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Veterinary Microbiology 105 (2005) 1928

www.elsevier.com/locate/vetmic

A real-time PCR assay for rapid detection and quantitation


of canine parvovirus type 2 in the feces of dogs
Nicola Decaroa,*, Gabriella Eliaa, Vito Martellaa, Costantina Desarioa,
Marco Campoloa, Livia Di Tranib, Elvira Tarsitanoa,
Maria Tempestaa, Canio Buonavogliaa
a

Department of Animal Health and Well-being, Faculty of Veterinary Medicine of Bari,


Strada per Casamassima Km 3, 70010 Valenzano, Bari, Italy
b
Istituto Superiore di Sanita`, viale Regina Elena 299, 00161 Rome, Italy

Received 14 April 2004; received in revised form 23 September 2004; accepted 29 September 2004

Abstract
We describe a rapid, sensitive and reproducible real-time PCR assay for detecting and quantifying canine parvovirus type 2
(CPV-2) DNA in the feces of dogs with diarrhea. An exogenous internal control was added to control the assay performance from
extraction to amplification. The method was demonstrated to be highly specific and sensitive, allowing a precise CPV-2 DNA
quantitation over a range of eight orders of magnitude (from 102 to 109 copies of standard DNA). The reproducibility of the CPV2 real-time PCR assay was assessed by calculating the coefficients of variation (CV) intra-assay and inter-assay for samples
containing amounts of CPV-2 DNA spanning the whole range of the real-time PCR standard curve. Then, fecal specimens from
diarrheic dogs were analyzed by hemagglutination (HA), conventional PCR and real-time amplification. Comparison between
these different techniques revealed that real-time PCR is more sensitive than HA and conventional gel-based PCR, allowing to
detect low viral titers of CPV-2 in infected dogs.
# 2004 Elsevier B.V. All rights reserved.
Keywords: Dog; Parvovirus; Real-time PCR; Diagnosis

1. Introduction
Canine parvovirus type 2 (CPV-2) is a pathogen of
dogs which causes acute gastroenteritis and lymphopenia in young pups. CPV-2 belongs to the feline
* Corresponding author. Tel.: +39 080 467 9833;
fax: +39 080 467 9843.
E-mail address: n.decaro@veterinaria.uniba.it (N. Decaro).

parvovirus (FPV) subgroup of the genus Parvovirus,


together with feline panleukopenia virus (FPLV),
mink enteritis virus (MEV), raccoon parvovirus
(RPV), raccoon dog parvovirus (RDPV) and blue
fox parvovirus (BFPV) (Berns et al., 2000). All these
viruses are highly related, showing a genome
conservation of 98% (Truyen et al., 1994a) and are
genetically and antigenically distinct from canine
parvovirus type 1 (CPV-1), also known as minute virus

0378-1135/$ see front matter # 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2004.09.018

20

N. Decaro et al. / Veterinary Microbiology 105 (2005) 1928

of canines (MVC), which is responsible for neonatal


death in dogs (Binn et al., 1970; Carmichael et al.,
1994). CPV mature virions are round, non-enveloped,
icosahedral particles, 26 nm in diameter, containing
two viral proteins (VPs) designated VP1 and VP2 and
formed by alternative splicing from the same RNA
(Reed et al., 1988). CPV genome consists of a
negative, single-stranded DNA, about 5200 nt in
length, with hairpin structures at both the 50 and 30
ends (Berns et al., 2000).
CPV-2 was first identified in 1978 after an epizootic
of gastroenteritis and myocarditis occurring worldwide in pups (Kelly, 1978; Appel et al., 1979;
Burtonboy et al., 1979; Johnson and Spradbrow,
1979), whereas FPV-induced disease in cats has been
known since the beginning of the 20th century (Verge
and Christoforoni, 1928). Evolutionary studies and
phylogenetic analyses strongly suggest that CPV-2 has
originated from a FPV-like virus infecting wild
carnivores (Truyen et al., 1995).
A few years after the emergence of CPV-2, two new
antigenic variants, designated type 2a and type 2b and
distinguishable by means of MAbs, arose consecutively (Parrish et al., 1985, 1991). Currently, the
antigenic variants have completely replaced the
original type 2 and are variously distributed in canine
population world-wide (Mochizuki et al., 1993a; De
Ybanez et al., 1995; Greenwood et al., 1996; Truyen et
al., 1996, 2000; Steinel et al., 1998; Sagazio et al.,
1998; Buonavoglia et al., 2000; Pereira et al., 2000).
Subsequently, the onset of CPV mutants has been
reported in different countries (Truyen et al., 1996,
2000; Ikeda et al., 2000; Steinel et al., 2000; Battilani
et al., 2001). Recently, a CPV-2 mutant with a change
(Asp-426 to Glu) occurring in a strategic residue for
the antigenicity of CPV-2 has been detected in Italy
(Buonavoglia et al., 2001). Molecular analysis of
several CPV-2 strains has shown that the Glu-426
mutant is widely distributed in Italy and it is currently
co-circulating together with type 2a and 2b CPVs
(Martella et al., 2004). In addition, such CPV-2 mutant
has been recently detected in other countries
(Nakamura et al., 2004).
Because of severity of parvovirus-induced disease,
several assays have been developed to detect CPV-2 in
the feces of infected dogs. Usually, feces from
diarrheic dogs are screened using ELISA or hemagglutination (HA) assays, but these techniques are

affected by low sensitivity (Mochizuki et al., 1993b;


Uwatoko et al., 1995). Conversely, methods based on
detection of CPV-2 DNA by PCR (Mochizuki et al.,
1993b; Hirasawa et al., 1994; Truyen et al., 1994b;
Schunck et al., 1995; Senda et al., 1995; Uwatoko et
al., 1995; Tempesta et al., 1998; Pereira et al., 2000;
Buonavoglia et al., 2001) have been shown to be more
sensitive. However, none of these PCR-based methods
were designed to be quantitative, although they are
time consuming and contain a certain risk of carryover
contamination, especially when a high sample
throughput is required. Accordingly, quantitative
assays using competitive or real-time PCR have been
established for the detection of human parvovirus B19
in biological samples (Gallinella et al., 1997; Gruber
et al., 1998, 2001; Fini et al., 1999; Aberham et al.,
2001). Real-time PCR assays have been found to be
more sensitive, rapid and reproducible than competitive PCR assays based on the detection of the PCR
products by gel electrophoresis and ethidium bromide
staining, allowing a precise DNA quantitation even
when a high sample throughput is screened (Aberham
et al., 2001; Gruber et al., 2001).
The aim of our study was to develop a real-time
PCR assay for the detection and quantitation of CPV-2
DNA in the feces of dogs with diarrhea. The assay is
based on TaqMan technology, which allows the
monitoring of the generation of PCR products by
measuring the increase of fluorescence caused by the
cleavage of a dual-labeled probe by the DNA
polymerase used for amplification. Signals are
regarded as positive if the fluorescence intensity
exceeded 10 times the standard deviation of the
baseline fluorescence (threshold cycle [CT]) (Holland
et al., 1991; Heid et al., 1996).

2. Materials and methods


2.1. Template DNA preparation
Fecal specimens were taken by a swab from the
anus of diarrheic dogs and homogenized (10% w/v) in
phosphate buffered saline (PBS). Fecal suspensions
were clarified by a brief centrifugation at high speed in
a microfuge and aliquots of 200 ml of the supernatants
were used for DNA preparation by boiling for 10 min
to inactivate PCR inhibitors and chilling on ice, as

N. Decaro et al. / Veterinary Microbiology 105 (2005) 1928

previously described (Schunck et al., 1995; Uwatoko


et al., 1995). This method was demonstrated to remove
inhibitors of the Taq polymerase (Schunck et al.,
1995). Residual inhibitors of DNA polymerase
activity (personal observation) were reduced to an
ineffective concentration by diluting 1:10 in distilled
water the DNA extracts prior to amplification by either
conventional or real-time PCR assays.
2.2. Design of primers and probe
The VP2 nucleotide sequences of some CPV-2
strains were retrieved from GenBank and aligned
using BioEdit software package (www.mbio.ncsu.
edu/BioEdit/bioedit.html). The strains and accession
numbers used for alignment were the following: CPV2: CPV-b, M38245 and CPV-Northern, M19296; CPV2a: CPV-15, M24003 and CPV-31, M24000; CPV-2b:
CPV-39, M74849 and CPV-133, M74852; CPV-2 Glu426 mutant: 56/00, AY380577.
Assay target region was first identified by visual
inspection of sequence alignment, and then exact
primer and probe sequences were selected by using a
primer design software (Beacon Designer, Bio-Rad
Laboratories Srl, Milan, Italy) in order to amplify a
conserved 93 bp fragment within the aligned VP2
sequences. Primers and probe were obtained from
MWG Biotech AG (Ebersberg, Germany). The
TaqMan probe was labeled with the fluorescent
reporter dye 6-carboxyfluorescein (FAM) at the 50
end and with the quencher dye 6-carboxytetramethylrhodamine (TAMRA) at the 30 end.
2.3. Standard DNA preparation
A pGEM1-3Z plasmid (Promega U.S., Madison,
WI) containing the nearly full-length genome (5076
nt) of a CPV-2 strain was kindly supplied by C.R.
Parrish (Cornell University, Ithaca, NY, USA). To
obtain large amounts of standard DNA, the plasmid
was amplified in Escherichia coli TOP10F (Invitrogen Srl, San Giuliano Milanese, Italy) and plasmid
DNA was purified from transformed cells using
Wizard Plus Midiprep (Promega Italia, Milan, Italy).
After DNA quantitation by spectrofotometrical analysis, 10-fold dilutions of the plasmid, representing
100109 copies of DNA/10 ml of template, were
carried out in a fecal suspension tested negative to

21

CPV-2 by HA and conventional PCR (and subsequently also by real-time PCR). Aliquots of each
dilution were frozen at 70 8C and used only once.
2.4. Real-time PCR
Real-time PCR was performed in an i-Cycler iQTM
Real-Time Detection System (Bio-Rad Laboratories
Srl) and the data were analyzed with the appropriate
sequence detector software (version 3.0). Duplicates
of the CPV standard dilutions and DNA templates
were simultaneously subjected to real-time analysis.
The 25-ml PCR mixture for one reaction contained
12.5 ml of IQTM Supermix (Bio-Rad Laboratories Srl),
600 nM of primer CPV-For (50 -AAACAGGAATTAACTATACTAATATATTTA-30 ) and CPV-Rev (50 AAATTTGACCATTTGGATAAACT-30 ), 200 nM of
probe CPV-Pb (50 -TGGTCCTTTAACTGCATTAAATAATGTACC-30 ) and 10 ml of DNA. The thermal
cycle protocol used was the following: activation of
iTaq DNA polymerase at 95 8C for 10 min and 40
cycles consisting of denaturation at 95 8C for 15 s,
primer annealing at 52 8C for 30 s and extension at
60 8C for 1 min.
2.5. Internal control
In order to verify the complete removal of the PCR
inhibitors after the easy and fast method of DNA
preparation, so that a precise quantitation of CPV-2
DNA could be ensured for all the fecal samples, an
internal control (IC) was added to each sample.
Recently, it has been proposed to introduce the seal
virus phocid herpesvirus type 1 (PhHV-1) as universal
internal viral control for real-time PCR (Niesters,
2004). Analogously, we used a DNA virus, ovine
herpesvirus type 2 (OvHV-2), the etiologic agent of
malignant catarrhal fever (MCF) of cattle, for which
real-time PCR is routinely carried out in our
laboratory. A sample of spleen, obtained from a
cattle affected by MCF (Decaro et al., 2004b), was
found to contain 2.26  107 OvHV-2 DNA copies/
mg, using the protocol established by Hu ssy et al.
(2001). A fixed amount of this virus was added to
each sample prior to DNA preparation, in order to
obtain an OvHV-2 DNA concentration of approximately 5000 DNA copies/ml of fecal suspension (able
to give a mean CT value in the real-time PCR assay of

22

N. Decaro et al. / Veterinary Microbiology 105 (2005) 1928

32.21 with a S.D. of 1.02, as calculated by 100


separate runs). Primers and probe for OvHV-2 DNA
amplification were derived from Hu ssy et al. (2001),
with the TaqMan probe labeled with the fluorescent
reporter dye Texas Red instead of FAM. The CPV-2
and OvHV-2 real-time PCR assays were carried out
as a duplex PCR in a single tube, with the same
conditions as the CPV-2 assay. Samples in which the
CT value for the IC was >34.25 (average plus 2 S.D.)
were excluded from analysis.
2.6. Conventional PCR
Conventional PCR was performed using AmpliTaq
Gold (Applied Biosystems, Applera Italia, Monza,
Italy) and primer pair Hfor/Hrev, which amplifies
a fragment of the capsid protein-encoding gene of
CPV-2 (Buonavoglia et al., 2001). Primers Hfor
(50 -CAGGTGATGAATTGCTACA-30 ) and Hrev
(50 -CATTTGGATAAACTGGTGGT-30 ), located at
nt position 35563575 and 41664185 of the CPV
genome, respectively, yield a 611 bp product. The
reaction was carried out in a total volume of 100 ml
containing PCR buffer 1 (KCl 50 mM, TrisHCl
10 mM, pH 8.3), MgCl2 2 mM, 200 mM of each
deoxynucleotide (dATP, dCTP, dGTP, dTTP), 1 mM of
primers Hfor and Hrev, 2 U of AmpliTaq Gold and
10 ml of DNA. The thermal conditions consisted of
activation of AmpliTaq Gold polymerase at 94 8C for
10 min and 40 cycles of denaturation at 94 8C for 30 s,
annealing at 50 8C for 1 min and polymerization at
72 8C for 1 min, followed by a final extension at 72 8C
for 10 min. The PCR products were detected by
electrophoresis through a 1.5% agarose gel and
visualization under UV light after bromide ethidium
staining.
2.7. Evaluation of analytical performances of the
CPV-2 real-time PCR assay
The analytical specificity of CPV-2 DNA detection
by real-time or conventional PCR was evaluated using
DNA preparations of several parvoviruses of dogs as
well, including CPV-2 (old type), CPV-2a, CPV-2b,
CPV-2 Glu-426 mutant, and MVC (Decaro et al.,
2002a), and other pathogens of dogs, including canid
herpesvirus 1 (CaHV-1) (Decaro et al., 2002b), canine
adenovirus type 1 (CAdV-1) (Pratelli et al., 2001) and

type 2 (CAdV-2) (Decaro et al., 2004a), or sterile


water.
Since no quantitative method has been previously
established for measuring CPV-2 DNA amounts in the
feces and a CPV-2 reference standard does not exist, to
evaluate the analytical sensitivity of conventional and
real-time PCR, 10-fold dilutions of the standard DNA,
ranging from 109 to 100 copies/10 ml, were tested by
both methods. In order to mimic realistic conditions,
the standard DNA was also diluted in fecal samples
tested negative to CPV-2 by HA and PCR and the
spiked samples were subjected to DNA preparation
and analysis by real-time and conventional PCR. In
addition, 10-fold dilutions in Dulbeccos minimal
essential medium (D-MEM) of the CPV-2b vaccinal
strain 29/97-40 (Buonavoglia et al., 1998), with a titer
of 105.50 TCID50/50 ml, determined by an immunofluorescence (IF) test on A-72 canine cell cultures,
were used. Each standard or virus dilution was being
quantified three times separately.
A CPV-negative fecal sample was spiked with
serial 10-fold dilutions of standard DNA (from 102 to
109 copies/10 ml of template) in order to generate a
standard curve to quantify parvovirus DNA in fecal
samples from naturally infected dogs. The standard
curve was created automatically by the i-Cycler IQ
Optical System Software, version 3.0 (Bio-Rad
Laboratories Srl), by plotting the CT values against
each standard dilution of known concentration.
Reproducibility of the method was established by
repeatedly testing samples containing several concentrations of CPV-2 DNA, spanning the whole range
covered by real-time PCR, with the DNA amounts
obtained for the same samples in different assays (10
consecutive runs) and within an assay (10 times in the
same assay), in order to calculate the inter-assay and
intra-assay coefficient of variation (CV), as previously
described (Stelzl et al., 2004).
2.8. Endpoint titration by IF, HA and
real-time PCR
To compare sensitivity of the IF test, HA and realtime PCR, A-72 cells were seeded in 24-well plates
and infected with 100 ml of 10-fold dilutions (101 to
1010) in D-MEM of the vaccinal strain CPV-2b 29/
97-40. Each dilution was inoculated in eight wells,
with four wells having slides for IF. After 5 days of

N. Decaro et al. / Veterinary Microbiology 105 (2005) 1928

incubation at 37 8C with CO2 5%, the slides were


subjected to IF using a monoclonal antibody specific
for CPV-2, and the cryolysates of the counterpart wells
were subjected to HA and real-time PCR.
2.9. Samples collection and typing of parvovirus
strains
A total of 50 fecal samples, collected from
diarrheic dogs between 1998 and 2004 and tested
positive by HA, were subjected both to conventional
and real-time PCR. The samples were characterized as
CPV-2a or CPV-2b by a hemagglutination inhibition
(HI) test using a panel of four MAbs (A4E3, B4A2,
C1D1 and B4E1), kindly provided by C.R. Parrish
(Cornell University, Ithaca, NY, USA), as previously
described (Sagazio et al., 1998; Buonavoglia et al.,
2001). Parvovirus strains from samples containing low
HA titers were typed after viral amplification in A-72
cells. All the strains characterized as type 2b by MAb
analysis were subjected to genetic screening by
restriction enzyme analysis to detect the newly
recognized CPV-2 Glu-426 mutants, as previously
reported (Buonavoglia et al., 2001; Martella et al.,
2004). Briefly, the PCR products obtained with primer
pair 555for/555rev, corresponding to the 30 end of
CPV open reading frame 2, were digested with the
enzyme MboII, whose restriction site is unique to the
Glu-426 mutants, yielding two fragments of about 500
and 80 bp.
In addition, 39 fecal specimens, collected from
CPV-2-infected dogs in the late stages of infection,
when viral shedding was no longer detectable by HA,
were subjected to both conventional and real-time
PCR. Real-time analysis was also carried out on 10
fecal samples collected from healthy dogs that had
tested negative to CPV-2 by both HA and conventional
PCR.

3. Results
3.1. Internal control performance
All the examined samples were positive for the IC,
giving CT values below the threshold value of 34.25.
Therefore, PCR inhibition did not occur in any sample
during the real-time PCR assay, enabling a precise

23

quantitation of the CPV-2 DNA in all the analyzed


samples. To investigate possible competition between
CPV-2 and OvHV-2 DNA amplifications, serial 10fold dilutions of CPV-2 DNA (102109 DNA copies)
were spiked with different amounts of OvHV-2 DNA
(102106 DNA copies). In all the experiments, no
significant interference between the amplifications of
CPV-2 and OvHV-2 DNA occurred (data not shown).
3.2. Analytical specificity, sensitivity and
reproducibility of the CPV-2 real-time assay
As expected on the basis of primer and probe
design, both conventional and real-time PCR amplified DNA from all the CPV-2 variants, including the
Glu-426 mutant, whereas neither MVC or other
pathogens were recognized. Specificity of the PCR
assays was confirmed by determining the sequence of
both the conventional and the real-time PCR products.
The detection limit of the real-time PCR was 1 log
higher than that of the gel-based conventional PCR,
ranging around 102 copies/10 ml and 101.50 TCID50/
50 ml for standard DNA and CPV vaccine, respectively, with a detection rate of 100% for each positive
dilution. The detection limit of 102 copies was also
obtained by diluting the standard DNA in CPV-2
Table 1
Results of intra-assay (a) and inter-assay (b) testing
Sample no.

Copy number of CPV-2 DNAa


Mean

S.D.

CV (%)

(a)
1
2
3
4
5
6
7
8

3.62  102
6.57  103
1.43  104
4.65  105
4.52  106
2.11  107
3.03  108
6.78  109

1.72  102
1.85  103
4.23  103
1.34  105
7.89  105
8.19  106
6.26  107
2.51  109

47.5
28.1
29.6
28.8
17.4
38.8
20.7
37.0

(b)
1
2
3
4
5
6
7
8

1.73  102
3.81  103
2.77  104
2.11  105
2.23  106
1.69  107
3.91  108
4.51  109

1.11  102
8.53  102
1.02  104
8.21  105
4.70  105
6.80  106
9.37  107
1.94  109

64.2
26.8
36.8
41.3
21.0
40.2
23.9
43.0

Copy numbers of CPV-2 DNA are referred to 10 ml of template.

24

N. Decaro et al. / Veterinary Microbiology 105 (2005) 1928

negative fecal samples instead of TE buffer, confirming that PCR inhibitors are reduced to ineffective
concentrations by means of the DNA preparation
procedure, consisting of boiling and diluting in
distilled water the fecal samples.
The standard curve, that was generated by the
standard dilutions, spanned eight orders of magnitude
and showed linearity over the entire quantitation range
(slope = 3.610), providing an accurate measurement
over a very large variety of starting target amounts.
The coefficient of linear regression (R2) was equal to
0.9995.
The CV between runs and within-run for samples
containing CPV-2 DNA amounts spanning the whole
range expected are reported in Table 1.
3.3. Endpoint titration by IF, HA and real-time PCR
As shown in Table 2, the virus titer was
105.50 TCID50/100 ml when determined by IF and
103.50 TCID50/100 ml (cryolysates of infected wells)
when determined by HA. Real-time analysis was able to
detect CPV-2 DNA in the cryolysates of all four
replicates at dilution 108 and even in the cryolysate
of one well of the dilution 109 (virus titer =
108.75 TCID50/100 ml).
3.4. Analysis of the fecal samples of dogs naturally
infected with CPV-2
By means of MAb characterization and restriction
analysis, 24 (48%) out of the 50 HA-positive samples,
Table 2
Viral titer by IF, HA and real-time PCR on cell cultures inoculated
with CPV-2
Dilutiona
1

10
102
103
104
105
106
107
108
109
1010

IF
+
+
+
+
+






HA
+
+
+
+
+






+
+
+
+
+






+
+
+
+
+






+
+
+








Real-time
+
+
+








+
+
+








+
+
+








+
+
+
+
+
+
+
+



+
+
+
+
+
+
+
+
+


+
+
+
+
+
+
+
+



+
+
+
+
+
+
+
+



(+) positive; () negative.


a
Dilutions are referred to CPV-2b vaccinal strain 29/97-40.

collected from dogs infected naturally with CPV-2,


were characterized as CPV-2a, 15 (30%) as CPV-2b,
and 11 (22%) as CPV-2 Glu-426 mutant.
All the HA-positive samples gave signals both in
conventional and real-time amplification. A comparison between the viral titers detected in the feces by
HA and real-time is reported in Table 3. By real-time
PCR, a wide range of parvovirus DNA amounts was
found in the samples, from 1.63  107 to 7.43  1011
copies/mg of feces. Analogously, the HA titers were
highly variable, ranging from 1:10 to 1:5120 per mg of
feces. However, no linear correlation was found

Table 3
Analysis of HA-positive fecal samples from diarrheic dogs by
conventional and real-time PCR
Sample

Origin

Year

HA
titera

PCRb

Real-time
titera

CPV-2a
102/01 (C)
103/01 (B)
104/01 (A)
255/01
273/01 (1)
273/01 (2)
288/01
297/01 (A)
307/01 (A)
307/01 (B)
133/02 (B)
256/02 (1)
18/03 (G)
133/03 (7)
197/03
300/03
312/03
366/03
28/04
51/04
62/04
67/04 (B)
74/04
105/04

Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Turkey
Apulia
Apulia
Apulia
Apulia
Hungary
Apulia
Apulia
Apulia
Apulia
Apulia

2001
2001
2001
2001
2001
2001
2001
2001
2001
2001
2002
2002
2003
2003
2003
2003
2003
2003
2004
2004
2004
2004
2004
2004

640
320
320
5120
320
2560
40
2560
80
2560
320
640
160
640
2560
40
2560
2560
1280
640
40
320
1280
320

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

9.82  1010
3.11  1010
1.85  1010
3.47  1010
2.29  1010
1.87  1011
2.06  108
7.43  1011
8.55  109
2.33  109
9.88  108
2.11  1010
1.12  109
5.02  109
2.25  1010
5.37  109
3.42  1010
8.19  1010
3.76  1010
4.89  108
3.24  109
1.77  109
2.24  1010
2.02  1010

CPV-2b
197/98
201/98
212/98
224/98
242/98 (B)
111/99
130/99 (A)
42/01

Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia

1998
1998
1998
1998
1998
1999
1999
2001

1280
320
80
2560
80
1280
320
2560

+
+
+
+
+
+
+
+

7.98  1010
6.87  109
9.62  108
3.59  1010
6.28  1010
9.13  108
8.11  109
9.68  1010

N. Decaro et al. / Veterinary Microbiology 105 (2005) 1928


Table 3 (Continued )
Sample
281/01
133/03
133/03
133/03
133/03
233/03
233/03

(B)
(11)
(12)
(28)
(38)
(19)
(29)

Glu-426 mutant
157/02 (2)
161/02
202/02 (1)
202/02 (2)
105/03
135/03
148/03
180/03
220/03
347/03
111/04 (2)

Origin

Year

HA
titera

PCRb

Real-time
titera

Apulia
Turkey
Turkey
Turkey
Turkey
Turkey
Turkey

2001
2003
2003
2003
2003
2003
2003

80
5120
2560
80
40
640
640

+
+
+
+
+
+
+

1.01  1010
9.44  109
4.19  1010
3.53  109
8.08  107
7.11  1010
1.31  1011

Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Basilicata
Apulia
Apulia

2002
2002
2002
2002
2003
2003
2003
2003
2003
2003
2004

2560
80
40
40
10
40
80
320
20
640
40

+
+
+
+
+
+
+
+
+
+
+

1.61  1011
4.01  108
1.63  107
9.98  108
9.79  108
5.71  108
3.44  108
2.10  1010
9.52  107
1.55  1010
4.33  107

(+) positive.
a
HA and real-time titers are referred to 1 mg of feces. HA titers
are expressed as the reciprocal of the fecal dilutions; real-time titers
are expressed as number of copies.
b
Buonavoglia et al. (2001).

between HA and real-time analysis, since several


samples containing high parvovirus DNA amounts, as
determined by real-time PCR, displayed low HA titers
(Table 3).
Out of the 39 HA-negative specimens, collected
from the dogs recovering from CPV-2 infection, 18
(46.15%) were found to be CPV-2 positive by
conventional PCR and 23 (58.97%) by real-time
analysis. Five samples were negative by conventional
PCR but they were found to contain CPV-2 DNA by
the real-time PCR assay. The amounts of CPV-2 DNA
in the HA-negative samples ranged from 3.65  103 to
2.83  105 copies/mg of feces. The results obtained
by conventional and real-time PCR assays were in
agreement with the DNA titers evaluated by the
quantitative assay. In conventional PCR, there was no
signal when the sample contained less than 103 copies/
10 ml of template, which corresponded approximately
to 104 CPV-2 DNA copies/mg of feces (Table 4), as
calculated by real-time analysis.
Totally, of the 89 specimens analyzed, 50 (56.18%)
tested positive by HA, 68 (76.40%) by conventional
PCR and 73 (82.02%) by real-time PCR (Table 5).

25

Table 4
Analysis of HA-negative fecal samples from diarrheic dogs by
conventional and real-time PCR
Sample
158/01 (A)
158/01 (B)
158/01 (1)
158/01 (4)
158/01 (5)
158/01 (6)
158/01 (7)
158/01 (8)
158/01 (10)
158/01 (12)
6/02 (A)
24/02
117/02 (7)
144/03 (B)
111/04 (1)
111/04 (3)
111/04 (4)
111/04 (5)
111/04 (7)
116/04 (1)
116/04 (3)
116/04 (4)
116/04 (5)

Origin
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia
Apulia

Year
2001
2001
2001
2001
2001
2001
2001
2001
2001
2001
2002
2002
2002
2003
2004
2004
2004
2004
2004
2004
2004
2004
2004

HA

PCRa
+

+
+
w
+
+
w
+

+
+
+
+
w

w
w
w

Real-time titer

6.91  10
7.33  103
1.92  105
1.89  104
1.44  104
3.88  104
5.21  104
6.06  103
5.41  104
5.88  103
2.83  105
5.32  103
2.33  104
8.21  103
6.50  104
8.11  104
4.79  104
1.27  105
1.55  104
3.65  103
1.23  104
1.85  104
2.06  104

() negative; (+) positive; w: weak signal.


a
Buonavoglia et al. (2001).
b
Real-time titers are expressed as number of copies/mg of feces.

Table 5
Summary of the results of CPV-2 detection in the fecal samples from
diarrheic dogs by HA, conventional and real-time PCR
Tested samples

89
a

Positive samples
HA

PCRa

Real-time

50 (56.18%)

68 (76.40%)

73 (82.02%)

Buonavoglia et al. (2001).

The 10 specimens collected from the healthy dogs


gave no signal when tested by real-time PCR,
confirming that non-specific amplifications do not
occur in the CPV-2 real-time PCR assay.

4. Discussion
Real-time quantitative PCR is based on continuous
optical monitoring of a fluorogenic PCR reaction
(Holland et al., 1991; Heid et al., 1996). In this study,

26

N. Decaro et al. / Veterinary Microbiology 105 (2005) 1928

we designed a simple, rapid TaqMan-based real-time


PCR assay for the detection and quantitation of CPV-2
DNA in the feces of dogs. The CPV-2 real-time assay
was demonstrated to be more sensitive than both HA
and conventional PCR based on detection by ethidium
bromide gel, being able to detect as few as 102 copies
of CPV-2 DNA. The established CPV-2 real-time
assay was highly reproducible and linear over a range
of eight orders of magnitude, from 102 to 109 copies,
allowing a precise calculation of CPV-2 DNA loads in
samples containing a wide range of viral DNA
amounts. Both intra-assay and inter-assay CVs were
satisfactorily low.
Real-time PCR has several advantages over
conventional PCR, allowing a large increase in
throughput and enabling simultaneous processing of
several samples. Real-time PCR is run in a 96-well
format, and many of the steps in the assay are
automated. Because of the inexpensive and quick
method used for DNA preparation, based on boiling of
fecal homogenates, the total time requested for
analysis of 2030 samples was about 6 and 3 h for
conventional and real-time PCR, respectively.
To date, methods allowing a rapid and accurate
quantitation of CPV-2 are not available. Virus titration
on cell cultures is laborious, time-consuming and not
applicable in the routine, since viral isolation on cell
cultures cannot be achieved for all the CPV-2 strains.
Furthermore, as several CPV-2 strains do not produce
evident cytopathic effect, the presence of viral
antigens in cell cultures must be evidenced by IF,
which implies additional loss of time. As confirmed by
the present study, HA is poorly sensitive, since high
viral amounts are required to determine a visible CPV2-induced hemagglutination. Therefore, by both
isolation on cell cultures and HA, CPV-2 infection
might be sometimes misdiagnosed and viral loads in
the feces of diarrheic dogs may not be quantified
precisely. Consequently, dogs infected subclinically or
recovering from CPV-2 infection may not be
identified, leading to environmental contamination
with CPV-2 and spread of the infection to other dogs.
Conversely, the high sensitivity and reproducibility
of the real-time PCR assay may allow for identification of dogs shedding CPV-2 at low titers in their
feces, helping to adopt adequate measures of
prophylaxis to prevent CPV infection, especially in
kennels and shelters, where CPV-2 is often responsible

for dramatic epizootics. The detection of the viral


DNA in the feces of low virus shedders, without
demonstration of the presence of infectious virus, does
not allow to modify or infer new data on the
epidemiology of CPV-2. However, if our results are
confirmed by subsequent in vivo experiments of CPV2 transmission by low virus shedders, real-time PCR
will help implement the CPV-2 prophylaxis programs.
In this study, specimens tested previously positive
by HA, were found to contain high amounts of CPV-2
DNA by real-time PCR, ranging from 1.63  107 to
7.43  1011 copies/mg of feces, while about 59% of
HA-negative feces were found to contain CPV-2 DNA,
thus providing additional evidence for the low
sensitivity of HA. The discrepancy between the
results of HA and real-time PCR may be due to the
fact that high viral titers are required to produce HA
and that specific antibodies in the intestinal lumen
frequently sequestrate most of the CPV-2 virions, thus
preventing or reducing parvoviral binding to erythrocytes. In fact, the CPV-2 strains which were adapted to
grow in vitro showed a good correlation between the
titers evaluated by HA and real-time analysis (data not
shown).
The CPV-2 real-time PCR assay will help to gain
new insights into the pathogenesis of CPV-2 infection,
with particular regard to the actual time extent and
amounts of CPV shedding in infected dogs or in
animals challenged during vaccine trials. The possibility to determine precisely the duration and the
amounts of viral shedding in the feces is of
fundamental importance to evaluate the efficacy of
vaccines. From this perspective, this novel assay will
be extremely useful to comprehend whether the
antigenic differences between CPV type 2a/2b and the
original type 2 may account for partial failure of the
old vaccines based on CPV-2 to protect pups against
the antigenic variants under either experimental or
field conditions.
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