Journal of Virological Methods 122 (2004) 171–178

Quantitation of porcine circovirus type 2 isolated from serum/plasma andtissue samples of healthy pigs and pigs with postweaning multisystemic

wasting syndrome using a TaqMan-based real-time PCR

Inger Marit Brunborga,∗, Torfinn Moldalb, Christine Monceyron Jonassena

a Section for Virology and Serology, National Veterinary Institute, P.O. Box 8156, N-0033 Oslo, Norwayb Section for Pathology, National Veterinary Institute, Oslo, Norway

Received 12 May 2004; received in revised form 13 August 2004; accepted 16 August 2004Available online 1 October 2004

Abstract

Porcine circovirus type 2 (PCV2) has been linked to several disease syndromes during the last decade. In this context, postweaningm terminationo titation ofP oding ORF2( esentericl two groups( sickP samplesf igh virall©

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ultisystemic wasting syndrome (PMWS) has emerged as a significant disease. As most pig herds are infected with PCV2, the def viral load in animals may be useful in discriminating between healthy and PMWS pigs. A TaqMan-based real-time PCR for quanCV2 in serum/plasma and tissue samples was established. A standard curve was created from serial dilutions of a plasmid enccapgene) of PCV2 and used to estimate the number of viral DNA copies in the analyzed samples. Comparison of viral load in mymph nodes and serum/plasma from healthy animals and PMWS animals showed statistical significant difference between thep < 0.01). No healthy pigs had viral load greater than 106 PCV2 genomes per ml serum or 500 ng tissue sample, while all clinicallyMWS pigs had PCV2 loads above 107 in both serum/plasma and in tissue samples. Furthermore, the estimated viral load in tissue

rom PMWS pigs was related to the immunohistochemical findings, with especially lymph nodes, ileum, and tonsil giving both hoad, and a high degree of staining by immunohistochemistry.

2004 Elsevier B.V. All rights reserved.

eywords:Porcine circovirus; PCV2; Quantitative PCR; Quantitation; Real-time PCR; Immunohistochemistry; PMWS

. Introduction

Infection with porcine circovirus type 2 (PCV2), but notCV1, has been associated with postweaning multisystemicasting syndrome (PMWS) in young weaned pigs. The dis-ase was first recognized in Canada in 1991 (Clark, 1997), andas since been described in the United States, South Amer-

ca, Europe, New Zealand, and South East Asia (Allan et al.,998a, 1998b, 1999b; Choi et al., 2000; Ellis et al., 1998;nuki et al., 1999; Sarradell et al., 2002). In Norway, PMWSas recently diagnosed in two herds by our laboratory.Serological surveys have shown that up to 100% of inves-

igated farms and up to 100% of individual pigs sampled inarts of Europe, the United States, and Canada were seropos-

∗ Corresponding author. Tel.: +47 23 21 64 08; fax: +47 23 21 63 01.E-mail address:inger-marit.brunborg@vetinst.no (I.M. Brunborg).

itive for PCV2 (Allan and Ellis, 2000; Atchinson et al., 199Cottrell et al., 1999; Walker et al., 2000). The fact that almost 70% of Northern Ireland archive pig sera from 1were PCV2 seropositive, and that seropositive pigs havefound in archive material from Belgium dating back to 19indicates that PCV2 has been common in pig populaseveral years before the disease became evident (Sanchez eal., 2001; Walker et al., 2000).

Usually PMWS appears in pigs aged 2–3.5 months, anfected pigs show wasting or unthriftiness, respiratory distenlarged lymph nodes, and occasionally, jaundice andrhoea (Harding, 1996; Segales and Domingo, 2002). Lym-phohistiocytic infiltration has been reported to occur ineral internal organs (Allan et al., 1998b; Ellis et al., 1998).Infection of pigs with PCV2 occurs more commonly thanobserved incidence of PMWS, and the factors pre-dispoto disease outbreak have not yet been determined. It has

166-0934/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2004.08.014

172 I.M. Brunborg et al. / Journal of Virological Methods 122 (2004) 171–178

speculated that co-infection with other pathogens could trig-ger PMWS (Allan et al., 1999a, 2000; Ellis et al., 2000;Kennedy et al., 1998; Krakowka et al., 2000). The associationof PCV2, detected in tissues by immunohistochemistry or insitu hybridization, with the PMWS characteristic histopatho-logical lesions, is therefore a necessary criterion for PMWSdiagnosis.

Several PCR methods have been described for the detec-tion of PCV2 in fresh and fixed material (Allan et al., 1999b;Kim et al., 2001; Kim and Chae, 2001; Larochelle et al.,1999; Mankertz et al., 2000; West et al., 1999). However,as PCV2 is so common within the swine population, quali-tative PCR is not suited for PMWS diagnosis. Quantitativereal-time PCRs have been described and mostly applied onserum samples (Ladekjaer-Mikkelsen et al., 2002; Olvera etal., 2004; Rovira et al., 2002).

The objectives of this study were: (1) to establish aquantitative PCR specific for PCV2 that could be used onserum/plasma and on tissue samples, and to compare theviral DNA load with immunohistochemical findings in thesame tissues, (2) to use this method on serum/plasma andtissue samples from healthy animals, as well as animals fromNorwegian PMWS outbreaks and, (3) to determine the or-gans that are most suited for quantitation of viral DNA as anindicator of PMWS.

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ufacturer’s instructions. For serum/plasma samples, 200�lwas used as starting material, while 5–25 mg tissue wereweighed and added to lysis buffer. Prior to incubation at55◦C for 1 h, the tissue samples were homogenised in ly-sis buffer with Proteinase K (20 mg/ml) using a Mixer Mill301 (Retsch) and tungsten carbide beads (Qiagen) to facili-tate complete disruption of the tissue before lysis. DNA waseluted in 125�l buffer for tissue samples and 60�l buffer forserum/plasma samples. Both the weight of the tissue used forextraction and yield of DNA were recorded.

2.3. Immunohistochemistry

Tissue samples from the heart, lung, liver, kidney,spleen, tonsils, mandibular glands, thymus, jejunum, ileum,mandibular, mesenteric, superficial inguinal, and subiliaclymph nodes were collected from the six pigs from herd A thatwere brought to the laboratory alive, while samples from alimited number of organs (including lymphoid organs) weretaken from the other pigs. The samples were fixed in 10%neutral buffered formalin, embedded in paraffin wax, andprocessed routinely for immunohistochemical investigation.A polyclonal rabbit anti-PCV2 antiserum (ER00/48 140) wasused at a dilution of 1:2000.

The results were grouped according to the number ofstained cells: no stained cells (ND), low number of stainedc um-b

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. Materials

.1. Animals and sample collection

During the spring and early summer of 2003, clinigns indicating PMWS were recognized in two farrowerds in Norway (herds A and B). Dead, clinically sind asymptomatic pigs, aged 6–9 weeks, from theseere transported to our laboratory for autopsy and paamplings for histopathological and virological investiions were carried out. Tissue samples from several orere collected from 14 pigs (11 from herd A and 3 frerd B). Of the 11 pigs from herd A, 6 were brough

he laboratory alive and euthanised shortly before autopleen and lymph nodes were sampled from all pigs, wther organs, such as liver, lung, kidney, ileum, jejunnd myocardium were included from some pigs. The tiamples were stored at−70◦C and serum/plasma sampere stored at−20◦C, until DNA isolation.Mesenteric lymph node samples from 20 healthy p

ged 5–6 weeks, were collected at an abattoir in late w004 and stored at−70◦C, until DNA isolation. Serum from0 healthy pigs, aged 5–20 weeks, from two herds waluded as controls. The serum samples were stored at−20◦C,ntil DNA isolation.

.2. DNA isolation

DNA was isolated from the tissue and serum/plasmales using DNeasy tissue kit (Qiagen) according to the m

ells (I), moderate number of stained cells (II), and high ner of stained cells (III) (Chianini et al., 2003).

.4. Design of primers and TaqMan probe

To obtain a type-specific PCR, primers were chosen iRF2 that encodes the capsid protein, as this region dis

he highest diversity between PCV1 and PCV2, and therore sequenced isolates available from ORF2 than Orepgene). Primers were chosen according to recommeions for efficient real-time PCR with TaqMan probe (Mackayt al., 2002). A TaqMan probe (TaqMan-1286-1314) arimers (PCV2-84-1256U21 and PCV2-84-1319L21),lifying an 84-base pair (bp) fragment of ORF2 of PCut not PCV1, were designed using Oligo Primer Analoftware, Version 6 (Molecular Biology Insights, Inc.),

ng the PCV2 strain from Canada (AF408635) as a mequence (Table 1).

As sequence information became available from Noian isolates, the effect of mismatches between the irimers/probe and template sequences was investigatedrimers and probes with various modifications (Table 1). Theorwegian isolates included both PCV2 from the two PMerds, named “Herd A” and “Herd B” as well as PCV2 frigs presented with other disease conditions, namedtrain 1” and “NO-strain 2” (Table 2).

.5. PCR and cloning of ORF2

Primers containing restriction sites forSalI andNotI wereesigned to amplify the complete ORF2 (capgene) of PCV2

I.M. Brunborg et al. / Journal of Virological Methods 122 (2004) 171–178 173

Table 1Primers and probes included in the study

TaqMan-1286-1314 5′-6-Fam-ATG TAA ACT ACT CCT CCC GCC ATA CAA TC-Tamra-3′TaqMan2-PCV2 5′-6-Fam-ATG TAA ACT ACT CCT CCC GCC ATA CCA TA-Tamra-3′TaqMan3-PCV2 5′-6-Fam-ATG TAA ACT ACT CCT CCC GCC ATA CCA TT-Tamra-3′PCV2-84-1256U21 5′-GTA ACG GGA GTG GTA GGA GAA-3′PCV-A-1256U21 5′-GTA GCG GGA GTG GTA GGA GAA-3′PCV-B-1256U21 5′-ATA GCG GGA GTG GTA GGA GAA-3′PCV-C-1256U21 5′-ATA GCG GGA GTG GTAAGA GAA-3′PCV2-84-1319L21 5′-GCC ACA GCC CTA ACC TAT GAC-3′PCV-D-1319L21 5′-GCC ACA GCC CAA ACC TAT GAC-3′PCV-E-1319L21 5′-GCC ACA GCC CTC ACC TAT GAC-3′PCV-F-1319L21 5′-GCA ACA GCC CTA ACC TAT GAC-3′

The names of the standard primers and probe in the assay are shown in bold. Differences in primer/probe sequences are shown in bold.

The amplicon was cloned into the TOPO TA vector (Invit-rogen), then cut withNotI and SalI, and the ORF2 frag-ment was sub-cloned into the pGEX-4T-3 vector (AmershamBiosciences) (for possible use in the production of recombi-nant protein). The recombinant pGEX-4T-3-ORF2 plasmidwas purified and the amount was determined by measuringOD260. Ten-fold dilutions were made in order to give 108–100

plasmids per 2.5�l sample for the PCR. The dilutions werestored at−20◦C, while stock plasmid was stored at−70◦C.Recombinant plasmids containing ORF2 sequences differentfrom the original clone were similarly made by amplifyingthe ORF2 from known Norwegian PCV2 isolates (“Herd A”,“Herd B” and “NO-strain 2” inTable 2).

2.6. Real-time PCR

TaqMan PCR was performed in a 25�l reaction volumeusing a LightCycler (Roche). For tissue samples, 500 ngDNA in an aliquot of 2.5�l was used as template, and forserum/plasma samples, 2.5�l of the DNA eluate was used astemplate. Viral load was expressed as copies per 500 ng DNA

Table 2Alignment of several sequences of porcine circovirus (PCV) in the primer/probe region of the TaqMan assay

S are No igs wio e Gen r/probeA n the G er/probe ari ted wit o sequence“ the as quence whend

or per ml serum/plasma, respectively. The procedure was op-timised with regard to concentrations of probe, MgCl2, andbovine serum albumin (BSA) (Fermentas). Optimisation wascarried out using three different concentrations of the recom-binant pGEX-4T-3-ORF2, and a modified Taguchi methodwas used to score the results in order to determine optimal re-action conditions (Cobb and Clarkson, 1994; Taguchi, 1986;Taguchi and Wu, 1980).

The best results were obtained with final concentrations of80 nM TaqMan probe, 4.5 mM MgCl2, and 0.1 mg/ml BSA.Later on, based on renewed recommendations from Invitro-gen, BSA concentration was increased to 0.25 mg/ml. Finalconcentrations were 200 nM of each primer, 1.5 U PlatinumTaq DNA polymerase, 20 mM Tris–HCl (pH 8.4), 50 mMKCl, 200�M of each dNTP using dUTP instead of dTTP,and 1 U uracil DNA glycosylase (UDG)) (Invitrogen). Cy-cling parameters were 50◦C for 2 min, 95◦C for 2 min, 45cycles of 95◦C for 10 s, and 62◦C for 30 s followed by cool-ing at 40◦C for 30 s at the end of the 45th cycle (LightCyclerOperator’s Manual, Version 3.0, Roche). The PCR was alsotried out successfully on an ABI 7900 thermocycler (Applied

equences called “NO-strain 1”, “NO-strain 2”, “Herd A” and “Herd B”ther disease conditions than PMWS. The other sequences are from thll but U49186 are PCV2 sequences. All PCV1 sequences available i

n the genome are given in bold, and orientation of the probe is indicaNO-strain 1” which is the one used as standard and positive control inesigning primers and probe.

rwegian strains, “NO-strain 1” and “NO-strain 2” were isolated from pthBank, and are chosen to visualise the sequence variations in the primeregion.enBank are identical in this region. The sequence spanning the primea

h FAM and TAMRA. Primers and probe are an almost perfect match tsay (pGEX-4T-3-ORF2). AF408635 (Canada) was used as master se

174 I.M. Brunborg et al. / Journal of Virological Methods 122 (2004) 171–178

Biosystems), with slight modifications both in cycling times(45 cycles of 95◦C for 15 s instead of 10 s, and 62◦C for45 s instead of 30 s), and in PCR mix (i.e., addition of Roxreference dye, and removal of BSA).

Three concentrations of pGEX-4T-3-ORF2 (107, 104, and102 copies per sample) were included in each run, and servedas positive controls as well as to derive the standard curve usedfor quantitation of PCV2 DNA in tissue and serum/plasmasamples. Each run included two negative controls (no tem-plate) as well.

3. Results

3.1. Detection and quantitation limit of the assay

In order to assess the linearity range and the detection limitof the assay, samples containing 107, 105, and 102 copiesof pGEX-4T-3-ORF2 per PCR sample were run in dupli-cates, and samples containing from 90 to 10 copies, preparedas a linear dilution in steps of 10 copies per PCR sample,were run in triplicates. Average Ct and standard deviationwere recorded (LighCycler software, Version 3.5, Roche).The testing resulted in detection and quantitation limits of10 and 100 copies per sample, respectively, as amounts be-t of thes

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3.3. Primer design and Fasta search for primersimilarities

The current assay leads to a PCR product of 84 bp, withprimers and probe covering 71 of the 84 bp (Table 2). Fastasearches with the chosen primers/probe showed that therewere up to six mismatches with some PCV2 strains. How-ever, when aligning all available PCV2 sequences from theGenBank in ORF2, no stretch of 100 bases meeting the crite-ria recommended for TaqMan PCR design were 100% con-served between all strains (Mackay et al., 2002). Besides, thepresent PCR efficiently amplified various Norwegian fieldstrains (data not shown) even if later nucleotide sequenc-ing showed up to six mismatches in the primers/probes. Al-though, some Norwegian strains have several mismatches inprimers and probe combined, there are also strains with al-most perfect match (Table 2), and even PCV2 sequences fromthe Norwegian PMWS cases with five mismatches proved tofunction well in the assay, as viral load was calculated to behigh (Fig. 2a).

3.4. Effect of mismatches between primers/probe andtemplate sequence on quantitation

Potential effect of mismatches in primers and/or probeo sam-p mersa f dif-f -s pyn s andp orig-i ersa berb int oftP fromt est yw

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ween 10 and 100 copies were outside the linear rangetandard curve (Fig. 1).

.2. Specificity of the assay

The specificity of the assay was examined with regaCV1. Duplicates of PCV1 DNA, isolated from 100�l of auspension of PK15 cells permanently infected with PCere run at the optimal conditions of the assay, and nrease of fluorescence was recorded.

ig. 1. Determination of detection and quantitation limit. Average Ct valotted against plasmid copy numbers with standard deviation given fooncentration. The line indicates the linear fit.

n the performance of the assay was tested by runningles in parallel with standard primers and probe, and prind probe designed to perfectly match sequences o

erent Norwegian isolates (“Herd A”, “Herd B”, and “NOtrain 2” inTable 2). Differences in Ct and calculated coumbers were recorded. The perfectly matched primerrobes gave higher estimates of copy number than the

nal primer/probe set. A total of five mismatches in primnd probe resulted in an underestimation of copy numy one log10 unit, while a total of six mismatches resulted

wo log10 units underestimation of copy number. Loweringhe annealing/elongation temperature from 62 to 60◦C in theCR reduced the effect of mismatches on quantitation

wo to one log10 unit for the template with six mismatchotal (sequence B inTable 2), without reducing specificitith regard to PCV1 (data not shown).

.5. Quantitation of viral load in different tissues anderum/plasma samples

The assay was tested on serum/plasma and sampleeveral tissues from both healthy and PMWS pigs (Fig. 2a).or both mesenteric lymph nodes and serum/plasma

istically significant differences between the PCV2 lrom healthy and PMWS pigs were found (p < 0.01)MicrocalTMOriginTM Version 5.0, Microcal Software, Incith a cut off between healthy and PMWS pigs at 106–107

CV2 genomes per ml serum/plasma or per 500 ng DNAal load differed slightly between tissues from the sameith lymphoid tissue, spleen, liver, thymus, lung, tonsil,

leum giving the highest yields, up to 1010 PCV2 genome

I.M. Brunborg et al. / Journal of Virological Methods 122 (2004) 171–178 175

Fig. 2. Box Plot of estimated viral load (copies per 500 ng DNA) in (a)mesenteric lymph nodes from healthy (N= 20) and PMWS (N= 11) animalsand serum or plasma (copies per ml) from healthy (N= 80) and PMWS (N=6) animals, respectively, (b) plasma and several organs from PMWS animals(N = 6). The horizontal lines in the box denote the 25th, 50th, and 75thpercentile values. The error bars denote the 5th and 95th percentile values.The two symbols below the 5th percentile error bar denote the 0th and 1stpercentile values. The two symbols above the 95th percentile error bar denotethe 99th and 100th percentiles. The square symbol denotes the mean of thecolumn of data. Some of the symbols denoting different percentiles coincidedue to low numbers of observations within each group.

per 500 ng DNA. Lymph nodes (mandibular, mesenteric, andsuperficial inguinal) and tonsil had consistently higher esti-mates of viral load than other organs tested. Estimated viralload in tissues and plasma were related (Fig. 2b).

3.6. Immunohistochemistry versus viral load

Viral load in tissues examined was related to the immuno-histochemistry findings (Fig. 3). A statistically significantdifference was observed between viral load in IHC groups IIand III, and IHC group ND (p< 0.05) (MicrocalTMOriginTM

Version 5.0, Microcal Software, Inc.). For most tissues an es-timated viral load of minimum 108 PCV2 genomes per 500 ng

Fig. 3. Box Plot of the correlation between estimated viral load by PCR(copies per 500 ng DNA) and immunohistochemical results grouped ac-cording to the number of stained cells, ND (not detected), I (+), II (++) andIII (+++). The horizontal lines in the box denote the 25th, 50th, and 75thpercentile values. The error bars denote the 5th and 95th percentile values.The two symbols below the 5th percentile error bar denote the 0th and 1stpercentile values. The two symbols above the 95th percentile error bar de-note the 99th and 100th percentiles. The square symbol in the box denotesthe mean of the column of data. Some of the symbols denoting differentpercentiles coincide due to low numbers of observations within each group.

DNA was required in order to give a visible staining in im-munohistochemistry. Lymph nodes had both high viral loadand strong immunohistochemistry staining in PMWS pigs.Still, even with viral load as high as 108 PCV2 genomes per500 ng DNA, no visible staining was observed in kidney andmyocardium of PMWS pigs.

4. Discussion

The current assay was applied to samples from bothhealthy and PMWS pigs. Most tissue samples tested byquantitative PCR were positive for PCV2, even from young,healthy pigs, while several serum samples were below thedetection limit of 1.2× 103 template copies per ml serum.The negative serum samples may be due to the low age of thepigs tested.

Mismatches between the target viral sequences and theprimers/probe set can influence the accuracy of quantita-tion. However, this is often difficult to avoid when designingprimers/probes for virus detection. In the currently describedassay, even samples that had some sequence heterogeneity inthe primers/probe region, yielded high estimates of viral load,comparable to those without mismatch. The presence of mis-matches was shown to affect the quantitation to some extent.E obe,t as-soo that

ven so, with a total of six mismatches in primers and prhe log10 difference did not exceed one, when running theay at an annealing/elongation temperature of 60◦C insteadf 62◦C. This difference is lower than the log10 differencebtained between healthy and PMWS pigs, suggesting

176 I.M. Brunborg et al. / Journal of Virological Methods 122 (2004) 171–178

the effect of these mismatches for discriminating betweenhealthy and sick pigs is not critical in the assay. Loweringthe annealing temperature to 60◦C did not lead to detec-tion of PCV1 in the assay, and it is therefore suggested thatfuture routine analyses should be undertaken with an anneal-ing/elongation temperature of 60◦C with the chosen primersand probe. PCV1 was otherwise shown to give high estimateson copy numbers when tested with the current probe in com-bination with PCV1-specific primers, even though there arefive mismatches in the probe. As shown inTable 2, PCV1has seven mismatches in one primer alone, and this is mostlikely the reason why PCV1 was not detected in the currentassay. Other researchers have reported a 3–5 log10 differencebetween perfectly matched primers and three mismatches inone primer in quantitative PCR (Markowski-Grimsrud et al.,2002). The annealing temperature in the assay in relation tothe Tm of the primer, as well as MgCl2 concentration, mightinfluence its efficiency. Another major factor influencing theeffect of mismatches in a quantitative assay is the locationof the mismatches in the primers and probe. Mismatches lo-cated in the 3′ end of primers, 5′ end of a TaqMan probe, or astretch of consecutive mismatches, are expected to have thegreatest influence on quantitation (Klein et al., 1999; Kwoket al., 1990; Smith et al., 2002).

Viral load in serum/plasma and tissue samples was pre-s A orp sedi bes dingD la-t ples.T bet ondi-t ter-n notg s. Inf ationt veryp highc parto parti-c anti-t sesh2 02;O y( ea e lat-tm tho 100t virall , asi dt als.T t be

due to the fact that their study was performed on experimen-tally infected pigs, allowing fresh collection of tissue samplesfrom the dead animals, while the present investigations arebased on cases of naturally infected pigs. Most pigs includedin the study were dead prior to shipping to the laboratoryand the collected tissue samples were in various states ofdegradation. This may have influenced the recovery of non-packaged viral DNA, and an underestimation of viral load intissues.

Even though most samples were positive for PCV2 byquantitative PCR, there was a statistically significant dif-ference between estimated viral load in mesenteric lymphnodes and serum/plasma when comparing healthy animalsto animals from PMWS herds (p < 0.01). Quantitative PCRas described here therefore seems to work as an indicatorof the relevance of PCV2 infection in an animal. A relationbetween PCV2 load and amount of antigen detected in as-sociation with lesions, as seen in immunohistochemistry inPMWS pigs, was observed in this study. This further corrob-orates that a high PCV2 load in tissues in an animal is a goodindicator of its PMWS status. Ranking of viral load in differ-ent animals was conserved for all analyzed tissues, and therelation between estimated viral load in plasma and tissuessupports further the proposal that viral load in serum/plasmais an indicator of PMWS. Significant differences in viral loado eend l.,2c sp ose ar mis-m osens n theG thesem mis-m 10P tissues pigs,a ey canb suest icatoro istryfi atesw ficiali bothc be-t

useda osis,a iatedw

asedr ndp atedv am-p sma.

ented as number of PCV2 genomes per 500 ng DNer milliliter serum/plasma. As no internal control was u

n this study, quantitation of viral load in tissues mightomewhat biased, depending on sample quality, incluNA degradation prior to analysis, efficiency of DNA iso

ion, and presence of potential PCR inhibitors in the samhe factor influencing most DNA recovery is expected to

ransport conditions and storage of samples, or other cions leading to tissue decomposition. Addition of an inal control upon arrival of samples in the laboratory willive any indications about these determinant yield factor

act, some tissue samples were in such state of degradhat most cellular DNA was degraded as assessed byoor DNA yield. Such samples, however, often showedopy numbers of target DNA, probably because a largef the virus genomes in the samples are packaged inles, and therefore protected from lytic processes. Quation of PCV2 and other previously described circoviruave been carried out on serum/plasma samples (Liu et al.,000; Markowski-Grimsrud et al., 2002; Moen et al., 20lvera et al., 2004; Rovira et al., 2002) in all but one stud

Ladekjaer-Mikkelsen et al., 2002), where the quantitativssay was performed on tissue samples as well. In th

er study, viral load was reported to be as high as 1010 perl serum and 3× 107 per cell. This corresponds well wiur findings in plasma samples, but is approximately 10–

imes more than our highest observations of estimatedoad of 1010 per 500 ng DNA in tissue samples. Howevern theLadekjaer-Mikkelsen et al. (2002)study, we observehe highest viral load in tissue samples from dead animhe difference in the highest estimated viral load migh

f PCV2 in serum from sick and healthy animals have bescribed by other authors (Liu et al., 2000; Olvera et a004; Rovira et al., 2002). Olvera et al. (2004)reported aut off for histopathological PMWS at 107 PCV2 genomeer ml serum, but, as in our assay, the researchers cheal-time PCR targeted to ORF2, and there were someatches in the primers/probe set, when comparing the ch

equences with an alignment of various PCV2 isolates ienBank. The researchers did not discuss the effects ofismatches on quantitation, but our results, even withatches, support a cut off for histopathological PMWS at7

CV2 genomes per ml serum. Serum was as suited asamples for discriminating between healthy and PMWSnd is less laborious to process than tissue samples. The used for screening of live animals as well. The tis

hat seemed most suited for quantitative PCR as an indf PMWS, based on the relation to immunohistochemndings and on the consistency of the viral load estimere lymph nodes (mesenteric, mandibular, and super

nguinal), tonsil, and ileum. Mesenteric lymph nodes areonvenient to sample and were suited for discriminationween healthy and PMWS pigs.

Quantitative PCV2-specific PCR can therefore bes a complementary test to histochemical PMWS diagnnd could help to identify other disease conditions associth PCV2.In conclusion, we have established a TaqMan-b

eal-time PCR for quantitation of PCV2 in tissue alasma/serum samples. A significant difference in estimiral load between healthy and PMWS pigs was found in sles from mesenteric lymph nodes as well as serum/pla

I.M. Brunborg et al. / Journal of Virological Methods 122 (2004) 171–178 177

This information supports the view that PCV2 load in theanimal is correlated with disease.

Acknowledgements

This study was supported by Grant No. 14328601 from theNorwegian Research Council through the strategic instituteprogramme No. 143286/140 “Virological investigations onemerging disease conditions in domestic animals and fish”.Dr. Gordon Allan at Department of Agriculture for North-ern Ireland, Veterinary Science Division, Belfast, UK, kindlyprovided the antibodies used for immunohistochemistry. Dr.Anette Bøtner at the National Veterinary Institute in Den-mark kindly provided the PK15 cells persistently infectedwith PCV1. We would also like to thank Professor EspenRimstad at the Norwegian School of Veterinary Science forconstructive reading of the article, while in process and Dr.Bjørn Lium for providing material from both negative con-trols and PMWS animals, and to Sheryl Asmus for proofreading the manuscript before final submission.

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