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Short
Communications
Indian Journal
of
rlnimal Sciences 76 (4): 324-325,
April
2006
Detection ofcanine parvovirus (CPV) DNA by polymerase chain reaction assay
and
its prevalence in dogs in and around Kolkata, West Bengal
S
BISWASl,
PJ
DAS2,
SK
GHOSH3
and
N R
PRADHAN
4
West Bengal University
of
Animal
and
FishelY Science, Kolkata, West
Bengal
700
037
India
Received:
10
May
2004;
Accepted:
30
January
2006
Key
words:
Canine
parvo
virus,
Diagnosis,
PCR,
Prevalence
Fig.
1.
PCR
product
ofCPV
in
ethidium
bromide
stained
1.2%
agarose
gel.
Lane
(2),
lane
(3)
and
lane
(5)
showing
amplified
CPY
DNA
product.
Lane
(1)
and
lane
(6)
negative
control,
lane
(5)
positive
control
using
DNA
from
Nobivac
Puppy
DP
vaccine
(Intervet),
lane
(2)
and
lane
(3)
positive
clinical
samples,
lane
(4)
negative
clinical
sample.
Ozkul
et
at. (2002)
by
using same specific primer.
In
few
cases there were 2
to
3extra bands nearly
of
0.2-0.3
kb,
which
has
been shown
in
lane 3indicated
by
star
in
Fig.
I,
that needed further study for conclusion.
On
the basis
of
the
results obtained
from
polymerase chain reaction,
103
dogs
were
found
to
be
CPY positive out
of
306 dogs examined
which
were
suffering
from
gastroenteritis. Therefore, 33.66%
were
positive for CPY infection amongst the gastroenteritis
cases
in
and
around Kolkata.
It
was also recorded that
49
(47.57%) male dogs were positive for CPY
and
54
bitches
(52.43%) were positive. The results indicated that there were
no
statistically significant variations
of
the occurrence
of
CPY
infection amongst the male
and
female dogs which
simulated with Banja
et
at. (2002). Regarding
age
wise
prevalence
the
highest prevalence
was
noted
at
0--6
months
of
age
group followed
by
6-12 months
of
age
group
and
12
Canine
parvo
viral disease (CPY) spreads rapidly through
the
susceptible canine population
and
is
responsible
for
higher
morbidity
and
about
50%
mortality. Mochizuki
et
al.
(1993)
and
Decaro et
al.
(2005) stated that,
PCR
is
as
sensitive
as
virus isolation
and
more
sensitive
than
the
haemagglutination
test
for
CPY
detection
from
the
faecal
samples
of
dogs.
Faecal
swab samples (306)
were
collected
from
dogs irrespective
of
age
and
sex
suffering
from
gastroenteritis
in
and
around
Kolkata
from
July 2002
to
June 2003,
and
immediately
immersed
in
500jll ofTE buffer
(pH
8.0)
and
transported
to
the laboratory
in
ice
box.
The samples were cleared by
centrifugation at
14
000
rpm
for
10
min
and
supernatants
were
taken
and
kept
in
the
ependorf
(1.5
ml)
separately
for
each sample
in
4°C
for
further processing. The
CPY
DNA
was
extracted
by
using phenol-chloroform method
as
per
Ozkul
el
al.
(2002).The capsid protein
YP
l/YP2 gene
was
amplified
based
on
the
method
described
by
Ozkul
el
al.
(2002)
with
slight
mod
ification. Aset 19-mer primers
as
designed
by
Mochizuki et
at.
(1993)
and
Ozkul et
al.
(2002)
(Forward: 5'-ATG
GCA
CCT
CCG
GCA
AAG
A-
3'
and
Reverse:
5'-TTT
CTA
GOT
GCT
AGT
TGA G-3')
were
used
for
amplification. Reaction product
(15
~tl)
along
with
AHind
III
marker
were
analyzed electrophoretically
in
1.2%
(w/v) agarose gel
in
1xT
AE.
The bands were visualized
after staining
with
ethidium bromide.
The
PCR
app
lication revealed
an
intensive product
in
the
faecal sample
in
parallel with the positive control
DNA
prepared
from
the
attenuated
live
vaccine.
In
the
Fig.
1,
lane-
2,3
and
5showing amplified
CPY
DNA
product, which
was
at
2.2
kb
(approximately)
was
considered
as
positive
for
CPY
infection amongst
which
lane-5
was
positive control using
DNA
from
Nobivac Puppy
DP
vaccine. Lane-l
and
6
were
negative control whereas
lane-4
was
negative clinical sample.
This
band
at
2.2
kb
was
agreed
with
the previous studies
of
Present
address:
'PhD
Scholar,
Department
of
Veterinary
Medicine,
Ethics
and
Jurisprudence;
2PhD
Scholar,
3S
en
ior
Lecturer,
Department
of
Animal
Genetics
and
Breeding;
4Professor
and
I-lead,
Department
of
Veterinary
Medicine,
Ethics
and
Jurisprudence.
2.32Kb~
2.03
Kb---+
M23 4 5 6
2.2
Kb
April
2006] DETECTION
AND
PREVALENCE
OF
CANINE
PARVOYIRUS
325
months
and
above
age
group
(Grigonis et al.
2002).This
might
be
the reason for
highest
OCCUlTence
of
CPV
amongst
the younger dogs
(0-6
months)
and
the highest mortality
rate might
be
due to the affinity
of
the
vims
towards the
cells
of
the
myocardium
resulting to cardiac failure.
Prevalence
of
CPV
infection
according
to
different
seasons (summer, from
March
to June; rainy, from July to
October; and winter,
from
November
to
February)
were
observed to
be
the highest in
the
sunm1er (53.4% -55/103)
followed
by
rainy
season
(34%
35/1
03)
and
winter
(12.6%-
13/103).
The
highest occurrence
ofCPV
infection insununer
might be due to the fact that majorities
oflitters
were
born
in
winter and therefore susceptible younger dogpopulationwere
more during the
summer
(Grigonis et
([1.
2002, Rypula et al.
2004).
It
may
be
concluded
that
PCR
can
be
used as aroutine
diagnostic tool for early detection
of
CPY. Its prevalence is
much
higher
among
the
young
aged
dog
population
(Hirayama et al.
2005)
during
summer
followed
by
rainy
season and winter.
SUMMARY
The
early detection
of
the canine
parvo
virus (CPV) is
of
paramountimportance. Canine faecal samples from 306 dogs
suffering from gastroenteritis
in
and around Kolkata were
collected, and
DNA
was
extracted
by
phenol-chloroform
method.
CPY
vaccine strain
was
used
as apositive control
for CPY. Polymerase
chain
reaction
(PCR) was carried out
to amplify
VPI/YP2
gene using aset
of
19-merprimers [CPV
-
PI
(Forward): 5'- ATG
GCA
CCT
CCG
GCA
AAG
A-3';
CPY
-
P,
(Reverse):
5'-TTT
CTA
GGT
GCT
AGT
TGA
G-
3')]. A
peR
product
of
approximately
2.2 kb was generated
with positive faecal samples
and
vaccine strain
CPY
vims.
After
screening, 103 dogs were found positive for CPV,
but
no sex variation was
noted
amongst
the
CPV
positive cases.
Dogs,
of
the age group
of
0-6
months were mostly susceptible
with highest mortality rate followed
by
6-12
months and 12
and
above months
of
age
and
highest occurrence was
noted
during
summer
followed
by
rainy
season
and winter.
REFERENCES
Decaro
N,
Elia
G,
Martella
Y,
Desario
C,
Campolo
M,
Trani
L
D,
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C.
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time
PCR
assay
for
rapid detection
and
quuntitation
of
canine
parvovirus type 2
in
the feces
of
dogs. Veterinary Microbiologr
105(1):
19-28.
Grigonis
A,
Macijauskas Yand Zamokas
G.
2002. Parvovirus
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V'etcrinurUa
ir
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K,
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T,
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K,
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K.
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wild- type
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