pi

pe

a, a Vito Martellaa, Costantina Desarioa,

We describe a rapid, sensitive and reproducible real-time PCR assay for detecting and quantifying canine parvovirus type 2

s internal control was added to control the assay performance from

penia in young pups. CPV-2 belongs to the feline fox parvovirus (BFPV) (Berns et al., 2000). All these

Veterinary Microbiology 105viruses 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

* Corresponding author. Tel.: +39 080 467 9833;

fax: +39 080 467 9843.

E-mail address: n.decaro@veterinaria.uniba.it (N. Decaro).

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

2 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 lympho-

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(CPV-2) DNA in the feces of dogs with diarrhea. An exogenouMarco Campoloa, Livia Di Tranib, Elvira Tarsitanoa,Maria Tempestaa, Canio Buonavogliaa

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

Strada per Casamassima Km 3, 70010 Valenzano, Bari, ItalybIstituto 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

AbstractNicola Decaro *, Gabriella Elia ,A real-time PCR assay for ra

of canine parvovirus tyd detection and quantitation

2 in the feces of dogs

www.elsevier.com/locate/vetmic

(2005) 1928

contamination, especially when a high sample

Microf 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 world-

wide 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 consecu-

tively (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 hemag-

N. Decaro et al. / Veterinary20glutination (HA) assays, but these techniques arethroughput 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 compe-

titive 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 supernatantswere used for DNA preparation by boiling for 10 minaffected 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

obiology 105 (2005) 1928to inactivate PCR inhibitors and chilling on ice, as

Micrpreviously 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: CPV-

2: CPV-b, M38245 and CPV-Northern, M19296; CPV-

2a: CPV-15, M24003 and CPV-31, M24000; CPV-2b:

CPV-39, M74849 and CPV-133, M74852; CPV-2 Glu-

426 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-carboxytetramethylr-

hodamine (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 (Invitro-

gen 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 ana-

lysis, 10-fold dilutions of the plasmid, representing

100109 copies of DNA/10 ml of template, were

N. Decaro et al. / Veterinarycarried out in a fecal suspension tested negative toCPV-2 by HA and conventional PCR (and subse-

quently 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 contained12.5 ml of IQTM Supermix (Bio-Rad Laboratories Srl),600 nM of primer CPV-For (50-AAACAGGAAT-TAACTATACTAATATATTTA-30) and CPV-Rev (50-AAATTTGACCATTTGGATAAACT-30), 200 nM ofprobe CPV-Pb (50-TGGTCCTTTAACTGCATTAAA-TAATGTACC-30) and 10 ml of DNA. The thermalcycle protocol used was the following: activation of

iTaq DNA polymerase at 95 8C for 10 min and 40cycles consisting of denaturation at 95 8C for 15 s,primer annealing at 52 8C for 30 s and extension at60 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 Hussy 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 approxi-

mately 5000 DNA copies/ml of fecal suspension (able

obiology 105 (2005) 1928 21to give a mean CT value in the real-time PCR assay of

Micr32.21 with a S.D. of 1.02, as calculated by 100

separate runs). Primers and probe for OvHV-2 DNA

amplification were derived from Hussy 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 atnt 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 mlcontaining PCR buffer 1 (KCl 50 mM, TrisHCl10 mM, pH 8.3), MgCl2 2 mM, 200 mM of eachdeoxynucleotide (dATP, dCTP, dGTP, dTTP), 1 mM ofprimers Hfor and Hrev, 2 U of AmpliTaq Gold and

10 ml of DNA. The thermal conditions consisted ofactivation of AmpliTaq Gold polymerase at 94 8C for10 min and 40 cycles of denaturation at 94 8C for 30 s,annealing at 50 8C for 1 min and polymerization at72 8C for 1 min, followed by a final extension at 72 8Cfor 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

N. Decaro et al. / Veterinary22adenovirus type 1 (CAdV-1) (Pratelli et al., 2001) andtype 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 byboth 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 immuno-fluorescence (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 astandard 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 con-

centrations 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 real-

time PCR, A-72 cells were seeded in 24-well plates

and infected with 100 ml of 10-fold dilutions (101 to1010) in D-MEM of the vaccinal strain CPV-2b 29/97-40. Each dilution was inoculated in eight wells,

obiology 105 (2005) 1928with four wells having slides for IF. After 5 days of

quantitation of the CPV-2 DNA in all the analyzed

samples. To investigate possible competition between

CPV-2 and OvHV-2 DNA amplifications, serial 10-

fold 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 ampli-

fied 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

Microbiology 105 (2005) 1928 23A 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 ofCPV 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 sampleincubation at 37 8C with CO2 5%, the slides weresubjected 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

N. Decaro et al. / Veterinaryduring the real-time PCR assay, enabling a precisehigher 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, respec-tively, 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 3.62 102 1.72 102 47.52 6.57 103 1.85 103 28.13 1.43 104 4.23 103 29.64 4.65 105 1.34 105 28.85 4.52 106 7.89 105 17.46 2.11 107 8.19 106 38.87 3.03 108 6.26 107 20.78 6.78 109 2.51 109 37.0

(b)

1 1.73 102 1.11 102 64.22 3.81 103 8.53 102 26.83 2.77 104 1.02 104 36.84 2.11 105 8.21 105 41.35 2.23 106 4.70 105 21.06 1.69 107 6.80 106 40.27 3.91 108 9.37 107 23.98 4.51 109 1.94 109 43.0

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

negative fecal samples instead of TE buffer, confirm-

ing 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 measurementover 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

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 compar-

ison 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 1011copies/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

N. Decaro et al. / Veterinary Microbiology 105 (2005) 192824

Table 3

Analysis of HA-positive fecal samples from diarrheic dogs by

conventional and real-time PCR

Sample Origin Year HA

titeraPCRb Real-time

titera

CPV-2a

102/01 (C) Apulia 2001 640 + 9.82 1010103/01 (B) Apulia 2001 320 + 3.11 1010104/01 (A) Apulia 2001 320 + 1.85 1010255/01 Apulia 2001 5120 + 3.47 1010273/01 (1) Apulia 2001 320 + 2.29 1010273/01 (2) Apulia 2001 2560 + 1.87 1011288/01 Apulia 2001 40 + 2.06 108297/01 (A) Apulia 2001 2560 + 7.43 1011307/01 (A) Apulia 2001 80 + 8.55 109307/01 (B) Apulia 2001 2560 + 2.33 109133/02 (B) Apulia 2002 320 + 9.88 108256/02 (1) Apulia 2002 640 + 2.11 101018/03 (G) Apulia 2003 160 + 1.12 109133/03 (7) Turkey 2003 640 + 5.02 109197/03 Apulia 2003 2560 + 2.25 1010300/03 Apulia 2003 40 + 5.37 109312/03 Apulia 2003 2560 + 3.42 1010

10range 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 and103.50 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 cryolysateof 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 IF HA Real-time

101 + + + + + + + + + + + +102 + + + + + + + + + + + +103 + + + + + + + + + + + +104 + + + + + + + +105 + + + + + + + +106 + + + +107 + + + +108 + + + +109 + 1010 (+) positive; () negative.a Dilutions are referred to CPV-2b vaccinal strain 29/97-40.366/03 Apulia 2003 2560 + 8.19 1028/04 Hungary 2004 1280 + 3.76 101051/04 Apulia 2004 640 + 4.89 10862/04 Apulia 2004 40 + 3.24 10967/04 (B) Apulia 2004 320 + 1.77 10974/04 Apulia 2004 1280 + 2.24 1010105/04 Apulia 2004 320 + 2.02 1010

CPV-2b

197/98 Apulia 1998 1280 + 7.98 1010201/98 Apulia 1998 320 + 6.87 109212/98 Apulia 1998 80 + 9.62 108224/98 Apulia 1998 2560 + 3.59 1010242/98 (B) Apulia 1998 80 + 6.28 1010111/99 Apulia 1999 1280 + 9.13 108130/99 (A) Apulia 1999 320 + 8.11 109

42/01 Apulia 2001 2560 + 9.68 1010

Micr

158/01 (A) Apulia 2001 + 6.91 104158/01 (B) Apulia 2001 7.33 103

158/01 (8) Apulia 2001 w 6.06 103158/01 (10) Apulia 2001 + 5.41 104158/01 (12) Apulia 2001 5.88 1036/02 (A) Apulia 2002 + 2.83 10524/02 Apulia 2002 5.32 103117/02 (7) Apulia 2002 + 2.33 104144/03 (B) Apulia 2003 8.21 103111/04 (1) Apulia 2004 + 6.50 104111/04 (3) Apulia 2004 + 8.11 104111/04 (4) Apulia 2004 + 4.79 104111/04 (5) Apulia 2004 + 1.27 105111/04 (7) Apulia 2004 w 1.55 104116/04 (1) Apulia 2004 3.65 103116/04 (3) Apulia 2004 w 1.23 104

4N. Decaro et al. / Veterinary

Table 3 (Continued )

Sample Origin Year HA

titeraPCRb Real-time

titera

281/01 (B) Apulia 2001 80 + 1.01 1010133/03 (11) Turkey 2003 5120 + 9.44 109133/03 (12) Turkey 2003 2560 + 4.19 1010133/03 (28) Turkey 2003 80 + 3.53 109133/03 (38) Turkey 2003 40 + 8.08 107233/03 (19) Turkey 2003 640 + 7.11 1010233/03 (29) Turkey 2003 640 + 1.31 1011

Glu-426 mutant

157/02 (2) Apulia 2002 2560 + 1.61 1011161/02 Apulia 2002 80 + 4.01 108202/02 (1) Apulia 2002 40 + 1.63 107202/02 (2) Apulia 2002 40 + 9.98 108105/03 Apulia 2003 10 + 9.79 108135/03 Apulia 2003 40 + 5.71 108148/03 Apulia 2003 80 + 3.44 108180/03 Apulia 2003 320 + 2.10 1010220/03 Basilicata 2003 20 + 9.52 107347/03 Apulia 2003 640 + 1.55 1010111/04 (2) Apulia 2004 40 + 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 titersbetween 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 to2.83 105 copies/mg of feces. The results obtainedby 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 approximatelyto 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).

are expressed as number of copies.b Buonavoglia et al. (2001).158/01 (1) Apulia 2001 + 1.92 105158/01 (4) Apulia 2001 + 1.89 104158/01 (5) Apulia 2001 w 1.44 104158/01 (6) Apulia 2001 + 3.88 104158/01 (7) Apulia 2001 + 5.21 104obiology 105 (2005) 1928 25

Table 4

Analysis of HA-negative fecal samples from diarrheic dogs by

conventional and real-time PCR

Sample Origin Year HA PCRa Real-time titer bThe 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,

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 Positive samples

HA PCRa Real-time

89 50 (56.18%) 68 (76.40%) 73 (82.02%)

a Buonavoglia et al. (2001).

116/04 (4) Apulia 2004 w 1.85 10116/04 (5) Apulia 2004 w 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.

Micrwe 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 CPV-

2-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 identifica-

tion of dogs shedding CPV-2 at low titers in their

feces, helping to adopt adequate measures of

prophylaxis to prevent CPV infection, especially in

N. Decaro et al. / Veterinary26kennels and shelters, where CPV-2 is often responsiblefor 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 CPV-

2 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 to7.43 1011 copies/mg of feces, while about 59% ofHA-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 erythro-

cytes. 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 possi-

bility 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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A real-time PCR assay for rapid detection and quantitation of canine parvovirus type 2 in the feces of dogsIntroductionMaterials and methodsTemplate DNA preparationDesign of primers and probeStandard DNA preparationReal-time PCRInternal controlConventional PCREvaluation of analytical performances of the CPV-2 real-time PCR assayEndpoint titration by IF, HA and real-time PCRSamples collection and typing of parvovirus strains

ResultsInternal control performanceAnalytical specificity, sensitivity and reproducibility of the CPV-2 real-time assayEndpoint titration by IF, HA and real-time PCRAnalysis of the fecal samples of dogs naturally infected with CPV-2

DiscussionReferences