Laboratory-scale inactivation of African swine fever virus and swine vesicular disease virus in pig slurry


Turner, C.W.lliams, S.

Journal of Applied Microbiology 87(1): 148-157

1999


Two methods were evaluated for the inactivation of African swine fever (ASV) and swine vesicular disease (SVD) viruses in pig slurry: chemical treatment and heat treatment. The addition of NaOH or Ca(OH)2 at different concentration/time combinations at 4 degrees C and 22 degrees C was examined, as was virus stability at different temperature/time combinations. ASF virus (ASFV) was less resistant to both methods than SVD virus (SVDV). In slurry from one source, ASFV was inactivated at 65 degrees C within 1 min, whereas SVDV required at least 2 min at 65 degrees C. However, it was found that thermal inactivation depended on the characteristics of the slurry used. Addition of 1% (w/v) of NaOH or Ca(OH)2 caused the inactivation of ASFV within 150 s at 4 degrees C; 0(.)5% (w/v) NaOH or Ca(OH)2 required 30 min for inactivation. NaOH or Ca(OH)(2) (1% (w/v)) was not effective against SVDV at 22 degrees C after 30 min, and 1(.)5% (w/v) NaOH or Ca(OH)2 caused inactivation of SVDV at both 4 degrees C and 22 degrees C. At higher chemical concentrations or temperatures, ASFV and SVDV inactivation was faster in slurry than in buffered medium.

Journal
of
Applied
Microbiology
1999,
87,
148-157
Laboratory-scale
inactivation
of
African
swine
fever
virus
and
swine
vesicular
disease
virus
in
pig
slurry
C.
Turner'
and
S.M.
Williams
2
1
Silsoe
Research
Institute,
Bedford,
and
2
lnstitute
for
Animal
Health,
Pirbright,
UK
7112/03/99:
received
3
March
1999
and
accepted
8
April
1999
C.
TURNER
AND
S.M.
WILLIAMS.
1999.
Two
methods
were
evaluated
for
the
inactivation
of
African
swine
fever
(ASV)
and
swine
vesicular
disease
(SVD)
viruses
in
pig
slurry:
chemical
treatment
and
heat
treatment.
The
addition
of
NaOH
or
Ca(OH)
2
at
different
concentration/time
combinations
at
4
°C
and
22
°C
was
examined,
as
was
virus
stability
at
different
temperature/time
combinations.
ASF
virus
(ASFV)
was
less
resistant
to
both
methods
than
SVD
virus
(SVDV).
In
slurry
from
one
source,
ASFV
was
inactivated
at
65
°C
within
1
min,
whereas
SVDV
required
at
least
2
min
at
65
°C.
However,
it
was
found
that
thermal
inactivation
depended
on
the
characteristics
of
the
slurry
used.
Addition
of
1%
(w/v)
of
NaOH
or
Ca(OH)
2
caused
the
inactivation
of
ASFV
within
150
s
at
4
°C;
0.5%
(w/v)
NaOH
or
Ca(OH)
2
required
30
min
for
inactivation.
NaOH
or
Ca(OH)
2
(1%
(w/v))
was
not
effective
against
SVDV
at
22
°C
after
30
min,
and
F5%
(w/v)
NaOH
or
Ca(OH)
2
caused
inactivation
of
SVDV
at
both
4
°C
and
22
°C.
At
higher
chemical
concentrations
or
temperatures,
ASFV
and
SVDV
inactivation
was
faster
in
slurry
than
in
buffered
medium.
INTRODUCTION
African
swine
fever
(ASF)
is
a
highly
contagious
viral
disease
of
wild
and
domestic
pigs.
In
endemic
areas
(the
southern
half
of
Africa),
wild
pigs
may
show
no
symptoms
of
the
disease.
However,
in
the
domestic
pig,
the
situation
is
quite
different.
Here,
ASF
can
be
very
serious,
with
some
strains
of
the
virus
causing
100%
mortality,
although
since
spreading
from
Africa
to
Europe,
the
virulence
of
some
isolates
of
ASF
has
decreased,
with
a
consequent
reduction
in
mortality
rates.
This
has
led
to
virus
being
carried
by
some
apparently
healthy
recovered
animals
(Wilkinson
1981)
and
these
may
pose
a
risk
to
healthy
pig
populations.
ASF
virus
(ASFV)
was
present
in
southern
Europe
from
its
arrival
in
Portugal
from
Africa
in
1957,
until
it
was
eradicated
from
Portugal
in
1993
and
from
Spain
in
1995.
It
is
still
present
in
Sardinia.
ASFV
is
a
large,
enveloped,
icosahedral
DNA
virus
of
approximately
200
nm
in
size.
It
is
generally
quite
resistant
to
inactivation,
and
can
survive
many
cycles
of
freezing
and
thawing.
It
is
resistant
to
pH
changes,
and
a
proportion
of
the
population
of
some
isolates
can
survive
at
pH
4
and
13
(Plowright
and
Parker
1967).
It
is,
however,
very
sensitive
to
Correspondence
to:
Dr
C.
Turner,
Silsoe
Research
Institute,
Wrest
Park,
Silsoe,
Bedford
MK45
4HS,
UK
(e-mail:
daire.turne
bsrc.ac.uk
).
drying,
and
is
readily
inactivated
by
lipid
solvents
because
of
its
envelope.
The
first
known
outbreak
of
swine
vesicular
disease
(SVD)
was
in
Italy
in
1966
(Nardelli
et
al.
1968),
although
it
may
have
had
another
origin.
Since
then,
there
have
been
many
outbreaks
across
Europe,
with
Great
Britain
and
Italy
being
particularly
affected
(Hedger
and
Mann
1989).
SVD,
while
not
usually
a
fatal
disease,
is
highly
contagious
and
produces
clinical
signs
that
are
indistinguishable
from
foot
and
mouth
disease
(mild
fever
and
vesicles
on
coronary
bands
of
foot
and
skin
of
limbs),
and
therefore
is
considered
a
serious
problem
for
differential
diagnosis
of
the
two
diseases.
SVD
virus
(SVDV)
is
a
member
of
the
Picornaviridae,
being
small
(about
30
nm),
icosahedral
and
lacking
an
envelope.
It
is
relatively
stable
over
a
wide
pH
range
(from
pH
2-12),
can
survive
many
days
without
loss
of
infectious
virus
titre
and
is
resistant
to
many
forms
of
inactivation.
In
the
UK,
in
the
interests
of
safeguarding
the
national
herd,
government
policy
is
that
outbreaks
of
either
disease
require
the
slaughter
of
pig
herds
and
the
decontamination
of
buildings
and
anything
that
has
come
into
contact
with
the
animals,
including
pig
slurry.
The
trend
towards
increasingly
large
and
intensively
reared
pig
herds
means
that
many
farms
have
insufficient
land
for
the
immediate
land
disposal
of
pig
slurry
and
manure.
This,
coupled
with
the
fact
that
winter
©
1999
The
Society
for
Applied
Microbiology
VIRUS
INACTIVATION
IN
PIG
SLURRY
149
slurry
spreading
carries
additional
pollution
risks,
means
that
pig
slurry
is
frequently
stored
for
4-6
months
prior
to
land
disposal.
The
Ministry
of
Agriculture,
Fisheries
and
Food
in
the
UK
(MAFF
1991)
strongly
recommend
a
storage
time
of
at
least
4
months.
As
a
result,
many
pig
farms
have
large
slurry
stores
(often
up
to
5000
tonnes),
and
should
a
disease
outbreak
occur,
the
slurry
stores
are
likely
to
be
contaminated
by
the
disease
agent.
Hence,
an
outbreak
of
ASF
or
SVD
is
likely
to
require
the
decontamination
of
large
quantities
of
animal
slurry
and/or
solid
manure.
Slurry
provided
the
initial
focus
for
the
investigation
of
suitable
decontamination
tech-
niques,
and
this
study
was
confined
to
investigations
into
the
inactivation
of
virus
in
slurry.
There
are
many
ways
of
inactivating
viruses.
These
include
physical
methods,
such
as
the
application
of
heat
(Herniman
et
al.
1973;
Monteith
et
al.
1986)
or
ionizing
radiation
(Vasl
et
al.
1983;
Farooq
et
al.
1993),
chemical
methods
using
chlor-
ine
(Lothrop
and
Sproul
1969;
Bosch
et
al.
1993),
ozone
(Warriner
et
al.
1985),
acids,
alkalis
etc.
(Herniman
et
al.
1973),
biological
methods
such
as
the
action
of
bacteria
or
proteases
(Deng
and
Cliver
1995),
or
the
use
of
aerobic
(Munch
et
al.
1987)
or
anaerobic
treatment
(Monteith
et
al.
1986).
Other
techniques
involve
physically
removing
the
virus
from
the
liquid
medium,
for
instance,
using
sand
col-
umn
filtration
(Powelson
and
Gerba
1994).
Although
nearly
all
of
these
methods
are
suitable
for
use
in
water
or
aqueous
solutions
with
low
dry
matter
(DM)
content,
only
a
limited
number
may
be
suitable
for
use
with
large
quantities
of
a
liquid
containing
substantial
levels
of
dry
matter,
such
as
animal
(pig)
slurry.
Others,
such
as
ozonation
or
u.v.
irradiation,
could
have
benefit
if
the
slurry
is
pre-clarified.
The
use
of
gamma
irradiation
is
limited
to
certain
viruses
(SVDV
is
considered
resistant)
unless
very
high
doses
are
used,
and
the
human
health
risks
at
large
scale
would
be
considerable.
The
same
concern
applies
to
ozonation
and
the
use
of
many
chemicals,
including
formalin,
which
is
highly
toxic.
Considering
the
factors
of
efficacy
and
reliability
plus
the
relative
costs
and
ease
of
scale-up
and
slurry
disposal
after
treatment,
two
techniques
were
identified
as
being
potentially
suitable
for
the
inactivation
of
ASFV
and
SVDV
in
pig
slurry:
heat
treatment,
and
dosing
with
an
alkaline
chemical,
specifically
sodium
hydroxide
or
calcium
hydroxide
(Turner
and
Burton
1997).
These
methods
are
relatively
easy
to
scale
up,
generally
inexpensive,
and
the
treated
slurry
(especially
after
heat
treatment)
can
be
disposed
of
in
the
usual
way,
i.e.
by
land
spreading.
Experimental
objectives
and
design
The
objectives
of
this
investigation
were
to
evaluate
two
alternative
methods
of
virus
inactivation:
heat
treatment
and
the
addition
of
calcium
or
sodium
hydroxide.
Both
approaches
were
applied
to
pig
slurries
inoculated
with
ASFV
or
SVDV,
to
determine
the
best
method
for
virus
inactivation,
and
to
identify
the
necessary
levels
of
treatment
to
meet
a
prescribed
standard
of
inactivation
under
practical
conditions.
The
level
of
inactivation
required
in
these
experi-
ments
was
set
at
a
10
4
-fold
reduction
of
infectious
virus
titre.
This
inactivation
level
was
set
because
it
is
the
standard
which
disinfectants
have
to
meet
in
order
to
be
certified
for
use
against
specific
viruses
in
the
UK.
It
would
have
been
imposs-
ible
to
verify
a
treatment's
efficacy
if
inoculating
with
low
levels
that
would
be
likely
to
occur
in
a
field
situation
(due
partly
to
the
relatively
high
minimum
detectable
levels),
so
a
high
titre
inoculum
was
necessary
to
reveal
the
effectiveness
of
the
inactivation
process.
Heat
treatment
and
experimental
design.
Virus
was
incu-
bated
in
slurry
at
various
temperatures
for
different
times
to:
(i)
determine
how
stable
ASFV
and
SVDV
were
at
different
temperatures
over
several
hours
in
both
slurry
and
EMEM;
(ii)
determine
at
which
temperatures
ASFV
and
SVDV
are
inactivated
in
slurry
within
15
min;
and
(iii)
determine
the
virus
inactivation
profile
of
ASFV
and
SVDV
in
slurry
at
different
temperatures
over
2-5
min.
Slurry
from
two
different
sources
was
used
in
the
heating
experiments.
Experiments
with
slurry
from
one
of
the
sources
(source
2)
were
performed
in
triplicate
and
in
this
case,
slurry
samples
were
diluted
by
tap
water
to
achieve
the
different
concentrations
of
total
solids.
Chemical
addition
and
experimental
design.
The
approach
here
was
to
find
a
range
of
chemical
concentrations
for
each
virus
which
would
lead
to
inactivation
within
30
min,
and
then
to
determine
the
concentrations
that
would
act
rapidly,
to
cause
inactivation
within
150
s.
All
chemical
inactivation
experiments
were
performed
using
slurry
from
a
single
source
(source
1).
MATERIALS
AND
METHODS
Virus
isolates
and
stock
preparation
ASFV
isolate
from
Malawi,
designated
Lilongwe
20/1
(Haresnape
1984),
was
obtained
from
infected
pig
spleen,
which
was
macerated
with
sterile
sand
and
added
to
90
ml
Eagle's
minimal
essential
medium
(EMEM)
containing
1%
ox
serum
and
1%
penicillin/streptomycin
(antibiotic)
solu-
tion
(containing
penicillin
at
10
000
U
m1
-1
and
streptomycin
at
10
pg
m1
-1
).
The
mixture
was
clarified
by
centrifugation
to
remove
gross
particles
and
stored
at
4
°C.
The
SVDV
strain
used
was
a
tissue
culture
adapted
strain
(UKG25/72)
grown
in
IB-RS2
(renal
swine)
cells.
©
1999
The
Society
for
Applied
Microbiology,
Journal
of
Applied
Microbiology
87,
148-157
150
C.
TURNER
AND
S.M.
WILLIAMS
Assays
Given
the
number
of
samples
generated
for
virus
assay,
a
detection
system
allowing
multiple
assays
was
required.
For
the
assay
of
ASFV,
a
microtitre
plate
system
was
chosen,
using
96
well
plates
seeded
with
100
µI
of
1.5
x
10
m1
-1
pig
bone
marrow
(PBM)
cells
in
Earle's
saline
solution.
A
plaque
assay
in
IB-RS2
(pig
kidney)
cells
was
used
to
detect
SVDV,
using
confluent
cell
sheets
on
6
well
plates.
ASFV
assay.
Samples
were
serially
diluted
10-fold
in
virus
diluent
(phosphate-buffered
saline
(PBS)
containing
1%
anti-
biotic
solution,
1%
ox
serum
and
0.1%
phenol
red).
Four
replicates
of
each
dilution
(50
µI
well
-1
)
were
added
to
PBM
cell
cultures.
The
plates
were
sealed,
shaken
gently
and
incu-
bated
for
6
d
at
37
°C
in
air
containing
5%
CO
2
.
At
the
end
of
this
incubation
period,
the
wells
were
examined
micro-
scopically
for
haemadsorption
(HAD)
and
any
well
containing
haemadsorbing
cells
was
designated
positive.
To
improve
the
sensitivity
of
the
assay,
20
µ1
of
a
fresh
1%
erythrocyte
suspension
was
then
added
and
the
plates
were
incubated
for
a
further
3
d
prior
to
final
examination.
The
infectious
virus
titre
(HAD
50
m1
-1
)
was
calculated
from
the
number
of
posi-
tive
wells
observed
at
each
dilution.
In
EMEM,
the
limit
of
detection
was
10
13
HAD
50
m1
-1
,
which
corresponds
to
two
out
of
four
positive
wells
at
zero
dilution.
Titres
below
this
where
haemadsorption
occurred
(one
positive
well
at
zero
dilution)
were
described
as
'trace'.
SVDV
assay.
Samples
were
serially
diluted
10-fold
in
virus
diluent,
and
200
µI
of
each
dilution
were
added,
in
duplicate,
to
a
confluent
monolayer
of
IB-RS2
cells
on
each
well
of
a
6
well
plate.
These
cells
were
incubated
in
an
incubator
pro-
viding
5%
CO
2
in
air
at
37
°C
for
1
h
and
were
then
overlaid
with
2
ml
Eagle's
overlay
medium
supplemented
with
2%
serum,
1%
antibiotics
solution
and
0.8%
Noble
agar.
After
incubating
for
a
further
48
h,
the
infectious
virus
titre
(pfu
m1
-1
)
was
determined
following
staining
with
2
ml
methylene
blue
solution,
counting
plaques
and
adjusting
to
get
an
infec-
tious
virus
titre
m1
-1
. In
EMEM,
the
limit
of
detection
was
10
07
pfu
m1
-1
.
Inoculation
of
slurry
with
virus.
Slurry
was
seeded
with
virus
to
give
a
10%
(v/v)
virus
suspension
and
mixed
gently.
Recovery
of
virus
from
slurry.
ASFV:
Equal
volumes
of
ox
serum
and
slurry
containing
ASFV
were
combined,
stirred
for
30
min,
centrifuged
at
7000
g
for
1
min
and
the
super-
natant
fluid
assayed
as
described
above.
The
lowest
detectable
level
for
virus
extracted
in
this
way
was
10
18
HAD
50
m1
-1
.
SVDV:
Equal
volumes
of
Freon
(1,1,2
trichlorofluoroethane)
and
slurry-virus
suspension
were
combined,
vigorously
shaken
for
a
few
minutes,
and
centrifuged
at
8000g
for
90
s.
The
top
layer
was
then
assayed
as
described
above.
The
lowest
detectable
level
for
virus
extracted
and
assayed
in
this
way
was
10
°7
pfu
m1
-1
.
Slurries
Two
sources
of
slurry
were
used
in
these
experiments
from
two
commercially
operated
pig
farms,
and
these
were
des-
ignated
Source
1
and
Source
2.
Slurry
from
Source
1
had
the
following
average
characteristics
(the
assays
according
to
APHA
(1985)).
Total
solids
(TS)
content:
2.3%;
chemical
oxygen
demand
(COD):
36
g1
-1
;
Kjeldahl
nitrogen
con-
centration:
2.8
g1
-1
;
ammoniacal
nitrogen
concentration:
g1
-1
.
Slurry
from
Source
2
had
the
following
average
characteristics
(prior
to
dilution).
TS
content:
5.0%;
COD:
60
gl
-1
;
Kjeldahl
nitrogen
concentration:
2.7
gl
-1
;
ammoniacal
nitrogen
concentration:
1.8
g1
-1
.
Slurry
from
Source
2
was
diluted
with
tap
water
in
experiments
(per-
formed
in
triplicate)
to
determine
the
effect
of
dilution
on
virus
inactivation
and
was
used
at
the
following
TS
con-
centrations:
5%,
2.5%,
1%
and
0.5%.
Uninfected
slurry
was
assayed
prior
to
use
for
the
presence
of
viruses
that
could
interfere
with
assays,
and
found
to
be
negative.
Heating
experiments
Slurry
or
EMEM
was
pre-heated
at
the
required
temperature
in
25
ml
glass
bottles
placed
in
a
water
bath
prior
to
inocu-
lation
with
virus.
When
the
temperature
had
equilibrated,
virus
was
added
to
the
slurry
or
EMEM
at
a
time
designated
time
zero
(t
=
0),
and
the
required
temperature
was
main-
tained.
Samples
were
removed
at
the
required
time
intervals,
extracted,
and
assayed
according
to
the
procedure
described
above.
Chemical
addition
experiments
Granular
NaOH
and
powdered
Ca(OH)
2
used
in
these
experi-
ments
were
obtained
from
BDH
Chemicals.
Each
sample
used
to
assess
the
effect
of
chemical
concentration
was
pre-
pared
by
adding
slurry
from
Source
1
to
an
appropriately
weighed
amount
of
either
chemical
in
a
25
ml
glass
bottle.
Each
experiment
was
initiated
when
virus
suspension
was
added
to
achieve
a
concentration
of
10%
v/v.
The
duration
of
each
experiment
was
measured
from
the
moment
of
virus
addition
(designated
t
=
0).
The
pH
of
the
slurry
was
mea-
sured
before
and
after
chemical
addition,
and
immediately
following
the
required
duration
of
chemical
treatment,
the
pH
of
the
suspension
was
restored
to
its
starting
value
by
adding
5
mol
1
-1
HC1
to
prevent
death
of
the
cell
sheets
used
in
the
virus
assays.
Control
treatments
were
performed
by
©
1999
The
Society
for
Applied
Microbiology,
Journal
of
Applied
Microbiology
87,
148-157
6
5
E
4
0
d
3
<
6
o
02
VIRUS
INACTIVATION
IN
PIG
SLURRY
151
adding
the
chemical,
then
slurry
or
EMEM,
then
restoring
the
starting
pH
before
addition
of
virus
to
determine
whether
the
5
mol
HC1
addition
had
any
effect
on
the
virus.
The
experiment
to
determine
the
efficacy
of
NaOH
or
Ca(OH)
2
against
ASFV
in
EMEM
used
the
following
con-
centrations
of
chemical
at
4
°C
or
22
°C:
NaOH
at
1%,
0.5%,
0.2%
and
0.1%
(w/v)
and
Ca(OH)
2
at
2%,
1%,
0.5%
and
0.2%
(w/v).
In
slurry
with
ASFV,
Ca(OH)
2
was
used
at
1%
and
0.5%
(w/v);
NaOH
was
used
at
1%,
0.5%
and
0.2%
(w/v)
at
4
°C
and
22
°C.
In
both
these
experiments,
the
chemical
was
allowed
to
act
for
a
period
of
30
min
before
neutralization.
The
inactivation
of
ASFV
over
150
and
300
s
was
also
examined,
where
NaOH
or
Ca(OH)
2
was
added
to
ASFV
in
slurry
containing
0.5%
(w/v)
of
either
chemical
at
22
°C,
and
1%
(w/v)
of
either
chemical
used
at
4
°C
with
samples
being
taken
after
150
and
300
s
before
neutralization.
For
SVDV
inactivation
in
EMEM,
NaOH
and
Ca(OH)
2
were
added
at
concentrations
of
0.2%,
0.5%
and
1%
(w/v)
to
vials
containing
EMEM,
mixed,
and
neutralized
after
30
min.
A
similar
experiment
was
performed
with
NaOH
and
Ca(OH)
2
at
1%,
0.5%
and
0.2%
(w/v)
in
slurry
for
30
min.
The
experiment
to
determine
the
effect
of
NaOH
and
Ca(OH)
2
on
SVDV
in
slurry
over
a
short
time
period
was
performed
as
follows:
slurry
containing
1%
and
1.5%
(w/v)
of
either
chemical
was
inoculated
with
10%
(v/v)
SVDV
at
22
°C,
and
samples
taken
at
150
s
and
300
s
and
immediately
neutralized
with
5
mol
1
-1
HC1.
RESULTS
Heat
inactivation
ASFV.
(i)
The
stability
of
ASFV
in
EMEM
at
4,
22,
40,
50
and
60
°C
over
24
h
was
examined
(Fig.
1).
It
can
be
seen
that
the
virus
was
relatively
stable
at
4,
22
and
40
°C,
losing
less
than
10
1
HAD
50
m1
-1
.
At
50
°C,
however,
the
virus
titre
declined
steadily,
so
that
after
5
h,
the
titre
was
10
1.8
HAD
50
m1
-1
and
after
24
h,
only
a
trace
of
virus
remained.
At
60
°C,
no
virus
could
be
detected
15
min
after
inoculation.
In
slurry
from
Source
1,
the
virus
titre
declined
more
rapidly,
and
the
titre
was
below
detectable
levels
after
only
4
h
at
40
°C
and
within
1
h
at
50
°C
(Fig.
2).
Again,
no
virus
could
be
detected
after
15
min
at
60
°C.
(ii)
ASFV
was
incubated
in
slurry
from
Source
1
at
50, 53,
56
and
60
°C.
Virus
was
only
detected
at
50
and
53
°C
up
to
15
min,
and
after
30
min,
no
virus
was
detected
at
any
of
the
temperatures.
These
results
demonstrated
that
virus
inac-
tivation
in
slurry
occurs
at
a
more
rapid
rate
than
it
does
in
EMEM.
(iii)
Having
ascertained
that
the
temperatures
at
which
ASFV
in
slurry
from
Source
1
was
inactivated
to
below
detectable
levels
within
15
min
was
between
53
and
60
°C,
ASFV
was
incubated
in
slurry
from
Source
1
at
56
and
60
°C
to
determine
the
lowest
temperature
at
which
ASFV
is
inactivated
within
90
s,
and
it
was
found
that
ASFV
was
inactivated
within
30
s
at
60
°C,
and
within
90
s
at
56
°C.
In
an
experiment
with
slurry
from
Source
2
at
different
TS
concentrations,
ASFV
was
incubated
at
56,
60
and
65
°C
for
up
to
5
min.
In
this
experiment,
it
was
found
that
ASFV
was
not
inactivated
as
readily
as
in
the
slurry
from
Source
1
(Table
1).
At
56
°C,
inactivation
to
below
detectable
levels
occurred
after
2-5
min.
At
60
°C,
inactivation
occurred
between
2
and
3
min
for
5,
2.5
and
1%
TS;
at
0.5%
TS,
it
occurred
between
3
and
4
min.
At
65
°C,
ASFV
was
inac-
tivated
at
all
TS
concentrations
within
60
s.
From
these
results,
it
seems
that
the
source
of
slurry
(and
hence
its
constituents)
affected
the
speed
of
ASFV
inactivation
at
dif-
ferent
temperatures.
There
also
appeared
to
be
a
marginally
6
5
E
4
E
0
>
p
3
<
o
2
+7.
.
.-
0
co
2
1
C
Limit
of
detection
Limit
of
detection
I I I I
0
5
10
15
20
25
Time
(h)
Fig.
I
Thermal
inactivation
of
ASFV
in
EMEM
after
24
h.
Initial
virus
titre:
IV
HAD
50
Titres
that
could
not
be
detected
are
shown
at
just
below
the
limit
of
detection.
A
trace
of
virus
was
detected
after
24h
at
50
°C.
(M),
4
°C;
(0),
22
°C;
(A),
40
°C;
(V),
50
°C;
(+),
60
°C
0
I I I I
0
5
10
15
20
25
Time
(h)
Fig.
2
Thermal
inactivation
of
ASFV
in
pig
slurry
after
24
h.
Initial
virus
titre:
IV
HAD
50
m1
-1
.
Titres
that
could
not
be
detected
are
shown
at
just
below
the
limit
of
detection.
(M),
4
°C;
(0),
22
°C;
(A),
40
°C;
(V),
50
°C;
(+),
60
°C
©
1999
The
Society
for
Applied
Microbiology,
Journal
of
Applied
Microbiology
87,
148-157
152
C.
TURNER
AND
S.M.
WILLIAMS
Table
I
Inactivation
of
ASFV
in
slurry
from
Source
2
at
5%,
2.5%,
1%
and
0.5%
TS
at
56,
60
and
65°C
%TS/Sample
time
(min)
56
°C
60
°C
65
°C
0.5%
0
5.4
(5.1-5.6)
5.6
(5.3-6.3)
4.5
(4.3-4.8)
0.5
5.5
(5.3-5.8)
NT
<1.8
(<1.8-<1.8)
1
54
(5.1-5.8)
5.6
(+6-6.3)
<1.8
(<1.8-<1.8)
2
5.2
(5.1-5.3)
5.0
(+6-5.8)
NT
3
NT
<3.3
(<1.8-3.3)
NT
4
NT
<1.8
(<1.8-<1.8)
NT
5
<1.8
(<1.8-trace)
<1.8
(
<1.8-<
1.8)
NT
1%
0
5.3
(5.1-5.6)
5.1
(+8-5.3)
4.6
(+3-4.8)
0.5
5.3
(5.1-5.6)
NT
<4.6(<1.8-46)
1
54
(5.1-5.6)
4.0
(3.1-5.1)
<1.8
(<1.8-<1.8)
2
5.2
(5.1-5.3)
<2.3
(<1.8-2.3)
NT
3
NT
<1.8
(<1.8-<1.8)
NT
4
NT
<1.8
(<1.8-<1.8)
NT
5
<1.8(<1.8-<1.8)
<1
.
8(<1
.
8-<1
.
8)
NT
2.5%
0
5.7
(5.3-6.1)
5.3
(5.3-5.3)
3.6
(2.6-4.6)
0.5
5.3
(5.1-5.6)
NT
<2.8
(<1.8-2.8)
1
5.4
(5.3-5.6)
4.3
(2.8-5.3)
<1.8
(<1.8-<1.8)
2
5.2
(+8-5.6)
<3.6
(<1.8-3.6)
NT
3
NT
<1.8
(<1.8-<1.8)
NT
4
NT
<1.8
(<1.8-<1.8)
NT
5
<24
(<1.8-2.1)
<1.8
(<1.8-<1.8)
NT
5%
0
5.4
(5.1-5.6)
5.4
(5.3-5.6)
4.3
(<1.8-+3)
0.5
5.4
(5.1-5.6)
NT
<1.8
(<1.8-<1.8)
1
5.2
(+8-5.6)
3.6
(2.8-4•1)
<1.8
(<1.8-<1.8)
2
5.2
(5.1-5.3)
<1.8
(<1.8-<1.8)
NT
3
NT
<1.8
(<1.8-<1.8)
NT
4
NT
<1.8
(<1.8-<1.8)
NT
5
<1.8
(<1.8-<1.8)
<1.8
(<1.8-<1.8)
NT
Three
replicates
were
tested
and
values
given
are
the
mean
(and
range).
Virus
titres
are
given
as
log
i0
HAD
50
Virus
added
had
a
titre
of
10
68
HAD
50
m1
-
',
and
was
added
at
a
10-fold
dilution.
NT
=
not
tested.
greater
stability
of
ASFV
in
slurry
at
0.5%
TS
compared
with
1,
2.5
and
5%
TS.
SVDV.
(i)
The
stability
of
SVDV
over
24
h
was
examined
in
both
EMEM
and
slurry
from
Source
1
at
the
following
temperatures:
4,
22,
40,
50
and
60
°C.
In
EMEM,
results
showed
that
SVDV
was
stable
over
24
h
at
4,
22
and
40
°C.
However,
the
titre
started
to
fall
after
1
h
at
50
°C
and
was
not
detectable
after
4
h
(Fig.
3).
In slurry
from
Source
1,
SVDV
was
stable
at
4
and
22
°C;
at
40
°C,
the
titre
had
started
to
decline
over
24
h.
No
virus
was
detectable
in
slurry
incubated
at
50
or
60
°C
(Fig.
4),
where
(apart
from
at
t
=
0)
the
first
sample
was
taken
after
1
h.
(ii)
In
a
separate
experiment
to
find
the
temperature
at
which
SVDV
was
inactivated
within
15
min,
SVDV
incu-
bated
in
slurry
from
Source
1
survived
for
up
to
1
h
at
50
°C,
although
the
decline
from
a
start
titre
of
10
7
pfu
m1
-1
was
rapid.
At
56
°C,
SVDV
survived
in
slurry
from
Source
1
for
less
than
15
min.
(iii)
Where
SVDV
was
incubated
in
slurry
from
Source
1
at
both
56
and
60
°C,
it
was
found
that
SVDV
at
56
°C
at
a
start
titre
of
10
795
pfu
m1
-1
survived
for
at
least
5
min
(although
the
titre
had
declined
to
10
15
pfu
m1
-1
).
At
60
°C,
a
trace
of
SVDV
was
detectable
after
90
s,
although
not
after
2
min.
These
experiments
indicated
that
SVDV
in
slurry
from
Source
1
is
inactivated
to
below
detectable
levels
within
2
min
at
60
°C.
In
slurry
from
Source
2,
where
slurry
samples
(in
triplicate)
at
different
TS
concentrations
were
heated
to
©
1999
The
Society
for
Applied
Microbiology,
Journal
of
Applied
Microbiology
87,
148-157
Limit
of
detection
VIRUS
INACTIVATION
IN
PIG
SLURRY
153
8
1
1
2
6
CO
a
4
co
o
o
2
CD
=
0
0
5
10
15
20
25
Time
(h)
Fig.
3
Thermal
inactivation
of
SVDV
in
EMEM
after
24
h.
Initial
virus
titre:
10'
pfu
Titres
that
could
not
be
detected
are
shown
at
just
below
the
limit
of
detection.
(M),
4
°C;
(0),
22
°C;
(A),
40
°C;
(V),
50
°C;
(+),
60
°C
7
Limit
of
detection
I I I I
0
5
10
15
20
25
Time
(h)
Fig.
4
Thermal
inactivation
of
SVDV
in
pig
slurry
after
24
h.
Initial
virus
titre:
10
7
pfu
Titres
that
could
not
be
detected
are
shown
at
just
below
the
limit
of
detection.
(M),
4°C;
(0),
22°C;
(A),
40°C;
(V),
50°C;
(+),
60°C
60
and
65
°C,
it
was
found
that
slurry
was
inactivated
within
5
min
at
60
°C
at
all
slurry
dilutions
except
0.5%
TS,
and
within
2
min
at
65
°C
except
in
one
replicate
at
5%
TS.
Results
are
given
in
Table
2.
These
experiments
indicated
that
as
with
ASFV,
SVDV
is
more
heat
labile
in
slurry
than
in
EMEM.
They
also
demonstrated
that
the
thermal
inactivation
of
viruses
is
stron-
gly
dependent
on
the
characteristics
of
the
slurry.
Chemical
inactivation
ASFV.
Addition
of
NaOH
and
Ca(OH)
2
to
virus
suspensions
in
EMEM
at
4
and
22
°C
showed
that
Ca(OH)
2
was
effective
at
both
temperatures
at
a
concentration
of
1%
(w/v);
NaOH
was
effective
at
0.5%
(w/v),
although
a
trace
of
virus
was
present
after
30
min
in
1%
NaOH
at
4
°C.
Temperature
had
an
effect
on
the
chemical
inactivation
of
ASFV,
with
Table
2
Inactivation
of
SVDV
in
slurry
from
Source
2
at
5%,
2.5%,
1%
and
0.5%
TS
at
60
and
65°C
%TS/Sample
time
(min)
60°C
65
°C
0.5%
0
6.8
(6.4-7.2)
+8
(4.4-5.0)
5.6
(5.5-5.9)
<0.7
(<0.7-<0.7)
2
3.6
(3.0-4.5)
<0.7
(<0.7-<0.7)
3
2.3
(2.0-2.7)
NT
4
2.4
(1.4-3.2)
NT
5
<2.0
(<0.7-2.4)
NT
1%
0
6.0
(5.6-6.3)
+1
(4.1-4.2)
5.1
(4.5-5.5)
<1.4
(<0.7-1.4)
2
+4
(2.9-5.4)
<0.7
(<0.7-<0.7)
3
2.4
(1.7-3.7)
NT
4
2.7
(1.7-3.3)
NT
5
<0.7
(<0.7-<0.7)
NT
2.5%
0
6.6
(6.2-6.8)
3.3
(3.2-3.6)
5.2
(5.0-5.4)
<0.7
(<0.7-<0.7)
2
2.8
(2.7-3.0)
<0.7
(<0.7-<0.7)
3
<0.7
(<0.7-<0.7)
NT
4
<0.7
(<0.7-<0.7)
NT
5
<0.7
(<0.7-<0.7)
NT
5%
0
7.2
(7.2-7.3)
5.7
(5.6-6.0)
1
6.6
(6.6-6.7)
1.8
(1.4-2.4)
2
6.0
(5.9-6.1)
2.1
(<0.7-2.1)
3
5.0
(4.3-5.4)
NT
4
4.3
(4.2-4.3)
NT
5
<0.7
(<0.7-<0.7)
NT
Three
replicates
were
tested
and
values
given
are
the
mean
(and
range).
Virus
titres
are
given
as
log
10
pfu
Virus
added
had
a
titre
of
l(r
pfu
at
a
10-fold
dilution.
NT
=
not
tested.
inactivation
occurring
at
lower
concentrations
of
chemical
at
22
than
at
4
°C
(Fig.
5).
In
slurry,
it
was
found
that
ASFV
was
inactivated
by
Ca(OH)
2
within
30
min
at
1%
and
0.5%
(w/v)
at
4
and
22
°C;
NaOH
was
effective
at
1,
0.5
and
0.2%
(w/v)
at
22
°C,
and
at
1
and
0.5%
(w/v)
at
4
°C,
0.2%
being
ineffective
at
this
temperature.
As
with
the
heat
inactivation
studies,
following
an
initial
evaluation
of
concentration
ranges
likely
to
be
effective,
it
was
necessary
to
find
a
concentration
of
chemical
that
could
act
rapidly.
A
study
of
the
inactivation
of
ASFV
in
slurry
(of
initial
titre
10
5.8
HAD
50
m1
-1
)
by
NaOH
or
Ca(OH)
2
after
150
and
300
s
demonstrated
that
1%
(w/v)
of
either
chemical
at
4
°C
rapidly
inactivated
ASFV
to
below
detectable
levels,
but
0.5%
(w/v)
at
22
°C
was
relatively
ineffective
after
5
min
u,
0.
o
-3
=
2
1
0
©
1999
The
Society
for
Applied
Microbiology,
Journal
of
Applied
Microbiology
87,
148-157
154
C.
TURNER
AND
S.M.
WILLIAMS
(a)
6—
d
5
co
E
4
-
2
0
5
co
a
3
.+7.
.
2
2
2
c- =
1
-
0
,3°V
,34‘\1'
cp
r
o
I I
0
ff'
0
ff'
0
ff'
0
.\,,
rt
o
(b)
6
5
CO
E
4
2
0
5
co
a
3
For
example,
the
addition
of
1%
(w/v)
NaOH
to
EMEM
changed
its
pH
from
74
to
12.9,
while
the
same
concentration
in
slurry
increased
the
pH
from
7.8
to
10.6.
This
implies
that
the
action
of
these
chemicals
was
not
a
pH
effect
alone.
SVDV.
NaOH
and
Ca(OH)
2
at
concentrations
effective
for
the
inactivation
of
ASFV
(0.2,
0.5
and
1%
(w/v))
were
used
against
SVDV
in
EMEM
22
°C
with
the
initial
SVDV
titre
being
10
7.1
pfu
m1
-1
.
NaOH
and
Ca(OH)
2
were
less
effective
at
these
concentrations
against
SVDV
than
against
ASFV
(Fig.
6).
The
effect
of
NaOH
and
Ca(OH)
2
against
SVDV
in
slurry
at
22
°C
over
30
min
was
examined
and
results
show
that
1%
(w/v)
of
NaOH
and
Ca(OH)
2
was
effective,
but
lower
concentrations
did
not
produce
sufficient
virus
inactivation
(Table
4).
Having
identified
chemical
concentrations
that
would
cause
inactivation
of
SVDV
over
30
min,
as
with
ASFV,
it
was
necessary
to
find
a
concentration
of
chemical
that
would
8—
0
cp
0 0
\Gr
0
0
0
0
A
o
A
o
0
\
0
r
t\
r
t%
4
5
c
l%
CY
CY
CY
CY
CY
Treatment
applied
Fig.
5
Inactivation
of
ASFV
in
EMEM
by
Ca(OH)
2
and
NaOH
at
(a)
22
°C
and
(b)
4
°C
(Table
3).
These
results
indicated
that
virus
inactivation
occurred
at
lower
concentrations
of
NaOH
or
Ca(OH)
2
in
slurry
than
in
EMEM.
This
is
in
spite
of
the
fact
that
slurry
buffers
the
system
much
better
than
does
EMEM,
and
addition
of
either
alkali
led
to
smaller
pH
changes
in
slurry.
d
6
co
'5
0.
42
co
4
o
o
4
1
2
=
2
d
01
,
01
,
01
,
d
z
,
z
,
°
d
e-
d
o
d
o
0
,
0
re\
o
Treatment
applied
Fig.
6
Inactivation
of
SVDV
in
EMEM
by
Ca(OH)
2
and
NaOH
at
22
°C
and
4
°C.
Control
virus
titre
shown
at
10
7
'
pfu
m1
-1
0
Table
3
The
inactivation
of
ASFV
in
slurry
by
chemicals
over
5
min
Chemical
and
temperature
Treatment
time
(s)
Virus
titre
(log
io
HAD
50
m1
-1
)
0.5%
NaOH,
22°C
150
41
300
3.6
0.5%
Ca(OH)
2
,
22
°C
150
3.1
300
3.3
1%
NaOH,
4
°C
150
51.8
300
<1.8
1%
Ca(OH)
2
,
4
°C
150
51.8
300
<1.8
Start
titre
=
10
6
'
7
pfu
m1
-1
©
1999
The
Society
for Applied
Microbiology,
Journal
of
Applied
Microbiology
87,
148-157
VIRUS
INACTIVATION
IN
PIG
SLURRY
155
Table
4
Inactivation
of
SVDV
after
30
min
in
slurry
with
chemicals
Chemical
treatment
Final
concentration
(%
w/v)
Virus
titre
(log
io
pfu
m1
-1
)
None-control
0
7.2
Ca(OH)2
1
<0.7
0.5
7.0
0.2
6.1
NaOH
1
0.7
0.5
6.3
0.2
7.1
act
rapidly,
i.e.
within
150
s.
Hence
the
effect
of
NaOH
and
Ca(OH)
2
at
a
concentration
of
1%
(w/v)
on
SVDV
in
slurry
over
a
short
time
period
(150
and
300
s)
was
studied.
After
300
s,
virus
still
persisted
in
the
slurry,
although
the
titre
was
reduced
(Table
5).
At
1.5%
(w/v),
either
chemical
at
4
and
22
°C
inactivated
SVDV
to
below
detectable
levels
within
150
s.
In
a
control
experiment
in
which
slurry
samples
containing
1.5%
(w/v)
NaOH
were
neutralized
with
5
mo11
-1
HC1
prior
to
SVDV
addition,
the
HCl
had
no
adverse
affect
on
the
virus,
indicating
that
it
was
not
responsible
for
virus
inactivation.
However,
some
loss
of
virus
titre
(10
15
pfu
m1
-1
)
occurred
when
slurry
samples
containing
Ca(OH)
2
were
neutralized
with
HCl.
DISCUSSION
The
results
presented
here
demonstrated
that
either
chemical
inactivation
or
heat
treatment
can
inactivate
both
ASFV
and
SVDV
in
pig
slurry
and
EMEM.
SVDV
was
more
resistant
to
both
treatments
than
ASFV,
requiring
a
greater
con-
centration
of
chemicals
or
higher
temperature
to
achieve
similar
inactivation.
SVDV
was
inactivated
to
below
detect-
able
levels
by
F5%
(w/v)
of
either
NaOH
or
Ca(OH)
2
whereas
ASFV
required
only
1%
(w/v)
of
either
chemical
to
achieve
similar
inactivation.
With
heat
treatment,
generally
higher
temperatures
or
longer
time
periods
were
required
for
the
inactivation
of
SVDV
compared
to
ASFV.
The
time
taken
for
treatment
to
be
effective
is
an
important
consideration
in
devising
a
method
for
large-scale
inactivation
of
virus
in
slurry.
If
the
treatment
method
is
to
be
applied
to
the
decontamination
of
a
large
volume
of
slurry,
the
faster
the
decontamination
can
be
achieved,
the
quicker
and
easier
the
treatment
of
the
entire
volume
of
slurry
will
proceed.
A
given
treatment
method
should
also
render
the
material
safe
within
a
quantifiable
safety
margin.
In
other
words,
it
should
not
only
be
possible
to
demonstrate
the
effectiveness
of
a
particular
treatment,
but
also
to
determine
the
minimum
level
of
treatment
to
achieve
a
particular
reduction
in
virus
titre.
Apart
from
identifying
suitable
safety
margins,
this
approach
will
also
prevent
a
process
from
being
'over-engi-
neered'
at
greater
expense.
An
interesting
feature
of
the
results
is
that
slurry,
or
com-
ponents
of
it,
appears
to
enhance
the
effects
of
the
virus
inactivation
treatment.
ASFV,
for
instance,
was
inactivated
to
below
detectable
levels
within
1
h
at
50
°C
in
slurry
from
Source
1,
whereas
similar
inactivation
in
EMEM
requires
more
than
24
h.
ASFV
levels
remained
stable
over
24
h
at
40
°C
in
EMEM
but
in
slurry
from
Source
1,
the
titre
declined
to
below
detectable
levels
within
4
h.
The
pattern
was
similar
for
SVDV.
These
results
imply
that
the
inac-
tivation
demonstrated
was
not
due
simply
to
the
effect
of
heat
alone,
but
may
have
been
assisted
by
the
release
of
Table
5
Stability
of
SVDV
in
slurry
at
chemical
concentrations
of
1%
at
22°C
after
150
and
300
s
Chemical
treatment
Duration
of
treatment
(s)
Virus
titre
(log
io
pfu
m1
-1
1%
NaOH
150
2.5
300
2.1
1%
Ca(OH)
2
150
2.8
300
3.0
Start
titre
=
10'
pfu
m1
-1
©
1999
The
Society
for
Applied
Microbiology,
Journal
of
Applied
Microbiology
87,
148-157
156
C.
TURNER
AND
S.M.
WILLIAMS
virucidal
agents,
possibly
ammonia,
in
the
slurry
when
it
was
heated.
The
mechanism
of
temperature
inactivation
of
ASFV
and
SVDV
appeared
to
be
different.
When
exposure
to
high
temperature
was
limited
to
90
s,
ASFV
was
inactivated
at
a
lower
temperature
than
SVDV.
However,
ASFV
appeared
to
survive
longer
at
sub-lethal
temperatures
(e.g.
50
°C)
than
SVDV.
This
can
be
seen
by
comparing
Figs
1
and
2
with
Figs
3
and
4.
Thermal
virus
inactivation
also
appeared
to
be
dependent
on
the
nature
or
source
of
the
slurry.
Results
obtained
with
both
ASFV
and
SVDV
showed
that
the
source
of
the
slurry
can
have
an
affect
on
the
inactivation
time.
ASFV
and
SVDV
were
both
inactivated
more
rapidly
and
at
a
lower
temperature
in
slurry
from
Source
1
than
in
slurry
from
Source
2.
The
reason
for
this
was
not
further
investigated,
and
conclusions
could
not
easily
be
drawn
from
the
differences
in
quoted
characteristics
of
the
slurries,
such
as
chemical
oxygen
demand,
total
solids
and
nitrogen
content.
However,
in
these
and
in
subsequent
experiments
(data
not
shown),
it
was
shown
that
both
ASFV
and
SVDV
were
consistently
inac-
tivated
at
higher
temperatures
in
cell
growth
medium
or
water
than
in
slurry.
Therefore,
in
order
to
be
confident
that
virus
inactivation
will
occur,
it
is
recommended
that
the
treatment
temperature
is
slightly
higher
than
that
shown
to
be
effective
in
any
of
the
media
tested.
Results
from
chemical
inactivation
were
also
not
entirely
predictable.
It
was
believed
that
the
inactivation
occurred
as
a
result
of
an
increase
of
pH
to
above
12,
which
was
the
case
when
NaOH
or
Ca(OH)
2
was
used
at
concentrations
above
0.5%
(w/v)
in
EMEM.
However,
the
slurry
had
a
strong
pH
buffering
effect,
and
the
pH
only
rose
to
10.6
when
1%
(w/v)
NaOH
was
added,
compared
with
a
pH
of
12.9
when
added
to
EMEM,
and
yet
higher
concentrations
of
chemicals
were
required
for
ASFV
inactivation
in
slurry
than
in
medium.
Therefore,
it
appeared
that
virus
inactivation
by
NaOH
or
Ca(OH)
2
was
not
merely
a
pH
effect.
Although
the
use
of
NaOH
or
Ca(OH)
2
and
the
application
of
heat
are
all
capable
of
inactivating
ASFV
and
SVDV
to
below
detectable
levels
in
pig
slurry,
chemical
treatment
is
likely
to
be
a
less
suitable
method
for
use
with
large
volumes
of
slurry
due
to
the
difficulties
in
ensuring
that
all
parts
of
the
slurry
are
adequately
mixed
and
come
into
contact
with
the
required
concentration
of
chemicals
for
the
duration
of
treatment.
Disposal
of
chemical-contaminated
slurry
is
also
a
potential
problem
when
large
volumes
are
involved.
For
small
volumes,
however,
either
method
is
adequate
for
ASFV
or
SVDV
inactivation.
Hence,
based
on
the
results
of
this
research,
the
rec-
ommendation
is
that
large
quantities
of
contaminated
pig
slurry
are
heat
treated
at
a
temperature
of
65
°C
for
a
mini-
mum
period
of
5
min.
This
treatment
will
ensure
that
a
reasonable
margin
of
safety
is
applied
to
the
treatment
process
to
account
for
differences
in
the
characteristics
of
the
slurry
which
have
been
shown
to
have
an
effect
on
virus
inactivation.
This
would
be
an
expensive
proposition
if
batch
processing
was
used.
However,
with
continuous
flow
processing,
engin-
eering
techniques
can
be
used
to
recover
much
of
the
heat
required.
The
differences
observed
in
inactivation
profiles
in
dif-
ferent
media,
and
even
in
similar
but
not
identical
media
(e.g.
slurry
from
two
sources)
under
different
conditions
demonstrate
that
the
process
of
inactivation
is
more
complex
than
a
mere
temperature
or
pH
effect.
Hence,
a
complete
picture
of
relevant
inactivation
data
needs
to
be
taken
into
account
when
designing
a
large-scale
inactivation
process
for
a
particular
virus.
ACKNOWLEDGEMENTS
This
work
was
funded
by
the
Ministry
of
Agriculture,
Fish-
eries
and
Food,
and
formed
part
of
an
Open
Contract.
The
authors
would
like
to
thank
Drs
T.R.
Cumby
and
P.J.
Wil-
kinson
and
Mr
C.H.
Burton
for
helpful
comments.
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