observation of canine parvovirus in wolves from. Europe. Key words: Parvovirus, wolf,. Canis lupus,. V1I11S isolation. , electron microscopy. The wolf population.
juiinwl
of
%til(1!if(’
:33(3). 1997.
I)ist’ase.s, © \Vikllife
Detection
of Canine
Parvovirus
in Wolves
from
Disease’
pp.
62S-631
1997
Associatioii
Italy
F. Martinello,1 F. Galuppo, F. Ostanello,2 V. Guberti,3 and S. Prosperl,1 ‘ Dipartimento di Sanit#{224} Pubblica Veterinaria e Patologia Animale, Universit#{224}di Bologna, 40064 Ozzano Emilia (BO), Italy; 2 Istituto di Patologia e Igiene Veterinaria, Universit#{224}di Padova, 35020 Legnaro (PD), Italy; Istituto Nazionale per Ia Fauna Selvatica, 40064 Ozzano Emilia (BO), Italy
ABSTIIA;1’:
One
wolf (CanEs lupus) 1994 to 1995 from of the north central
The
aly.
samples
vovirus
by virus
in
by
wolves
The
for
canine
as
samples
confirmed
elec-
population
in
lupus,
1992).
The
presence
diseases
could
interfere
the
et
canids al.
the
,
(Eugster
1979;
wolf
cause
et al.,
Mann
et
(Canis
lupus)
gastroenteritis
(Mech and transmitted
al.,
natural
in
and
Goyal, via
and
by
the
the
(Swango, presence
the
States
indirectly in
fecal-oral
sera
finding
(Goyal
et
,
has
in
central
Italy;
never
been
directly
however, ascertained
of
11#{176}48’E)
species
in
in
to as-
(Mattioli
et
and Orecchiella with dogs was the
Casentino
in
the
spring of 1994 65), respectively. packs them
stored
at
sample
same
areas
and
(n = 50) Samples
during
and 1995 belonged
but it was impossible at the individual level. -20
C until
was
examined
of
(DAS-ELISA), hemagglutination and virus isolation (VI) on permissive
both by
antibody unosorbent
line These
embryonic procedures
fibroblast were
time
to
the
increase
used
sensitivity
to All
by three
ferent methods: enzyme-linked
car-
double imm
(n to
testing.
by the in the
and
the
collected
Each
dif-
sandwich assay (HA), fe-
cells (FEA). at the same of the
in-
vestigation.
detection of the virus in wolf feces (Muneer et al., 1988). Fico et al. (1996) found CPV antibodies in four wolves captured during 1993 to 1994
of the
whereas
were
is
antibodies
1986)
samples
in order
negligible;
and
assessed CPV
al.
habits
the same identifie
1983; Thomas et al., of CPV in wolves in been
collected
In the Gigante risk of contact
=
puppies
is secured resistance
food
were
may
route,
(43#{176}56’N,
were
al., 1995). parks, the
the
disease
presence
has
through
wolf
of
The
possible
rier animals 1984). The United
death
fifteen
populations in north Italy (Fig. 1), were sub-
Val Parma parks this risk was possible. Moreover, the Casentino hosts one of the largest wolf population of Italy. Samples
Fletcher it
four
Alta
including
which
1993).
maintenance of infection high environmental viral feces
1978; 1980)
and
Park feces
sess
demography of this species which has been close to the threshold of extinction. Canine parvovirus (CPV) infects domestic dogs all over the world (Appel et al., 1978) as well as captive and free-ranging wild
detection the north
wolves were radio-collared; Alta Val Preserve (44#{176}30’N, 09#{176}56’E) and
which
of infectious with
countries.
direct from
Italy.
hundred
Casentino
of
about 400 individuals ranging throughout the Apennines Mountains (Francisci and Guberti,
European
Apennines,
three Parma
consists
Italy
other
mitted for testing for CPV. There were four collection sites: Gigante Regional Park (44#{176}25’N, 10#{176}16’E) and Orecchiella Natural Park (44#{176}11’N, 10#{176}23’E) in which
Europe.
Canis
in
wolf feces from central Apennines,
were by
or
we describe the in feces of wolves
One
All positive samples were Park in Tuscany. This is the observation of canine parvovirus Parvovirus, wolf, , electron microscopy.
wolf
im-
hemagglutination,
of these
Italy
Herein, of CPV
par-
enzyme-linked
isolation
from
words: isolation
V1I11S
central
tested
Four
virus
microscopy. Casentino definitive
Key
were
isolation.
positive
tron from first
in
antigen-capture assay (ELISA),
munosorbent
and
hundred fifteen samples of feces were collected during four free-living populations Apennines Mountains, It-
The Madrid,
SA
for
technique
DAS-ELISA
Spain) is an CPV antigen;
(Ingenasa,
antigen-capture both the coating
ELIan-
tibodies as well as those conjugated with the enzyme are a mixture of highly specific monoclonal antibodies that allow detection
virus either 628
SHORT
trophotometer Eflab,
(Titertek
Helsinki,
wavelength;
I
greater
tive (18)
than
control The HA
terial (49)
with
or
to
Vail
Parma (28)
Preaerve
Oretchiella
Park
Natural
(20)
using
1.
FI(;UIIE
c.’es were
Sites
in
c’ollecte(l.
miortlwrn
Number
Italy
of
wolf
where
samliples
is in
fe-
1)areml-
were
inhibition
serum.
With
samples
specifically
w/v
in
ing
1,000 any
of
dogs
in
1990).
the
All
wells
CPV
Europe
and
strains
samples the
isolated
from
(Rimmelzwaan were
results
were
run
et in
read
al.,
duplicate by
a spec-
for
x
in
GO2,
and
fect
(CPE)
similar
100
(‘roscopv.
Bar
1(X)
1)artic’leS
Ncgatiselv mmmii.
isolate(l staine(l
iii
cell
electron
ciiimiii-
FEA
pA of
cells freshly USA). C, 5%
cytopathic
hr.
Each
sample
passages and
fol-
using
respective
passage
ef-
control cell this generally
blind the
Laho-
York, at 37
procedure
preceding
as
culture
which
had
thawed. 2 and
oculation. The third-passage sample was tested SA and HA-HI.
supernatant of each for CPV by DAS-ELIPositive samples were
for
2 mm by adding phosphotungstate Stained
samples
Sonic
been
rapidly frozen and had CPE between
supernatant.
feces.
for
to 96
further
pore
treated
(Corning
daily
72
two
nm
1986)
the negative confluent;
was
at super-
each
of
plates
until
a
450
p.1 of
Co., New incubated
observed
within
stained potassium
Itrsoviral
1:10 contain-
solution
Louis,
Hashimoto,
24-wells
inoculum
2.
diluted 7.4)
through
and
underwent
sol1
consid-
Missouri,
filtered
monolayer
UIIE
were
St.
,
added 100 a suspension
occurred
from
anti-GPV
agglutinating
antimycotic Go.
ratories Sciences Cell cultures were
FI(
all
(pH
We to
seeded
tore
an
were
was
the
hemagglu-
using
buffer
filters. sample
at
erythrocytes
After shaking and centrifuging G for 15 mm at 4 C, the
(Mochizuki
mawells
Agglutinating
inhibited
natant
lowing
posi-
V-bottom
specific
test,
antibiotic
the
positive. to fecal
1980).
this
absorption of
swine
(HI)
Chemical
USA). of
al.,
tested
405-nm
Labor-Technik,
1%
phosphate
1%
an
96
positive for CPV. For VI, fecal samples
(Sigma
theses.
et
tination
200 km
in
Plus,
a
20%
(Greiner
by
samples
ered
equal
plates
(Carmichael AIta
at
samples
conducted
Germany)
629
Multiskan
Finland)
were considered technique applied
was
plastic Park
Casentino
COMMUNICATIONS
4
samples after
days
50 to
in-
al of 2% 50 il of
were
trans-
ferred to colloid carbon-coated copper grids, blotted, dried in air and examined at 126,000 x in a Zeiss-109 (Zeiss, Oherkochen, (Fig. 2).
Germany)
electron
microscope
630
JOURNAL
TABLE
I.
OF WILDLIFE
Detection
DISEASES,
of canine
VOL.
from
parvovinis
(;igante
Regional
Alta
Park
Natural
Val
Parma
Casentino
Park
Preserve
Park
fecal
Number
positive/number
I) NI)
not
=
all
three
were
SA, HA.
one was positive The discordance
positive
SA
and
HA
0/18
0/18
NDb
0/20
0/20
0/20
ND
1994
0/12
0/12
0/12
ND
1995
0/16
0/16
0/16
ND
1995
2149
3/49
4149
4149
2/115
3/115
4/115
4/49
techniques; by
CARMICHAEL,
HA
but
not
in
three
by
fecal
methods:
poor
in the sample and presence
ELI-
of CPV in the is established. Further research
mine
the
distribution
Italy
and
to
virus
the
is
needed
J.
(;.,
B.
J.
E. CARMICHAEL. enteritis.
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the
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(Mech 1994).
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F
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Canine Veterinary
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1994
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by
two
and
1994
sampled.
ples
the
in Italy,
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