Localization of African swine fever viral antigen, swine IgM, IgG and C1q in lung and liver tissues of experimentally infected pigs


Fernandez, A.; Perez, J.; Martin de las Mulas, J.; Carrasco, L.; Dominguez, J.; Sierra, M.A.

Journal of Comparative Pathology 107(1): 81-90

1992


An immunohistological study was carried out on lungs and livers of pigs experimentally infected with two different African swine fever virus (ASFV) isolates. ASFV antigen, swine immunoglobulins (IgM and IgG) and (Clq) complement were demonstrated in both organs at different stages of infection. The ASFV antigen was mainly found in mononuclear phagocytic system (MPS) cells. Immunoglobulins and complement were observed in plasma, infected and non-infected phagocytic cells and cell debris. These findings suggest the presence, in acute infection, of immune complexes which may be involved in immunopathogenic mechanisms.

J.
Comp.
Path.
1992
Vol.
107,
81-90
Localization
of
African
Swine
Fever
Viral
Antigen,
Swine
IgM,
IgG
and
Chi
in
Lung
and
Liver
Tissues
of
Experimentally
Infected
Pigs
A.
Fernandez,
J.
Perez,
J.
Martin
de
las
Mulas,
L.
Carrasco,
J.
Domineuez*
and
M.
A.
Sierra
Departments
of
Histology
and
Pathological
Anatomy,
Veterinary
Faculty,
University
of
Cordoba
and
*Department
of
Virology,
INIA,
Madrid,
Spain
e
..--
AAAAAA
y
An
immunohistological
study
was
carried
out
on
lungs
and
livers
of
pigs
experimentally
infected
with
two
different
African
swine
fever
virus
(ASFV)
isolates.
ASFV
antigen,
swine
immunoglobulins
(IgM
and
IgG)
and
(C
1
q)
complement
were
demonstrated
in
both
organs
at
different
stages
of
infection.
The
ASFV
antigen
was
mainly
found
in
mononuclear
phagocytic
system
(MPS)
cells.
Immunoglobulins
and
complement
were
observed
in
plasma,
infected
and
non-infected
phagocytic
cells
and
cell
debris.
These
findings
suggest
the
presence,
in
acute
infection,
of
immune
complexes
which
may
be
involved
in
immunopathogenic
mechanisms.
Introduction
African
swine
fever
(ASF)
is
an
important
disease
caused
by
a
deoxivirus
of
intracytoplasmic
development
affecting
domestic
pigs
and
wild
boars
(Hess,
1971)
.
The
ASF
virus
replicates
mainly
in
mononuclear
phagocytic
system
(MPS)
cells
(Maurer,
Grisemer
and
Jones,
1958;
Colgrove,
Haelterman
and
Coggins,
1969;
Coggins,
1974;
Pan,
1987).
The
interaction
with
and
destruction
of
this
system
seems
to
be
a
central
feature
of
the
disease
(Mebus,
1988).
However,
virus
replication
has
also
been
reported
in
c,ther
cell
types
(Goggins, 1974;
Casal,
Enjuanes
and
Vinuela,
1984;
Edwards,
Dodds
and
Slauson,
1985).
There
are
different
clinical
and
pathological
forms
of
the
disease
associated
with
virus
virulence,
which
is
itself
dose-dependent
(Pan
and
Hess,
1984).
Thus,
highly
virulent
virus
can
cause
high
mortality
with
specific
antibodies
npppn
r
i
ng
at
4.
tr,
1
(-lays
pnet-innrillatinn
(Wardley
and
Willeinenn
1
CiStfl;
Mebus,
1988).
Sera
from
pigs
infected
in
this
way
and
from
other
virus-
resistant
animal
species
inoculated
with
ASFV
do
not
neutralize
the
virus
(Hess,
1971),
although
they
can
fix
complement
and
mediate
some
immuno-
pathological
reactions,
such
as
complement-
and
antibody-dependent
cellular
cytotoxicity
(Norley
and
Wardley,
1982,
1983).
The
presence
of
immunocomplexes
in
chronic
ASFV-infected
tissues
has
0021-9975/92/050081
+
10
$08.00/0
(e)
1992
Academic
Press
Limited
82
A.
Fernandez
et
al.
been
reported
(Moulton,
Pan,
Hess,
DeBoer
and
Tessler,
1975;
Slauson
and
Sanchez-Vizcaino,
1981;
Pan,
1987).
However,
such
studies
have
not
been
carried
out
in
acute
African
swine
fever.
In
this
study,
we
show
the
distribution
of
ASFV
antigen,
immunnglnhudins
(Tg1\4
and
IgG)
and
complement
(C:1
q)
in
lung
and
liver
tissues
from
pigs
experimentally
infected
with
two
ASFV
isolates
of
different
virulence.
Meth^As
Eighteen
Large
White
X
Landrace
male
pigs
were
used,
each
with
a
live
weight
of
approximately
20
kg
at
the
beginning
of
the
experiment
and
free
from
parasitic
and
infectious
diseases.
They
were
divided
into
two
groups
(I
and
II)
each
of
nine
animals,
which
were
inoculated
with
the
different
strains
of
the
ASFV.
Groups
I
and
II
were
divided
into
three
subgroups,
each
consisting
of
three
animals;
two
animals
from
each
subgroup
received
intramuscular
inoculations
of
5
X
10'
50
per
cent
haemadsorbing
doses
(HAD„)
of
either
the
highly
virulent
E70
isolate
(group
I)
or
the
attenuated
E75
isolate
(group
II)
of
ASF
virus
(both
supplied
by
Instituto
Nacional
de
Investigaciones
Agrarias
de
Madrid);
the
third
animal
from
each
subgroup
(non-inoculated
pig)
served
as
a
control.
Animals
were
killed
at
3,
5
and
7
days
post-inoculation
(dpi)
(group
I)
and
at
9
and
11
dpi
(Group
II).
Two
animals
remaining
from
group
II
scheduled
to
be
killed
at
day
13,
were
found
dead
on
day
12
dpi.
Animals
were
killed
by
anaesthetizing
with
azaperone
and
thiopentone
followed
by
vascular
perfusion
(Sierra,
Carrasco,
Gomez-Villamandos,
Martin
de
las
Mulas,
Mendez
and
Jover,
1990).
Organs
were
fixed
by
perfusion
in
2.5
per
cent
glutaraldehyde
in
0.1
i
phosphate
buffer;
samples
of
lung
and
liver
were
embedded
in
paraffin
wax
and
araldite.
For
structural
study,
sections
of
various
thicknesses,
dependent
on
the
type
of
embedding,
were
cut.
Staining
techniques
used
were
haematoxylin
and
eosin
(HE),
and
toluidine
blue.
Biotinylated
swine
IgG-anti
ASF'V
(kindly
supplied
by
the
Virology
department
of
INIA,
Madrid)
was
used
to
detect
ASFV
antigen,
as
described
elsewhere
(Fernandez,
Sierra,
Carrasco,
Wohlsein
and
Jover,
1990).
In
order
to
demonstrate
swine
immunoglobulins
and
complement
(Clq),
two
polyclonal
antisera
directed
against
swine
IgG
and
swine
Clq
(both
raised
in
rabbits)
were
utilized
by
a
peroxidase—antiperoxidase
method
(PAP).
A
monoclonal
anti-swine
IgM
antibody
(supplied
by
the
Virology
department
of
INIA,
Madrid)
was
used
with
the
avidin-biotin-peroxidase
complex
(ABC)
method.
For
negative
controls,
uninfected
pig
tissues
were
used,
as
well
as
other
routine
negative
controls
as
described
elsewhere
(Fernandez
et
al.,
1990).
Results
African
Swine
Fever
Virus—E70
isolate
Liver.
In
the
liver,
only
scattered
pyknotic
cells
were
observed
in
the
sinusoids
at
a
dpi.
However,
a
progressive
multifocal
parenchymal
necrosis
was
evident
at
5
and
7
dpi.
At
3
dpi,
the
ASFV
antigen
was
only
observed
in
the
cytoplasm
of
both
Kupffer
cells
and
circulating
monocytes.
Some
of
these
cells
showed
the
haemadsorption
phenomenon.
A
C
T
s:
+1.
7
:
4
a..L.gen
was
more
iiequeutiy
ooser
-
VLA.1.
kJ
a
1
ill
1
Lip.1
3
since
was
only
found
in
Kupffer
cells
and
circulating
monocytes,
but
also
in
hepatocytes
AST
virus
antigen
and
immunoglobulins
/33
and
interstitial
histiocytes.
A
doubtful
reaction
was
observed
in
some
neutro-
phils
and
endothelial
cells.
At
3,
5
and
7
dpi,
a
positive
specific
immunoreaction
against
IgM,
IgG
and
rlq
was
r^nstqntly
nhserveri
in
pinsma
Scattered
rirridnting
ninrinrytPs,
Kupffer
cells
and
interstitial
macrophages
showing
degenerative
changes
were
also
IgM
and
C
1
q
immunoreactive.
At
7
dpi,
some
interstitial
plasma
cells
showed
cytoplasmic
positivity
against
either
IgM
or
IgG.
Luna
.
The
main
rhanges
in
the
lung
were
ennfined
to
the
interstitial
tissues
Thus,
an
increase
in
the
number
of
septal
cells
was
observed
3
dpi,
while
abundant
necrotic
cells
were
seen
by
5
and
7
dpi.
ASFV
antigen
was
observed
only
in
the
interstitial
tissue.
A
large
number
of
intravascular
mononuclear
cells
showed
intracytoplasmic
immunoreactivity
at
1
5
and
7
flpi
These
cells
showed
different
degrees
of
necrosis,
with
the
highest
number
of
infected
cells
seen
at
5
dpi.
At
3
dpi,
IgM
and
C
1
q
were
found
in
the
cytoplasm
of
some
intravascular
mononuclear
cells,
viable
and
necrotic
as
well
as
in
intravascular
plasma.
No
IgG
was
observed
in
these
cells
but
it
was
present
in
plasma.
At
5
dpi,
an
intense
positive
specific
immune
reaction
against
IgM
and
Clri
was
seen
in
mononuclear
cells
and
plasma.
IgG
was
also
present
in
the
same
locations
but
associated
with
a
lower
number
of
mononuclear
cells
and
cell
debris.
In
bronchiolar
lymph
follicles,
some
plasma
cells
showed
cytoplasmic
IgM
or
IgG.
The
immune
reaction
at
7
dpi
was
very
intense
for
IgM
(Fig.
6)
and
for
C
lq,
but
was
of
lower
intensity
for
IgG.
This
was
found
in
plasma,
cytoplasm
of
mononuclear
cells
and
cell
debris.
The
intracytoplasmic
reaction
was
either
diffuse
or
granular.
African
Swine
Fever
Virus—E75
Isolate
Liver.
Kupffer
cell
hyperpiasia
showing
haemadsorption
was
present
at
9
dpi.
At
11
dpi
abundant
intrasinusoidal
necrosis
associated
with
neutrophilic
and
lymphocytic
cells
was
observed.
Equally,
multifocal
necrosis
and
severe
interstitial
tissue
oedema
were
the
most
significant
liver
lesions.
At
9
dpi,
several
cell
types
displayed
haemadsorption
and
showed
cytoplas-
mic
ASFV
antigen
(Fig.
1).
At
11
dpi,
a
marked
decrease
in
the
number
of
cells
showing
cytoplasmic
ASFV
antigen
was
observed.
This
included
macrophages,
neutrophils
and
cell
debris.
Only
some
mononuclear
cells
showed
ASFV
antigen
at
12
dpi
(animals
that
were
found
dead).
At
9
dpi,
immune
complexes
were
observed
particularly
in
those
Kupffer
cells
showing
haemadsorption
(Fig.
2).
immunoglobulins
and
complement
stained
within
cell
cytoplasm.
Extracytoplasmic
staining
was
diffuse
within
vessels,
as
well
as
in
the
interstitial
tissues.
At
11
dpi
and
12
dpi,
a
few
mononuclear
cells
and
neutrophilic
cells
containing
immunoglobulins
and
complement
in
the
form
of
small
intracyto-
plasmic
globules
were
seen.
The
reaction
was
also
associated
with
cell
necrosis
and
with
plasma.
84
A.
Fernandez
et
al.
Lung.
At
9
dpi
the
inter-alveolar
septa
showed
abundant
mononuclear
cells.
At
11
dpi
a
diffuse
intravascular
cell
necrosis
and
alveolar
macrophages
associated
with
cell
debris
and
fibrin
were
present.
These
lesions
were
increased
at
12
dpi.
ASFV
antigen
was
particularly
abundant
in
intravascular
macrophages
at
9
dpi,
however,
it
decreased
substantially
in
these
cells
at
11
and
12
dpi,
at
which
time,
the
alveolar
macrophages
showed
cytoplasmic
ASFV
antigen
(Fig.
3)
Immunoglobulins
and
complement
were
visible
within
and
outside
cells
at
9
dpi.
By
11
and
12
dpi
they
were
observed
within
alveolar
macrophages
(Figs
4
and
5),
especially
in
those
showing
clear
signs
of
necrosis.
Neutrophils,
intravascular
and
alveolar
macrophages
were
found
to
contain
small
intracyto-
plasmic
granules
which
reacted
positively
to
immunoglobulins
and
comple-
ment.
Discussion
The
liver
and
the
lung
are
considered
as
secondary
viraemic
organs
in
ASFV
infection
(Maurer
et
al.,
1958;
Hess,
1971;
Coggins,
1974).
Both
organs
contain
a
large
number
of
MPS
cells,
which
are
the
natural
primary
target
cells
for
ASFV
(Colgrove
et
al.,
1969;
Hess,
1971;
Pan,
1987),
as
we
have
also
seen
in
the
present
work
using
two
ASFV
isolates
of
different
virulence.
An
early
and
rapid
destruction
of
MPS
cells
is
the
major
pathogenic
mechanism
in
the
development
of
acute
African
swine
fever
(Moulton
and
Coggins,
1968;
Mebus,
1988),
which
is
characterized
by
liver
necrosis,
conges-
tion,
acute
alveolar
oedema,
and
hepatic
and
pulmonary
haemorrhage
(Maurer
et
al.,
1958;
Moulton
and
Coggins,
1968;
Coggins,
1974).
The
early
Kupffer
cell
necrosis
probably
allows
the
entry
and
replication
of
the
virus
in
hepatocytes
which
have
been
observed
in
highly
virulent
ASFV
infections
(Coggins,
1974;
Sierra,
Bernabe,
Mozos,
Mendez
and
Jover,
1987).
In
the
lung,
the
same
ASFV
isolate
(E70)
infects
intravascular
macrophages
(Pan,
1987;
Sierra
et
al.,
1990),
which
in
contrast
to
Kupffer
cells,
were
hyperplastic
and
showed
a
lower
necrosis
rate
in
all
stages
of
infection.
This
may
be
due
to
a
different
behaviour
of
the
virus-macrophage
interaction
in
each
organ
(Pan,
1987).
At
7
dpi,
the
ASFV
antigen
observed
was
enough
to
confirm
the
diagnostic
value
of
both
organs
by
immunohistological
methods.
A
similar
number
of
infected
cells
was
also
seen
at
9
dpi
(ASFV-E75),
but
at
11
dpi
and
thereafter
a
marked
decrease
of
antigen
is
frequently
found
(Pan
and
Hess,
1984;
Edwards
et
al.,
1985).
This
could
be
due
to
either
the
masking
of
the
antigen
by
Fig.
1.
ASFV
antigen
in
Kupffer
cells
and
circulating
monocytes
showing
haemadsorption
(arrows)
(9
dpi
ASFV-E75).
Avidin-biotin-peroxidase
complex
(ABC)
x
300.
Fig.
2.
Kupffer
cells
showing
haemadsorption
and
cytoplasmic
swine
Clq
immune
reactivity
(arrows)
(9
dpi
ASFV-E75).
PAP
x
300.
Fig.
3.
ASFV
antigen
in
alveolar
macrophages
at
11
dpi.
These
cells
show
cytoplasmic
immunoreactive
inclusions
(arrows).
(ASFV-E75).
ABC
x
300.
ASF
virus
antigen
and
immunoglobulins
85
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ASF
virus
antigen
and
immunoglobulins
87
endogenous
specific
antibodies,
since
at
this
time
both
circulating
and
tissue
anti-virus
antibodies
are
detected
(Heuschele,
Goggins
and
Stone,
1966;
Edwards
et
al.,
1985;
Fernandez
et
al.,
1990),
and/or
to
the
poor
sensitivity
of
the
technique
to
demonstrate
small
amounts
of
ASFV
antigen
immunohisto-
logically
(Fernandez,
Perez,
Carrasco,
Sierra,
Sanchez-Vizcaino
and
Jover,
1992).
The
role
of
immunoglobulins
and
complement
in
acute
African
swine
fever
is
still
unclear.
It
has
been
observed
that
animals
which
died
most
quickly
showed
the
earliest
antibody
response
at
3
to
4
dpi,
found
by
radioimmuno-
assay
(RIA)
(Wardley
and
Wilkinson,
1980).
Complement
fixing,
but
not
precipitating,
antibodies
were
also
found
in
the
acute
disease
(Dardiri
and
Hess,
1970).
The
presence
of
IgM,'
C
1
q
and
IgG
associated
with
plasma,
ASF
virus
antigen-positive
and
-negative
cells
and
cell
debris
in
both
infections
could
represent
an
antibody
response
to
ASFV
and,
therefore,
the
possibility
of
forming
immune
complexes.
The
ability
of
such
immunocomplexes
to
mediate
infection
is
difficult
to
establish,
since
in
vitro
tests
have
shown
that
the
Fc
receptor
does
not
mediate
ASFV
replication
in
macrophages
(Alcami
and
Villuela,
1991).
However,
the
involvement
of
IgM,
complement
and
comple-
ment
receptors
has
not
been
demonstrated
and
should
be
investigated,
as
in
other
viral
diseases
(Sissons
and
Borysiewicz,
1985).
Hypergammaglobulinaemia
and
immunocomplexes
have
been
found
in
chronically
infected
ASFV
lung
and
renal
tissues
(Pan,
DeBoar
and
Heuschele,
1970;
Pan,
Moulton
and
Hess,
1975;
Slauson
and
Sanchez-Vizcaino,
1981).
We
have
also
found
that
IgM
participates,
as
well
as
IgG
and
complement,
in
the
early
alveolar
pneumonic
lesions
at
11
dpi,
with
a
massive
destruction
of
intravascular
macrophages
and
scarce
ASFV
antigen.
At
this
time
of
infection
the
immune
complexes
were
particularly
associated
with
alveolar
macro-
phages,
which
were
also
infected.
In
contrast,
the
ASFV
antigen
in
intravascu-
lar
macrophages
was
significantly
higher
at
7
dpi
(ASFV-E70)
than
at
11
dpi
(ASFV-E75).
These
observations
might
establish
a
hypothetical
limit
between
acute
and
subacute-chronic
African
swine
fever
lung
lesions
in
which
the
pulmonary
intravascular
macrophages
could
play
a
central
role
(Sierra
et
al.,
1990).
In
this
way,
the
immunocomplexes
would
stimulate
the
phagocytic
ability
of
the
intravascular
macrophages
at
5
and
7
dpi
(Smith,
Jachimowicz
and
Bingham,
1986;
Mims,
1987)
resulting
in
a
higher
production
and
secretion
of
active
substances
that
mediate
many
circulatory
mechanisms,
such
as
alveolar
oedema
(Bertram,
1986;
Bertram,
Eling
and
Brody,
1989),
the
most
frequently
found
lung
lesion
in
acute
African
swine
fever.
Fig.
4.
Swine
Clq
in
alveolar
macrophages
(arrows)
at
11
dpi
(ASFV-E75).
Some
of
them
show
evident
signs
of
necrosis
and
negative
alveolar
macrophages
are
also
present.
PAP
x
300.
Fig.
5.
Swine
IgM
positive
cells
distributed
throughout
the
lung
parenchyma
at
11
dpi
in
the
same
locations
as
Clq
(arrows).
(ASFV-E75).
ABC
x
150.
Fig.
6.
Swine
IgM
associated
with
plasma,
intravascular
mononuclear
cells
and
cell
debris
at
7
dpi.
(ASFV-E70).
ABC
x
375.
00
0 0
A.
Fernandez
et
al.
On
the
other
hand,
the
destruction
of
most
of
the
intravascular
macro-
phages
(Sierra
et
al.,
1990),
observed
at
11
dpi
with
the
moderately
virulent
virus
isolate
confirmed
that
the
antibodies
created
failed
to
prevent
cell
lysis,
or
to
inhibit
haemadsorption,
although
these
phenomena
have
been
observed
in
vitro
by
other
authors
(Ruiz
Gonzalvo,
Caballero,
Martinez
and
Carnero,
1986).
The
massive
destruction
o
n
f
intravascular
macrophages
may
ti_--
ye
particularly
due
to
the
cytopathic
effect
of
the
virus
replication.
However,
the
possibility
cannot
be
ruled
out
of
an
immune-mediated
cytotoxicity,
such
as
antibody-
dependent
cytotoxicity
(ADCC)
and/or
complement-mediated
cell
lysis
observed
in in
vitro
studies
of
ASFV
infection
(Norley
and
Wardley,
1982,
1983
1
since
at
11
dpi
neutrophils,
lymphocytes,
monocytes
and
macrophages
were
observed
associated
with
cell
necrosis.
These
cells
might
mediate
cytotoxic
phenomena
(Forman,
Wardley
and
Wilkinson,
1983;
Mims,
1987).
It
may
thus
be
concluded
that
immune
complexes
can
be
present
in
acute
African
swine
fever
infections,
especially
when
moderately
virulent
virus
is
involved,
probably
playing
a
role
in
different
immunopathogenic
mechanisms
that
should
be
more
closely
investigated
in
vivo.
Acknowledgments
Work
on
this
paper
was
supported
by
grants
from
"ComisiOn
Asesora
de
Investigacion
Cientifica
y
Tecnica"
and
"Junta
de
Andalucia".
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