Detection of Canine Parvovirus in Wolves from Italy

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.

Medical

free

the

Association

search 41: 1978.

elsewhere et al.

deter-

H.

in

impact

of wolf

pop-

(Mech 1994).

and

Parvovirus

R.,

F

Status of the

report: American

15 16-1518.

Canine Veterinary

H.

canine

diagnostic

ap-

of Veterinary

Re-

BENDELE,

AND

L.

in dogs.

Medical

MARSILIO, sulla

della

parvovirosi

l’Ehrlichia dell’Italia

P. JONES.

Journal

of the

Association

173:

G.

TISCAR.

di anticorpi

canina, cane,

del

in

cimurro,

sieri

di

K.

C.,

SCHMITT.

A.

Journal

Association

FRANCISCI,

F,

dehl’epatite

cane

(Canis

e

lupus)

alle Ricerche

di

137-143.

EUGSTER,

R.

AND

E.

Parvovirus infection in maned of the American Veterinary Med-

1979.

wolves.

K.

In-

ii virus

del

lupo

centrale. Supplemento della Selvaggina 24:

FLETCHER,

1996.

contro

coronavirus

il

canis

Biologia

ical

AND

presenza

del

175:

897-900.

AND V. GUBERTI.

1992.

Recent

trends

of wolves in Italy as apparent from kill figures and speciments. In Wolves in Europe, status and perspectives, C. Promberger and W. Schroeder (eds.). Munich Wildlife Society, Ettal, Germany, pp. 91-102. GOYAL,

S. M.,

L. D.

KHAN,

AND

canine

American

189:

1092-1094.

M.

ER.

1994.

R.,

and

(Cams

lupus)

canine

1975

pup

AND

M.

J.

L.

virus

infection

vi-

of

American

D.

APPEL, 1980.

in South

H.

American

Veterinary

As-

PLETSCH-

relation

parto wolf

Journal

G.

1985.

Medical

in

R. J. MONTALI.

Minne-

of canine

mortalities.

BUSH,

of

through

Veterinary

BOYD,

M.

Antibodies

in wolves from

distemper

M.

P. C.,

1986.

investigations

270-273.

AND

MATHYS,

K.

30:

LER, the

D.

Serologic

vovirus

SEAL.

parvoviruses

of the

JOHNSON,

R. A. RADEMACHER,

S.

study

sociation

177:

MECH, U.

A serologic

Diseases

AND

V

by and

infection

Veterinary

dagine

MANN,

(;REISEN,

K. , R. A.

Journal

CITED

COoPER,

173:

,

R.

AND

784-791.

A.

against

in wolves

possible

dynamics

1978.

Journal

studies

Journal

A.

Casentino to

of CPV

as reported 1993; Johnson

M.

Serologic

sota:

evaluate

on

of

JOUBERT,

American

infettiva

the HA. The positive samvirus titer as-

wolves

LITERATURE

ral

of

C.

Hemagglutination

plications.

FICO,

by

with clinical disease (Mathys et Based on these results, the pres-

ence Park

APPEL,

by

1340-1341.

affecting the of antibodies

in feces which would affect presence of two DAS-ELISA ples is evidence for a high

ulations Goyal,

isolation

parvovirus:

American

samples

presence

J.

E., 1980.

EUGSTER,

sam-

but not DAS-ELI-

L.

POLLOCK.

posOf one

two

by ELISA between

tests

viral antigen DAS-ELISA,

of the

Viral

Confirmed electron microscopy

0/18

could not be explained by false positive reactions, because the virus was detectable by VI and electron microscopy (EM). Differences could be due to low sensitivity of

sociated al., 1983).

Hemagglutination

1995.

1994

parvovirus was isolated in FEA four samples (Table 1); all were in the spring of 1995 in different

by

two

and

1994

sampled.

ples

the

in Italy,

1994

of the Casentino Park. The CPV fecal samples were soft in nature. the virus was detected from

sample

of wolves

done.

Canine cells from collected areas itive these,

samples

ELISA

Total .1

1997

Year

Area

Orecchiella

33, NO. 3, JULY

of Wildlife B. A. BEEH-

Canine

paivo-

canids.

Journal

Medical

Association

779-783.

A.,

R. MUELLER,

N.

C.

PEDERSEN,

AND

G.

SHORT

H. THEILEN. nation

and

sorbment

1983. Comparison of hemaggluticompetitive enzyme-linked immunoassay procedures for detecting canine

parvovirus

in feces.

Research

44:

MArnou, E.

L.,

American

M.

ungulate

tinesi

National

Veterinary

V

1995.

MAZZARONE,

Wolf

availability

food

in the

Foreste

Acta

Thenologica

Park,

Italy.

S. M.

GOYAL.

AND

habits

and

Casen40:

387-402. MECH,

L.

D.,

AND

effect

vovirus survival.

1993.

on wolf population

Journal

of

Wildlife

Canine

change Diseases

par-

and pup 29: 330-

MOCHIZUKI,

M.,

feline

in vitro.

ence

48: M.

AND

T HASHIMOTO.

panleukopenia

virus

MUNEER,

E.

Japanese

virus

1986. and

canine

of

Veterinary

Journal

Growth parvoSci-

D.

G.

F,

F. G. C.

by

Diseases

24: 170-172. AND

Evaluation

immunosorbment

assays

THOMAS,

CPV

American

Norden

News

N. J., W. J. FORm, AND

prevalence from

disease. F

of canine

Texas,

Utah Medical

I. 0.

FARAH,

K. E.

POMEROY,

S. M.

on

parvovirus

Receivedfor

publication

4-10.

Association 15

May

1984.

in wild

Idaho.

Journal 185: 1996.

deVet-

questions

58:

KNOWLTON.

and

M.

mono-

antigen The

J. F EVERMANN,

F

841-844. A.,

J.

A. D.

of enzymebased

clonal antibodies for the serology and tection in canine parvovirus infections. erinaiy Quarterly 12: 14-20. SWANGO, L. J. 1983. Frequently asked about

of

microsco-

B. KLINGEBORN,

UYTDEHAAG,

1990.

OSTERHAUS.

electron

N. JUNTFI, M.

631

Detection

1988.

feces

of Wildlife

Journal

linked

MECH.

in wolf

WINDBERG,

333. of

py.

GROEN,

APOLLONIO,

L.

AND

RIMMELZWAAN,

152-154.

CENTOFANTI.

wild

Journal

GOYAL, parvoviruses

COMMUNICATIONS

L. A.

Serocoyotes of

1283-1287.

the