens probably play important roles in protective
immunity.
kd.
The largest glycoprotein detected for ILTV is 205
Such large glycoproteins also occur on other
herpesviruses, including a 129 kd glycoprotein on herpes
simplex virus I, a 350/300 kd glycoprotein on Epstein-Barr
virus (Edson & Thorley-Lawson, 1981), and a 250 kd
glycoprotein on human cytomegalovirus (Farrar & Oram, 1984).
Purified glycoproteins and whole virus gave the same profile
in western blotting, indicating that ILTV glycoproteins are
both antigenic and immunogenic (York, et al., 1987).
Two viral bands, 205 kd and 90 kd, were visible in
extracts of ILTV-infected CELC after Coomassie staining.
Only the 90 kd band was seen in Coomassie-stained ILTVinfected CELC extracts treated with tunicamycin.
Other
viral proteins that also may have been present either were
masked by more abundant cellular proteins or had similar
molecular weights to cellular proteins.
As at least 1 Ag
95
protein per band is required for visualization by this
method according to the gel manufacturer's instructions
(Mini-Protein II Ready Gels, Bio-Rad), viral proteins
present in lower concentrations may not have been detected.
96
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Cremer, K.J., M. Mackett, C. Wohlenberg, A. L. Notkins, and
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Takatsuki, A., K. Arima, G. Tamura.
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van Drunen Littel-van den Hurk, S., J. V. van den Hurk, J.
Gilchrist, V. Misra, L. A. Babiuk. Interactions of
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Villegas, P. Procedure for primary chicken embryo liver
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diseases (AM 805). College of Veterinary Medicine,
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Enzyme-linked
Voller, A., D. E. Bidwell, and A. Bartlett.
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Wachsman, M., L. Aurelian, C. C. Smith, B. R. Lipinskas, M.
E. Perkus, and E. Paoletti. Protection of guinea pigs from
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York, J. J., and K. J. Fahey.
Humoral and cell-mediated
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York, J. J., S. Sonza, M. R. Brandon, and K. J. Fahey.
Antigens of infectious laryngotracheitis herpesvirus defined
by monoclonal antibodies. Arch. Virol. 115:147-162. 1990.
York, J. J., S. Sonza, and K. J. Fahey.
Immunogenic glycoproteins of infectious laryngotracheitis
herpesvirus. Virology. 161:340-347. 1987.
York, J. J., J. G. Young, and K. J. Fahey.
The appearance
of viral antigen and antibody in the trachea of naive and
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laryngotracheitis virus. Avian Pathol. 18:643-658. 1989.
100
CHAPTER III
Use of the Polymerase Chain Reaction and a Non-Radioactive
DNA Probe to Detect Infectious Laryngotracheitis Virus
Ferhat Abbas,A James R. Andreasen, Jr.,A
and Mark W. JackwoodB
ACollege of Veterinary Medicine,
Oregon State University, Corvallis OR 97331
BDepartment of Avian Medicine,
College of Veterinary Medicine,
University of Georgia, Athens, Georgia, 30602-4875
101
SUMMARY
The polymerase chain reaction (PCR) was developed as a
diagnostic technique for detection of infectious
laryngotracheitis virus (ILTV) using primers made from a
portion of the ILTV thymidine kinase gene.
DNA extracted
and purified from a variety of field isolates, from the USDA
challenge strain of ILTV, and from commercial ILTV vaccines
was specifically amplified.
No amplification occurred using
template DNA extracted and purified from uninfected chickenembryo liver cells (CELC), from several non-avian
alphaherpesviruses, from Mycoplasma gallisepticum,
Mycoplasma synoviae, Pasteurella hemolytica, Escherichia
coli, avian adenovirus strain 301, fowlpox virus, or a
parrot herpesvirus.
The 647-base-pair amplified ILTV PCR
product was labeled to create a non-radioactive,
biotinylated DNA probe.
Hybridization was performed using
the probe to detect ILTV.
Both PCR and hybridization
detected ILTV, and neither hybridization nor PCR gave
positive results with any other pathogen.
Hybridization was
specific for ILTV using a very high stringency salt solution
for a 30-min wash step, or a somewhat less stringent salt
solution for a 60-min wash step.
However, slight
hybridization occurred with CELC DNA when the less stringent
salt solution was used in a 30-min wash step.
disadvantage of this probe.
This is a
102
INTRODUCTION
Infectious laryngotracheitis virus (ILTV) is an
alphaherpesvirus of the herpesviridae family.
ILTV causes
respiratory disease in chickens characterized by depression,
conjunctivitis, sneezing, rales, and nasal exudate.
In
severe cases, birds may also show signs of gasping, dyspnea,
and death (Jordan, 1990).
The disease caused by this virus
is of great economic importance, causing significant
mortality and loss of productivity.
Besides the production
of acute disease in chickens, like other alphaherpesviruses,
ILTV may establish a latent carrier state in recovered birds
(Bagust, 1985).
Various techniques including virus isolation (VI),
direct fluorescent antibody test (DFAT) and indirect
fluorescent antibody test (IFAT), and histopathology have
been used to confirm ILTV infection.
Shortcomings of those
procedures, including delay in obtaining results or lack of
specificity, have kindled interest in developing more rapid,
specific diagnostic techniques such as the polymerase chain
reaction (PCR).
ILTV has been detected in tracheas,
turbinate tissue, and trigeminal ganglia using PCR
(Williams, et al., 1992).
Latent virus was detected in the
trigeminal ganglia of recovered chickens 61 days
postinfection using PCR, after VI failed to detect virus
(Williams, et al., 1992).
ILTV also has been detected in
joint and bone samples by PCR and VI, possibly reflecting
103
infection of bone marrow macrophages (Jones, et al., 1993;
Von Bulow & Klasen, 1983).
DNA hybridization using
nonradioactive ILTV probes also has detected expei'imentally-
infected chickens, field outbreaks and latent ILTV
infections (Keam, et al., 1991; Key, et al., 1994).
The purpose of this study was to develop PCR as'a
diagnostic technique for the detection of ILTV, and also to
investigate hybridization as a diagnostic technique using a
nonradioactively-labelled, amplified PCR product as a probe.
MATERIALS AND METHODS
Template DNA.
ILTV DNA from Georgia field isolates
including 88-1453, 86-1169, 88-1059, 88-1668, 88-1194, 881387, 85-304, and 88-1301; from California field isolate
S8800224; from Oregon field isolate 94-11221; from the USDA
challenge strain of ILTV; from commercial ILTV vaccines
including Laryngo-vac (Salsbury Laboratories, Inc., Charles
City, Iowa), Trachivax and LT-ivax (American Scientific
Laboratories, Schering Corp., Cream Ridge, N.J.),
Broilertrake (Sterwin Laboratories, Millsboro, Del.), and
Trachine (Intervet America, Inc., Millsboro, Del.); and the
DNA from uninfected chicken-embryo liver cells (CELC) was
extracted, purified as previously described (Andreasen, et
al., 1990), held at -70 C, and was used as template in the
PCR.
Other USDA challenge strain DNA also was freshly
extracted and purified for use as template.
104
An avian adenovirus isolate 301 and a parrot
herpesvirus isolate from the liver of an acutely dead Amazon
parrot were grown in CELC, and the viral DNA was similarly
extracted and purified.
A standard phenol-chloroform
extraction (Sambrook, et al., 1989) was done on a
lyophilized commercial fowipox virus (FP Blen, CEVA
Laboratories, Inc., Overland Park, Kansas) and on cultures
of avian isolates of Escherichia coli, Pasteurella
hemolytica, Mycoplasma gallisepticum (MG), and Mycoplasma
synoviae (MS) to obtain purified DNA from those organisms.
DNA was also extracted and purified from several other
alphaherpesviruses including infectious bovine
rhinotracheitis virus (IBRV), feline herpesvirus (FHV),
equine herpesvirus 1 (EH1), equine herpesvirus 2 (EH2),
pseudorabies virus (PRV), and bovine herpesvirus D 599 (BHV)
using the described method (Andreasen, et al., 1990).
Polymerase chain reaction (PCR).
The PCR was performed
with a commercial kit (GeneAmp PCR Reagent Kit, Perkin Elmer
Cetus, Norwalk, Conn.) using a DNA thermal cycler (Perkin
Elmer Cetus) in accordance with kit instructions.
The
primers, ILTV/PCR 5'primer = 5'-ACGATGACTCCGACTTTC-3', and
ILTV/PCR 3' primer = 5'-CGTTGGAGGTAGGTGGTA-3' were made from
the published sequence data of the thymidine kinase (TK)
gene (Griffin & Boursnell; Keeler, et al., 1991) at the
Center for Gene Research at Oregon State University.
The
ILTV/PCR 5' primer starts at base 222 in the Keeler sequence
105
and base 4501 in the Griffin sequence.
The ILTV/PCR 3'
primer starts at base 847 in the Keeler sequence and base
5130 in the Griffin sequence (Griffin & Boursnell; Keeler,
et al., 1991).
A PCR run consisted of 35 cycles of 95 C for
1 min, 50 C for 1 min, and 35 C for 1.5 min.
Strict precautions were taken to avoid cross
contamination.
facilities.
DNA extraction and PCR were done in separate
Negative controls, such as DNA extracted from
uninfected CELC, were used for each set of reactions.
Also,
a PCR master mixture was used that did not contain template
DNA to verify that there was no nonspecific amplification.
The sensitivity of the PCR was determined by using
different concentrations of ILTV USDA challenge strain DNA
template ranging from 1 Ag to 0.0625 Ag.
PCR products were examined by agarose gel
electrophoresis using standard techniques (Ausubel, et al.,
1993) and the sizes of PCR products were determined by
comparison with molecular weight standards (Sigma Molecular
Biology, St. Louis, MO.).
Sequencing of PCR product.
Ethanol precipitated
(Sambrook, et al., 1989) PCR product produced from the USDA
challenge strain of ILTV was sequenced (Center for Gene
Research, Oregon State University) to confirm that the PCR
product corresponded to the expected fragment of the ILTV TK
gene.
A complete sequencing of the PCR product was obtained
using both 5' and 3' primers, sequencing from opposite ends,
106
The sequenced product was
to produce overlapping sequences.
compared with the published sequence for the ILTV TK gene
(Griffin & Boursnell, 1990).
DNA probe.
2% agarose gel.
ILTV PCR product was electrophoresed in a
The desired ethidium bromide-stained
amplified band was cut with a scalpel from the gel and
placed in a 1.5 ml Eppendorf tube with sufficient Tris EDTA
(TE) buffer (10 mM Tris-HC1, pH 7.5; 1 mM Na2 EDTA) to bring
the agarose percentage under 0.4%.
The sample was kept at
65 C until the agarose completely melted.
The DNA was
purified by phenol extraction and ethanol precipitation
using standard techniques (Ausubel, et al., 1993).
To
reduce the inhibition of enzymatic reactions by residual
agarose, bovine serum albumin (BSA) was added at the rate of
500 µg /ml.
The DNA concentration was determined by
spectrophotometry.
DNA was labeled using a commercial kit (BioPrimen4 DNA
Labeling system, Gibco BRL, Life Technologies, Inc.,
Gaithersburg, Md.) following the manufacturer's
instructions.
This labeling system produces non-
radioactive, biotinylated probes by annealing random primers
to denatured DNA and extending by Klenow fragment in the
presence of biotin-14-dCTP.
Unincorporated nucleotides were
removed from the resultant biotinylated DNA probe by
repeated ethanol precipitation.
The DNA concentration was
107
determined by spectrophotometry and the probe was stored at
-20 C.
Hybridization.
Hybridization was performed using a
commercial kit (PhotogeneTM Nucleic Acid Detection System,
Gibco BRL) following the manufacturer's instructions with
slight modification.
Ten-fold dilutions (10° = 1 Ag/A1 of
DNA) of DNA samples were made and 1 Al of each sample was
transferred to a 10 x 10 cm nylon membrane.
The nylon
membrane was then treated with ultraviolet light for 30 min
to fix the DNA, avoiding dust particles or other
contaminants.
Prehybridization was performed for 2 hours
using 10 ml of prehybridization solution (1 M NaC1, 1% SDS
in double-distilled water) in a preheated hybridization oven
maintained at 65 C.
After denaturing in boiling water, the
calculated amount of biotinylated DNA probe (500 ng) was
mixed with 10 ml prewarmed, 65 C hybridization solution (10%
Dextran sulfate [Sigma Molecular Biology], 1M NaC1, 1% SDS
in double-distilled water, 100 µg /ml denatured salmon sperm
DNA [Sigma Molecular Biology]).
Prehybridization solution
was removed, hybridization solution was added, and
hybridization was performed for 18 hours at 65 C.
After
hybridization, washings with stringent salt solutions were
done according to the instructions provided with the kit.
Two additional stringent washings, a 2 X sodium chloride
sodium citrate dihydrate (SSC), 1% sodium dodecyl sulfate
108
(SDS) wash and a 0.5 X SSC, 1% SDS wash (both stringent salt
solutions were made according to the kit instructions) were
performed for 30 min each, following a 5 X SSC, 0.5% SDS
wash.
All other steps including blocking of nylon membrane,
incubation with streptavidin-alkaline phosphatase conjugate,
washings with Tween-tris buffered saline (TTBS) and final
wash buffer, incubation with detection reagent, and exposure
to X-ray films were performed according to the kit
instructions.
Hybridization was attempted with DNA from the ILTV
strains, including vaccine strains and field isolates, with
CELC DNA, and with DNA from the other pathogens listed as
PCR templates, but not with mammalian alphaherpesviruses.
A computer search for homologous sequences was
performed to help evaluate the hybridization results
(Altschul, et al., 1990).
RESULTS
PCR.
PCR performed using the USDA challenge strain of
ILTV as template produced a product of exactly 647 base
pairs (Fig. 3.1) based on the results obtained from
sequencing of the PCR product.
Comparison of the sequenced
PCR product with the published corresponding sequence of the
ILTV TK gene (Griffin & Boursnell, 1990) demonstrated the
sequences to be identical with the exception of 14
mismatches and 9 insertions of a single nucleotide each
109
(data not shown).
Most of the mismatches occurred in
sequences close to the primer binding sites.
PCR produced a
product of similar size using template DNA concentrations
ranging from 1 Ag to 0.0625 Ag (data not shown).
Additional PCR experiments using other ILTV strains and
isolates consistently produced a product of approximately
647 base pairs (Fig. 3.1).
sequenced.
Those products were not
ILTV isolates amplified specifically include
Georgia field isolates 88-1668, 88-1194, 88-1387, 85-304,
and 88-1301, California field isolate S8800224, Oregon field
isolate 94-11221 (data not shown), and Georgia field
isolates 88-1453, 88-1169, and 88-1059, (Fig. 3.1).
ILTV
vaccine strains amplified include LT-ivax, Laryngo-vac (Fig.
3.1), Trachivax, Broilertrake, and Trachine (data not
shown).
Control DNA templates including DNA extracted from
uninfected CELC DNA failed to amplify (Fig. 3.1).
PCR experiments using template DNA from other agents
including E. coli, P. hemolytica, MG, MS, IBRV, FHV, EH1,
EH2, PRV, and BHV, also failed to amplify (Fig. 3.2), as did
template DNA from parrot herpesvirus, avian adenovirus
strain 301, and fowl pox virus (data not shown).
110
1
2
3
4
5
6
7
8
L
by
1353
1078
872
603
310
Fig. 3.1.
PCR products electrophoresed on a 4% agarose
gel, stained with ethidium bromide, and viewed under
ultraviolet radiation.
Lane 1: ILTV USDA challenge strain;
lane 2: ILTV isolate 88-1453; lane 3: ILTV isolate 86-1169;
lane 4: CELC mitochondrial DNA; lane 5: uninfected CELC DNA;
lane 6: Laryngo-Vac ILTV vaccine; lane 7: ILTV isolate 881059; lane 8: LT-ivax ILTV vaccine; lane L: ladder, a tX174RF Hae III digest (bands of 1,353, 1,078, 872, 603, and 310
bp).
111
L
1
2
3
4
5
6
7
8
9
10
11
12
L
Op
by
1353
1078
872
1353
1078
872
603
603
310
310
Fig. 3.2.
PCR products electrophoresed on a 4% agarose
gel, stained with ethidium bromide, and viewed under
ultraviolet radiation.
Lane 1: ILTV USDA challenge strain;
lane 2: Escherichia coli; lane 3: Pasteurella hemolytica;
lane 4: Mycoplasma gallisepticum; lane 5: Mycoplasma
synoviae; lane 6: ILTV USDA challenge strain; lane 7:
Infectious bovine rhinotracheitis virus (IBR); lane 8:
Feline herpesvirus (FHV); lane 9: Equine herpesvirus 1
(EH1); lane 10: Equine herpesvirus 2 (EH2); lane 11:
Pseudorabies virus; lane 12: Bovine herpesvirus DN599; lane
L on both left and right side: ladder, a (1X174-RF Hae III
digest (bands of 1,353, 1,078, 872, 603, and 310 bp).
112
Hybridization.
The biotinylated ILTV PCR product DNA
probe hybridized with the DNA from ILTV isolates and vaccine
strains including Georgia field isolates 88-1453, 88-1169,
and 88-1059; Oregon field isolate 94-11221; LT-ivax, and
Laryngo-vac (Figs. 3.3, 3.4); and Georgia field isolates 881668, 88-1194, 88-1387, 85-304, and 88-1301; California
field isolate S8800224; Trachivax, Broilertrake, and
Trachine (data not shown).
No hybridization occurred with
When
any other virus, bacteria, or mycoplasmas (Fig. 3.4).
only 0.5 X SSC, 1% SDS was used for washing for 30 min, the
probe appeared to hybridize very slightly with CELC DNA,
producing a questionable reaction (data not shown).
When
0.5 X SSC, 1% SDS was used for 60 min to create conditions
less favorable for nonspecific hybridization, the probe did
not hybridize with CELC DNA, and similarly when 0.1 X SSC,
1% SDS was used for 30 min following 0.5 X SSC, 1% SDS wash
for 30 min, no nonspecific hybridization to CELC DNA
occurred (Fig. 3.5).
Under those conditions the probe still
hybridized with ILTV DNA but the intensity of hybridization
was slightly reduced (Fig. 3.5).
The hybridization was very
readable with 1 Ag DNA but almost negative with 0.1 gg DNA.
Hybridization was negative with less than 0.1 gg of DNA
(Fig. 3.5) fixed on the nylon membrane.
Computer searches for homologous sequences were
performed to evaluate hybridization results.
The CELC TK
gene has 36.6% homology with the 647-base-pair ILTV PCR
113
product (Altschul, et al., 1990).
Other similar sequences
include human cytomegalovirus strain AD169 with 66.2%
homology over an 80 base pair overlap portion of the PCR
product, vaccinia virus Copenhagen strain DNA clone VC-2
with 70.3% homology in a 37 by overlap, variola major
Bangladesh strain with 80.6% homology in a 36 by overlap,
variola virus DNA with 79.3% homology in a 29 by overlap,
and equine herpesvirus 1 with 80.0% identity in a 35 by
overlap (Altschul, et al., 1990).
114
1
Fig. 3.3.
with ILTV DNA.
2
3
4
5
6
7
8
Hybridization of a biotinylated DNA probe
Lane 1: ILTV USDA challenge strain; lane 2:
ILTV isolate 88-1453; lane 3: ILTV isolate 86-1169; lane 4:
Laryngo-Vac ILTV vaccine; lane 5: ILTV isolate 88-1059; lane
6: LT-ivax ILTV vaccine; lane 7: CELC mitochondrial DNA;
lane 8: Uninfected CELC DNA; lane 9: Infectious bovine
rhinotracheitis virus (IBRV).
DNA concentrations: a = 1 Ag
DNA; b = 0.1 Ag DNA; c = 0.01 Ag DNA.
115
1
2
3
4
5
6
a
Fig. 3.4.
Hybridization of biotinylated ILTV DNA probe
with DNA extracted from other viruses, bacteria, and
mycoplasmas.
a: Lane 1: Pasteurella hemolytica; a: lane 2:
Escherichia coli; a: lane 3: ILTV isolate 94-11221; a: lane
4: Mycoplasma gallisepticum; a: lane 5: Mycoplasma synoviae;
a: lane 6: LT-ivax ILTV vaccine; b: lane 1: Avian
adenovirus; b: lane 2: Parrot herpesvirus; b: lane 3: Avian
reovirus; b: lane 4: Fowlpox virus; b: lane 5: Uninfected
CELC DNA; b: lane 6: ILTV USDA challenge strain.
116
1
2
3
4
5
6
7
8
a
C
Fig. 3.5.
Hybridization of a biotinylated ILTV DNA
probe with ILTV DNA at 2 different stringencies.
Lane 1:
ILTV USDA challenge strain; lane 2: CELC mitochondrial DNA;
lane 3: Uninfected CELC DNA; lane 4: LT-ivax ILTV vaccine;
(lanes 1 to 4 are results of a single wash with 0.5 X SSC,
1% SDS for 60 min).
Lane 5: ILTV USDA challenge strain;
lane 6: CELC mitochondrial DNA; lane 7: Uninfected CELC DNA;
lane 8: LT-ivax ILTV vaccine; (lanes 5 to 8 are results of a
wash with 0.1 X SSC, 1% SDS for 30 min following a 0.5 X
SSC, 1% SDS wash for 30 min). DNA concentrations: a = 1 gg
DNA; b = 0.1 gg DNA; c = 0.01 lig DNA.
117
DISCUSSION
PCR based on primers made from the ILTV TK gene offers
a specific means of detecting various strains of ILTV,
including both vaccine strains and field isolates.
strains that were tested amplified specifically.
DNA consistently failed to amplify.
All ILTV
Non-ILTV
Other workers have used
a different set of primers based on the ILTV TK gene and
have obtained an amplified product of 673 by (Williams, et
al., 1992).
They found PCR very effective in detecting ILTV
in the trigeminal ganglia of chickens in which virus
isolation and organ culture failed to detect the virus
(Williams, et al., 1992).
Thus, PCR can be used to diagnose
acute as well as latent ILTV infections in carrier birds.
PCR did not amplify CELC DNA, suggesting the primers
used in this study are very specific for ILTV and do not
produce false positive results from host chicken DNA.
Failure of amplification of template DNA from bacteria,
mycoplasmas and other viruses, including other
alphaherpesviruses, is verification of the specificity of
this PCR.
The PCR described here specifically amplified ILTV DNA
from the USDA challenge strain, chicken-embryo-origin
vaccines, a tissue-culture-origin vaccine, and several
Georgia field isolates, including ILTV genomes that were
shown to differ from each other by analysis of restriction
endonuclease fragments (Andreasen, et al., 1990).
That
118
result, along with amplification of California and Oregon
field isolates of ILTV, supports the use of this PCR as a
diagnostic tool capable of detecting any strain of ILTV.
This result also suggests there is considerable similarity
between the TK genes of different ILTV strains.
The nonradioactively-labeled, amplified PCR product DNA
probe hybridized with ILTV DNA, including that from both
vaccine strains and field isolates.
Under very stringent
conditions (a 0.5 X SSC, 1% SDS wash plus a 0.1 X SSC, 1%
SDS wash for 30 min each or a 0.5 X SSC, 1% SDS wash for 60
min) the probe was specific for ILTV.
Under comparatively
less stringent conditions (a 0.5 X SSC, 1% SDS wash for 30
min), the probe hybridized weakly with CELC DNA.
More
stringent conditions, however, eliminated nonspecific probe
hybridization with CELC DNA (Fig. 3.5).
The CELC TK gene
has 36.6% homology with the probe, which probably explains
why the probe hybridizes at certain stringencies.
Such
nonspecific hybridization to CELC DNA is a disadvantage of
this probe.
Other, shorter probes could probably be
designed that would not hybridize nonspecifically with host
chicken genomic DNA.
No hybridization was detected using less than 0.1 gg
DNA fixed on the nylon membrane with the probe used in this
study.
Others have described a much more sensitive 1.1 kb
non-isotopically labeled DNA probe and have detected 40 pg
of ILTV DNA extracted from purified ILTV (Key, et al.,
119
1994).
A 1.4 kb non-radioactive DNA probe also has been
reported to hybridize with a minimum of 500 ng ILTV DNA
(Keam, et al., 1991).
120
REFERENCES
Altschul, S. F., W. Gish, W. Miller, E. W. Meyers, and D. J.
Lipman.
Basic local alignment search tool. J. Mol. Biol.
215:404-410. 1990.
Andreasen, J. R., J. R. Glisson, and P. Villegas.
Differentiation of vaccine strain and Georgia field isolates
of infectious laryngotracheitis virus by their restriction
endonucleases fragment patterns. Avian Dis. 34:646-656.
1990.
Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G.
Seidman, J. A. Smith, and K. Struhl. Preparation and
analysis of DNA. In: Current Protocol in Molecular Biology.
Massachusetts General Hospital, Harvard medical School.
Green Publishing Associates, Inc., and John Wiley and Sons,
Inc. pp. 2.5-2.8.1. 1993.
Bagust, T. J.
Infectious laryngotracheitis herpesvirus
(ILT): latency and detection and in vitro reactivation of
hemorrhagic and vaccine strains. Proceedings of the VIIIth
International Congress of the World Veterinary Poultry
Association, Jerusalem, Israel. pp.23. 1985.
Griffin, A. M., and M. E. G. Boursnell. Analysis of the
nucleotide sequence of DNA from the region of the thymidine
kinase gene of infectious laryngotracheitis virus; potential
evolutionary relationships between the herpesvirus
subfamilies.
J. Gen. Virol. 71:841-850. 1990.
Jones, R. C., R. A. Williams, C. E. Savage.
Isolation of
infectious laryngotracheitis virus from proximal femora of
lame broiler chickens. Res. Vet. Sci. 55:377-378. 1993.
Jordan, F. T. W.
Infectious laryngotracheitis. In: poultry
Diseases, 3rd edn., F. T. W. Jordan. London: Bailliere
Tindall. pp. 154-158. 1990.
Keam, L., J. J. York, M. Sheppard, and K. J. Fahey.
Detection of infectious laryngotracheitis virus in chickens
using a non-radioactive DNA probe. Avian. Dis. 35:257-262.
1991.
Keeler, C. L., D. H. Kingsley, and C. R. Adams Burton.
Identification of thymidine kinase gene of infectious
laryngotracheitis virus. Avian Dis. 35:920-929. 1991.
Key, D. W., C. B. Gough, J. B. Derbyshire, and E. Nagy.
Development and evaluation of a non-isotopically labeled DNA
121
probe for the diagnosis of infectious laryngotracheitis.
Avian Dis. 38:467-474. 1994.
Sambrook, J., E. F. Fritsch, and T. Maniatis. Commonly Used
Techniques in Molecular Cloning. In: Molecular Cloning: A
Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. pp. E.1-E.12. 1989.
Effects of avian
Von Bulow, V., and A. Klasen.
viruses on cultured chicken bone marrow-derived macrophages.
Avian Pathol. 12:179-198. 1983.
Williams, R. A., M. Bennet, J. M. Bradbury, R. M. Gaskell,
R. C. Jones, and F. T. W. Jordan. Demonstration of sites of
latency of infectious laryngotracheitis virus using the
J. Gen. Virol. 73:2415-2420.
polymerase chain reaction.
1992.
122
CHAPTER IV
Comparison of Diagnostic Tests for
Infectious Laryngotracheitis
Ferhat Abbas and James R. Andreasen, Jr.
College of Veterinary Medicine, Oregon State University,
Corvallis, OR 97331
123
SUMMARY
Diagnostic tests to detect ILTV in tracheas of
experimentally-infected chickens, including the indirect
fluorescent antibody test (IFAT), immunoperoxidase (IP),
virus isolation (VI), histopathology, polymerase chain
reaction (PCR), and DNA hybridization, were performed and
compared.
Using virus isolation as a reference, the
sensitivity and specificity of the tests were calculated.
The sensitivity of IP, IFAT, histopathology, PCR, and
hybridization was 100%, 93%, 7%, 27%, and 0% respectively,
and the specificity of IP, IFAT, histopathology, PCR, and
hybridization was 93%, 93%, 100%, 100%, and 100%
respectively.
Histopathology, PCR, and hybridization were
more specific but lacked sensitivity compared to IP and.
The IP test and IFAT were equally specific, but the
IFAT.
IP test was more sensitive than IFAT.
Based on these
results, the IP test performed better than any other test
used in this study for the detection of ILTV in tracheal
samples of experimentally-infected chickens.
INTRODUCTION
Infectious laryngotracheitis virus (ILTV) is an
alphaherpesvirus which causes laryngotracheitis, an
important disease of chickens that can produce high
mortality and decreased egg production (Hanson & Bagust,
1991).
ILTV may cause both severe and mild forms of disease
in layers and broilers (Curtis & Wallis, 1983).
The mild
124
form of disease is characterized by depression,
conjunctivitis, sneezing, rales, and nasal exudate.
In the
severe form of the disease, commonly observed signs are
gasping, dyspnea, and death by asphyxiation (Jordan, 1990).
Classical ILTV can be diagnosed based on history,
signs, and gross lesions, but laboratory diagnostic workup
is required for confirmation of mild disease.
Because of
its regulatory importance, rapid and accurate diagnosis of
ILTV is critical.
Various laboratory diagnostic techniques have been
described for detection of acute cases of ILTV, including
the agar gel immunodiffusion test (AGID) (Jordan & Chubb,
1962), direct fluorescent antibody test (DFAT) (Hitchner, et
al., 1977), virus isolation (VI)
(Hughes and Jones, 1988),
histopathology for the detection of syncytia and
intranuclear inclusion bodies in tissues (Seifried, 1931),
immunoperoxidase procedures (IP)
(Guy, et al., 1992), DNA
probe hybridization (Abbas, et al., 1994b; Keam, et al.,
1991;
(PCR)
Key, et al., 1994), and the polymerase chain reaction
(Abbas, et al., 1994b; Williams, et al., 1992).
Detection of antibody by serum neutralization (SN)
(Hithchner, et al., 1958) or ELISA (York, et al., 1983) may
occasionally be used diagnostically but serological tests do
not provide a timely diagnosis.
Each diagnostic test has advantages and disadvantages.
The AGID test is relatively insensitive (Adair, et al.,
125
1985).
The DFAT is rapid, but suffers from nonspecific
reactions and also requires a fluorescent microscope.
techniques are labor intensive and time-consuming.
Most
Virus
isolation and identification is widely used and provides
satisfactory results but is time-consuming.
Histopathology
is very useful, but requires skilled labor for fixation,
processing, and evaluation of tissue.
PCR and hybridization
are very specific techniques for detection of ILTV, but
extraction or purification of DNA may be required and the
techniques are labor intensive.
The IP technique is simple,
rapid, and sensitive compared to other techniques, provides
a more permanent record, and requires only a conventional
light microscope (Guy, et al., 1992).
One source reports
detection of ILTV in chickens from day 1 to day 10
postinoculation (PI) for VI, from day 1 to day 9 PI for IP,
from day 2 to day 9 PI for IFAT, and from day 2 to day 7 PI
for histopathology (Guy, et al., 1992).
The main objective of this study was to evaluate
diagnostic tests for ILTV using monoclonal antibodies (MCA)
produced in our laboratory (Abbas, 1992).
We also compared
the sensitivity and specificity of diagnostic tests for
ILTV.
MATERIALS AND METHODS
Chickens.
130 day-old broiler-type chickens were
obtained from a commercial source.
The chickens were housed
126
in floor pens in a poultry house where ILTV has never been
known, and fed a standard grower diet.
At 4 weeks of age,
the chickens were divided into a group of 50 controls and
another of 80 birds to be infected with ILTV.
Each group
was placed in separate rooms of an isolation facility.
Challenge and sample collection. The USDA challenge
strain of ILTV (National Veterinary Services Laboratory,
USDA, Ames, Iowa) was grown in chicken-embryo liver cells
(CELC) using a described procedure (Abbas, 1992).
After 24
hours growth, characteristic cytopathic effect (CPE) of ILTV
was observed, and virus was collected by scraping the cells
from tissue culture plates and freezing at -70 C.
titer was 106 TCID50 /ml measured in CELC.
The ILTV
For challenge, the
ILTV was thawed in a 37 C water bath, diluted in minimal
essential medium (MEM) (Sigma Chemical Company, St. Louis,
Mo.) to bring the titer to 105 TCID50 /m1 and placed on ice.
On day 0, chickens were inoculated intratracheally with 0.1
ml of diluted ILTV, giving a challenge dose of 104
TCIDm/bird.
The control chickens were inoculated
intratracheally with 0.1 ml of MEM without ILTV.
On days 2,
4, 6, 8, 10, 12, and 14 PI, 6 birds were arbitrarily
selected first from the control group and then from the
infected group.
Birds were humanely sacrificed and their
tracheas were collected separately for each group, avoiding
cross-contamination.
A portion of each trachea was saved at
127
-70 C for use in the indirect FAT (IFAT) and IP tests, and
for VI, hybridization and PCR.
About 2 cm of trachea from
each bird also was put into neutral-buffered formalin
solution.
Monoclonal antibody (MCA) to ILTV.
been described (Abbas, et al., 1994a).
The mouse MCA has
MCA C ascites fluid
was used for IFAT and for the IP test at a dilution of
1:100.
IFAT.
Tracheal mucosal cells were separately scraped
from control and infected tracheas and washed three times in
neutral phosphate-buffered saline (PBS) at 500 x g for 5
min.
The supernatant was discarded and the cell pellet was
suspended in 100 Al of 0.01M FA buffer (containing Na2HPO4,
NaH2PO4.H20, and NaCl) with 2% bovine serum albumin (BSA).
Five Al of cells from each trachea were smeared onto each
top-row well of teflon-coated slides (Cel-line Associates,
Inc., Newfield, N.J.).
Cells from uninfected tracheas were
smeared onto the bottom-row wells of each slide to serve as
negative cell controls.
On each slide, one well also was
smeared with cells from an ILTV-infected CELC culture as a
positive control.
for 10 min.
Slides were airdried and fixed in acetone
The IFAT was performed according to a described
procedure (Abbas, 1992).
Briefly, 20 Al of MCA C in FA
buffer was overlayered on each well and incubated for 20
minutes at 37 C.
After washing slides with FA buffer, the
128
slides were soaked in FA buffer for 20 minutes.
Slides were
Twenty Al of
washed with distilled water, rinsed and dried.
diluted (1:200) rabbit anti-mouse conjugate (Sigma) was
added to each well, incubated, and washed as before.
Slides
were counterstained with 0.001% Evan's blue, washed, dried,
and examined under a fluorescent microscope.
A positive
score was recorded if specific immunofluorescence was
detected.
IP test.
The IP test was performed according to the
manufacturer's instructions using the Vectastain, ABC Kit,
designed for mouse IgM, (Vector Laboratories Inc.,
Burlingame, Cal.).
Briefly, tracheal samples from control
and infected birds were thawed, cells were scraped from
tracheal mucosa, and smeared onto slides, exactly as
described for IFAT.
Slides were fixed in acetone, and
soaked in PBS for 20 min at room temperature.
Each well was
incubated for 20 min at room temperature in blocking serum
(provided with the kit) then slides were washed with
distilled water, and 20 Al of MCA C in phosphate-buffered
saline (PBS), pH 7.5, was added to each well and incubated
for 30 min at room temperature.
Slides were washed, soaked
in buffer, then washed with distilled water, dried, and
incubated with 20 Al diluted secondary antibody
(biotinylated, affinity-purified anti-immunoglobulin M,
provided with the kit).
Slides were washed, soaked in
buffer and incubated in Vectastain ABC reagent (provided
129
with the kit) according to the kit instructions.
were washed and soaked in buffer as before.
Slides
The substrate
solution was made according to the kit instructions and
slides were incubated in substrate solution for 5 min.
Slides were washed in tap water for 5 min, counterstained
with Mayer's hematoxylin for 5 min, washed again in tap
water for 5 min, then dipped 10 times in 0.5% ammonium
hydroxide.
Slides were washed again in tap water for 5 min,
dried, mounted, and examined with a light microscope.
A
positive score was recorded if epithelial cells showed a
reddish-brown staining.
PCR.
Cells were scraped from about 2 cm of tracheal
mucosa using a separate scalpel for each trachea.
The cells
were washed 3 times in PBS by centrifuging at 500 x g for 10
min at room temperature.
the cell pellet
The supernatant was discarded and
from each trachea was suspended in 100 Al
PBS containing gentamicin and fungizone.
DNA was extracted
from the cells by a described procedure (Andreasen, et al.,
1990).
The DNA was resuspended in 50 Al distilled water,
and the entire 50 Al DNA solution was used as a template
after measuring DNA concentration by spectrophotometry
.
PCR was performed using a kit (Perkin Elmer Cetus,
Norwalk, Conn.) and a thermal cycler (Perkin Elmer Cetus) in
accordance with the manufacturer's instructions, as
described, using described PCR primers (Abbas, et al.,
1994b).
Negative controls included the uninfected tracheal
130
samples.
Samples containing DNA template extracted from
ILTV-infected CELC were included as positive controls.
A
total of 35 cycles were run, each cycle consisting of 95 C
then 50 C for 1 min each then 35 C for 1.5 min.
Amplification products were separated, stained, and
visualized as described (Ausubel, et al., 1993).
A positive
PCR produced an amplified fragment of approximately 647 base
pairs.
Hybridization.
After electrophoresis on a 4% agarose
gel and staining with ethidium bromide of the PCR product
obtained using DNA from USDA challenge strain of ILTV as a
template, the amplified DNA fragment was removed from the
gel and labeled using a BioPrime DNA Labeling kit (Gibco
BRL, Life Technologies, Inc., Gaithersburg, Md.) according
to the kit instructions as described (Abbas, et al., 1994b).
Cells were collected from each trachea, DNA was
extracted as for PCR, and the DNA pellet was redissolved in
10 gl of TE buffer (10 mM Tris-HC1, 1 mM Sodium EDTA, pH
7.5).
Hybridization was performed as described (Abbas, et
al., 1994b) using a kit, PhotogeneTM Nucleic Acid Detection
System (Gibco BRL), which uses a streptavidin-alkaline
phosphatase system.
Briefly, the 10 gl DNA sample was fixed
on a nylon membrane by ultraviolet light treatment.
In some
reactions, 10-fold dilutions of the DNA were also made and
131
fixed onto the nylon membrane.
After the completion of the
prehybridization and hybridization steps, the nylon membrane
was washed with solutions of two different stringencies, a
less stringent solution containing 0.5 X sodium chloride
sodium citrate dihydrate (SSC)
(25 ml of 20 X SSC (3 M NaC1,
0.3 M sodium citrate dihydrate, pH, 7.0] in 1000 ml
distilled water), 1% sodium dodecyl sulfate (SDS) and a
comparatively more stringent solution containing 0.1 X SSC
and 1% SDS.
All other hybridization steps were performed
according to the described procedure (Abbas, et al., 1994b).
The USDA challenge strain of ILTV was used as a positive
control for hybridization reactions.
Negative controls
consisted of DNA samples extracted from the tracheas of
uninfected chickens.
A positive score was recorded if
hybridization occurred with a sample.
Histopathology.
Formalin-fixed tracheal segments were
processed, embedded, sectioned at 5 Am, stained with
hematoxylin and eosin, and examined under a light
microscope.
A tracheal section received a positive score if
syncytial cells and typical intranuclear inclusion bodies
were detected in tracheal epithelium or in epithelial
debris.
VI Procedure.
VI was performed using CELC grown in 24-
well plates according to a described procedure (Abbas,
1992).
The mucosa was scraped from approximately 2 cm of
132
each trachea, using a separate scalpel for each trachea,
mixed into 0.5 ml MEM, and used to infect the CELC
monolayers.
Three separate wells were infected with
material from each trachea, using a separate 0.45 Am filter
and a separate syringe for each tracheal sample.
One well
in each row of the 24-well plate was not infected, serving
as a cell control.
The 24-well plate was examined every 24
hours for CPE characteristic of ILTV.
The cells were
incubated for a maximum of 7 days if no CPE was detected.
CELC that did not show CPE were collected and used to infect
freshly-grown CELC.
A total of 3 passages were made with
those samples not showing CPE before assigning them a
negative score.
To confirm the presence of ILTV, PCR and
IFAT were performed on CELC cultures that showed viral CPE.
Statistics.
Sensitivity and specificity for different
diagnostic techniques were calculated as described
(Fletcher, et al., 1982).
RESULTS
Response to ILTV challenge.
The chickens infected
intratracheally with ILTV showed respiratory signs 48 hours
PI, including depression, coughing, gasping, extension of
the neck during breathing, dyspnea, and rales.
severe from days 2 through 6 PI.
signs were observed.
Signs were
After day 8 no respiratory
Mortality totalled 7 birds of the 80
inoculated, all dying between days 3 and 6.
All dead birds
133
had hemorrhagic exudate in their tracheas and blood on their
beaks.
Dead birds were not used in diagnostic tests.
Bloody exudate was found in tracheas of chickens only on day
4 PI.
However, streaks of blood were seen in tracheas 6
days PI.
No gross lesions were observed 8 days PI or later
in tracheas of any infected chickens.
No control group
chickens had any abnormal respiratory signs during the
trial.
Diagnostic test results.
Table 4.1 presents the
results of diagnostic tests performed on ILTV-infected
chickens.
With VI using ILTV-infected tracheas, viral CPE
consisting of formation of typical syncytia was observed
after 24 hours in CELC monolayers, but CPE after 24 hours
was not as extensive as it was after 48 hours of infection.
VI detected virus from tracheas on days 2 through 6 PI, but
not after day 6.
In each case, positive VI results were
confirmed by both PCR and IFAT analysis of the infected CELC
(data not shown).
Histopathology, based on the presence of typical
syncytia and intranuclear viral inclusion bodies, detected
ILTV only on day 2 PI (Table 4.1).
However, other
pathological changes such as formation of syncytia, presence
of inflammatory cells (predominantly degranulated
heterophils but also lymphocytes and plasma cells) thickened
mucosa and tunica propria, loss of cilia, collapsed mucus
134
glands, edematous epithelial cells, cellular destruction,
separation of mucosa, and hemorrhage were consistently
present, but were usually insufficiently diagnostic to make
a specific diagnosis.
Inclusion bodies were seen primarily
in epithelial cells which still remained intact.
No
inclusion bodies were seen in detached cell clumps, which
were infiltrated with hetrophils and heterophil granules.
Tracheal sections 2 days PI often still contained normal
structures such as intact cilia and epithelial cells.
Pathological changes were especially severe on days 4 and 6
PI.
By 8 days PI, tissues contained fewer heterophils and
ciliated epithelial cells had reappeared.
tracheas appeared normal.
By day 10 PI,
All tracheal sections from
uninfected chickens were normal on each day of the trial.
Antigens of ILTV were detected in tracheal cells both
by IFAT and the IP test on days 2 through 8, but not after
day 8 PI (data not shown).
The intensity of staining in
infected tracheas with IP and IFAT was most intense on day 4
PI.
IP and IFAT both detected infected chickens longer than
any other test.
PCR yielded positive results only on days 2 through 6
PI, but not after day 6 (data not shown).
Hybridization
failed to detect virus from any infected trachea on any day.
VI and PCR both detected virus until day 6 PI, but VI
detected more infected birds than PCR.
detected after day 10 PI by any test.
Virus was not
135
In tests performed with tracheas from control chickens,
no positive result was obtained from any trachea in any test
at any time.
Table 4.1. Results of the virus isolation (VI),
histopathology, indirect fluorescent antibody (IFAT),
immunoperoxidase (IP), polymerase chain reaction (PCR), and
hybridization diagnostic tests for detection of ILTV in
tracheas of experimentally-infected chickens.
Days postinoculation'
VI
Histopathology
IFAT
IP
PCR
Hybridization
2
3/6b
1/6
4/6
4/6
2/6
0/6
4
6/6
0/6
6/6
6/6
1/6
0/6
6
6/6
0/6
5/6
6/6
1/6
0/6
8
0/6
0/6
1/6
1/6
0/6
0/6
'Only data for days 2, 4, 6, and 8 postinoculation are
shown.
No positive results were obtained from any
diagnostic test on days 10, 12, or 14 postinoculation.
bNumber of positive test results/number of tracheas tested.
The specificity and sensitivity of each test,
calculated using VI as a reference, are presented in Table
4.2.
Sensitivity and specificity were 100% and 93% for
IFAT, 93% and 93% for *IP, 7% and 100% for histopathology,
27% and 100% for PCR, and 0% and 100% for hybridization.
136
Table 4.2.
Sensitivity* and specificityb of the
histopathology, indirect fluorescent antibody (IFAT),
immunoperoxidase (IP), polymerase chain reaction (PCR), and
hybridization diagnostic tests for detection of ILTV in
tracheas of experimentally infected chickens, based on virus
isolation (VI) results as a standard.
Diagnostic
test results
Histo-
VI results
+
-
1
0
pathology
14
27
IFAT
14
2
1
25
15
2
0
25
4
0
11
27
Hybrid-
0
0
ization
15
27
IP
PCR
Agreement
Sensi-
Speci-
with VI
tivity
ficity
67%
7%
100%
93%
93%
93%
95%
100%
93%
74%
27%
100%
64%
0%
100%
'Sensitivity = number of true-positive results/(number of
true-positive results + number of false-negative results).
bSpecificity = number of true-negative results/(number of
false-positive results + number of true-negative results).
`The 15 positive and 27 negative VI results are considered
to be true-positive and true-negative results.
137
DISCUSSION
The IP test was more sensitive than any other
diagnostic technique in this study.
Other workers also have
found IP to be more sensitive than other diagnostic
techniques (DeLellis, et al., 1979; Smith, et al., 1989).
The high sensitivity of IP is due to enzymatic amplification
of the antigen-antibody reaction and decreased background
staining (DeLelis, et al., 1979).
sensitive test.
IFAT was the second most
IFAT has detected virus in tracheal tissues
from day 2 to day 14 PI (Wilks & Kogan, 1979), although
shorter periods (6-8 days PI) are more often reported
(Bagust, et al., 1986; Hitchner, et al., 1977).
ILTV
antigens also have been detected in tracheal samples with an
antigen-capture ELISA, and it has been reported that such an
ELISA is a rapid diagnostic technique that is more sensitive
than IFAT (York & Fahey, 1988).
In this study such an ELISA
was not compared to other diagnostic tests.
VI was less
sensitive than IP and IFAT, suggesting that some tracheal
samples may have contained viral antigens but not infectious
ILTV.
The sensitivity of histopathology was 7%.
The specific
diagnostic combination of syncytial cells and virus
inclusion bodies was seen only in one tracheal section, on
day 2 PI.
Inclusion bodies may have been undetectable in
other tracheal sections because of the severe inflammatory
response.
Other workers have reported a higher sensitivity
138
for histopathology (based on the presence of ILTV inclusion
bodies) as an ILTV diagnostic procedure (Guy, et al., 1992).
As necrosis and desquamation destroy infected epithelium,
ILTV inclusion bodies can be detected in tracheal sections
only during the early stages of disease (day 1 to day 5
postinoculation) (Hanson & Bagust, 1991).
In one study,
ILTV inclusion bodies were seen in 57% of 60 specimens, but
virus was isolated from 72% of the same specimens (Hanson &
Bagust, 1991).
PCR was less sensitive than IP, IFAT, and virus
isolation, but more sensitive than histopathology and
hybridization.
PCR can detect latent infections, as it
detected latent virus in the trigeminal ganglia of recovered
birds up to 61 days PI while other tests failed to detect
the virus (Williams, et al., 1992).
This suggests the
superiority of PCR over other tests for detection of
latently-infected carrier birds.
IP and IFAT may be more
sensitive than PCR because they detect ILTV antigens even if
no viral DNA is present.
PCR in our hands.
VI was much more sensitive than
VI depends on the presence of infectious
viral particles while PCR depends on the presence of ILTV
DNA, so PCR in theory should be at least as sensitive as VI.
As PCR was performed by extracting DNA from ILTV-infected
tracheas, it is possible that DNA was lost in that
procedure.
Spectrophotometry results support that
possibility, because in most cases that were negative by PCR
139
there was little or no DNA in extracted samples.
The
primers used in this study have amplified 0.0625 Ag of ILTV
DNA (Abbas, et al., 1994b), suggesting that less than that
amount of ILTV DNA was obtained from tracheal samples.
ILTV-infected CELC were used as a control for the DNA
extraction procedure, and in all cases viral DNA was
obtained from ILTV-infected CELC.
Infected tracheas had a
very severe inflammatory response associated with many
heterophils, especially on days 4 and 6 PI.
Factors
associated with inflammation may have destroyed viral DNA.
Also, inflammatory exudate or blood may contain Tag
polymerase inhibitors.
That IP and IFAT were positive on
some tracheas that were negative by PCR suggests that there
still were viral antigens capable of detection by IP and
IFAT, even in the presence of phagocytosis.
Hybridization did not ever detect ILTV in infected
tracheas, possibly because the samples contained
insufficient target DNA.
The DNA probe used in this study
does not detect less than 0.1 Ag of ILTV DNA (Abbas, et al.,
1994b).
Two different stringencies including a low
stringency salt solution (0.5 X SSC, 1% SDS) and a
comparatively high stringency salt solution (0.1 X SSC, 1%
SDS) were used in hybridization attempts.
Even using more
probe, specific hybridization did not occur.
In certain tracheas ILTV antigens were detected by IP
and IFAT but virus was not isolated.
These cases suggest
140
the presence of viral antigens but no infectious virus
particles, that could be a result of suppression of virus
growth in those tracheas by the immune system (production of
specific antibodies against virus) as has been suggested by
others (Guy, et al., 1992).
Since the chickens involved
were infected, these may represent false-negative VI
results.
The IP test performed better than any other diagnostic
test including VI in this study.
The sensitivity of the IP
test was 100%, more than any other diagnostic test in this
study, and its specificity was 93%.
No false-negative
results occurred with the IP test, while one false-negative
result occurred with the IFAT, compared to VI.
The IP test
is as simple as IFAT, but is more sensitive than IFAT and
does not require a fluorescent microscope.
In addition,
results obtained with the IP test can be saved longer than
results obtained by IFAT in which the fluorescence is lightsensitive and fades rapidly.
141
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