AN ABSTRACT OF THE THESIS OF
Hsuan-Jen Huang for the degree of Doctor of Philosophy in
Comparative Veterinary Medicine presented on Jan 31,
2000.
Escherichia
Title: Non-Specific Innate Immunity against
coil Infection in White Leghorn Chickens
Redacted for Privacy
Abstract approved:
Ikazu Matsumoto
This
research was
problem
mortality
chickens
in
initiated
occurring
market
in
field
Initial
Oregon.
investigate
to
age
a
high
broiler
investigations
revealed that mortality was due to the systemic bacterial
infections.
The
subsequent
experiments
laboratory
suggested that suppressed short-term, non-specific innate
immunity rather than pathogenic properties of bacteria
caused the systemic bacterial infection.
The objectives
of research described were to analyze and characterize
the type of innate immunity in chickens by developing a
model; analyzing this immunity with artificial induction
by inactive agents; and demonstrating the involvement of
the type of immunity in viral infection.
Escherichia
coli
strain
Ol:Kl
systemic
causes
infection in chickens after the intra-airsac inoculation.
Levels
of
immunity
can
be
determined
by
the
viable
organism count in the internal organs of infected birds.
Bacterial counts were significantly lower in the liver or
spleen of vaccinated birds
inoculation than in controls.
at
6,
12
or
24
hrs
after
An oil-adjuvanted vaccine
showed
some
deterioration
in
its
immunogenicity after
prolonged storage or heating at 100 C.
Non-specific
innate
immunity induced by intravenous
injection of inactivated bacteria or LPS, or subcutaneous
injection of silver nitrate induced significant immunity
in
24
viable
infection with
hours against
bacterial
counts
in
the
E.
coli
cell suspension
(FSA)
Nonspecific
spleen.
Staphylococcus
immunity induced by formalin-inactivated
aureus
based on
was comparable to specific
immunity induced by a specific vaccine as determined by
cumulative
bacterial
during
mortality
count
in
the
7
spleen
days
after
and
the
infection.
nonspecific immunity appeared as early as
viable
This
hours and
6
lasted for less than 72 hours after stimulation.
Birds vaccinated with NDV vaccines induced significant
protection against challenge exposure with Ol:Kl strain
for a period of 2-8 days post vaccination.
NDV vaccination administered
14
days
later
Secondary
failed
to
induce immunity against E. coli when infected 1 or 5 days
after the vaccination.
Treatment with cold stress or
corticosterone suppressed the
induction of
nonspecific
immunity by FSA or NDV vaccination.
These results indicate that nonspecific innate immunity
against E.
coli in chickens can be induced by injection
of killed bacteria or primary NDV vaccination.
Non-Specific Innate Immunity Against Escherichia coli
Infection in White Leghorn Chickens
by
Hsuan-Jen Huang
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented January 31, 2000
Commencement June 2000
Doctor of Philosophy of Hsuan-Jen Huang presented on
31, 2000
APPROVED:
Redacted for Privacy
Major Pro'ssor, Representing Comparative Veterinary
Medicine
Redacted for Privacy
Redacted for Privacy
Dean c 7Grduate School
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
Hsuan-Jen Huang, Aut
r
ACKNOWLEDGEMENT
I
would
like
thank
to
my major
advisor,
Dr.
M.
Matsumoto, for his guidance, encouragement, and critical
reviews during my research and papers.
Appreciation is also extended to my committee members:
Drs.
Thomas G Chastain,
Jerry Heidel,
Paul
Reno,
and
Anthony Vella for their helpful suggestions on my work.
A special
note
of
thanks
goes
to
my parents
and
parents in law for their support and encouragement during
these years.
Last but not least, my deep appreciation goes to my
wife,
Ching-Chaun,
for
her
making this thesis possible.
love
and encouragement
in
TABLE OF CONTENTS
Page
Chapter
1.
INTRODUCTION
Chapter
2.
LITERATURE REVIEW
1
-------------------
7
-------------------2.1 Innate immune response
2.1.1 Major component in innate immunity
2.1.2 The linking of innate and adaptive
immunity
7
2.2 Acute phase response ---------------------2.2.1 Introduction
2.2.2 Initiation of acute phase response
2.2.3 Acute phase protein -----------------2.2.4 Function of major acute phase protein:
CRP, SAA, SAP ----------------------2.2.5 Interspecies and sex differences
-----------------2.2.6 Regulation of APP5
2.2.7 Resolution of acute phase response
10
10
ii
13
2.3 Other acute phase phenomena --------------2.3.1 Neuroendocrine changes
--------------2.3.2 Hematopoietic changes
---------------------2.3.3 Hepatic changes
22
22
22
23
2.4 Glucocorticoids and immunity
--------------------2.4.1 Bioavailability
2.4.2 Effects on cytokines
23
24
25
-------------------------------
26
2.5 References
7
9
13
16
18
21
Chpater 3. Immunity against Escherichia Coli Infection
in Chickens Assessed by Viable Bacterial Counts in
34
Internal Organs ---------------------------------3.1 Abstract -----------------------------------
35
----------------------------
36
3.2 Introduction
3.3 Materials and methods
3.4 Results
---------------------- 38
-----------------------------------
44
TABLE OF CONTENTS (CONTINUED)
Page
3.5 Discussion
51
3.6 References
55
Induction of Short-term, Non-specific
Chapter 4.
Immunity against Escherichia Coil Infection in
Chickens Is Suppressed by Cold Stress or
Corticosterone Treatment
57
4.1 Abstract
58
4.2 Introduction
59
4.3 Materials and methods
60
4.4 Results
67
4.5 Discussion
-------------------------------
4.6 References
73
77
Non-specific Innate Immunity against
Coil Infection in Chickens Induced by
Vaccine Strains of Newcastle Disease Virus
Chapter 5.
Escherichia
79
5.1 Abstract -----------------------------------
80
5.2 Introduction
81
5.3 Materials and methods
82
5.4 Results
88
5.5 Discussion
--------------------------------
94
5.6 References
--------------------------------
100
Chapter
6.
BIBLIOGRAPHY
CONCLUSIONS
-----------------------------------
102
104
LIST OF FIGURES
Figure
3.1
Page
The number of viable bacteria (mean ± s.d.)
detected in the blood, lung, spleen, or
liver at different times after the intraairsac
with
inoculation
Escherichia
of
coli
5-week-old chicks
strain 01:1(1; the
vaccinated or control group
Experiment 1) were tested.
(
N= 16 /group
p < 0.05.
*
45
3.2
3.3
effect of various adjuvants on the
immunogenicity
of
inactivated
bacterial
suspensions against challenge-exposure by
homologous
the
strain
of
E.
coli
*
(Experiment 3)
See the
p < 0.05.
abbreviation list and text for details.
The
47
The effect of storage and heating on the
immunogenicity of inactivated vaccines as
determined by the number of E. coli strain
01:1(1
3.4
4.1
4.2
in the spleen or lung 24 hrs after
the intra-airsac inoculation; F, fresh; 0,
old;
F
+
H,
heated fresh vaccine; AC,
adjuvant control; C, non-treated control
------------(Experiment 2). * p < 0.05.
49
The effect of pretreatment with inactivated
01:1(1 cells or Salmonella LPS on the number
of viable Escherichia coli strain 01:1(1 in
the
spleen
24
hrs
after
intra-airsac
*
inoculation (Experiment 4)
p < 0.05.
See details under Experiment 4 described in
thetext. --------------------------------
50
The
assay
procedure
for
nonspecific
-------------------immunity in chickens.
62
The effect of various agents
antibacterial
immunity
in inducing
indicated
by
the
number of viable E. coli in the spleen 24
hours after challenge infection (Experiment
Groups with differing letters indicate
1)
significant (P < 0.05) differences.
.
64
LIST OF FIGURES (Continued)
Figure
4.3
4.4
Page
The number of viable E. coli in the spleen
after challenge
detected
exposure
at
various time periods after stimulation with
the
formalin-treated
Staph.
aureus
--------------------------(Experiment 3)
The number of viable E. coli in the spleen
after
challenge
detected
exposure
at
various days after stimulation with the
formalin-treated Staph. aureus (Experiment
4)
5.1
71
.
The number of viable E. coli in the spleen
chickens at
24
of
hr
after
challenge
infection.
The chickens were previously
exposed to MDV, Roakin strain, with time
period (days) indicated (* p < 0.05, ** p
<0.01)
5.2
70
90
.
The number of viable E. coli in the spleen
chickens
at
infection.
The
of
24
hr
after
challenge
chickens were previously
exposed to MDV, La Sota strain, with time
period (days) indicated (* p < 0.05, ** p
<0.01)
5.3
.
The number of viable E. coli in the spleen
of chickens at 24 hr after infection.
The
chickens were previously exposed to MDV (5
days), injected with inactivated S. aureus
or vaccinated
twice
with an
(24
hr),
inactivated homologous vaccine before the
challenge infection (** p <0.01)
91
93
LIST OF TABLES
Table
Page
--------------------
2.1
Acute phase proteins
2.2
Major interspecies differences in APPs
17
3.1
Different adjuvants given in Exp. 3
41
3.2
Different vaccine
chicks in Exp. 2.
4.1
preparation
given
14
to
43
The effect of nonspecific immunity induced
by
inactivated
Staph.
aureus
or
that
of
specific immunity induced by a homologous
vaccine on the E. coli viable count in the
4.2
5.1
5.2
spleen at 24 hours, cumulative mortality and
weight
gain during
7
days,
or
lesion
frequency
at
7
days
after
challenge
infection
with
E.
coli
in
5-week-old
------------------------------chickens
68
Suppressive effects of corticosterone, ACTH,
or cold stress treatment on the induction of
nonspecific
immunity
against
E.
coil
infection
----by Staph. aureus cells (Experiment 5)
73
The absence of nonspecific immunity against
E. coli in chickens at 1 or 5 days after the
secondary vaccination with La Sota strain
86
effect
of
cortitorsterone
on
the
nonspecific immunity against E. coli induced
by NDV vaccination ------------------------
88
The
NON-SPECIFIC INNATE IMMUNITY AGAINST ESCHERICHIA
COLI INFECTION IN WHITE LEGHORN CHICKENS
Chapter 1
INTRODUCTION
research of
The
investigation
of
a
thesis was
the
initiated with the
high mortality problem of
broiler
chickens during the last two weeks of the growing period
in Oregon. A field study from 3 broiler farms indicated
that
mortality
the
infection
(Awan
was
and
due
to
Matsumoto,
a
systemic
1998).
bacterial
The
systemic
infections were found not to be caused by any predominant
bacterial
pathogenic
There
species.
were
some
significant relationships between the isolated bacterial
and
species
organisms
particular
sampling
were
abundant
poultry
suggesting
sites,
house.
the
in
Since
that
environment
adaptive
appeared normal in these affected chickens,
certain
of
a
immunity
it suggests
that systemic infections in market age broilers might be
suppression
caused
by
innate
immunity rather than the pathogenic
the
microorganisms.
of
short-term,
nonspecific
factors
of
2
Innate immunity, believed to be phylogenetically older
is present in all multicellular
than acquired immunity,
It is defined as the first line host defense
organisms.
against pathogens and works under different mechanisms
from those involved in adaptive immunity (Hoffann, 1999).
Innate
immunity may also play an
determining
respond
and
Locksley,
which
to
the
1996)
of
in
immunity
may
(Fearon
and
response
the
role
It generally provides non-specific and
.
immunity
short-term
adaptive
antigens
nature
instructive
rather
than
specific
the
and
efficient adaptive immunity, and its protective role was
identified
1997)
in
bacterial
(Naiki,
1999)
,
viral
(Welsh,
or parasitic infection (Diefenbach, 1999)
,
Common criteria used to evaluate immunity against
coli
differences
are
lesion scores,
loss
after
control
Panigrahy,
treated
1984) .
vaccine
evaluating
effectiveness
animals
to
mortality
pathological
rates,
isolation frequencies, and/or weight gain
artificial
and
in
E.
but
obtain
challenge
groups
(Deb
methods
These
products
requires
exposures
a
reproducible
in
and
between
Harry,
are
terms
the
1978;
useful
of
for
their
substantial
number
results.
Biomedical
of
research investigators are being pressured to avoid or
improve procedures that may involve pain in experimental
3
For example, the animal care committee of this
animals.
institution has
recently adopted
government
recommendation
should not
be
used
a
that
1984)
(NIH,
an endpoint
as
rule based on
in
the
lethality
vertebrate
all
Since mortality has been used traditionally for
species.
evaluating the virulence of E.
coli
(Sicardi,
1966)
and
for determining efficacy of vaccines (Panigrahy, 1984), a
reliable, alternative method should be available to study
infection
coli
E.
(Pourbakhsh,
of E.
1997)
in
Recently
poultry.
study
reported that inoculation of a strain
coli into the airsac resulted consistently in the
systemic
of viable
Predictable numbers
infection.
E.
coli were detected in internal organs up to 48 hrs after
the inoculation.
thesis
The
first part
evaluate
includes
(Chapter
the
3)
level
3
parts.
The
objective
of
the
was to examine whether one can
antibacterial
of
immunity
by
determining viable bacterial counts in internal organs in
an E.
coli infection model in white leghorn chicks.
We
found that the air-sac inoculation of lO6lO7 cfu of E.
coli
01:
septicemic
immunity
Ki
results
infection
against
E.
a
predictable
white
leghorn
in
in
coli
infection
quantitated by the viable count method.
pattern
chickens
can
of
and
precisely
4
The second part of studies
(Chapter 4)
was to induce
the non-specific innate immunity against E. coli infection
We found
in chickens by administering inactive agents.
that intravenous injection of homologous or heterologous
bacteria
inactivated
protect
significantly
The anti-bacteria
coli challenge infection.
against E.
chickens
effect induced by killed S. aureus appears as early as 6
Cold stress
hr and lasts 2-3 days after the stimulation.
and
corticosterone
treatment
suppress
can
this
non-
specific immunity.
In the last part
(Chapter 6),
we evaluated if mild
viral infection such as NDV vaccination can induce the
non-specific immunity against
results
showed
vaccination
for
vaccination,
strain,
immunity.
that
the
E.
coli
immunity was
period
of
infection.
by
induced
2
to
Secondary vaccination with NDV,
however,
failed
to
induce
this
NDV
the
days
8
The
post
La Sota
nonspecific
This immunity is also suppressed by the stress
or corticosterone treatment.
This research was intended to reduce the economic loss
of
the broiler
industry,
and
to understand nature
innate immunity involved in bacterial infection.
of
References
Awan, M.A. & Matsumoto, M. Heterogeneity of Staphylococci
and other bacteria isolated from six-week-old broiler
chickens. Poultry Science, 77, 944-949. 1998.
R.
and E. G. Harry. Laboratory trials with
J.
inactivated vaccines
against
Escherichia
coli
02:Kl
infection in fowls. Res. Vet. Sci. 24:308-313. 1978.
Deb,
Diefenbach,
A.,
H.
Schinfler,
M.
Rollinghoff,
W.
M.
Yokoyama, and C. Bogdan.
Requirement for type 2 NO
IL-12
synthase
for
signaling
in
innate
immunity.
Science. 284: 951-955. 1999.
Fearon, D.T., and R. M. Locksley.
The instructive role
of
innate immunity in the acquired immune response.
Science. 272: 50-54. 1996.
Hoffann, J.A., F.C. Kafatos, C. A. Janesway Jr.,
and
R.A.B.
Ezekowitz.
Phylogenic
perspectives
in
innate
immunity.
Science 284: 1313-1318, 1999.
Mishimura,
T.
Kawano,
Y.
Tanaka,
S.
Taniguchi, and Y. Yoshikai. Regulatory role
of peritoneal NK 1.1 + a T cells in IL-12 production
during Salmonella infection. J. Immunol. 163: 2057-2063.
Y.,
Naiki,
Itohara, M.
H.
1999.
National Institute of Health.
and use committee guideline.
Institutional animal care
NIH Publication No. 92-
3415. 1992.
Panigrahy,
B.,
J.
E.
Gyimah,
C.
F.
Hall and J. D.
Williams.
Immunogenic
potency
of
an
oil-emulsified
Escherichia coli bacterin. Avian Dis. 28:475-481. 1984.
Pourbakhsh, S. A., M. Boulianne, B. Martineau-Doize, C.
M. Dozois, C. Desautels, and J. M. Fairbrother. Dynamics
Escherichia
coli
infection
of
in
experimentally
Avian Dis. 41:221-233. 1997.
inoculated chickens.
Sicardi,
ability
F.
of
J.
Identification and disease producing
Escherichia coli associated with E.
coli
infections of chickens and turkeys.
of Minnesota, St. Paul, MN. 1966.
M.
S.
Thesis. Univ.
M-Y Lin, B.L. Lohman, S.M. Varga, C.C.
Zarozinski, and L.J. Selin. a13 and yö T-cell networks and
their roles in natural resistance to viral infections.
Immunol Rev. 159: 79-93. 1997.
Welsh,
R.
M.,
7
Chapter 2
LITERATURE REVIEW
2.1 Innate immune system
Innate
referred
immunity,
to
as
basic
the
resistance mechanism against microorganism invasion,
immediately
triggered
breached
(Kuby,
specific
and
when
This response
1994).
does
protective
not
require
a
barriers
is
host
is
are
not antigen-
prolonged period
of
induction.
2.1.1 Major components in innate immunity
Anatomic
barriers.
Physical
and anatomic
barriers
that tend to protect the entry of pathogens are the first
line of defense against infection.
The skin and mucous
membrane are examples in this category.
Physiologic barriers.
temperature,
factors.
pH,
oxygen
Physiologic
tension,
and
barriers
include
various
soluble
Many species are resistant to certain pathogens
simply because their body temperature inhibits pathogen
growth.
For example,
high body temperature
of
chicken
8
A variety of soluble
inhibits the growth of anthrax.
interferon,
lysozyme,
1994)
contribute
also
factors
These
.
to
innate
their
have
others
and
complement,
factors
including
immunity,
own
(Kuby,
mechanisms
to
destroy the microorganisms.
Endocytic and Phagocytic barriers.
through two processes:
endocytosis
digested,
pinocytosis or receptor-mediated
(Besterman,
macromolecules
and
1983)
particles
and
eliminated
process.
Phagocytosis
particular
material,
from
Extracellular
.
can
be
the
cells
involves
phagocytic
induces
whole
cells
microorganisms,
only
not
antigen
also
but
during
the
ingestion
of
pathogenic
include
blood
The process of
monocytes, neutrophils, and macrophages.
phagocytosis
internalized,
the
including
These
microorganisms.
Endocytosis occurs
killing
invading
processing
and
presentation for adaptive immunity (Aderem, 1999).
Barriers
created
by
inflammatory
the
Following a wide variety of stimuli,
trauma
or
injury,
a
complex
series
response.
such as infection,
of
reactions
are
executed by the host to prevent ongoing tissue damage,
destroy, dilute, or localize the injurious agent and to
facilitate
repair
of
the
cumulative
process
is
known
damaged
as
tissues.
inflammation,
This
and
the
9
early and immediate sets of reactions that are induced
are called acute phase response (APR)
(Kushner, 1982)
2.1.2 The linking of innate and adaptive immunity
immunity
Innate
is
defined
broadly
as
immune
or
protective features of the host that are generally not
included
in
a
category
(Hoffman, 1999).
of
adaptive
specific
immunity
However, it may have an additional role
in determining which antigens adaptive immunity responds
to
and
the
nature
the
of
response
(Fearon,
1996)
Cellular and soluble components of innate immunity may
provide instruction for the acquired immune response to
select appropriate antigens and the strategies for their
elimination.
The human homologue of Toll,
factor
for
regulating
lipopolysaccharide
expression
of
in
an important signaling
the
recognition
innate
immunity,
NF-B-controlled
cytokine
process
of
induces
the
genes
for
interlukin (IL)- 1, 6, or 8, as well as the expression of
the costimulatory molecule B7.1,
deliver the
cells
secondary signal
(Medhzhitov,
1997;
for
Janeway,
which
the
is
required to
activation of
1997)
.
T
Srivastava
(Srivastava, 1994,1995) used the tumor-derived heat shock
10
proteins
cancer
(HSPs)
to
tumor-specific
induce
Their
immunotherapy.
results
immunity
showed
in
that
incubation of macrophages with exogenously added antigens
chaperoned by HSPs results in the loading of the major
histocompatibility complex
theses specific antigens,
restricted
presentation
(MHC)
class
molecules with
I
as illustrated by their MHCcytotoxic
to
T
cells.
It
suggested that macrophages may be able to utilize the
uptake of HSP-chaperoneed peptides derived from the host
organism for the loading of NRC molecules, thus providing
the ligand for T-cell receptor recognition and delivery
of the first signal for T-cell activation.
These
results
provide
some
evidences
that
innate
immunity may also play an instructive role in determining
to which antigens adaptive immunity may respond and the
nature of the response.
2.2 Acute phase response
2 .2 .1 Introduction
Acute phase response
orchestrated sequence
(APR),
of
a predetermined and well-
process,
is
initiated at
the
11
site of infection or injury,
soluble mediators that
leading to the release of
regulate the metabolic response
through the whole organism.
The initial recognition of
acute phase response can be attributed to ancient Greeks,
who recognized that red blood cells from a sick person's
peripheral blood sedimented more rapidly than the normal
This increase of erythrocyte sedimentation
individuals.
rate has been found to result largely from an increase in
plasma concentration of fibrinogen and other acute-phase
proteins (Mackiewicz, 1993)
2.2.2 Initiation of acute phase response
The mammalian acute phase response is characterized by
fever,
changes
changes
in
the
in
vascular
biosynthetic,
profiles of many organs
is
permeability,
metabolic
(Kushner,
initiated and coordinated by
inflammatory
mediators,
anaphylatoxins,
APR involves
1982)
.
along
and
catabolic
This response
a variety of
including
and glucocorticoids.
with
diverse
cytokines,
Initiation of the
a highly complex mechanism,
including the
release of various mediators, binding to the respective
receptor(s),
transduction of
the
signal
from the
cell
membrane to the nucleus where gene transduction is up- or
12
the processing of mRNA,
down-regulated,
the
change
in protein
synthesis
and
and ultimately
export
(Kushner,
1982)
The tissue macrophages and blood monocytes are the most
important
whole
associated with the
cells
cascade
events
of
(Baumann,
initiation of
1994)
the
Activated
.
macrophages can release a broad spectrum of cytokines.
Interleukin-1
family,
(IL-i)
and
Tumor-necrosis-factor
which appear early after the
(TNF)
stimulation,
are
considered the "alarm" cytokines which are important for
initiation
the
series
next
the
reactions.
of
Interleukin-1 and TNF can stimulate the adjacent stroma
cells,
such as fibroblasts and/or endothelial cells,
to
induce the second wave of cytokines, which can augment
the homeostatic
signal
and initiates
the
cellular and
cytokine cascade that are involved in the complex process
of the APR (Gabay,
For example,
1999) .
IL-1,
IL-6, and
TNF can stimulate the production of acute phase proteins
(APP5) from hepatocytes.
Interleukin-1, MCP and IL-8 can
induce the expression of important adhesion and integrin
molecules,
more
inflammatory cells
1992; Rot,
and
like ICAM from endothelial cells and attract
1992; Laskey,
aggregation
induced
to
the
1992)
.
local
sites
(Williams,
Mast cell degranulation
platelet
activation
can
also
13
induce the release of mediators that are chemotactic for
monocytes and macrophases.
2.2.3 Acute phase protein
An acute phase protein is defined as one whose plasma
concentration increases (positive acute phase protein) or
decreases (negative acute phase protein) by at least 25 %
during the inflammatory disorder
(Morley,
1982)
Those
.
proteins whose concentrations increase are referred to as
positive APP, while those whose levels decline are termed
negative APP.
A partial list of human APP is seen in
Table 2.1.
2.2.4 Function of major acute phase protein:
CRP,
SAP,
SAP
CRP.
C-reactive protein
(CRP)
was originally named
for its ability to bind the C-polysaccharide of the cell
wall in Pneurnococcus and has been shown to have a number
of
1982)
calcium-dependent
.
material
binding
specificities
C-reactive protein can
like
chromatin.
This
also bind
(Kushner,
to
nuclear
CRP-chromatin
complex
helps the removal of exposed nuclear DNA by complement-
14
Table 2.1 Acute phase proteins
Positive acute phase proteins
Type-i APP5
C-reactive protein (CRP)
Serum amyloid A
Serum amyloid P
al-Acid glycoprotein
Complement C3
Complement B
Ferritin
Heptoglobin (rat)
Hemopexin (rat)
Type-2 APP5
Fibrinogen
al Anti-trypsin
a2 Macroglobulin
al Anti-chymotrypsin
Ceruloplasmin
Heptoglobin (human)
Hemopexin (human)
Albumin
Transferin
a2-HS glycoprotein
a-Fetoprotein
Thyroxin-binding globulin
Insulin-like growth factor I
Factor XII
Negative acute phase proteins
dependent
solubilization
Shephard, 1986)
ligands,
.
copmplement
phagocytes
1985;
(Robey,
Complex of CRP with a variety of other
including
naturally
of
occurring
(Seigel,
chromatin,
several
polycations
1974;
Seigel,
can
1975;
synthetic
also
Claus,
and
activate
1977)
15
C-reactive protein acts as opsonin for phagocytic cells.
purified
Using
and
CRP
unfractionated
washed
blood
leukocytes as a source of polymorphonuclear leukocytes,
it was clearly shown that CRP enhances the phagocytosis
of a variety of Gram-positive and Gram-negative pathogens
This opsonic property of
(Ganrot, 1969; Kindmark, 1971).
CRP were
found
to
depend on
ability to
its
activate
complement and on the presence of PCh in the bacterial
capsule (Edwards,
1982; Mold,
1982)
C-reactive protein
.
has also been reported to induce macrophage tumoricidal
activity (Barna, 1984; Zahedi, 1986; Barna, 1987)
BAA.
number
The
serum amyloid A
of
differentially
acute-phase SAks
(A-SAA5)
family comprises
expressed
a
apolipoproteins,
and constitutive SA1s
(Uhlar and Whitehead, 1999).
reactants,
(SAA)
(C-SAA5)
They are major acute-phase
the in vivo concentrations of which increase
by as much as 1000-fold during inflammation.
Although
the liver is the primary site of synthesis of both A-SAA
and C-SA7, extrahepatic production has been reported for
most
family members
studied.
in most
of
the mammalian species
Although the precise role of A-SAA in host
defense during inflammation has not been defined,
potential
proposed
clinically
for
important
individual
SAA
functions
family
have
members.
many
been
These
16
involvement
include
(Husebekk,
1987),
degrading
enzymes,
metabolism/transport
lipid
in
induction
extracellular-matrix-
of
chemotactic
and
recruitment
inflammatory cells to sites of inflammation
of
(Baldolato,
1994; Xu, 1995)
amyloid
Serum
SAP.
protein
plasma
preserved
P
component
named
for
(SAP),
a
highly
ubiquitous
its
presence in amyloid deposits, belongs to the same family
of pentraxins as CRP, and may have comparable activities.
Studies showed that SAP has the ability to bind to either
or
single
double
strand
DNA
in
dependent manner (Pepys and Butler,
postulated that
this
ability of
calcium-
specific
a
1987).
SAP
It has been
could provide
an
efficient means of preventing that initiation of nuclearantigen
specific
autoimmunity
(Steel
and
Whitehead,
1994)
2.2.5
Interspecies and sex differences
Production
of
different species.
APP
also
In man,
shows
great
variiety
in
CRP and SAA are the major APP
while SAP and a2-M do not have significant change in APR.
In rabbit,
CRP
and SAA show similar response,
however,
17
serum
level
(Mackiewicz,
of a-M and
1988) .
SAA
however,
is
transferrin
increase
in
APR
a2-M is the major APP,
In the rat,
non-detectable
and
evidence
no
of
transcription of SAA mRNA is found in liver (Sipe, 1982)
A summary of interspecies differences in APP is shown in
Table 2.2.
Table 2.2
Major interspecies differences in APPs
SAP
a2-M
AGP
Man
0
0
++
Rabbit
?
++
?
CRP
Species
SALk
Mouse
+
+++
++
?
++
Rat
+
ND
0
+++
++
o
+
++
+++
?
ND
no significant difference
increase about 2 fold
increase about 2-10 fold
increase more than 100 fold
not konwn
not detected in plasma
Sex is also believed a factor for the APP response.
Considerable differences in changes of plasma x1-M and a2M
have
been
reported
in
male
and
female
rats
after
injection with cortisol and turpentine (Bosanquet, 1976)
18
Female protein, belong to the pentraxin family (together
with CRP and SAP),
plasma
the
of
is present in high concentration in
hamsters
female
and
relative
in
low
During APR, its concentration may
concentration in male.
increase three fold in males, while it decrease by 50
%
in female (Coe, 1981)
2.2.6 Regulation of APP5
which regulate
Mediators,
the production of APP by
hepatic cells can be divided into following categories;
cytokines,
type
IL-i
IL-6
cytokines,
type
glucocorticoids, growth factors.
IL-i type cytokines,
IL-i type cytokines.
including
IL-la, IL-1, TNF-a, and TNF-13, are characterized by their
ability to predominantly stimulate the expression of
certain set
CRP,
SA1,
of APP5,
and so on.
type-i APP5
1990;
cells in vitro.
1),
including
The pattern of the induction of
these APP5 was observed in rat
(Falus,
(Table
a
Perlmuttr,
1986;
(Bauman,
Rogers,
1993)
1990)
and human
hepatoma
19
IL-6
type
including
cytokines,
factor
(LIF),
factor
(CNTF),
another
cytokines.
IL-6,
set
fibrinogens,
of
gene
the
expression
named
APPs,
type-2
antitrypsin,
a2M,
inibitory
ciliary neurotrophic
(OSM),
stimulate
can
type
(IL-6)
lekemia
IL-il,
onco-statin M
specific
including
Interleukin-6
and
of
APP5,
others.
These proteins regulated by IL-6 type cytokines have been
described in different
rat
(Andus,
1988;
Andus,
1988;
Koj, 1991) and human (Koj, 1991; Castell, 1989; Heinrich,
heptocyte cell lines in vitro.
1990)
Interleukin-6
is
believed to be the chief stimulator for most acute phase
It induces not only the type-2 APP5 production
proteins.
but also type-i APPs in certain condition,
the
CRP
1989).
production
in
Combination of
human
hepatoma
for example,
cells
(Ganter,
IL-6 and either IL-113 or TNF-a
regulate synergistically the express of type-i APPs in
rat and human hepatoma cells
Andus, 1988; Steel,
in culture
1991; Baumann,
1987)
.
1990;
(Falus,
However,
IL-i
type cytokins have minor or no direct stimulatory effects
on the expression of type-2 APPs, nor do they enhance the
effects
of
IL-6
type
cytokines
and,
if
influence, it is inhibitory (Gauidie, 1987)
there
is
any
20
Glucocorticoids
Glucocorticoids.
stimulate
example,
Van
the
of
some
APPs
the al acid glycoprotein in rat
Gool,
1984)
synthesis
most
However,
.
substantial
demonstrate
APP5
expression
(Kushner,
effects
1993;
of
able
to
directly,
for
are
(Prowse,
failed
to
glucocorticoids
on
studies
Baumann,
principal action of glucocorticoids
1988;
is
The
1987).
to
enhance
the
effect of IL-i and IL-6- type cytokines synergistically
on many APPs.
Growth factors,
Growth factors.
hepatocyte
factor
growth
(FGF),
factor
(HGF)
and
including insulin,
fibroblast
and transforming growth factors
growth
(TGF),
not have direct effect on the production of APPs,
do
but
have the potential to modulate the liver response to IL-i
or IL-6 type cytokines.
Insulin has no direct effect on
APP gene expression in rat and human hepatoma cells when
administered alone,
IL-6
type
cytokines
however,
it can attenuate IL-i and
stimulation of
most APP genes
human and rat hepatoma cells (Campos, 1992).
in
21
2.2.7 Resolution of acute phase response
In some respects, because of the short half life of
many of the cytokines and RNA species involved in the
APR, active inhibitory mechanism may not as important as
initiation factors.
However,
some factors are believed
to be involved in the resolution process.
which can enhance
Glucocorticoisteroid hormone,
induced by other
response
hepatic
cytokins,
can
the
also
interfere the APR by inhibiting the production of some
secondary cytokines
and
initiating
stromal cell
(Ray,
by macrophages
and
This negative feedback loop
1990).
may play an important role on the damage control in APR.
primarily released by Th2
Interleukin-4,
IL-i, and IL-B, as well as the
can downregulate the TNF,
release
and
PGE2
of
apoptosis
monocytes,
accumulation
tissues
of
(Mangan,
lymphocytes,
these
1992).
monocytes,
shown
been
also
has
of
anion
superoxide
Interleukin-4
lymphocyte,
therefore
important
(Bauman,
enhance
to
leading
cells
1994)
to
the
reduced
inflammatory
in
Interleukin-lO, produced by TH2
macrophages
inhibit the synthesis of IL-1,
colony-stimulating factors
TNF,
(CSF5),
and
IL-6,
B
cells,
IL-8,
can
and the
and up-regulate IL-i
PA (de Waal Malefyt, 1992; Howard, 1992)
22
2.3 Other acute phase phenomena
2.3.1 Neuroendocrine changes
Fever
hypothalamus
through
neuroendocrine
the
the
intraperitoneal
1991)
vagotomy
transmission in the
for
febrile response
Some behavioral changes,
cytokines
That
after
the
neural
(Goldbach,
including anorexia,
TNF
(PGE2)
implicates
LPS
of
E2
fever.
fever
blocks
injection
and
IL-6,
However,
.
inducers
only
subdiaphragmatic
IL-1,
prostaglandin
of
Dinarella,
1988;
not
mediated by
is
induction
the
(Dinarella,
are
of
The alteration of the temperature set points in
changes.
the
representative
the
is
1997).
somnolence,
and lethargy, often accompany with APR.
2.3.2 Hematopoietic changes
These
inflammation
implicated
disease,
in
for
erythrocyte
the
associated
pathogenesis
example,
precursors
cytokines
of
decreased
to
production of erythropoietin,
anemia
have
in
chronic
responsiveness
erythropoietin,
been
of
decreased
and impaired mobolization
of iron from macrophages (Means, 1995)
23
2.3.3 Hepatic changes
inflammation-associated
These
some
intracellular
inducible
hepatic
oxide
nitric
cytokines
alter
also
constituents,
including
manganese
superoxide
synthase,
dismutase, and microsomal heme oxygenase.
2.4 Glucocorticoids and immunity
Glucocorticoids are widely used in medical fields as
anti-inflammatory
1982; Munck,
and
1984;
immunosuppressive
1994; Chrousos,
1995)
agents
.
(Cupps,
However,
the
role of glucocorticoids in physiology is oversimplified
because its therapy was well established long before the
mechanisms that regulate its bioavailability and receptor
activation were determined (Jefferires, 1994) .
clear
that
glucocorticoids
the
differences
produced
synthetic glucocorticoids
by
the
in terms
between
adrenal
of
It is now
endogenous
grand
and
their regulatory
mechanisms are crucial for their biological actions.
For
example, synthetic glucocorticoids differ from endogenous
glucocorticoids
globulin,
in binding to the corticostroid-binding
tissue specific metabolism,
the affinity for
24
the diverse glucocorticoid receptors, and the interaction
with transcription factors
(Wilckens,
1995)
.
Since the
various effects of glucocorticoids on immune cells and
cytokine productions,
conclusions
on
it
is
effect
its
to draw general
difficult
on
immunity
1998;
(Davis,
Vieira, 1998)
2.4.1 Bloavailability
A
large
proportion
the
of
(90-97%)
endogenous
glucocorticoids in circulation is bound to corticosteroid
binding protein (CBP) and a lesser extent to albumin.
only
contrast,
glucocorticoids
Thus,
small
a
such
as
of
effects
synthetic
bound
dexamethasone
"physiological"
the
proportion
In
to
CEG.
glucocorticoids
of
observed in vitro do not necessarily accurately reflect
free
physiological
bioactive
cortisol
concentrations
(Mendel,
1989)
of
.
bioavailable
It
is
not
and
known
precisely how free corticosterone levels are regulated in
inflammation
response.
or
during
other
immunological
stress
25
2.4.2 Effects on cytokines
Glucocorticoids have been shown suppressive effect on
cytokines produced by monocytes or moacrphages, including
TNF-
1997; Han,
(Joyce,
(Ereuniger,
1993)
1997)
(Blotta,
1990)
IL-8
,
,
(Antilla,
1992)
1988)
,
and
,
also
Glucocorticoids
.
CLew
IL-113
IL-6
,
IL-12
inhibit
the
production of IL-S, and IFN-y by helper T cells (Brinkmann
and Kristofic,
1995) .
However, other studies have also
been shown that glucocorticoids can induce the production
of
some
of
above
the
For
cytokines.
corticosterone induce the production of
when administered at either basal
example,
IL-6 and TNF-a
or stress-
(35 ng/ml)
related (350 ng/ml) levels in an in situ liver perfusion
system in the absence of other stimuli.
infused
with
together
potenciated
cytokine
acted
related
dose
finding
clearly
endotoxin,
production,
suppressively
contrasts
with
certain immune
basal
the
whereas
(Liao,
the
the
reactions depend not
dose
stress-
1995) .
sole
effects of glucocorticoids reported earlier.
that
However, when
This
suppressive
It suggests
only on
the
presence of basal glucocorticoid levels but also on a
dynamic glucocorticoid response (Wilckens, 1997)
26
2.5 References
Aderem A, D. M. Underhill. Mechanisms of phagocytosis in
macrophages. Annu Rev Immuno. 17: 593-623, 1999.
Andus, T., T. Geiger, T. Hirano, T. Kishimoto, T.A. Tranand
P.C.
Heinrich.
Regulation
of
Decker,
Thi,
K.
synthesis and secretion of major rat acute-phase proteins
human
interleukin-6
(BSF-2/IL-6)
in
recombinant
by
hepatocyte primary cultures. Eur. J. Biochem. 173: 287293, 1988.
Andus, T., T. Geiger,
Action of
Heinrich.
T. Hirano,
recombinant
Kishimoto, and P.C.
human interleukin 1,
T.
interleukin 113 and tumor necrosis factor-cL on the mRNA
induction of acute phase proteins. Eur. J. Immnol. 18(5):
739-746, 1988.
Antilla, H.S., S. Reitamo, M. Ceska, and M. Hurme. Signal
transduction pathways leading to the production of IL-S
differentially regulated by
are
by human monocytes
dexamethasone. Clin. Exp. Immunol. 89(3) : 509-512, 1992.
Badolato,
J.M.
R.,
Wang,
W.J.
Murphy,
A.R.
Lloyd,
D.F.
Michiel, L.L. Bausserman, D.J. Kelvin, and J.J.Oppenheim.
Induction of
a
chemoattractant:
Serum amyloid A is
migration, adhesion, and tissue infiltration of monocytes
and polymorphonuclear leukocytes. J. Exp. Med. 180: 203209, 1994.
Barna,
B.P.,
S.D.
Deodhar,
S.
Gautam,
B.
Yen-Lieberman,
and D. Roberts. Macrophages activation and generation of
tumoricidal activity by liposome associated human Creactive protein. Cancer Res. 44: 305, 1984.
and S. D. Deodhar. Activation of
human monocyte tumoridal activity by C-reactive protein.
Cancer Res. 47: 3959-3963, 2987.
Barna,
B.P,
K.
James,
Baumann, H., and J. Gauldie. The acute phase response.
Immunology today 15 (2) : 74-80, 1994.
Baumann, H., K.K. Morella, and G.H.K. Wong. TNF-a, IL-lf3,
and hepatocyte growth factor cooperate in simulating
27
specific acute phase plasma protein genes in rat hepatoma
cell. J. Immunol. 151: 4248-4257, 1993.
H.,
C.
Richards, and J. Gauldie. Interaction
among hepatocyte-stimulating factors, interleukin 1 and
glucocorticoids for regulation of acute phase plasma
proteins in human hepatoma (HepG2) cells. J. Immunol.
139: 4122-4128, 1987.
Baumann,
Besterman JM and R.
B.
mechanisms and plasma
210(1): 1-13, 1983.
Endocytosis: a review of
membrane dynamics.
Biochem J.
Low:
R.H.
DeKruyff,
and
D.T.
Umetsu.
N.H.,
inhibit
IL-12
production
in
human
Corticosteroids
monocytes and enhance their capacity to induce IL-4
synthesis in CD4+ lymphocytes. J. Immunol. 158: 55895595, 1997
Blotta,
Bosanquet,
A.G.,
A.M.
Chandler,
H.
Gordon.
Effects
of
injury on the concentration of alpha-I-Macroglobulin and
alpha-2-macroglobulin in he plasmas of male and female
rats. Experientia 32(10): 1348-1349, 1976.
Breuninger, L.M., W.L. Dempsey, J. Uhl, and D.M. Murasko.
regulation
of
interleukin-6
protein
Hydrocortisone
production by a purified population of human peripheral
blood monocytes. Clin. Immunol. Immunopathol. 69: 205214, 1993.
Regulation
and
C.
Kristofic.
by
corticosteroids of Thl and Th2 cytokine production in
human CD4+ effector T cells generated from CD45R0 and
Brinkmann,
V.
CD45RO subsets. J. Immunol. 155: 3322-3328, 1995
Insulin is a prominent
S.P., and H. Baumann.
modulator of the cytokine-stimulated expression of acutephase plasma protein genes. Mol. Cell Biol. 12: 1789-
Campos,
1797, 1992.
Castell,
J.V.
T.
Andus,
D.
Kunz,
and
P.C.
Heinrich.
Interleukin-6. The major regulator of acute phase protein
synthesis in man and rat. Ann.NY Acad. Sci. 557: 87-101,
1989.
28
Chrousos, G.P. The hypothalamic-pituitary-adrenal axis and
immune-mediated inflammation. New Engi. J. Med. 332(20):
1351-1362, 1995.
Coe, J.E., S.S. Margossian, H.S. Slayter, and J.A., Sogn.
Hamster female protein, a new pentraxin structuarally and
functionally similar to C-reactive protein and amyloid P
component. J. Exp. Med. 153: 977-991, 1981.
R.
and A.S.
Fauci.
Corticosteroid-mediated
T.
immunoregulation in man. Immunol. Rev. 65: 133-155, 1982.
Cupps,
Davis, S.L. Environmental modulation of the immune system
endocrine
system.
Domestic
Animal
the
via
Endocrinology.15, 283-289,1998.
Malefyt,
R.,
H.
Yssel,
and M-G Roncarolo.
Waal
Interleukin-lO. Curr. Opin. Immunol. 4: 314-320, 1992.
de
Dinarello, C.A., J.G. Cannon, and S.M. Wolff. New concepts
on the pathogenesis of
fever.
Rev.
Infec.
Dis.
10(1):
168-189, 1988.
Dinarello, C.A., J.G. Cannon, J. Mancilla, I. Bishai, J.
Interleukin-6
an
Coceani.
as
endogenous
Lees,and F.
pyrogen: induction of prostaglandin E2 in brain but not
in peripheral blood mononuclear cells. Brain res. 562:
199-206, 1991.
Edwards, K. M., H. Gewurz, T. F. Lint, and C. Mold. A role
C-reactive
protein
in
the
complement-mediated
for
stimulation of human neutrophils by type 27 Streptococcus
pneumoniae. J. immunol. 128: 2493, 1982.
Falus, A. H. Rokita, E.Walcz, et al. Hormonal regulation
biothesis
in
human
complement
cell
lines.-II.
of
Upregulation of the biosynthesis of complement components
factor B and Cl inhibitor by interlukin-6 and
C3,
interlukin-1 in human hepatoma cell line. Mol. Immunol.
27: 197-201, 1990.
Fearon D. T. and R. M. Locksley. The inductive role of
in
the
innate
immunity
acquired
immune
response.
Science. 272: 50-54, 1996.
Fearon D. T. Seeking wisdom in innate immunity.
388: 323-324, 1997.
Nature
29
p. 0. and C.-O. Kindmark. C-reactive protein:
1
factor.
Scand.
Cull.
phagocytosis-promoting
J.
Lab.
Invest. 24: 215, 1969.
Ganrot,
Toniatti, G. Morrane, and G.
of
C-reactive protein gene
Ganter, U., R. Arcane, C.
Dual
control
Ciliberta.
expression by interleukin-1 and interleukin-6. EMBO J.
3773-3779, 1989.
8:
Gauldie, J., C. Richards, D. Harnish, P. Lansdorp, and H.
Baumann. Interferon beta 2/B-cell stimulatory factor type
identity with monocyte-derived
hepatocyte2
shares
stimulating factor and regulates the major acute phase
protein response in liver cells. Proc. Natl. Acad. Sci.
USA. 84: 7251-7255, 1987.
Goldbach,
J.M.,
J.
Roth,
and
E.
Zeisberger.
Fever
suppression by subdiaphragmatic vagotomy in guinea pigs
depends on the route of pyrogen administration. Am J.
Physiol. 272: R675-681, 1997.
J., P. Thompson, and B. Beutler. Dexamethasone and
pentoxifylline inhibit endotoxin- induced cachectin/tumor
necrosis factor synthesis at separate points in the
signaling pathway. J. Exp. Med. 172(1): 391-394, 1990.
Han,
Heinrich, P.C., J.V. Castell, T. Andus. Interleukin-6 and
the acute phase response. Biochem. J. 265: 621-636, 1990.
and A. O'Garra.
Biological properties
interleukin 10. Immunol. Today. 13: 198-200, 1992.
Howard,
M.
Husebekk,
A.,
B.
Skogen,
G.
Hurby.
of
Characterization of
amyloid proteins AA and SAA as apolipproteins of high
density lipoprotein (HDL). Displacement of SAA from the
HDL-SAA complex by apo Al and apo All. Scand. J. Immunol.
25: 375-381, 1987.
McK.
Mild
adrenocortical
W.
deficiency,
chronic allergies, autoimmune disorders and the chronic
fatigue syndrome: a continuation of the cortisone story.
Med. Hypotheses. 42: 183-189, 1994.
Jefferies,
Joyce,
D.A.,
Glucocorticoid
Steer,
modulation of
J.H.
and
human
Abraham.
monacyte/macrophage
L.
J.
30
function: control of TNF-alpha secretion.
46: 447-451, 1997.
Inflamm.
Res.
Kindmark, C. 0. Stimulating effect of C-reactive protein
phagocytosis
of
various
on
species
of
pathogenic
bacteria. Clin. Exp. Immunol. 8: 941, 1971.
Koj,
A.,
H.
Role
Travis.
Rokita, T. Kordula, A. Kurdowska,
of cytokines and growth factors
and
in
J.
the
induced synthesis of proteinase inhibitors belonging to
acute phase proteins. Biomed. Biochim. Acta. 50: 421-425,
1991.
Immunology.,
Company. 1-10, 1994.
Kuby,
J.
2nd edition.
W.
H.
Freeman and
Regulation of the acute phase response by
cytokines. Perspect Biol. Med. 36: 611-622, 1993.
Kushner,
I.
Kusherner, I. The phenomenon of the acute phase response.
Ann. New York Acad. Sci. 389, 39-48, 1982.
I., J.E. Volanakis, and H. Gewurz. Ann. New Yor
Acad. Sci. 389: 235-274, 1982.
Kushner,
Laskey, L.A. Science. 128: 964-969, 1992.
Lew, W., J.J. Oppenheim, and K. Matsusshima. Analysis of
the suppression of IL-i and IL-i p roduction in huaman
mononuclear
peripheral
blood
adherent
cells
by
a
glucocorticoid hormone. J. Immunol. 140: 1895, 1988.
Liao, J., J.A. Keiser, W.E. Scales, S.L. Kunkel. And M.J.
Kluger. Am. J. Physiol.
268: R699-R706, 1995.
Regul.
Integr.
Comp.
Physiol.
Mackiewicz, A., M.K. Ganapathi, D. Schultz, D. Samols, J.
Reese, and I. Kushner. Regulation of rabbit acute phase
protein biosynthesis by monokines.
Biochem.
J.
253:
851, 1988.
Mackiewicz, A., I. Kushner, and H. Baumann.
Acute phase
proteins
molecular biology, biochemistry, and clinical
Applications. Boca Raton: CRC Press, 3-20, 1993.
:
31
Mangan, D.F., B. Robertson, and S.M. Wahl. IL-4 enhances
in stimulated human
programmed cell death (apoptosis)
monocytes. J. Immunol. 148: 1812-1816, 1992.
Pathogenesis of the anemia of chronic
disease: a cytokine-mediated anemia. Stem Cells. 13: 32-
Means,
R.T.
Jr.
37, 1995.
Medhzhitov, R,
P. Preton-Hurlburt, and C.A. Janeway Jr. A
human homologue of the Drosophila Toll protein signals
activation of adaptive immunity. Nature. 388: 394-397,
1997.
hormone
hypothesis:
a
model.
Endocr.
Rev.
physiologically based mathematical
10: 232-274, 1989.
Mendel,
The
C.M.
free
M.
Edwards, and H. Gewurz. Effect of CK.
C.,
reactive protein on the complement-mediated stimulation
of human neutrophils by Streptococcus Pneumoniae serotype
Mold,
3 and 6, Infect. Immun. 37: 987-992, 1982.
Serum C-reactive protein
Morley, J.J., and I. Kushner.
Ann. N.Y. Acad. Sd. 389: 406-418.
levels in disease.
1982.
A., P.M. Guyre, and N.J. Holbrook, Physiological
functions of glucocorticoids in stress and their relation
to pharmacological actions. Endocr. Rev. 5: 23-44, 1984.
Munck,
and A. Naray-Fejes-Toth. Glucocorticoids and
A.
stress: permissive and suppressive actions. Ann. New York
Acad. Sci. 746: 115-130, 1994.
Munck,
Pepys, M.B. and Butler, P.J.G. Serum amyloid P component
specific DNA binding
major calcium-dependent
the
is
protein of the serum. Biochem Biophys. Res. Commun. 148:
308-313, 1987.
Perlmutter,
D.H.,
C.A.
Dinarello,
P.1.
Punsal,
et
al.
Cachectin/tumor necrosis factor regulates hepatic acutephase gene expression. J. din. Invest. 78: 1349-1354,
1986.
Prowse,
factor,
K.R.,
32
and
H
Baumann.
interferone,
and
Hepatocyte-stimulating
interleukin-1
enhance
32
expression of the rat a1-acid glycoprotein gene via a
distal upstream regulatory region. Mol. Cell Biol. 8: 4251, 1988.
A., S. LaForge, and P.B. Sehgal. On the mechanism
for efficient repression of the interleukin-6 promoter by
glucocorticoids: enhancer, TATA box, and RNA start site
(mr motif) occlusion. Mol. Cell. Biol. 10: 5746-5746,
Ray,
1990.
Robey, F.A., K.D. Jones, and A. D. Steinberg. C-reactive
protein mediated the solubilization of nuclear DNA by
complement in vitro. J. Exp. Med. 161: 1344-1356, 1985.
Rogers, J.T., K.R. Bridges, G.P. Durmowicz, J. Glass, P.E.
Auron, and H.N. Munro. Translational control during the
acute phase response. Ferritin synthesis in response to
interlukin-l. J. Biol. Chem. 265:14572-14578, 1990
Endothelial cell binding of NAP-l/IL-8: role in
neutrophil emigration. Immunol. Today. 13: 291-294, 1992.
Rot,
A.
Shephard,
E.
G.,
P.D.
VanHelden,
M.
Strauss,
L.
Bohm.,
and F. C. DeBeer. Functional effect of CRP binding to
nuclei. Immunology. 58: 489, 1986.
and H. Gewurz. Interactions of Creactive protein with the complement system. I. Protamininduced consumption of complement in acute phase sera. J.
Exp. Med. 140: 631, 1974.
Siegel,
J.
R.
Rent,
Siegel,
J.
R.
Rent,
Gewurz. Interactions of Ccomplement system. II.
Cconsumption by poly-L-lysine
and H.
reactive protein with the
protein-mediated
polymers and other polycations.
reactive
Exp.
Med.
142:
709,
J. H. Rokita, M. Mackiewicz, S. Szla, and A.
Fed. Proc., Fed. Am. Soc. Exp. Biol. 45: 1587, 1986.
Koj.
J.
1975.
Sipe,
P.K.,
and H. Udono.
Heat shock proteinSrivastava,
in cancer
immunotherapyCurr.
Opin.
peptide complexes
Immunol. 6: 728-732, 1994.
Steel, D.M., and A.S. Whitehead. Heterogenous modulation
of acute-phase-reactant mRNA levels by interleukin-113 and
33
interleukin-6
in
the
human
Biochem.J. 277: 477-482, 1991.
cell
line
PCL/PRF/5.
and A.S. Whitehead. The major acute phase
reactants: C-reactive protein, serum amyloid P component,
D.M.,
Steel,
and serm amyloid A protein.
Immunol.
Today.
15:
81-88,
1994.
Suto,
and
R.
P.K.
Srivastava.
A mechanism
for
the
specific immunogenicity of heat shock protein-chaperoned
peptides.Science. 269: 1585-1588, 1995.
Uhiar, C.M. and A.S.Whitehead. Serum amyloid A, the major
vertebrate
acute-phase
reactant.
Eur.
J.
Biochem.
265(2) :501-23, 1999.
Van Gool, J., W. Boers, M. Sala, and N.C. Ladiges.
Glucocoticoids and catecholamines as mediators of acutephase
proteins,
especially
rat
a-macrofoetoprotein.
Biochem. J. 220: 125-132, 1984.
Vieira, P.1., Kalinski, P., Wierenga, E.A., Kapsenberg,
M.L. & de Jong, E.C. Glucocorticoids inhibit bioactive
IL-12p70 production by in vitro-generated human dendric
cells
without
affecting
their
T
cell
potential. J. Immunol, 161, 5245-5251, 1998.
stimulatory
Glucocorticoids
and
immune
function:
relevance
and pathogenic
potential
of
hormonal dysfunction.Trends Pharmacol. Sci. 16: 193-197,
Wilckens,
T.
physiological
1995.
Williams,
and P.
G., Hellewell.
Endothelial cell
biology. Adhesion molecules involved in the microvascular
inflammatory response. Am. Rev. Respir. Dis. 146: S45T.
J.
S50. 1992.
Badolato,
W.J.
Murphy.,
et
al.
A novel
biologic function of serum amyloid A. Induction of T
lymphocyte migration and adhesion. J. Immunol. 155: 11841190, 1995.
Xu,
L.,
R.
and R.F. Mortensen. Macrophage tumoricidal
activity induced by human C-reactive protein. Cancer Res.
46: 5077-5083, 1986.
Zahedi,
K.,
34
Chapter 3
Immunity against Escherichia coli Infection
in Chickens Assessed by Viable Bacterial Counts
in Internal Organs
35
3.1 Abstract
Septicemic strains of E. coil cause systemic infection
We have
in chickens after the intra-airsac inoculation.
investigated whether levels of immunity can be determined
by the viable organism count in the internal organs of
The intra-airsac inoculation of Ol:K1
infected birds.
strain caused acute systemic infection in
at
The
The count was significantly (p
liver, spleen and blood.
<
hrs.
lung followed by the
in the
viable count was highest
6
0.05) lower in the liver or spleen of vaccinated birds
12 or 24 hrs after inoculation than in controls.
6,
Vaccines containing various adjuvants were tested in this
system,
significant
(p
<
adjuvants
oil-based
three
and
0.05)
demonstrated
while
immunity,
an
alum-
precipitated vaccine or one without an adjuvant failed to
do
An oil-
so compared with non-vaccinated controls.
vaccine
adjuvanted
showed
deterioration
some
in
its
immunogenicity after prolonged storage or heating at 100
C.
The
acute
injection of
Salmonella
significant
phase
killed Ol:K1
typhimurium
immunity
induced by
response
in
cells
or LPS purified from
aqueous
against
the
intravenous
suspension
E.
coli
induced
infection.
36
These results indicate that the method referred to as "in
vivo viable count method" produces quantitative results
in a reproducible manner and suggest that it may be used
as an alternative method to mortality measurement.
3.2 Inroduction
Escherichia
pathogens
coli
conditions
septicemia
coil
E.
the
of
most
respiratory
other
or
criteria
Common
(3) .
immunity against
one
causing
poultry,
in
cellulitis,
remains
important
infections,
diverse
used
to
clinical
evaluate
in mortality
are differences
rates, pathological lesion scores, isolation frequencies,
weight
and/or
gain
after
loss
exposures between control
artificial
and treated groups
challenge
(5,
10)
These methods are useful for evaluating vaccine products
in
terms
of
substantial
results.
al.
under
(2)
their
number
of
effectiveness
animals
to
requires
but
obtain
a
reproducible
A passive transfer method described by Arp et
offers
defined
an
accurate
conditions
determination
although
humoral components of immunity.
it
of
immunity
measures
only
37
Biomedical research investigators are being pressured
to avoid or improve procedures that may involve pain in
experimental
For
animals.
example,
animal
the
care
committee of this institution has recently adopted a rule
based on the government recommendation (8)
should not
species.
used
be
as
endpoint
an
in
that lethality
vertebrate
all
Since mortality has been used traditionally for
evaluating
the
virulence
efficacy
determining
of
coli
E.
vaccines
of
(15)
a
(10),
and
for
reliable,
alternative method should be available to study E.
coil
infection in poultry.
Pourbakhsh
Recently
inoculation
resulted
of
a
strain
consistently
et
of
in
al.
E.
the
Predictable numbers of viable E.
(11)
coil
reported
into
systemic
the
that
airsac
infection.
coii were detected in
internal organs up to 48 hrs after the inoculation.
The
objective of the present study was to examine whether one
can
evaluate
the
level
of
antibacterial
immunity
by
determining viable bacterial counts in internal organs in
an E. coli infection model in White Leghorn chicks.
38
3.3 Material and methods
One
Chickens.
Leghorn
White
day-old
male
chicks
without any vaccination or injection were obtained from a
Birds were raised in battery cages with
local hatchery.
a starter ration
(9)
containing 0.01% decoquinate.
Feed
and water were given ad. lib.
Escherichia coli strain Ol:K1,
Bacteria.
-hemolytic
and glucose-fermenting, was a gift from Dr. B. Panigrahy
of National Veterinary Services Laboratory,
A
on
culture
Laboratories,
5%
Detroit,
MI)
phosphate buffered saline
stored
and
frozen.
blood
sheep
A
was
(0.85%)
frozen
Ames,
plates
agar
harvested
in
Iowa.
(Difco
0.01
solution, pH 7.2
culture
was
M
(PBS)
recovered,
transferred and grown on blood agar plates for 4 hours at
41
C
and
harvested
in
enumerated by dilution
cold
in
PBS.
bacteria
The
brain heart
infusion
was
(BHI)
broth (Difco) and plating out onto MacConkey agar plates
(Difco)
.
added to E.
For inactivated vaccines,
0.3%
formalin was
coli suspensions containing 1.0 x
forming-units
(CFU)/ml.
Sterility
was
colony-
checked
by
inoculating 1 ml of the vaccine into 100 ml BHI broth for
3
consecutive days.
An inoculum for challenge exposure
was a bacterial suspension originated from 4-hr growth on
39
blood agar followed by dilution in PBS to give an optical
value
density
of
0.2
at
600
approximately 1 x 108 CFU/ml.
corresponding
nm,
to
Colony counts were made for
each inoculum.
Experimental
infection
and
bacterial
isolation.
Chicks at 5-weeks of age were moved to an isolation unit
coil strain Ol:Kl into the left
and inoculated with E.
caudal thoracic air sacs as follows: At the right lateral
recumbency,
hypodermic needle was inserted at
ga.
22
a
the fourth intercostal space immediately ventral to the
uncinate process in a quarter of an inch in depth, and,
after free movement of the nedie was assured,
0.1 ml of
the inoculum was deposited wth an attached syringe of
ml
At
capacity.
Experiment
1
or at
various
times
post
hrs
after
inoculation in other
24
approximately 3
experiments,
inoculation
1
in
ml of blood was collected
with heparin (10 units/mi) when required, and birds were
euthanatized by CO2 asphyxiation.
bacterial
growth,
the
carcasses
To minimize possible
were
running water for approximately 10 mm
soaked
in
cold
immediately after
asphyxiation followed by storage at 4 C for maximum of 90
Organs were isolated by sterile scissors and
minutes.
forceps, placed in sterile plastic bags and immediately
soaked
in
ice
water.
Each
sample
except
blood
was
40
weighed and cold PBS was added (9 mug) and homogenized
with Stomacher Lab-Blender 80
(Brinkman,
Westbury,
NY).
Serial 10 fold dilutions were made in tryptose phosphate
broth (Difco).
One milliliter of undiluted homogenate or
blood was mixed with 14 ml MacConkey agar and plated out
One hundred
in 100 x 15 mm sterile plastic Petri dishes.
microlitters of 10, i0, or i0
duplicates
in
MacConkey
onto
dilutions, were plated
plates
agar
subsequently incubated at 41 C for 24 hours.
which
were
The number
of colonies was counted to determine the CFU/g or ml in
each
Random
organ.
colonies
hemolysis on blood agars,
were
examined
for
1-
positive glucose fermentation
and positive slide agglutination with antiserum against
01: Kl strain.
Experiment
into 3 groups,
group,
(Sigma)
group,
1.
Forty-eight
6,
14
and
28
were
distributed
In the immunosuppressed
16 birds/group.
each bird was given
at
chicks
100
mg/kg cyclophasphamide
In the vaccinated
day-old.
each bird was given formalin-inactivated vaccine
emulsified 1:1 with incomplete Fruend adjuvant (Difco) at
14
and 28
-day-old.
At
35
days
of
age,
birds were
inoculated with strain 01:Kl via an intraairsac route.
Four birds per group were examined at 0.5,
hr postinoculation.
6,
12, and 24
41
Sixty chicks were distributed into 6
Experiulent 2.
groups, 10 birds/group (Table 3.1).
Different adjuvants given in Experiment 3.
Table 3.1
Group
N
Adjuvant
Inactivated
O1:Kl cells
14-day-old
28-day-old
PBS
10
Yes
PBS
PBS
Alum+Quil-A
10
Yes
Alum+Quil-A
Alum+Quil-A
IFA
10
Yes
IFA
IFA
MDP+IFA
10
Yes
MDP+IFA
IFA
MPL+TDM
10
Yes
MPLTDM
MPLTDM
Control
10
No
None
None
See the text for abbreviations and details.
Birds
injections
in
each
group
of vaccines
received
containing
2.5
two
x
subcutaneous
108
formalin-
inactivated cells with different adjuvants or PBS at 14
and 28 days of age.
In Alum
+
Quil-A group, a bacterial
suspension adsorbed onto aluminum hydroxide gel according
the method specified by the manufacturer
to
Co.,
Frydenlundsvej, Denmark;
(Superfos
and Quil-A saponin was
1)
added to the final concentration of 12.5 ig/ml (Superfos;
14) .
In IFA group, a bacterial suspension was emulsified
in incomplete Fruend adjuavant
group,
In MDP
(Sigma).
+
IFA
birds were primed by vaccine emulsified in IFA
containing
N-acetylmuramyl-alanyl-D-
peptide,
a
isoglutamine;
(MDP; Sigma; 7)
In MPL group,
2
ml
and boostered by IFA only.
of inactivated vaccine in PBS was
added to the vial containing monophosphoryl lipid A in
trehalose
dicorynomycolate
(TDM;
and
Sigma)
manually for 3 mm. to form emulsion
(12)
.
shaken
Birds were
challenge-exposed as described above, and spleen and lung
samples were examined for E.coli counts.
Experiment 3.
5
groups,
9
Forty-five chicks were distributed into
birds/group.
immunized subcutaneously at
Birds
of
each
group
were
the neck region twice with
different inactivated vaccines in IFA at 14 and 28 days
of age (Table 3.2).
43
Table 3.2
Different vaccine preparations given to chicks
in Experiment 2.
Ki cells
Group
N
F
9
Fresh, stored at 4 C for 7 days
IFA
0
9
Old, stored at 4 C for 5 months
IFA
F + H
9
'F' heated at 100 C for 5 mm
IFA
AC
9
None
IFA
C
9
None
None
Inactivated 01
:
Adjuvant
See the text for abbreviations and details.
(fresh), a vaccine was prepared as described
For group F
above and stored totally for 7 days at 4 C.
For group 0
(old), a similar preparation was stored for 5 months at 4
C.
For F+H group, freshly made vaccine was heated at 100
C for 5 mm.
treatment
Group AC and C were an adjuvant and non-
control
challenge-exposed
group,
and
the
respectively.
samples
were
Birds
were
collected
as
described under experiment 2.
Experiment 4.
Thirty chicks were distributed into
groups, 10 birds/group.
3
At age of 35 days, each bird was
44
inoculated intravenously with formalin inactivated E.coli
CFU or with 5 mg LPS of
Ol:Kl cells corresponding to iü
Salmonella
Lyphimurium
The
(Difco).
birds
were
challenge-exposed as described above with Ol:K1 strain at
24 hrs after the injections and the spleen samples were
collected
afterwards
hr
24
and
assayed
as
described
above.
Difference of bacterial counts
Statistical analysis.
between
different
treatments
and
control
groups
was
analyzed by the Students t-test.
3.4 Results
Bacterial counts in organs.
E.
was
To examine the extent of
coli multiplication in internal organs, Ol:Kl strain
inoculated
CFU/bird).
organs
into
the
thoracic
airsac
(1.2
x
The organism multiplied rapidly in all the
tested
plateau around
in
6
the
control
birds,
hrs post inoculation,
reaching
to
the
and the plateau
level was maintained for 24 hrs (Experiment 1; Fig. 3.1).
45
I
10
8
Blood
I Control
Inactivated vaccine
Cyclophosphamide
6
4
*
2
0
10
I-
8
0,
6
U-
4
0
00
2
C,
0
-J
18
8
6
4
2
1
8
6
4
2
0
0.5
24
12
6
Hrs after intra-airsac inoculation
Fig.
3.].
The number of viable bacteria
m
or
±
s.d.
liver
at
detected in the
different times after the intra-airse. ii culation of 5strain O1:K1; the
week-old chicks with Escherichia coJ
roup
N= 16
Experiment
vaccinated or control group
*
p < 0.05.
1) were tested.
blood,
lung,
(
spi eu,
:
46
The highest counts were observed with the lung containing
108
and
CFU/g,
the
rest
of
the
organ
showed
samples
approximately 100-fold or 2 log10 fewer viable counts per
g or ml.
In the vaccinated group,
significantly
(p
<
12, or 24 hrs post
.05) lower numbers were observed at 6,
inoculation in the liver and spleen than in the control
group, but such difference was observed in the blood or
The treatment with
lung only at 24 hrs post inoculation.
known
cyclophosphamide,
resulted
no
in
an
immunosuppressive
agent,
in
general
a
as
effects
except
few
significantly low numbers at 6 and 12 hrs in the spleen
The results indicated that the antibacterial
and liver.
immunity
induced
vaccination
by
suppressed
bacterial
multiplication significantly in the spleen and liver and
that the level of immunity may be determined by assessing
in viable bacterial
the difference
counts
in
internal
organs between the vaccinated and control group.
Different adjuvants.
Formalin-inactivated 01 :Kl cells
were mixed with different adjuvants or PBS,
were
immunized
(Experiment
CFU/bird.
including
lower
2;
twice
Fig.
3.2),
with
MDP
preparations
and infected with 1.7 x
Administration of
IFA,
various
and birds
and MPL
1O7
three oil-based adjuvants
resulted
in
significantly
viable counts in the spleen, while alum + quil A
adjuvant and PBS suspension failed to do so.
no significant differences (p >
.05)
There was
in the antibacterial
effect among the three oil-based adjuvants.
7
6
I
5
III
UC-)
0
-J
2
I
0
-
PBS AIum+Quil-A IFA
Fig.
3.2
The
MDP+WlO MPL Control
of
various
effect
adjuvants
on
the
inactivated
of
bacterial
suspensions
immunogenicity
against challenge-exposure by the homologous strain of B.
*
p < 0.05.
coli (Experiment 3)
See the abbreviation
list and text for details.
48
Some variability in immunogenicity
Storage or heating.
was
between
noted
vaccines
inactivated
prepared
freshly
containing
cold-stored
and
Ol:Kl
The
strain.
immunizing effect of a freshly prepared vaccine lot was
tested with another lot stored for
heat-treated
lot
mm.
(5
at
5 months at
100
C
),
and
only the fresh lot showed significant
from all
difference
the
other group,
.
In the
p <
(
while
a
adjuvant
controls by E.coli infection (9.8 x 106 CFU/bird)
lung,
C,
4
,
in
.05)
the
the fresh as well as old and heat-treated lot
spleen,
showed significant differences compared with the control
group
(Fig.
3.3).
The adjuvant control did not prevent
bacterial multiplication significantly.
Acute
phase
immunity
response.
induced
by
To
test
acute
whether
phase
non-specific
response
has
a
significant effect in antibacterial activity, birds were
given
inactivated
01:1(1
cells
or purified
LPS
of
S.
typhimurium intravenously 24 hrs prior to the infection
with the live 01:1(1 strain (1.5 x 106 CFU/bird)
4()
8
7
Spleen
*
6
5
*
4
3
2
C)
I
U-
0
C)
8
0
0
-J
7
Lung
6
*
5
4
3
2
I
0
F
Fig.
3.3
The
0
effect
F+H
of
storage
AC
C
and heating on
the
immunogenicity of inactivated vaccines as determined by
the number of E. coli strain O1:K1 in the spleen or lung
24 hrs after the intra-airsac inoculation; F, fresh; 0,
old; F + H, heated fresh vaccine; AC, adjuvant control;
C, non-treated control (Experiment 2)
*
p < 0.05.
50
As shown in Fig. 3.4, both treatments significantly (p <
05) prevented
bird
died
in
E. coli
each
multiplication in the spleen.
treatment
group
before
One
challenge-
exposure.
0)
(I)
6
.!
4-I
C
C)
a
U)
C)
C
0)
*
0
*
ui
fl
Inactivated S. typhimurium
E. coli Ol:Kl
LPS
Control
The effect of pretreatment with inactivated
cells or Salmonella LPS on the number of viable
Escherichia coli strain 01:1(1 in the spleen 24 hrs after
*
intra-airsac inoculation (Experiment 4)
p < 0.05.
Fig.
3.4
01:1(1
See details under Experiment 4 described in the text.
51
3.5 Discussion
Strain Ol:K1 consistently induced systemic infection in
5-week-old chicks
inoculation
(Fig.
The inoculum dose was adjusted to between 6 x 106
3.1) .
and
intra-airsac
after
3
x
i07
Preliminary experiments showed that
CFU.
doses lower than that
infection
level
consistently;
yield detectable
some
levels
caused
did not
organisms
internal
inoculation (data not shown)
in
Doses higher than 5 x l0
mortality
some
induce systemic
inoculated birds
the
of
organs other than the lung.
CFU/bird
failed to
.
hrs
24
after
the
The results shown in Fig.
1 generally confirmed those obtained by Pourbaksh et al.
(11);
in control
the organism was detected in
chicks,
highest numbers in the lung followed by approximately 100
fold fewer numbers in the liver and spleen during a 6 to
24 hr period after the inoculation of the strain into the
thoracic airsac.
Differences
in viable
counts
between
spleen were not significant in control
vaccinates
the
liver
and
(p = 0.43), or in
(p = 0.6) except for samples taken at
.5 hrs.
The results are consistent with those of a previous study
with
E.
coil
(11),
but
different
from
those
with
52
Pasteurella multocida,
in which the multiplication of the
organism was inhibited only in the liver but not in the
spleen of immune turkeys
In the current study,
(16) .
both organs were examined in initial
studies,
but
the
spleen was used for the rest of experiments due to the
ease of producing homogenate.
The effect of an oll-adjuvant vaccine was significant
only at the 24 hr post inoculation with lung and blood
samples,
but
showed
significant
decrease
numbers in spleen and liver samples at 6,
compared with the
multiplication
was
control group
inhibited
more
(Fig.
viable
in
12 and 24 hrs.
3.1)
.
Bacterial
efficiently
in
the
liver or spleen where limited numbers of the organisms
entered via bloodstream, while overwhelming numbers of E.
coil may have interfered with the bactericidal mechanisms
in
the
lung.
These
results
indicate
that
the
antibacterial immunity induced by vaccination suppressed
bacterial multiplication significantly in the spleen and
liver and that the level of immunity may be determined by
differences in viable bacterial counts in internal organs
between the vaccinated and control group.
Vaccines containing three oil-based adjuvants induced
significant antibacterial immunity in the spleen,
while
vaccines without an adjuvant or with alum did not
(Fig.
53
3.
2)
results
The
.
with
consistent
are
those
of
Panigraphy et al. (10) with Ol:Kl strain and those of Deb
and Harry
with 02:K1
(5)
strain based
on
mortality,
In the Experiment
lesion scores and/or weight gain loss.
2, adjuvant controls were not tested, but an oil adjuvant
alone did not induce significant immunity as examined in
Experiment 3
(Fig 3.3)
.
The significance of MDP or MPL is
unknown as corresponding oil adjuvant alone controls were
not
However,
examined.
differences
were
significant
no
detected
inhibitory
in
(p
>
.05)
activities
induced by the three oil-based adjuvants.
Some deterioration of immunogenicity was detected after
prolonged storage at 4 C or heating at 100 C
(Fig. 3.3).
Such deterioration caused a significant difference
(p
<
.05) only in the lung although some decreases in immunity
were also noted in the spleen; in the latter organ, the
stored
and heat-treated
lot
still
induced
significant
Further investigations
immunity compared with controls.
are needed to confirm the existence and mechanism of such
deterioration in the immunogenicity.
Intravenous administration of inactivated O1:K1 cell
suspension
or
LPS
purified
from
S.
typhimuri urn
significantly activated host antibacterial activity in 24
hrs
(Fig.
3.4)
.
A series of complex reactions occur
54
after inflammatory stimuli known as acute phase response
(4) .
The response is characterized by the synthesis of
acute phase proteins
in
liver
the
protein and complement components
such
(6)
.
Some acute phase
proteins have been associated with direct
destruction of bacterial pathogens
C-reactive
as
(13),
or
indirect
but supporting
in vivo and in vitro evidences are lacking.
The present
results indicate that the acute phase response induced by
killed homologous cells or LPS from unrelated bacteria is
effective in inducing antibacterial immunity against E.
coli in 24 hrs.
The present results demonstrate that immunity against
E.
coli may be measured by determining differences in
viable numbers in the spleen or liver between immune and
control
chicks after they are
challenge-exposed at
week-old with a septicemic strain.
Compared with other
methods of evaluating immunity against E.
mortality or lesion scores,
5-
this method,
coli such as
which may be
referred to as "in vivo viable count method", requires a
relatively small number
(N=1O)
of subjects and produces
quantitative results in a reproducible manner.
measures
the
outcome
of
immunity
components such as antibody titers.
rather
than
Yet,
it
immune
This method may be
useful for developing new vaccines or treatments or for
55
routine
quality
control
In addition,
products.
of
vaccine
or
therapeutic
it may serve as an alternative
method to lethality determination.
3.6 References
1. Aihydrogel for selective adsorption. Technical Manual
by Superfos a/s, available through Accurate Chemical &
Scientific Corp,. Westburg, N.Y.
2.
Arp,
L.
Effect
H.
of
passive
immunization
on
phagocytosis of blood-borne Escherichia coil in spleen
and liver of turkeys. Am. J. Vet. Res. 43:1034-1040.
1982.
Barnes, H. J. and W. B. Gross. Colibacillosis In
"Diseases of Poultry" 10th ed., Calnek B. W. et al.,
eds., Iowa State University Press, Ames, Iowa. pp. 131141. 1997.
3.
4. Baumann, H. and J. Gauldie. The acute phase response.
Immunol. Today 15:74-80. 1994.
Deb, J. R. and E. G. Harry. Laboratory trials with
inactivated
vaccines
against
Escherichia
coli
02:K1
infection in fowls. Res. Vet. Sci. 24:308-313. 1978.
5.
Gewurz and M. D. Benson. C-reactive
protein and the acute phase response. J. Lab. Clin. Med.
6.
Kushner,
I.,
H.
97:739-749. 1981.
7.
Lafrancier, P., M. Derrien, X. Jamet, and J. Choay.
Apyrogenic,
adjuvant-active
N-acetylmuramyl-dipeptides.
J. Med. Chem. 25: 87-90. 1982.
National Institute of Health.. Institutional animal
8.
care and use committee guideline.
NIH Publication No.
92-3415. 1992.
9.
National Research Council. Nutrient
requirement of poultry. 8th ed.
National Academy Press,
Washington D.C. 1984.
56
10. Panigrahy, B., J. E. Gyimah, C. F. Hall and J. D.
Immunogenic
potency
of
an
oil-emulsified
Williams.
Escherichia coli bacterin. Avian Dis. 28:475-481. 1984.
11.
C.
Pourbakhsh,
Dozois,
M.
S.
M. Boulianne, B. Martineau-Doize,
Desautels,
and J.
M.
Fairbrother.
A.,
C.
Dynamics of Escherichia coli infection in experimentally
inoculated chickens. Avian Dis. 41:221-233. 1997.
12. Qureshi, N., K. Takayama, and E. Ribi. Purification
and structural determination of nontoxic lipid A obtained
from the lipopolysaccharide of Salmonella typhimurium. J.
Biol. Chem. 257: 11808-11815. 1982.
13.
Ratnam,
S.
and
S.
Mookerjea.
The
regulation
of
superoxide generation and nitric oxide synthesis by Creactive protein. Immunology. 94: 560-568. 1998.
Ronnberg, B., M. Fekadu, and B. Morein. Adjuvant
14.
non-toxic
Quillaja
saponaria
Molina
of
activity
Vaccine.
13:1375ISCOM
matrix.
for
use
in
components
1382. 1995.
15. Sicardi, F. J. Identification and disease producing
coil
ability of Escherichia coil associated with E.
M. S. Thesis. Univ.
infections of chickens and turkeys.
of Minnesota, St. Paul, MN. 1966.
16. Tsuji, M. and M. Matsumoto. Immune defense mechanism
against blood-borne Pasteurella multocida in turkeys.
Res. Vet. Sci. 48:344-349. 1990.
57
Chapter 4
Induction of Short-term, Nonspecific Immunity
against Escherichia coli Infection in Chickens
Is Suppressed by Cold Stress or Corticosterone
Treatment
58
4.1
bstract
Nonspecific
immunity
against
E.
infection
coil
chickens was studied with white leghorn chickens of
weeks
of
Intravenous
age.
injection
in
5
inactivated
of
bacteria or silver nitrate induced significant
(p < 0.05)
immunity in 24 hours against infection with Escherichia
coil Ol:Kl strain based on viable bacterial counts in the
spleen.
Nonspecific
immunity
induced
inactivated Staphylococcus aureus cell
by
formalin-
suspension
(FSA)
was comparable to specific immunity induced by a specific
vaccine as determined by cumulative mortality during
7
days and the viable bacterial count in the spleen after
infection.
Nonspecific
immunity was
induced by FSA as
early as 6 hours and lasted for less than 72 hours after
Treatment
stimulation.
corticosterone
suppressed the
immunity by FSA.
with
cold
induction
stress
of
or
nonspecific
The results indicate that
inducible
nonspecific immunity against E. coli may be suppressed by
stress in chickens.
59
4.2 Introduction
immunity
Innate
broadly
is
defined
as
immune
or
protective features of the host that are generally not
included
in
category of
a
specific
adaptive
immunity.
Generally, immunity is non-specific, appears in the early
hours and is of short duration. it is, however, believed
to
developed
have
considerably
earlier
the
in
evolutionary process than adaptive immunity for animal
species
to
infections
the
cause
protect
themselves
(Hoffmann,
high
of
microbiological
against
In investiqatinq
et al.,1999).
mortality
in
commercial
broiler
chickens, our past study suggested that immunosuppression
induced systemic bacterial infection including bacteremia
(Awan
& Matsumoto,
1998)
.
Since bacteremia was
transient in a given flock of chickens,
speculated that
presence
innate
of
intact
demonstrated
that
immunity was
acquired immunity
anti-bacterial
found
it was further
suppressed in
(Awan,
immunity
1997)
can
the
.
We
be
determined in a systemic infection model in chickens by
comparing viable bacterial counts in the spleen between
control and treated groups at periodic intervals after
challenge infection with an E.
coli strain.
Using this
60
system, we have shown that the intravenous administration
formalin-inactivated
of
lipopolysaccharide
against systemic
bacteria
non-specific
induces
(LPS)
coil infection
E.
purified
or
immunity
& Matsumoto,
(Huang
This paper reports further characterization of
1999).
the inducible innate immunity with the chicken E.
infection
model
to
as
protective
its
coil
efficiency,
nonspecific nature, duration, and the negative effect of
cold stress or glucocorticosteroid on the immunity.
4.3 Materials and methods
Escherichia
Bacteriology.
obtained from Dr.
Dr.
B.
Allan
O1:K1
Iowa, and E.
Veterinary
of
typhimurium was a gift of Dr.
Univ.
of
was
coli, 078 strain from
Massachusetts.
G.
Disease
Infectious
Organization, Saskatoon, Saskatchwan, Canada.
of
strain
Panigrahy of National Veterinary
B.
Services, USDA, Ames,
coli,
H.
Salmonella
Snoeyenbos formerly
Staphylococcus
aureus
was
isolated from an arthritic joint of a broiler chicken at
a local farm.
were
grown on
Lyophilized cultures of bacterial strains
the
5%
sheep blood agar
at
37
C
for
overnight, harvested in a distilled water containing 3%
61
skim milk powder (Difco) and 5% glucose, pH7.4, and kept
For inactivated bacteria used to induce non-
at -70 C.
specific immunity, frozen stock cultures of these strains
were
recovered and grown on the
plates at 37 C
overnight and harvested with 5 ml of cold
0.05 M phosphate buffered saline,
(PBS)/plate
sheep blood agar
5%
0.85%
at pH 7.2
(w/v)
Viable counts were made by
(100 x 15 mm).
the dilution and plating-out method onto the blood agar
Formalin
plates.
was
added
0.3%
to
(v/v)
and
the
concentration was adjusted to 5 x 108 colony-forming-units
based
(CFU)/ml
on
the
viable
Sterility
count.
checked daily by inoculating 1.0 ml of
the suspension
into 100 ml of brain-heart infusion broth
Detroit, MI, U.S.A.) .
was
(BHI:
Difco,
The suspension was judged sterile
when the BHI cultures showed negative bacterial growth
for
three
consecutive
Experiment 2,
1.0 X l0
days.
For
the
vaccine
used
in
formalin-inactivated 01:1(1 cells
were emulsified in an equal amount of incomplete Freunds
adjuvant (Difco).
Systexnic
reared
in
described
Briefly,
infection model with E.
isolation
in
detail
and
infected
(Huang
and
coli.
with
Chicks were
E.
Matsumoto,
coli
as
1999)
battery-reared white leghorn male chicks of
5
weeks of age were moved to isolation units and inoculated
62
with E.
coli,
O1:K1 strain via an airsac route
(Figure
4.1).
Determination of Nonspecific Anti-E.Coli Immunity
IV injection of IPS, killed S. areus,
or PBS (control) into five week-old
chickens
24 hrs.
Inoculated with 1 O'organisms of
E. Coli via the air sac.
t 24 hrs.
--
The spleen was isolated.
Broth
Diluted
Homogenized
Plated onto
MacConkOy
41 C overnight
I
.
Colony counts
Figure 4.1
The assay procedure for nonspecific immunity
in chickens.
63
Actual viable numbers of inocula were determined by the
dilution and plating out
after the
inoculation,
Twenty-four hours
method.
birds were euthanatized by
Carcasses were immediately chilled,
asphyxiation.
the viable colony count of
E.
CO2
and
coli in the spleen were
determined by the dilution and plating out onto MacConkey
agar plates in duplicates. Representative colonies were
checked for b-hemolysi,s on blood agars, positive glucose
antiserum
positive
and
fermentation,
against
Ol:Kl
agglutination
slide
strain.
Various
with
were
agents
intravenously or subcutaneously injected to induce innate
immunity 24 hr prior to E.
coli challenge infection as
described below.
Statistics.
Colony count in log10 and weight gain data
in per cent were analyzed with the one-way ANOVA,
and
those of mortality and positive lesion rates by the chi-
square test with the program software
(JMP IN 3.2,
SAS
Institute Inc., Cary, North Carolina, U.S.A.).
Experiment
against
chickens
E.
coli,
coil
were
Twenty-four
Induction
1.
infection by various
distributed
hours
nonspecific
of
before
to
6
agents.
groups
challenge
immunity
of
10
infection
Sixty
birds.
with
E.
birds in each of the 4 groups were intravenously
injected
with
1
ml
of
one
of
the
four
inactivated
64
bacterial suspensions as shown in figure 4.2.
Birds in
the fifth group were subcutaneously injected with 1 ml of
3% silver nitrate in distilled water and those
control group with 1 ml of PBS.
in the
Challenge dose was 1.8 x
CFU/bird.
El
7
C,
al
C,
C)
0
C)
0
e
3
ui
'I-
0
0
z
-
I
1
-
- -1
E.coli S.aureus E.coli S.typhi AgNO3 Control
O1:K1
078
Figure 4.2
The effect of various agents in inducing
antibacterial immunity indicated by the number of viable
E. coli in the spleen 24 hours after challenge infection
(Experiment 1)
Groups with differing letters indicate
.
significant (P < 0.05) differences.
65
Comparison between vaccinal inimunity
Experiment 2.
and nonspecific iimuunity induced by
FSA.
chickens were distributed to
A group of thirty
groups.
3
Ninety-two
0.5 ml per
birds were twice injected subcutaneously with
bird of the vaccine made of homologous Ol:Kl strain as
described above at 14 and 28 days of age.
Another group
(5 X 108)
of 31 birds were injected intravenously with FSA
24 hours prior to challenge infection
(34 days of age)
The control group of 31 birds was left untreated.
all the birds were weighed and inoculated
days of age,
with 1.8 X l0
post
CFU of Ol:Kl strain.
infection,
from
birds
10
Twenty-four hours
each
euthanatized and the viable numbers of
E.
spleen were determined as described above.
the birds were observed for
recorded.
birds
necropsy.
7
days,
group
coli
weighed,
euthanatized,
were
in the
The rest of
and mortality was
At the seventh day post infection,
were
At 35
and
surviving
examined
at
Pericarditis, hepatitis, and/or airsacculitis
were counted as positive lesions.
Liver swab samples
were streaked onto Macconkey agar plates to detect the
presence of E. coli.
Experiment
3
Duration of nonspecific ilmilunity
and 4.
induced by Staph. aureus.
were distributed to
5
In Experiment 3,
groups of
10
birds.
50 chickens
They were
66
injected intravenously with 5 X 108 of FSA in 1 ml at 3,
6,
9, or 12 hours before challenge infection with 3 X i0
Ten birds in the control group were
CFU of O1:K1 strain.
infected
without
Experiment 4,
X i0
prior
treatments.
Similarly,
in
30 chickens were challenge exposed with 1.7
CFU of Ol:Kl strain at
1,
2,
or 5 days after
4,
injection of the Staph. aureus suspension.
Experiment
The
5.
effect
cold
of
stress
or
corticosterone on the induction of nonspecific immunity
against E. coli.
Five-week-old
distributed
to
5
leghorn
white
male
groups
of
10
chickens
birds.
were
They
were
intravenously injected with 5 X 108 FSA 24 hours prior (34
days of age)
to E.
coli infection except for those in
Group 5 which received intravenous injection of 1 ml PBS.
Birds in group 1 were fed corticosterone (Sigma Chemical
St. Louis, MO, U.S.A.) mixed in the feed (40 mg/kg)
Co.,
at
day 32,
Group
2
33
birds
and
were
34
of
age
injected
(Davison et
with
al.,
1983).
adrenocorticotropic
hormone (ACTH) of porcine origin (Sigma)
in a dose of 20
i.u./kg body weight at 24,
16,
coil challenge infection.
Birds in Group 3 were moved
from a warm brooder
and 8 hours prior to E.
(average temperature
=
20.2
C)
to
67
cold environment
of
immediately after the injection
(9.7 C)
FSA and kept there for 24 hrs until
Group
challenge.
additional
4
treatment,
received
birds
and
FSA
group
those
the bacterial
without
were
5
an
PBS
controls.
4.4 Results
Experiment
1.
Induction
of
nonspecific
against E. coli by various agents.
tested
agents
induced
highly
Injection of all the
significant
reduction in the viable counts of E.
immunity
(p
<
0.01)
coli in the spleen
compared with the PBS control group (Figure 4.2). Ranges
of the reduction varied from approximately 100-fold with
AgNO3 to more than 1000-fold with Staph. aureus or E. coli
cells.
No adverse clinical signs were observed with any
of the treatments.
Experiment
2.
Comparison between vaccinal immunity
and nonspecific immunity.
Nonspecific immunity induced
by FSA was as effective as specific immunity induced by
the homologous vaccine as evidenced by significant
(p
<
0.05) suppression of E. coli viable counts in the spleen
at 24 hours and significant reduction in mortality during
68
7 days post infection (Table 4.1).
significant
however,
vaccinated group.
among
the
three
isolation of
E.
reduction
For the lesion score,
was
seen
only
in
the
There was no significant difference
groups
in
the
weight
gain.
Cultural
coli was uniformly negative with liver
swab samples obtained from the survivors at necropsy.
Table 4.1 The effect of nonspecific immunity induced by
inactivated Staph. aureus or that of specific immunity
induced by a homologous vaccine on the E. coli viable
count in the spleen at 24 hours, cumulative mortality and
weght gain during 7 days, or lesion frequency at 7 days
after challenge infection with E. coli in 5-week-old
chickens (Experiment 2)
Group
1
(n=30)
2
(n=31)
3
(n=3l)
Teatment
vaccinea
E.coli
counts a
Weight
Log10 (CFU)
Mortaity
323207A
0120A
Lesion gain % in
7 days
7120A
21.1±9.1
(n = 10)
S.aureusc 280164A
9120B
15.0±6.0
(n =10)
Untreated 487126B
5/6B
14.0±8.6
(n =10)
aMean ± standard deviation; differing superscripts within
a column indicate significant (p <0.05) difference.
bFormalintreated homologous E. coli strain emulsified in
Freunds incomplete adjuvant
cFormalintreated Staphylococcus aureus, given at 5 X 108
cells/bird
69
Experiments 3 and 4.
induced by Staph.
0.05)
Duration of nonspecific immunity
aureus.
A significant level
of immunity was detected 6,
9,
and 12 hours,
(p
<
but
not at 3 hours after injection of FSA suspension (Figure
4.3).
similarly, significant immunity was observed at 1,
but not at
4.4) .
3,
4,
or 5 days after the injection (Figure
This experiment was repeated two more times with
slightly different
schedules,
and the
results
experiments indicated that significant levels
of
immunity were detected at
(data not shown)
2
days post
of both
(p <
0.05)
stimulation
70
9
C
8
a.
7
C)
C)
C')
a)
0
6
a)
0
5
.-
4
0
()
L
3
'4-
0
a
z
2
I
[!I
I
1
-
3
I
4
5
Control
Days after injection of S. aureus
The number of viable E. coli in the spleen
Figure 4.3
after challenge exposure at
various time
detected
with
the
formalin-treated
after
stimulation
periods
Staph. aureus (Experiment 3)
71
7,
C
6
C)
.EU,
2
0
5.
bc
ab
ab
3.
()
LU
4-
0
d
z
2
I
1
I
I
I
3
6
9
I
12
I
Control
Hrs after injection of S. aureus
Figure 4.4 The number of viable E. coli in the spleen
detected after challenge exposure at various days after
stimulation with
(Experiment 4).
the
formalin-treated
Staph.
aureus
72
Experiment
The
5.
effect
cold
of
stress
or
corticosterone on the induction of nonspecific immunity
against
coli.
E.
the
In
process
of
determining
the
duration of nonspecific immunity, experimental birds were
inadvertently
exposed
electricity
exposure
poultry
the
of
coldness
to
environment
after
a worker accidentally turned off
stimulation with FSA;
the
cold
to
A
facility.
(approximately
10
sudden
lower
C
than
normal brooding temperature) and darkness for at least 18
hours
blocked
the
induction
of
nonspecific
immunity
To test the hypothesis that cold stress
against E. coli.
or a treatment with stress-related hormone inhibits the
induction
of
pretreated
with
nonspecific
chickens
immunity,
corticosterone
or
ACTH
were
before
the
stimulation with FSA, or exposed to cold temperature for
24
hours after the
with
E.
induction
resulted
coli
of
stimulation.
in
nonspecific
infection by the
The cold stress
Subsequent
inhibition
significant
immunity
against
corticosterone treatment
(group 3)
infection
E.
(Table
in
coli
4.2)
also inhibited the induction,
but to a lesser extent. Some inhibition in the induction
was
noted
with
significant level
ACTH
the
(p
>
control group (group 4).
0.05)
(group
2),
but
not
at
a
compared with the positive
73
Table 4.2
Suppressive effects of corticosterone, ACTH,
or cold stress treatment on the induction of nonspecific
immunity against E. coli infection by Staph. aureus cells
(Experiment 5)
E. coli
S. aureusa
in the spleen'
Treatmentc
IV injection
1
Corticosterone
Yes
6.27
2
ACTH
Yes
3.62 ± 162BC
3
Cold stress
Yes
4.58
4
None
Yes
2.59 ±
5
None
No (PBS)
Group
Log10 (CFU/g)d
±
089BD
±
5.64
±
053A
l.l6
aFormalintreated cell suspension given at 24 hours before
E. coli infection
bViable counts at 24 hours after infection in spleen
homogenate
cSee text for details.
dDiffering superscripts in the column indicate significant
(p < 0.05) difference.
4.5 Discussion
The
E.
coil
immunity was
infection model
to
described previously
detect
(Huang
nonspecific
&
Matsumoto,
1999). The model was developed in an attempt to replace
traditional mortality testing to evaluate immunity.
All
74
the scientists at the authors' institution were urged to
stop any experiments,
consistent
be
in which death is an endpoint,
with
government
the
Institute of Health, 1992)
.
policy
to
(National
The results shown in Table 1
indicate that the viable counts detected in the spleen at
24 hrs correlate well with cumulative mortality during 7
after
days
challenge
there was
demonstrate
that
difference
between
inactivated Staph.
Both
infection.
parameters
significant
no
nonspecific
immunity
(p
>
also
0.05)
induced
by
aureus and specific immunity by the
homologous vaccine.
was found important
It
in the assay procedure that
chicken carcasses should be cooled down with ice or cold
water
soon
as
as
they
killed
are
to
minimize
multiplication of the organism in the internal organs.
absence
The
of
such immediate cooling procedure
in
consistent manner leads one to erroneous results as
coil
multiplies
rapidly
without
any
host
especially during the hot summer months.
against
such
a
possibility
it
is
a
E.
defense,
To safeguard
necessary to
run
a
positive and negative control group to make sure that the
results are valid.
Inactivated bacterial cells of both Gram-positive and
-negative
origin were
capable
of
inducing
nonspecific
75
protection
chickens
against
(Figure
immunity may be
proteins
of
non-specific
caused by production
of
acute
.
C-reactive,
phagocytes.
repeated
vitro
in
amyloid
C3,
P
protein,
attempts
bactericidal
failed
to
activity
in
activated serum with or without avian complement.
other
when
hand,
stimulation
with
transferred
to
nonspecific
pooled
recipients after
taken
sera
inactivated
unstimulated
protective
E.
Staph.
challenge
Matsumoto, unpublished data).
after
aureus
were
3
chickens,
detected
was
On the
hrs
at
recipient
activity
coli
phase
or nonspecific activation
1999),
Our
any
5-week--old
of
including
demonstrate
in
Such elevation
4.2)
(Gabay & Kushner,
etc.
infection
coli
E.
infection
in
the
(Huang
&
These preliminary results
favor the direct activation of phagocytes as a mechanism
of nonspecific immunity against E. coli.
Nonspecific immunity against E. coli was induced at 6,
but not at 3 hours, and valid for less than 3 days after
a single stimulation with staphylococcal cells
(Figures
4.3 and 4.4) .
Experiment 4 was repeated twice with some
modifications,
and the results showed that
levels
(p
<
0.05)
significant
of immunity was detected at 48 hours
after the stimulation (data not shown)
.
Therefore,
the
inactivated bacterial cells induced effective nonspecific
76
immunity against E. coli infection between 6 and at least
48 hours after stimulation.
Induction of nonspecific immune response was inhibited
by administration of
cold stress
corticosterone or by exposure
(Table 4.2). The mechanisms involved in the
inhibition are unknown.
corticosterone was
consecutive days
In the present investigation,
administered
(Davison,
Staph. aureus cells.
feed for three
the
in
1983) before the injection of
Glucocorticosteroid is widely used
in medical fields as an anti-inflammatory agent.
has
various
productions,
conclusions
to
effects
on
immune
and
is
difficult
on
it
effect
its
on
cells
and
It also
cytokine
draw
to
immunity
general
(Davis,
1998;
The results presented here support
Vieira et al., 1998).
that innate immunity is suppressed by host exposure to
stress
treatments.
Gross
(1992),
on
the
other
hand,
reported that feeding 40 or 60 mg/kg of corticosterone to
chickens
increased
infection with E.
their
coli.
resistance
to
subsequent
The author infected chickens
without prior activation of nonspecific immunity, and his
method of determining "resistance" was based on mortality
"combined with"
lesion
differences
methodology
in
score.
In
between
addition
Gross's
to
and
these
the
present study, another obvious difference between the two
77
studies is the fact that Gross's conclusion was based on
his observation during a relatively short period (3 to 10
hours)
after the start of corticosterone administration,
while,
in the present study,
observed
after
consecutive
3
Corticosterone
feeding.
the inhibitory effect was
days
may
corticosterone
of
have
dual
effects
of
stimulation and inhibition depending on the timing and
dosages
of
its
administration.
Further
studies
are
needed to clarify the effect of glucocorticosteroid on
innate immunity.
Nonspecific immunity or innate immunity has neither
been investigated in detail nor utilized for prevention
or treatment of health problems.
this area progress,
immunity
may
be
the role and importance of
elucidated,
technology utilizing
As investigations in
this
promoting
arm of
host
innate
development
defense
in
of
the
future in avian medicine.
4.6 References
M.A.
(1997)
Systemic bacterial
infections
in
broiler chickens. M. S. Thesis, Oregon State University,
Corvallis, Oregon, U. S. A.
Awan,
.
M.A.
&
Matsumoto,
M.
(1998).
Heterogeneity of
Staphylococci and other bacteria isolated from six-weekold broiler chickens. Poultry Science, 77, 944-949.
Awan,
78
Davis, S.L. (1998). Environmental modulation of the immune
endocrine
system.
Domestic
Animal
the
via
system
Endocrinology, 15, 283-289.
Effects of dietery corticosterone on
the growth and metabolism of immature Gallus domesticus.
General and Comparative Endocrinology, 50, 463-468.
Davison, T.F.
(1983) .
C & Kushner, I. (1999). Acute-phase proteins and
other systemic responses to inflammation. New England
Journal of Medicine, 340, 448-454.
Gabay,
Gross,
W.B.
to
chickens
(1992).
Effect of short-term exposure of
corticosterone on resistance to challenge
exposure with Escherichia coli and antibody response to
Veterinary
American
Journal
of
erythrocytes.
sheep
Research, 53, 291-293.
Janesway Jr.,
C.A.,
&
Kafatos,
F.C.,
J.A.,
Hoffman,
in
Phylogenic perspectives
R.A.B.
(1999) .
Ezekowitz,
innate immunity. Science 284, 1313-1318.
& Matsumoto, M. (1999). Immunity against
Escherichia coli infection in chickens assessed by viable
bacterial counts in internal organs. Avian Diseases, 43,
Huang,
H.J.,
(in print).
Huff, G.R., Huff, W.E., Balog, J.M. & Rath, N.C. (1999).
The effect of a second dexamethasone treatment on turkeys
previously challenged in an experimental Escherichia coli
osteomyelitis
complex.
model
of
turkey
respiratory
Poultry Science, 78, 1116-1125.
National
Institute
of
Health.
(1992) .
animal care and use committee guideline.
Institutional
NIH (U.S.A.)
Publication No. 92-3415.
Vieira, P.1., Kalinski, P., Wierenga, E.A., Kapsenberg,
M.L. & de Jong, E.C. Glucocorticoids inhibit bioactive
IL-12p70 production by in vitro-generated human dendric
affecting
their
T
cell
without
cells
potential. J Immunol, 161, 5245-5251, 1998.
stimulatory
79
Chapter 5
Non-specific Innate Immunity against Escherichia
coli Infection in Chickens Induced by Vaccine
Strains of Newcastle Disease Virus
80
5.1 Abstract
The objective of this study was to test the hypothesis
that vaccine strains of NDV induce nonspecific immunity
against subsequent infection with E. coli.
chickens at
White leghorn
5 weeks of age were vaccinated with a NDV
vaccine at various days before challenge exposure with
Ki
01:
strain of
E.
coli via an intra-air sac route.
Immunity was determined based on the viable number of E.
coli in the spleen 24 hr after the infection.
Roakin
strain induced significant immunity against E. coli at 4,
6,
and 8 days,
and La Sota strain at 2,
4,
and 8 days
Secondary NDV vaccination administered
post vaccination.
14 days later failed to induce immunity against E.
when
infected
or
1
5
days
after
the
coil
vaccination.
Suppression of this nonspecific immunity was observed in
birds treated with corticosterone,
given for
3
40
mg/ kg in
feed,
consecutive days immediately prior to the
bacterial exposure but not in those treated prior to that
period.
The
results
indicate
that
innate
immunity
induced by the primary MDV vaccination can significantly
suppress the multiplication of E.
period of
2
to
8
coli in chickens for a
days post vaccination and that MDV-
81
induced
immunity
can
inhibited
be
by
stress
or
corticosterone treatment.
5.2 Introduction
Innate immunity, believed to be phylogenetically older
than
acquired
the
immunity,
multicellular organisms.
present
is
in
all
It is defined as the first line
host defense against pathogens and works under different
mechanisms from those involved in adaptive immunity
generally
It
provides
non-specific
(8)
short-term
and
immunity rather than the specific and efficient adaptive
immunity,
and
bacterial
(10,11),
(5)
.
protective
its
viral
role
identified
was
or parasitic
(14),
in
infection
Innate immunity may also play an instructive role
in determining to which antigens adaptive immunity may
respond
and
investigating
broiler
suppressed
infection
nature
the
a
high
chickens,
innate
(1)
.
mortality
our
past
immunity
By
the
problem
study
was
use
response
the
of
of
.
In
commercial
in
suggested
resulted
a
(6)
in
systemic
that
systemic
E.
coil
infection model in chickens, anti-bacterial immunity was
determined by comparing the viable bacterial counts in
82
internal organs between treatment and control groups
(9)
With this system,
coli
infection was
nonspecific immunity against
induced by
successfully
administration
of
inactivated
immunity appeared as early as
the
bacteria
E.
intravenous
(10)
.
This
6 hr and lasted 2-3 days
after the injection.
The objective of the study was to determine whether
similar anti-E.
coil
immunity can be
induced by mild
viral infections such as NDV vaccination.
5.3 Materials and methods
One-day-old male white leghorn chicks were
Chickens.
obtained
from
a
local
hatchery,
vaccinated against Marek's disease.
heated battery cages with a
0.01% decoquinate.
where
they
were
Birds were raised in
starter ration containing
Feed and water were given ad.
lib.
Birds were bled at 21 days of age to check the absence of
maternal antibodies against NDV by the hemagglutionationinhibition (HI) test
Bacteria.
from Dr.
B.
(3)
Escherichia coli strain 01:
Kl was a gift
Panigrahy of National Veterinary Services
Laboratory, Ames. IA.
Staphylococcus aureus was isolated
83
from an arthritic joint of a broiler chicken at a local
Preparation
farm.
vaccination or
induction
described in detail
inactivated
of
for
immunity was
non-specific
of
(10) .
bacteria
Briefly, frozen stock cultures
were recovered and grown on 5% sheep blood agar plates
(heart-infusion agar; Difco Lab, Detroit, MI).
Bacteria
were harvested with cold phosphate-buffered saline
(PBS;
0.85% NaC1)
solution, pH 7.2 and viable bacterial counts
were
by
made
the
dilution
and
Formalin was added to 0.3%
(v/v)
plating
method.
out
and the concentration
was adjusted to 5 X 108 colony-forming-units (CFU) /ml for
induction of non-specific immunity by S.
1.0
X
CFU/ml
10
for
the
preparation
aureus,
of
an
or to
E.
coli
inactivated vaccine, which was subsequently emulsified in
incomplete
Freund's
adjuvant
(Difco)
checked by broth inoculation.
The E.
Sterility
.
was
coli inoculum for
challenge exposure was prepared as follows
(9) ;
four-hour
bacterial growth on blood agar was harvested and diluted
1:10 in PBS to give an optical density value of 0.2 at
600
nm,
corresponding to approximately
1
X
108 CFU/ml.
Viable counts were made for each inoculum by the dilution
and plating out method.
Virus.
National
Strain La Sota of NDV was obtained from the
Veterinary Service
Laboratory,
Ames,
IA,
and
84
Roakin
strain
was
gift
a
from
Dr.
D.
J.
King,
the
Southeast Poultry Research Laboratory, ARS, USDA, Athens,
GA.
The strains were propagated in the allantoic chamber
of 10-days-old chicken embryos.
Candling was done twice
a day and embryos which died during the first 24 hr were
discarded.
Allantoic
fluid
harvested
was
once
the
embryos were dead or 7 days post-inoculation, pooled and
stored at
The frozen stock was titrated in 10-
70 C.
days-old chicken embryos and 50 % embryo-infection dose
(EID50)
was determined
A hemagglutination test was
(3).
performed to determine the titer of
the frozen stock.
Sterility was checked by inoculating
1
fluid into 100 ml
ml of allantoic
of brain-heart infusion broth.
For
vaccination, 0.1 ml containing iO E1D50 of La Sota, or 106
E1D50 doses of Roakin strain was given to chicks via an
Blood samples were obtained from
intra-tracheal route.
vaccinated chickens
infection to make
at
the
sure
time
of
the negative
E.
coli
titer
challenge
in control
birds and positive in vaccinated birds by the HI assay.
Infection with E.
coli.
Chickens were moved to an
isolation unit for NDV vaccination at various days before
E.
coli challenge infection (10).
with E.
coil strain,
thoracic air sac.
01:
Ki,
Birds were inoculated
in 0.1 ml via the caudal
At 24 hr post infection, birds were
85
euthanatized by CO2 and carcasses were cooled immediately
by cold running water.
The
spleen was
in broth under aseptic
homogenized
viable counts of E.
isolated and
condition and
the
coli were determined by the dilution
and plating out method onto MacConkey agar plates (Difco)
in duplicates.
Colony counts in log10 were analyzed by
Statistics.
the students' t-test with Statgraphics Plus V4.0 program
(Statistical Graphics, Corp., Englewood Cliffs, NJ).
Experiment 1 and 2.
against E.
Induction of nonspecific immunity
coli infection by NDV vaccination.
In the
first experiment, 60 chicks were distributed to 6 groups.
Birds were vaccinated with a dose containing 106 E1D50 of
Roakin strain intratracheally at
before the E.
of
4,
2,
coli challenge infection.
age with
6,
or
7
X
i05
processed as described above.
days
A control group
CFU of
coli/
E.
bird and
In the second experiment,
the procedure was repeated with La Sota strain (iO
doses/bird) and birds were vaccinated at
14
8
All the birds were infected at 35
was left untreated.
days
1,
days before the E.
coli infection
1,
(1.8
2,
X
4,
106
E1D50
8,
CFU
or
/
bird).
Experiment
3.
The absence of nonspecific immunity
after the secondary NDV vaccination.
Fifty birds were
86
distributed to 5 groups
(Table 5.1).
La Sota strain in
i05 E1D50 dose was inoculated intra tracheally at 21 and
35 days of age in groups 1 and 4.
In group 2, birds were
given primary vaccination only at 21 days of age.
3 and 5 were untreated controls.
Birds in groups 1,
and 3 were infected with 2.1 X 106 CFU of E.
36 days of age.
Birds
in groups
Group
4
2,
coli/ bird at
and 5 were infected
CFU of E. coli/ bird at 40 days of age.
with 1.7 X
Table 5.1
The absence of nonspecific immunity against
E. coli in chickens at 1 or 5 days after the secondary
vaccination with La Sota strain
Primary
Group
Secondary
NDV
Age in days Age in days
NDVA
2
21
21
3
No
No
No
4
21
35
5
No
No
1
35
E. coli in
E. coli
infection
Age in days
spleenB
Log10 (CFU /g)C
36
36
36
3.50 ± 1.30
5.15 ± 1.40
4.49 ± 1.63
40
40
5.46 :1: 1.32
A
5.53 ± 0.68
Birds were infected with 10
E1D50 per bird of NDV La
Sota strain via an intra-trachea route.
B
At 24 hr after E. coli infection.
C
Mean ± 95% c.i.
87
Experiment 4.
formalin-inactivated
vaccine,
coli induced by a
Immunity against E.
aureus
S.
NDV
or
Forty birds were distributed to 4 groups.
vaccination.
Ten birds were vaccinated subcutaneously with inactivated
coil vaccine twice at
homologous E.
age,
intravenously
injected
inactivated
aureus
S.
intravenously with iü
day-old.
with
108
X
5
34-day-old,
at
and 28 days of
14
formalin-
vaccinated
or
E1D50 doses of LaSota strain at 30-
Control birds were left untreated.
All the
birds were infected with the E. coil strain (1.4 X 106 CFU
/ bird) at 35 days of age.
Experiment 5.
nonspecific
The effect of corticorsterone on the
immunity
against
E.
by NDV
induced
coli
vaccination.
Sixty birds were distributed to
(Table 5.2).
Birds in the NDV groups were given
6
groups
E1D50
doses of La Sota strain at 35 days of age.
Groups with
corticosterone were given
feed
at
40
mg/kg
in
consecutive days as indicated in the table.
for
Birds
3
in
group 5 received corticosterone at 60 mg/kg feed for a 4
hr period before E.
coil infection
(7)
.
At 40 days of
age, all were infected with 5 X 106 CFU of E.
coii/ bird
via the air sac route and the viable count of E. coil was
determined as described above.
88
Table
5.2
nonspecific
vaccination
A
The
effect
of
immunity against
Group
NDV
1
Yes
Yes
Yes
Yes
No
No
2
3
4
5
6
corticorsterone
on
the
E.
coil
induced by NDV
Corticosterone
E. coii in spleen
given at days after
Log10 (CFU /g) D
NDV vaccination
No
-1,
0,
3.76
4.31
4.35
5.59
1
1,
2,
3
3,
4,
5
±
±
±
±
0.70 a
1.11 ab
1.00 ab
0.82 C
5.24±0.80'
No
5.40 + 0.74
bc
A
Five-weeks-old chickens were intratracheally vaccinated
with La Sota strain.
At 5 days post vaccination, they
were infected with E.
coli
via an air sac route.
Corticosterone
was
given
in
the
feed
for
periods
indicated.
B
Feed containing 40 mg/kg corticosterone was given in
group 2, 3, and 4.
C
Birds were infected with 5 X 10
strain O1:K1, at day 5.
D
Mean ±
95%
c.i.
6
per bird of E. coii,
Different superscripts within the
column indicate significant (p < 0.05) difference.
E
Feed containing 60 mg/kg corticosterone was given for 4
hr before E. coil infection.
5.4 Results
Induction
of
nonspecific
immunity
against
infection by NDV vaccination (Experiment
E.
1 and 2).
test whether nonspecific immunity against E.
coli
To
coil can be
89
induced by virus infection,
MDV,
at various days before the E.
Roakin strain,
challenge
Antibodies
infection.
negative
without
birds
in
birds were vaccinated with
or
before
viable
1,
counts
of
E.
coil
were
vaccination,
became positive 4 days after vaccination.
Fig.
NDV
against
in
coil
and
As shown in
spleen was
the
significantly (p < 0.05) lower at 4 days group and highly
significantly
(p
<
infection groups
significant
group
and
lower at
0.01)
than
the
difference was
the
control
6
and
non-treatment
found between
control.
or
No
2
days
results
were
1
Similar
group.
days post
8
obtained when birds were vaccinated with La Sota strain.
Viable
counts
of
E.
coli
in
the
spleen
was
highly
significantly (p < 0.01) lower at 2 and 8 days group and
significantly
group
(Fig.
between 1
indicate
(p <
2).
0.05)
lower at 4 days post infection
No significant difference was observed
or 14 days and control group.
that
non-specific
immunity
These results
against
E.
coli
infection can be induced by the NDV vaccination for the
period of 2-8 days post vaccination.
a)
*
0
**
**
0
C.)
1
2
4
6
8
Days Post Vaccination
Control
Figure 5.1 The number of viable E. coli in the spleen of
24
hr after challenge
chickens at
infection.
The
chickens were previously exposed to NDV, Roakin strain,
with time period (days) indicated (* p < 0.05, ** p
<0.01)
91
El
a,
*
**
0)
**
0
0)
0
C.)
0
0
z
4
2
1
8
Control
14
Days Post Vaccination
Figure 5.2 The number of viable E. coli in the spleen of
chickens at 24 hr after challenge
infection.
The
chickens were previously exposed to NDV, La Sota strain,
with time period (days) indicated (* p < 0.05, ** p
<0.01)
The
absence
secondary
whether
NDV
the
of
nonspecific
vaccination
secondary
(Experiment
NDV
after
ilrimunity
3)
vaccination
To
.
can
nonspecific immunity as well as the primary one,
the
test
induce
birds
were given one or two La Sota vaccination before the E.
92
coli infection (Table 5.1)
Viable counts in the spleen
.
of birds with secondary vaccination at
before the E.
from the control
(p= 0.299)
significant
No
.
(group
1)
coli challenge was slightly lower but not
significant different
3)
day
1
difference
was
0.496)
(p=
(group
found
between birds received primary vaccination 15 days before
E.
coil infection (group 2)
Viable counts
and control.
in birds received secondary vaccination at 5 days (group
4)
before
coli
E.
infection
were
significantly
not
different (p= 0.92) from control birds (group 5)
Immunity
against
E.
formalin-inactivated
S.
counts of E.
group
received
compared with
a
NDV
or
vaccine,
vaccination
coil in spleen were observed in the
homologous
intravenously
injected
aureus
by
As shown in Fig. 5.3, lower viable (p <
(Experiment 4).
0.01)
induced
coli
those
in
inactivated
with
the
vaccine
inactivated
non-treatment
and
aureus
S.
group.
No
significant difference was observed in the viable count
between
control
birds
and
birds
received
La
Sota
vaccination
at
infection.
It was observed, however, that birds in the
5
days
before
the
E.
coli
challenge
NDV group were accidentally prevented from eating for at
least 48 hr immediately prior to the E. coil infection.
93
The
cage
ceiling was
accidentally dropped on
the
two
sides.
6
7
a,
a
If)
**
j2
0
C.)
**
c1
z
0
Killed
Killed
S. aureus E. coli
Vaccine
NOV
Control
Figure 5.3
The number of viable E. coli in the spleen of
chickens at 24 hr after infection.
The chickens were
previously
exposed to NDV
days),
(5
injected with
inactivated S. aureus (24 hr)
or vaccinated twice with
an inactivated homologous vaccine before the challenge
,
infection (** p <0.01)
The
effect
of
corticorsterone
nonspecific immunity against E.
(Experiment 5).
on
the
induction
of
coli by NDV vaccination
In experiment 4,
birds vaccinated with
94
NDV
were
stressed
immunity against
and
the
did
coli
E.
not
develop
non-specific
To test
infection.
the
hypothesis that stress can inhibit the induction of the
non-specific
immunity
treated
with
between
the
5.2) .
NDV
by
corticosterone
vaccination and
vaccination,
at
E.
different
coli
birds
were
time
period
infection
(Table
Birds treated with corticosterone for a period
between 3
significant
and 5 days post vaccination
(p <
0.05)
(group 4)
showed
suppression in inducing immunity.
Other groups including one treated with a high dose for a
short period (group 5)
failed to demonstrate significant
effects.
5.5 Discussion
Two common vaccine strains
immunity against
E.
of NDV induced systemic
infection in chickens.
coli
nonspecific or innate immunity appeared at
and lasted until around
8
1
or
days post vaccination.
Such
2
days
Our
previous study showed that the intravenous injection of
formalin-inactivated
S.
aureus induced similar immunity
against E. coli between 3 and 48 hr after the stimulation
(10)
.
The viral
infection,
therefore,
induced innate
95
immunity of longer duration than the inactivated bacteria
Since adaptive immunity, both cellular and humoral,
did.
commonly become effective at 4 or 5 days post infection,
innate immunity appears to play an important protective
role in the early stage of infection, although it is not
clear from the present study that
immunity against ND
infection itself was
far
concerned,
the
study
As
induced.
as
coli
E.
is
it was our consistent observation throughout
that
1
or
2
out
the
of
control
10
birds
frequently showed 10 or fewer organisms/g in the spleen
at 24 hr after the challenge infection with 106 or
CFU.
In these birds,
effective innate immunity may be
any prior
induced without
stimulation.
The
rate
of
protection due to innate immunity increased when lower
challenge
doses
were
or
used,
in
other
words,
lower
challenge doses failed to induce a consistent systemic
infection
normal
due
to
5-week-old
interpreted that
interference
chickens
the
(9)
level
of
of
innate
The
.
innate
immunity
results
may
in
be
immunity against
multiple microbial pathogens is significantly elevated by
mild NDV infections in chickens.
The
mechanism leading
to
the
activation
of
innate
immunity has not been elucidated and is the subject of
our current investigation.
The production of acute phase
96
proteins
such
which
components,
bacterial
growth
protein
C-reactive
as
directly
of
inactivate
organisms
like
but sera obtained at
suspected,
with killed
S.
suppressive
such
produced
cytokines
cells
release
inducing
failed to show any signs
activities
macrophages,
is
in
vitro
that
some
dendritic
cells,
indirectly activate phagocytes
promote
to
was
(2),
hr after stimulation
6
biological
by
and/or granulocytes
other
coli
E.
Another possibility
(unpublished data).
suppress
or
which were capable of
aureus,
innate immunity in recipients,
of
complement
or
antibacterial
bacterial
substances
major infection in mice,
killing
(11)
and/or
In
.
and
to
Leishmania
induction of IL-l2 as well as
nitric oxide synthase was found to be a prerequisite for
cytokine
signaling
in
immunity
innate
(5).
Innate
immunity appears to consist of multiple components which
interact constantly among themselves to regulate effector
From this context,
mechanisms.
it
is interesting that
secondary vaccination, known to have a prominent booster
effect in adaptive immunity, failed to induce nonspecific
innate immunity
(Table 5.1).
responses
cellular
different
and
between
stimulation.
primary
The dynamics of cytokine
interactions
and
may
secondary
be
quite
immunological
97
induced
NDV
by
a
the
vaccination
innate
according
response
immune
to
results
the
In our previous study, a sudden exposure
presented here.
to
with
interferes
Stress
cold environment or corticosterone administration
was found to block innate immune response against E. coli
in chicken
(10)
Corticosterone or physical stress has
.
been known to alter adaptive immune response
(4)
Gross
.
(7), using an E. coli infection model in chickens similar
to one used here,
corticosterone
reported that short-term exposure to
could
with non-treated controls.
exposure
method
enhanced immunity compared
induce
was
Although a similar dose and
followed,
his
results
were
not
reproduced in the current study (Table 5.2). In fact, the
results presented here and in the previous study
consistently
showed
suppressive,
effects
stress
and
of
however,
immunity.
did
not
rather
corticosterone.
differentiate
innate
(10)
than enhancing
Gross
from
(7),
adaptive
The effect of stress and corticosterone on
innate immunity should be studied in a systematic manner
with a well defined infection model.
Viral infection in the respiratory or digestive tract
often
induces
colisepticemia
secondary
in
bacterial
chickens.
infection
Damage
done
such
on
as
the
epithelial surface by virus infections allows pathogenic
98
bacteria
to multiply locally and to
stream.
Immunosuppression induced by virus, bacteria, or
the blood
invade
environmental stress is known to amplify the pathogenic
process
(12)
Innate immunity against both viruses and
.
bacteria may play an important role in determining the
course
infection.
of
immunity
innate
If
is
induced
systemically within hours after the onset of infection,
the
pathogen
entering
bloodstream
the
efficiently
is
inactivated by internal organs such as the liver, spleen,
or bone marrow, restricting the infection to local areas,
and,
inflammation
likewise,
On the other hand,
level.
immunity
suppressed
is
may
remain
at
minimum
a
if the induction of innate
by
stress,
pathogens
spread
quickly to many organs and tissues, resulting in grossly
exaggerated
such
as
inflammatory
edema,
response
hemorrhage,
and
throughout
a
large
body
the
influx
of
heterophils; these conditions, in turn, enhance bacterial
or viral
and uncoordinated
multiplications
release
of
cytokines, chemokines, nitric oxide, oxygen radicals and
other harmful
substances,
resulting
in various
damages and clinical conditions including death.
time
adaptive
immunity
is
induced
after
4-5
tissue
By the
days
of
infection, the condition is too grave to cause immediate
resolution.
The
latter possibility may explain high
99
in broilers at market age due
mortality incidences
cardiomyopathy, ascites or arthritis.
to
The current series
of studies were initiated to investigate this mortality
problem in broilers
pathological
ascites
(1)
studies
A recent report resulting from
.
dead
of
broilers
cardiomyopathy cases
and
indicate
that
common hepatic
share
pathological lesions (13).
immunity
Innate
immune
is
mechanisms
throughout
immunity
developed
evolutionary
process.
efficient
of
seems
microbial
were
that
the
development
conglomeration of many natural
a
play
to
pathogens
adaptive
minor
a
vertebrate
in
preserved
and
With
immunity,
role
in
the
innate
combating
Recent
species.
discoveries, however, indicate that vertebrates including
humans
rely
still
recognizing
and
efficient manner
efficient
on
innate
eliminating
(8)
.
immune
mechanisms
microbial
invaders
in
for
an
Some innate immune mechanisms are
in recognizing common pathogens through pre-
formed receptors and subsequent immediate activation of
specific
effector
mechanisms
although
specificity may be limited in scope.
the
receptor
There is another
kind of innate immunity in which recognition of microbial
pathogens leads to a rapid release of "alarm" cytokines,
resulting
in
systemic
elevation
of
inactivating
100
mechanisms against bacteria,
reported
results
latter
in
They
category.
induced by NDV
the
fungi and parasites.
present
study belong
suggest
that
infection plays
innate
important
an
The
to
immunity
role
chickens
in deciding the ultimate outcome such as
severity
of
studies
secondary
should be
bacterial
carried out
to
infections.
elucidate
the
in
the
Future
the
exact
mechanisms involved in the observations presented here
including the inhibitory effect of stress.
5.6 Refereces
Heterogeneity of
staphylococci and other bacteria isolated from six-weekold broiler chickens. Poult. Sci. 77: 944-949. 1998.
1.
Awan,
M.A.,
and
M.
Matsumoto.
2. Baumann, H., and J. Gauldie. The acute phase response.
Immunol. Today 15:74-80. 1994.
Beard C.W. Serologic procedures. In: Isolation and
identification of avian pathogens,
2nd edition.
The
American Association of Avian Pathologists.
129-135.
3.
1980.
Dhabhar, F. S.,
and B. S. McEwen.
Acute stress
enhances while chronic stress suppresses cell-mediated
immunity
in
vivo:
a
potential
role
for
leukocyte
trafficking. Brain. Behav., Immun. 11: 286-306. 1997.
4.
Diefenbach, A., H. Schinfler, M. Rollinghoff, W. M.
Yokoyama, and C. Bogdan.
Requirement for type 2 NO
synthase
for
IL-12
signaling
in
innate
immunity.
Science. 284: 951-955. 1999.
5.
101
Fearon, D.T., and R. N. Locksley.
The instructive
role of innate immunity in the acquired immune response.
Science. 272: 50-54. 1996.
6.
7.
Gross,
chickens
W.
to
B.
Effect
corticosterone
of
on
short-term exposure of
resistance to challenge
exposure with Escherichia coli and antibody response to
sheep erythrocytes.
Am
J. Vet. Res. 53 (3) : 291-293.
.
1992.
Hoffann, J.A., F.C. Kafatos, C. A. Janesway Jr., and
R.A.B.
Ezekowitz.
Phylogenic
perspectives
in
innate
immunity.
Science 284: 1313-1318, 1999.
8.
and M.
Matsumoto.
Immunity against
infection in chickens assessed by viable
bacterial counts in internal organs. Avian Dis. 43: 4699.
Huang,
H.J.,
Escherichia coli
475. 1999.
10.
Matsumoto, M.,
nonspecific
term,
and H.J.
immunity
infection in chickens
Huang. Induction of shortagainst
Escherichia coli
suppressed by cold stress or
is
corticosterone treatment. Avian Pathol.
(Accepted)
Naiki, Y., H. Nishimura, T. Kawano, Y. Tanaka, S.
Itohara, M. Taniguchi, and Y. Yoshikai. Regulatory role
11.
of peritoneal NK 1.1
+
a13
during Salmonella infection.
T cells in IL-12 production
Immunol.
J.
163: 2057-2063.
1999.
Nakamura,
Yuasa,
and
Effect of
infectious bursal disease virus on infections produced by
12.
Escherichia
K.,
N.
F.
Abe.
coli of high and low virulence in chickens.
Avian Pathol. 19: 713-721. 1990.
Nakamura,
K.,
Y.
Shibahara.
Comparative
Ibaraki,
Mitarai,
and T.
pathology of heart and liver
lesions of broiler chickens that died of ascites, heart
failure, and others. Avian Dis. 43:526-532. 1999.
13.
Z.
14. Welsh, R. N., M-Y Lin, B.L. Lohman, S.M. Varga, C.C.
Zarozinski, and L.J. Selin. a3 and y T-cell networks and
their roles in natural resistance to viral infections.
Immunol Rev. 159: 79-93. 1997.
102
Chapter 6
CONCLUS IONS
The air sac
strain
Ki
01:
inoculation of
lO61O7 cfu of
results
predictable
in
a
pattern
septicemic infection in white leghorn chickens.
against
infection
coli
E.
quantitated
by
the
viable
can
be
of
Immunity
precisely
more
method
count
coli,
E.
with
smaller
number of subjects than other methods.
Nonspecific
intravenous
innate
injection
inactivated bacteria,
silver
nitrate
immune
response
homologous
heterologous
or the subcutaneous
injection of
solution
effect induced by killed S.
6
by
or
of
can
significantly
chickens against E. coli infection.
early as
induced
protect
This anti-bacterial
aureus injection appears as
hours and lasts 2-3 days after stimulation.
Such immunity, when induced in a timely manner, can be as
effective
as
specific
immunity
induced by
a
vaccine.
Cold stress or corticosterone treatment can suppress this
nonspecific innate immunity.
Non-specific innate immunity can also induced by the
mild virus
infection such
as NDV vaccination for the
103
period
of
2
to
8
days
vaccination with NDV,
this
nonspecific
post
vaccination.
Secondary
La Sota strain failed to induce
immunity.
This
immunity
is
suppressed by the stress or corticosterone treatment.
also
104
BIBLIOGRAPHY
Alhydrogel for selective adsorption. Technical Manual by
Superfos a/s,
available through Accurate Chemical
&
Scientific Corp,. Westburg, N.Y.
Aderem A, D. M. Underhill. Mechanisms of phagocytosis in
macrophages. Annu Rev Immuno. 17: 593-623, 1999.
Andus, T., T. Geiger, T. Hirano, T. Kishimoto, T.A. TranThi,
K.
Decker,
and
P.C.
Heinrich.
Regulation
of
synthesis and secretion of major rat acute-phase proteins
by
recombinant
human
interleukin-6
(BSF-2/IL-6)
in
hepatocyte primary cultures. Eur. J. Biochem. 173: 287293, 1988.
Andus, T., T. Geiger, T. Hirano, T. Kishimoto, and P.C.
Heinrich.
Action of recombinant human interleukin 1,
interleukin 1
and tumor necrosis factor-a on the mRNA
induction of acute phase proteins. Eur. J. Immnol. 18(5):
739-746, 1988.
Antilla, H.S., S. Reitamo, M. Ceska, and M. Hurme. Signal
transduction pathways leading to the production of IL-8
human monocytes
by
are
differentially
regulated by
dexamethasone. Clin. Exp. Immunol. 89(3): 509-512, 1992.
Arp, L. H. Effect of passive immunization on phagocytosis
of blood-borne Escherichia coli in spleen and liver of
turkeys. Am. J. Vet. Res. 43:1034-1040. 1982.
Awan,
M.A.
Systemic bacterial infections
chickens.
M.
S.
Thesis,
Oregon
State
Corvallis, Oregon, U. S. A., 1997.
broiler
University,
in
and
Matsumoto,
M.
Heterogeneity
of
Staphylococci and other bacteria isolated from six-weekold broiler chickens. Poult. Sci., 77, 944-949, 1998.
Awan,
M.A.
Badolato,
R.,
J.M.
Wang,
W.J.
Murphy,
A.R.
Lloyd,
D.F.
Michiel, L.L. Bausserman, D.J. Kelvin, and J.J.Oppenheim.
Serum amyloid A is a chemoattractant:
Induction of
migration, adhesion, and tissue infiltration of monocytes
105
and polymorphonuclear leukocytes.
209, 1994.
Barna,
B.P.,
S.D.
Deodhar,
S.
J.
Exp. Med.
Gautam,
B.
180: 203-
Yen-Lieberman,
and D. Roberts. Macrophages activation and generation of
tumoricidal activity by liposome associated human Creactive protein. Cancer Res. 44: 305, 1984.
and S. D. Deodhar. Activation of
human monocyte tumoridal activity by C-reactive protein.
Cancer Res. 47: 3959-3963, 1987.
Barna,
B.P,
K.
James,
H.
J.
and W.
B.
Gross.
Barnes,
Colibacillosis
In
"Diseases of Poultry" 10th ed., Calnek B. W. et al.,
eds., Iowa State University Press, Ames, Iowa. pp. 131141. 1997.
Baumann, H., and J. Gauldie. The acute phase response.
Immunology today 15 (2) : 74-80, 1994.
Baumann, H., K.K. Morella, and G.H.K. Wong. TNF-a, IL-1f3,
and hepatocyte growth factor cooperate in simulating
specific acute phase plasma protein genes in rat hepatoma
cell. J. Immunol. 151: 4248-4257, 1993.
Gauldie. Interaction
among hepatocyte-stimulating factors, interleukin 1 and
glucocorticoids for regulation of acute phase plasma
proteins in human hepatoma (HepG2) cells. J. Immunol.
139: 4122-4128, 1987.
Baumann,
H.,
C.
Richards,
and
J.
Beard C.W.
Serologic
procedures.
In:
Isolation and
identification of avian pathogens,
2nd edition.
The
American Association of Avian Pathologists.
129-135.
1980.
Besterman JM and
R.
B.
mechanisms and plasma
210(1): 1-13, 1983.
Low: Endocytosis:
a review of
membrane dynamics.
Biochem J.
DeKruyff,
and
D.T.
Umetsu.
Corticosteroids
inhibit
IL-12
production
in
human
monocytes and enhance their capacity to induce IL-4
synthesis in CD4+ lymphocytes. J. Immunol. 158: 55895595, 1997
Blotta,
M.H.,
R.H.
106
Bosanquet,
A.G.,
A.M.
Chandler,
H.
Gordon.
Effects
of
injury on the concentration of alpha-I-Macroglobulin and
alpha-2-macroglobulin in he plasmas of male and female
rats. Experientia 32(10): 1348-1349, 1976.
Breuninger, L.M., W.L. Dempsey, J. Uhi, and D.M. Murasko.
Hydrocortisone
regulation
of
interleukin-6
protein
production by a purified population of human peripheral
blood monocytes. Clin. Immunol. Immunopathol. 69: 205214, 1993.
V.
and
C.
Kristofic.
Regulation
by
corticosteroids of Thl and Th2 cytokine production in
human CD4+ effector T cells generated from CD45R0 and
CD45RO subsets. J. Immunol. 155: 3322-3328, 1995
Brinkmann,
S.P.,
and H. Baumann. Insulin is a prominent
modulator of the cytokine-stimulated expression of acutephase plasma protein genes. Mol. Cell Biol. 12: 1789-
Campos,
1797, 1992.
Castell,
J.V.
T.
Andus,
D.
Kunz,
and
P.C.
Heinrich.
Interleukin-6. The major regulator of acute phase protein
synthesis in man and rat. Ann.NY Acad. Sci. 557: 87-101,
1989.
Chrousos, G.P. The hypothalamic-pituitary-adrenal axis and
immune-mediated inflammation. New Engl. J. Med. 332(20):
1351-1362, 1995.
Coe, J.E., S.S. Margossian, H.S. Slayter, and J.A., Sogn.
Hamster female protein, a new pentraxin structuarally and
functionally similar to C-reactive protein and amyloid P
component. J. Exp. Med. 153: 977-991, 1981.
T.
R.
and A.S.
Fauci.
Corticosteroid-mediated
immunoregulation in man. Immunol. Rev. 65: 133-155, 1982.
Cupps,
Davis, S.L. Environmental modulation of the immune system
via
the
endocrine
system.
Domestic
Animal
Endocrinology.15, 283-289,1998.
Davison, T.F. Effects of dietery corticosterone on the
growth and metabolism of immature Gallus domesticus.
General and
Comparative Endocrinology,
50,
463-468,
1983.
107
Waal
Malefyt,
R.,
H.
Yssel,
and M-G Roncarolo.
Interleukin-lO. Curr. Opin. Immunol. 4: 314-320, 1992.
de
J.
R.
and E. G. Harry. Laboratory trials with
inactivated vaccines
against
Escherichia
coli
02:Kl
infection in fowls. Res. Vet. Sci. 24:308-313. 1978.
Deb,
F. S., and B. S. McEwen.
Acute stress enhances
while chronic stress suppresses cell-mediated immunity in
vivo: a potential role for leukocyte trafficking.
Brain.
Behav., Immun. 11: 286-306. 1997.
Dhabhar,
Diefenbach, A.,
H.
Schinfler,
M.
Rollinghoff,
W.
M.
Yokoyama, and C. Bogdan.
Requirement for type 2 NO
synthase
for
IL-12
signaling
in
innate
immunity.
Science. 284: 951-955. 1999.
Dinarello, C.A., J.G. Cannon, and S.M. Wolff. New concepts
on the pathogenesis of
fever.
Rev.
Infec.
Dis.
10(1):
168-189, 1988.
Dinarello, C.A., J.G. Cannon, J. Mancilla, I. Bishai, J.
Lees,and F.
Coceani.
Interleukin-6
as
an endogenous
pyrogen: induction of prostaglandin E2 in brain but not
in peripheral blood mononuclear cells. Brain res. 562:
199-206, 1991.
Edwards, K. M., H. Gewurz, T. F. Lint, and C. Mold. A role
for
C-reactive
protein
in
the
complement-mediated
stimulation of human neutrophils by type 27 Streptococcus
pneumoniae. J. immunol. 128: 2493, 1982.
Falus, A. H. Rokita, E.Walcz, et al. Hormonal regulation
of
complement
biothesis
in
human
cell
lines.-II.
Upregulation of the biosynthesis of complement components
C3,
factor B and Cl inhibitor by interlukin-6 and
interlukin-1 in human hepatoma cell line. Mol. Immunol.
27: 197-201, 1990.
Fearon D. T. and R. M. Locksley. The indtructive role of
innate
immunity
in
the
acquired
immune
response.
Science. 272: 50-54, 1996.
Fearon D. T. Seeking wisdom in innate immunity. Nature
388: 323-324, 1997.
108
& Kushner, I. Acute-phase proteins and other
systemic responses to inflammation. New England Journal
of Medicine, 340, 448-454, 1999.
Gabay,
C
Ganrot, p. 0. and C.-0. Kindmark. C-reactive protein: I
phagocytosis-promoting
factor.
Scand.
J.
Clin.
Lab.
Invest. 24: 215, 1969.
Ganter, U., R. Arcone, C.
Ciliberto.
Dual
control
Toniatti, G. Morrone, and G.
of
C-reactive
protein gene
expression by interleukin-1 and interleukin-6. EMBO J.
3773-3779, 1989.
8:
Gauldie, J., C. Richards, D. Harnish, P. Lansdorp, and H.
Baumann. Interferon beta 2/B-cell stimulatory factor type
shares
identity with monocyte-derived
2
hepatocyte-
stimulating factor and regulates the major acute phase
protein response in liver cells. Proc. Natl. Acad. Sd.
USA. 84: 7251-7255, 1987.
Goldbach,
J.M.,
J.
Roth,
and
E.
Zeisberger.
Fever
suppression by subdiaphragmativ vagotomy in guinea pigs
depends on the route of pyrogen administration. Am J.
Physiol. 272: R675-681, 1997.
Gross, W.B. Effect of short-term exposure of chickens to
corticosterone on resistance to challenge exposure with
Escherichia
coli
and
antibody
response
to
sheep
erythrocytes. Am. J. Vet. Res. 53, 291-293, 1992.
J., P. Thompson, and B. Beutler. Dexamethasone and
pentoxifylline inhibit endotoxin- induced cachectin/tumor
necrosis factor synthesis at separate points in the
signaling pathway. J. Exp. Med. 172(1): 391-394, 1990.
Han,
Heinrich, P.C., J.V. Castell, T. Andus. Interleukin-6 and
the acute phase response. Biochem. J. 265: 621-636, 1990.
J.A.,
Kafatos,
Hoffman,
F.C.,
Janesway Jr.,
C.A.,
&
R.A.B.
Phylogenic
Ezekowitz,
perspectives
in
innate
immunity. Science 284, 1313-1318, 1999.
M.
and A. O'Garra. Biological properties
interleukin 10. Immunol. Today. 13: 198-200, 1992.
Howard,
and
Matsumoto,
of
Immunity
against
Escherichia coli infection in chickens assessed by viable
Huang,
H.J.
M.
109
bacterial counts in internal organs. Avian Diseases, 43:
469-475, 1999.
Huff, G.R., Huff, W.E., Balog, J.M. & Rath, N.C. (1999).
The effect of a second dexamethasone treatment on turkeys
previously challenged in an experimental Escherichia coli
respiratory
model
of
turkey
osteomyelitis
complex.
Poultry Science, 78, 1116-1125.
Husebekk,
A.,
B.
Skogen,
G.
Hurby.
Characterization of
amyloid proteins AA and SAA as apolipproteins of high
density lipoprotein (HDL). Displacement of SAA from the
HDL-SAA. complex by apo Al and apo All. Scand. J. Immunol.
25: 375-381, 1987.
Jefferies,
W.
McK.
Mild
adrenocortical
deficiency,
chronic allergies, autoimmune disorders and the chronic
fatigue syndrome: a continuation of the cortisone story.
Med. Hypotheses. 42: 183-189, 1994.
Joyce,
D.A.,
Steer,
modulation of
and
human
Abraham.
Glucocorticoid
monocyte/macrophage
function: control of TNF-alpha secretion. Inflamm. Res.
46: 447-451, 1997.
J.H.
L.
J.
Kindmark, C. 0. Stimulating effect of C-reactive protein
on
phagocytosis
of
various
species
of
pathogenic
bacteria.
Clin. Exp. Immunol. 8: 941, 1971.
Koj,
A.,
Travis.
Rokita, T. Kordula, A. Kurdowska,
Role of cytokines and growth factors
H.
and J.
in
the
induced synthesis of proteinase inhibitors belonging to
acute phase proteins. Biomed. Biochim. Acta. 50: 421-425,
1991.
Immunology.,
Company. 1-10, 1994.
Kuby,
J.
2nd edition.
W.
H.
Freeman and
I.
Regulation of the acute phase response by
cytokines. Perspect Biol. Med. 36: 611-622, 1993.
Kushner,
Kushner,
I.
The phenomenon of the acute phase response.
Ann. New York Acad. Sci. 389, 39-48, 1982
Kushner,
Gewurz
and
Benson.
C-reactive
protein and the acute phase response. J. Lab. Clin. Med.
I.,
H.
97:739-749. 1981.
M.
D.
110
Kushner, I., J.E. Volanakis, and H. Gewurz. Ann. New Yor
Acad. Sd. 389: 235-274, 1982.
Lafrancier, P., M. Derrien, X. Jamet, and J. Choay.
Apyrogenic,
adjuvant-active
N-acetylmuramyl-dipeptides.
J. Med. Chem. 25: 87-90. 1982.
Laskey, L.A. Science. 128: 964-969, 1992.
Lew, W., J.J. Oppenheim, and K. Matsusshima. Analysis of
the suppression of IL-i
and IL-i p roduction in huaman
peripheral
blood
mononuclear
adherent
cells
by
a
giucocorticoid hormone. J. Immunol. 140: 1895, 1988.
Liao, J., J.A. Keiser, W.E
Kluger. Am. J. Physiol.
268: R699-R706, 1995.
Scales, S.L. Kunkel. And M.J.
Regul.
Integr.
Comp.
Physiol.
Mackiewicz, A., M.K. Ganapathi, D. Schultz, D. Samols, J.
Reese, and I. Kushner. Regulation of rabbit acute phase
protein biosynthesis by monokines.
Biochem.
J.
253:
851, 1988.
Mackiewicz, A., I. Kushner, and H. Baumann.
Acute phase
proteins
molecular biology, biochemistry, and clinical
Applications. Boca Raton: CRC Press, 3-20, 1993.
:
Mangan, D.F., B. Robertson, and S.M. Wahl. IL-4 enhances
programmed cell death (apoptosis) in stimulated human
monocytes. J. Immunol. 148: 1812-1816, 1992.
and H.J.
Induction of short-term,
nonspecific immunity against Escherichia coli infection
in
chickens
is
suppressed
by
cold
stress
or
corticosterone treatment. Avian Pathol. (Accepted)
Matsumoto,
N.,
Huang.
R.T. Jr. Pathogenesis of the anemia of chronic
disease: a cytokine-mediated anemia. Stem Cells. 13: 32-
Means,
37, 1995.
Medhzhitov, R,
P. Preton-Hurlburt, and C.A. Janeway Jr. A
human homologue of the Drosophila Toll protein signals
activation of adaptive immunity. Nature. 388: 394-397,
1997.
111
Mendel,
The
hormone
hypothesis:
a
physiologically based mathematical model. Endocr. Rev.
10: 232-274, 1989.
C.M.
free
C.,
K.
M.
Edwards, and H. Gewurz. Effect of Creactive protein on the complement-mediated stimulation
of human neutrophils by Streptococcus Pneumoniae serotype
Mold,
3 and 6, Infect. Immun. 37: 987-992, 1982.
Morley, J.J., and I. Kushner.
Serum C-reactive protein
levels in disease.
Ann. N.Y. Acad. Sd. 389: 406-418.
1982.
Munck, A., P.M. Guyre, and N.J. Holbrook, Physiological
functions of glucocorticoids in stress and their relation
to pharmacological actions. Endocr. Rev. 5: 23-44, 1984.
A.
and A. Naray-Fejes-Toth. Glucocorticoids and
stress: permissive and suppressive actions. Ann. New York
Acad. Sd. 746: 115-130, 1994.
Munck,
Nishimura,
T.
Kawano,
Y.
Tanaka,
S.
Taniguchi, and Y. Yoshikal. Regulatory role
of peritoneal NK 1.1 + a T cells in IL-12 production
during Salmonella infection. J. Immunol. 163: 2057-2063.
Naiki,
Y.,
Itohara, M.
H.
1999.
Nakamura, K., N. Yuasa, and F. Abe.
Effect of infectious
bursal
disease
virus
on
infections
produced
by
Escherichia coli
of high and low virulence in chickens.
Avian Pathol. 19: 713-721. 1990.
Nakamura, K., Y. Ibaraki,
Comparative pathology of
Z.
Mitarai, and T.
heart
and
liver
Shibahara.
lesions of
broiler chickens that died of ascites, heart failure, and
others. Avian Dis. 43:526-532. 1999.
National Institute of Health.. Institutional animal care
and use committee guideline.
NIH Publication No. 923415. 1992.
National
Research
Council.
Nutrient
requirement
of
poultry. 8th ed.
National Academy Press, Washington D.C.
1984.
112
Panigrahy, B., J.
E.
Gyimah,
C.
F.
Hall and J. D.
Williams.
Immunogenic
potency
of
an
oil-emulsified
Escherichia coli bacterin. Avian Dis. 28:475-481. 1984.
Perimutter,
D.H.,
C.A.
Dinarello,
P.1.
Punsal,
et
al.
Cachectin/tumor necrosis factor regulates hepatic acutephase gene expression. J. din. Invest. 78: 1349-1354,
1986.
Pepys, M.B. and Butler, P.J.G. Serum amyloid P component
the major
is
calcium-dependent
specific DNA binding
protein of the serum. Biochem Biophys. Res. Commun. 148:
308-313, 1987.
Pourbakhsh, S. A., M. Boulianne, B. Martineau-Doize, C.
M. Dozois, C. Desautels, and J. M. Fairbrother. Dynamics
of
Escherichia
coli
infection
in
experimentally
inoculated chickens.
Avian Dis. 41:221-233. 1997.
Prowse,
factor,
K.R.,
P2
and
H
Baumann.
interferone,
and
Hepatocyte-stimulating
interleukin-1
enhance
expression of the rat a1-acid glycoprotein gene via a
distal upstream regulatory region. Mol. Cell Biol. 8: 4251, 1988.
Qureshi, N., K. Takayama, and E. Ribi. Purification and
structural determination of nontoxic lipid A obtained
from the lipopolysaccharide of Salmonella typhimurium. J.
Biol. Chem. 257: 11808-11815. 1982.
Ratnam, S. and S. Mookerjea. The regulation of superoxide
generation and nitric oxide synthesis by C-reactive
protein. Immunology. 94: 560-568. 1998.
A., S. LaForge, and P.B. Sehgal. On the mechanism
for efficient repression of the interleukin-6 promoter by
glucocorticoids: enhancer, TATA box, and RNA start site
(mr motif) occlusion. Mol. Cell. Biol. 10: 5746-5746,
Ray,
1990.
Robey, F.A., K.D. Jones, and A.
D.
Steinberg. C-reactive
protein mediated the solubilization of nuclear DNA by
complement in vitro. J. Exp. Med. 161: 1344-1356, 1985.
Rogers, J.T., K.R. Bridges, G.P. Durmowicz, J. Glass, P.E.
Auron, and H.N. Munro. Translational control during the
113
acute phase response. Ferritin synthesis in response to
interlukin-1. J. Biol. Chem. 265:14572-14578, 1990
Ronnberg, B., M. Fekadu, and B. Morein. Adjuvant activity
of non-toxic Quillaja saponaria Molina components for use
in ISCOM matrix. Vaccine. 13:1375-1382. 1995.
Rot,
Endothelial cell binding of NAP-l/IL-8: role in
A.
neutrophil emigration. Immunol. Today. 13: 291-294, 1992.
Shephard,
E.
G.,
P.D.
VanHelden,
M.
Strauss,
L.
Bohm.,
and F. C. DeBeer. Functional effect of CRP binding to
nuclei. Immunology. 58: 489, 1986.
Sicardi,
F.
J.
Identification and disease producing
ability of Escherichia coli associated with E.
coil
infections of chickens and turkeys.
M. S. Thesis. Univ.
of Minnesota, St. Paul, MN. 1966.
R.
J.
Rent, and H. Gewurz. Interactions of Creactive protein with the complement system. I. Protamininduced consumption of complement in acute phase sera. J.
Exp. Med. 140: 631, 1974.
Siegel,
Siegel,
J.
R.
Rent,
and H.
Gewurz.
reactive protein with the complement
Interactions of Csystem.
II.
C-
reactive protein-mediated consumption by poly-L-lysine
polymers and other polycations. J. Exp. Med. 142: 709,
1975.
H. Rokita, M. Mackiewicz, S. Szla, and A. Koj.
Fed. Proc., Fed. Am. Soc. Exp. Biol. 45: 1587, 1986.
Sipe,
J.
P.K.,
and H. Udono. Heat shock proteinSrivastava,
peptide complexes
in cancer
immunotherapyCurr.
Opin.
Immunol. 6: 728-732, 1994.
Steel, D.M., and A.S. Whitehead. Heterogenous modulation
of acute-phase-reactant mRNA levels by inter1eukin-1
and
interleukin-6
in
the
human
Biochem.J. 277: 477-482, 1991.
cell
line
PCL/PRF/5.
D.M., and A.S. Whitehead. The major acute phase
reactants: C-reactive protein, serum amyloid P component,
and serm amyloid A protein. Immunol. Today. 15: 81-88,
Steel,
1994.
114
Suto,
R.
and
P.K.
Srivastava.
A mechanism
for
the
specific immunogenicity of heat shock protein-chaperoned
peptides.Science. 269: 1585-1588, 1995.
M.
and M. Matsumoto.
Immune defense mechanism
against blood-borne Pasteurella multocida in turkeys.
Res. Vet. Sci. 48:344-349. 1990.
Tsuji,
Uhiar, C.M. and A.S.Whitehead. Serum amyloid A, the major
vertebrate
acute-phase
reactant.
Eur.
J.
Biochem.
265(2) :501-23, 1999.
Van Gaol, J., W. Boers, M. Sala, and N.C. Ladiges.
Glucocoticoids and catecholamines as mediators of acutephase
proteins,
especially
rat
a-macrofoetoprotein.
Biochem. J. 220: 125-132, 1984.
Vieira, P.1., Kalinski, P., Wierenga, E.A., Kapsenberg,
M.L. & de Jong, E.C. Glucocorticoids inhibit bioactive
IL-12p70 production by in vitro-generated human dendric
cells
without
affecting
their
T
cell
potential. J Immunol, 161, 5245-5251, 1998.
stimulatory
M-Y Lin, B.L. Lohrnan, S.M. Varga, C.C.
Zarozinski, and L.J. Selin. c4 and yö T-cell networks and
their roles in natural resistance to viral infections.
Immunol Rev. 159: 79-93. 1997.
Welsh,
R.
M.,
Wilckens,
T.
physiological
Glucocorticoids
and
immune
function:
relevance
and
pathogenic potential
of
hormonal dysfunction.Trends Pharmacol. Sci. 16: 193-197,
1995.
Williams,
and P. G., Hellewell.
Endothelial cell
biology. Adhesion molecules involved in the microvascular
inflammatory response. Am. Rev. Respir. Dis. 146: S45S50. 1992.
Xu,
L.,
T.
J.
Badolato,
Murphy.,
et
al.
A novel
biologic function of serum amyloid A. Induction of T
lymphocyte migration and adhesion. J. Immunol. 155: 11841190, 1995.
R.
W.J.
115
K.,
and R.F. Mortensen. Macrophage tumoricidal
activity induced by human C-reactive protein. Cancer Res.
46: 5077-5083, 1986.
Zahedi,