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1 Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
2 Center for Molecular Medicine and Infectious Diseases, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
3 Animal Health Laboratory, Laboratory Services Division, University of Guelph, PO Box 3612, Guelph, ON N1H 6R8, Canada
Correspondence
T. Opriessnig
tanjaopr{at}iastate.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the complete genomic sequences of PCV2 isolates Can-17639 and NC-16845 are respectively EU340257 and EU340258.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) that belongs to the genus Circovirus of the family Circoviridae (Todd et al., 2005). To date, two types of PCV have been recognized (Allan et al., 1998; Hamel et al., 1998; Morozov et al., 1998) in pigs: the non-pathogenic PCV type 1 (PCV1) and the pathogenic PCV type 2 (PCV2), which is the aetiological agent of porcine circovirus-associated disease (PCVAD). Previously, phylogenetic analyses have shown that PCV2 isolates can be further divided into two main clusters (Larochelle et al., 2002; Mankertz et al., 2000; Olvera et al., 2007) now commonly referred to as PCV2a and PCV2b (Gagnon et al., 2007).
Systemic PCV2 infection, which is also known as post-weaning multisystemic wasting syndrome (PMWS), is characterized clinically by wasting or decreased weight gain, enlarged lymph nodes and dyspnoea (Harding & Clark, 1997; Opriessnig et al., 2007). The hallmark microscopic lesions of systemic PCV2 infection are lymphoid depletion and granulomatous lymphadenitis associated with the presence of PCV2 antigen or nucleic acids (Sorden, 2000). Systemic PCV2 infection or PMWS was initially observed in a Canadian high-health-status herd in 1991 (Harding & Clark, 1997) and was later recognized worldwide (Allan & Ellis, 2000) and associated with major losses in Europe (Harding, 2004). PCVAD essentially became quiescent in Canada shortly after its initial recognition in the early 1990s except for sporadic case reports. In 2004, a marked increase in the incidence and severity of PCVAD was observed in eastern Canada (Carman et al., 2006; DeLay et al., 2005). The severe outbreaks of PCVAD in Canada, followed by similar outbreaks in North Carolina and the Midwest United States, have raised concerns over the introduction of a new and more virulent PCV2 variant into North America. Several research groups found the PCV2b cluster, previously not recognized in North America, to be associated with the majority of the recent severe PCVAD outbreaks (Carman et al., 2006; Cheung et al., 2007; DeLay et al., 2005; Gagnon et al., 2007; Horlen et al., 2007). Similar observations have been made in Europe (Dupont et al., 2008; Grau-Roma et al., 2008).
It is still debatable whether differences in virulence between PCV2 clusters and isolates exist. PCV2 has been detected in both severely affected herds and clinically non-affected herds by PCR and ELISA. Interestingly, nearly identical PCV2 genomes have been found in healthy pigs and in diseased pigs from affected and unaffected herds (de Boisseson et al., 2004; Larochelle et al., 2002). In contrast, a PCV2a field isolate from a case with moderate-to-severe PCV2-associated lesions was compared with a PCV2a isolate from a case with no PCV2-associated lesions and significant differences in expression of lesions were seen in experimentally inoculated pigs (Opriessnig et al., 2006c). To our knowledge, with the exception of one small study done in germ-free pigs (Lager et al., 2007) and one study using conventional PCV2-seropositive pigs (Fort et al., 2008), a thorough head-to-head comparison of the virulence of PCV2a and PCV2b isolates has not yet been done. The first objective of this study was to compare the virulence of recent PCV2b isolates with well-characterized US PCV2a isolates in the conventional specific-pathogen-free (SPF) pig model.
Allan et al. (2002) demonstrated that piglets with passively acquired anti-PCV2-antibodies were protected from developing PCVAD after PCV2 challenge. Similarly, Fenaux et al. (2004a) demonstrated that pigs vaccinated with PCV2 and challenged with the same strain were protected. McKeown et al. (2005) found that protection against PCV2 infection conferred by maternal antibodies was titre-dependent. However, although PCV2 is widespread and essentially all pig herds have anti-PCV2-antibodies (Larochelle et al., 2003; Liu et al., 2002; Mankertz et al., 2000; Walker et al., 2000) in certain geographical regions such as the UK (Woodbine et al., 2006), Canada (Carman et al., 2006; DeLay et al., 2005), Denmark (Vigre et al., 2005) and the USA (Cheung et al., 2007), PCVAD appears to have spread, implying that the antibodies present in the pigs were not protective. Nevertheless, it has been shown that the presence of PCV2-neutralizing antibodies correlates with decreased PCV2 replication and development of clinical PCVAD under experimental conditions (Meerts et al., 2005, 2006) and in the field (Fort et al., 2007). Interestingly, Lefebvre et al. (2008) identified differences in affinity and neutralization ability of monoclonal antibodies directed against the PCV2 capsid protein when tested against PCV2 isolates with different genetic or clinical backgrounds. The second objective of this study was to determine whether infection with PCV2a isolates induces protective immunity against a recent PCV2b isolate.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and has been well characterized genetically (Fenaux et al., 2000) and in the conventional SPF pig model (Fenaux et al., 2002, 2003, 2004a, b; Opriessnig et al., 2003, 2004a, b, 2006a, b). Isolate ISU-40895 was found to be capable of inducing characteristic PCV2-associated microscopic lesions and clinical disease under experimental conditions (Opriessnig et al., 2004a, b, 2006a).
Isolate ISU-4838 (cluster a; GenBank accession no. DQ397521
[GenBank]
) was recovered from a subclinically infected pig on an Iowa farm in 2003 and is of low virulence, based on experimental inoculations (Opriessnig et al., 2006c).
Isolate Can-17639 (cluster b) was recovered from a clinically affected pig fram North Carolina with systemic PCV2 infection during the PCVAD outbreak in 2006 in Canada. Affected pigs had clinical signs of wasting and coughing, with bronchointerstitial pneumonia and histiocytic lymphadenitis, and were found to be negative for swine influenza virus (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae. Abundant PCV2 antigen was found by immunohistochemistry (IHC) in tonsil and lymph nodes.
PCV2 isolate NC-16845 (cluster b) was recovered from a clinically affected 9-week-old pig with systemic PCV2 infection from a group of pigs from North Carolina with a history of severe respiratory disease in 50 % of the pigs and 
20 % mortality in the group. The pigs had severe necrotizing lymphadenitis with lymphoid depletion of follicles and severe bronchointerstitial pneumonia and were positive for PCV2 (abundant staining) by IHC and for PRRSV by PCR.
Amplification and genetic and phylogenetic analyses of the complete genomic sequence of the PCV2 isolates.
PCV2a isolates ISU-40895 and ISU-4838 have been sequenced in previous studies (Fenaux et al., 2000; Opriessnig et al., 2006c). To determine the complete genomic sequence of the PCV2b isolates NC-16845 and Can-17639, DNA was extracted from pooled lung and lymph node homogenates according to the protocol of the QIAamp DNA Mini kit (Qiagen). Primers F-PCV2SacII and R-PCV2ScacII were used as described previously (Opriessnig et al., 2006c) to amplify the entire genome of both isolates. The PCR products, 1.7 kb, were purified by gel extraction (QiaQuick gel extraction kit, Qiagen) and ligated into plasmid pSKII+ (Stratagene) after SacII digestion of both plasmid and inserts. The pSKII+/PCV2b constructs were sequenced as described previously (Opriessnig et al., 2006c). Sequence assembly was carried out using Lasergene6 (DNASTAR). Sequences were analysed and percentage identities were determined using the CLUSTAL alignment program in the MacVector computer software package. Phylogenetic analysis was conducted with the PAUP program using the maximum-parsimony method with 100 bootstrap replicates (David L. Swofford, Smithsonian Institution, Washington, DC; distributed by Sinauer Associates). The heuristic search protocol with bootstrap analysis was used to generate a phylogenetic tree.
Generation of infectious DNA clones of PCV2a and PCV2b and production of virus inocula.
Construction of infectious DNA clones for the PCV2a isolates ISU-40895 and ISU-4838 has been reported previously (Fenaux et al., 2002; Opriessnig et al., 2006c). The infectious DNA clones of the PCV2b isolates NC-16845 and Can-17639 were constructed essentially as described by Fenaux et al. (2000) and Opriessnig et al. (2006c). The infectivity of all infectious DNA clones was confirmed by transfection of PK-15 cells followed by immunofluorescence detection of virus capsid antigen and the generation of infectious stock of the PCV2 isolates was carried out as described previously (Fenaux et al., 2002, 2003, 2004a, b; Opriessnig et al., 2006c) with a minor modification. DNA quantification for transfection was carried out using the Nanodrop spectrophotometer (Thermo Fisher Scientific). For comparison, the infectious titres of all four viral stocks were adjusted to the same titre, 104.0 50 % tissue culture infectious dose (TCID50) per ml. Each pig received 5 ml of the respective PCV2 inoculum intranasally (3 ml) and intramuscularly (2 ml).
Animals and housing.
One-hundred-and-thirteen crossbred, 21-day-old, conventional SPF pigs were purchased from a herd that is free of PCV2, PRRSV and SIV. Blood samples taken from a portion of pigs on the day of arrival and at 11 weeks of age were negative for porcine parvovirus (PPV), PRRSV, H1N1 SIV, H3N2 SIV and Mycoplasma hyopneumoniae. On the day of arrival, the pigs were randomly assigned to one of 13 rooms (Table 1). Rooms contained 2.5x3.6 m raised wire decks and were equipped with one nipple drinker and a self-feeder per pen. All groups were fed a balanced, pelleted, complete feed ration free of animal proteins and antibiotics (Nature's Made) ad libitum.
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In vivo characterization and virulence comparison of PCV2a and PCV2b isolates.
This part of the experiment compared the PCV2a and PCV2b isolates in an established conventional SPF pig model (Opriessnig et al., 2006c). Pigs were inoculated with ISU-40895, ISU-4838, Can-17639 or NC-16845 at 4 or 9 weeks of age (Table 1). Six pigs per group were necropsied 16 days post-inoculation (p.i) and six to eight pigs per group were necropsied at 51 days p.i. The amount of PCV2 DNA in serum samples, the levels of anti-PCV2-antibodies, and the average scores of overall PCV2-associated lymphoid lesions were compared at 16 and 51 days p.i.
Evaluation of cross-protection between PCV2a and PCV2b isolates.
Pigs were inoculated with ISU-40895, ISU-4838 or NC-16845 at 4 weeks of age followed by challenge with the heterologous cluster at 9 weeks of age (Table 1). All pigs were necropsied 16 days after the challenge (51 days after initial inoculation). The amount of PCV2 DNA in serum samples, the levels of anti-PCV2 antibodies and the average scores of overall lymphoid lesions in heterologously challenged pigs were compared with those of pigs singly inoculated with either PCV2a (ISU-40895 and ISU-4838) or PCV2b (NC-16845) at 9 weeks of age. The presence and amount of neutralizing PCV2-antibodies in heterologously challenged pigs were compared with those for pigs singly inoculated with either PCV2a or PCV2b at 4 weeks of age.
Serology.
Blood samples were collected upon the arrival of the pigs at the research facility and weekly thereafter until necropsy. For anti-PCV2 IgG antibodies, serum samples were tested by an ORF2-PCV2 IgG ELISA as described previously (Nawagitgul et al., 2002). Samples were considered positive if the calculated sample-to-positive (S : P) ratio was 0.2 or greater. For neutralizing PCV2 antibodies a fluorescence focus neutralization assay was done on serum samples collected at 0 (inoculation 1), 35 (inoculation 2) and 49 days p.i. in order to determine the presence of neutralizing antibodies against PCV2 according to the Iowa State University Veterinary Diagnostic Laboratory standard operating protocol (Pogranichnyy et al., 2000). The assay was performed twice with PCV2a isolate ISU-98-15237 and with PCV2b isolate ISU-07-6594. In addition, serum samples from one randomly selected pig in each group taken on arrival at the research facility and at necropsy were tested for the presence of antibodies to PRRSV by PRRSV-ELISA (IDEXX Laboratories), PPV by haemagglutination inhibition (HI) assay (Mengeling et al., 1988), Mycoplasma hyopneumoniae by ELISA (Bereiter et al., 1990) and H1N1 SIV and H3N2 SIV by HI assays according to the protocol used at the Veterinary Diagnostic Laboratory of Iowa State University.
Clinical evaluation.
Following PCV2-inoculation, the pigs were evaluated daily for clinical signs including wasting, behavioural changes such as lethargy and inappetence.
PCV2 DNA quantification.
DNA extraction from serum samples collected at 0, 7, 14, 21, 28, 35, 42 and 49 days p.i. was performed using the QIAamp DNA Mini kit (Qiagen). DNA extracts were used for quantification of PCV2 genomic DNA copy numbers by real-time PCR (Opriessnig et al., 2003).
Necropsy.
Necropsies were performed on randomly selected pigs at 16 days p.i. and on the remainder at 51 days p.i. The total numbers of macroscopic lung lesions were estimated in a blinded fashion as described elsewhere (Opriessnig et al., 2004b). Sections of lung, lymph nodes (superficial inguinal, mediastinal, tracheobronchial and mesenteric), tonsil, thymus, ileum, kidney, colon, spleen and liver were collected at necropsy and fixed in 10 % neutral-buffered formalin and processed routinely for histological examination.
Histopathology.
Microscopic lesions in lungs, heart, liver, kidney, brain, ileum and colon were evaluated in a blinded fashion as described previously (Opriessnig et al., 2004b). Lymphoid tissues including lymph nodes, tonsil and spleen were evaluated for the presence of lymphoid depletion, ranging from 0 (normal) to 3 (severe), and histiocytic inflammation and replacement of follicles, ranging from 0 (normal) to 3 (severe), (Opriessnig et al., 2004b).
Immunohistochemistry.
IHC for detection of PCV2-specific antigen was performed on formalin-fixed and paraffin-embedded sections of lymph nodes (superficial inguinal, mediastinal, tracheobronchial and mesenteric), tonsil, spleen, Peyer's patches and thymus using a rabbit polyclonal antiserum (Sorden et al., 1999). PCV2 antigen scoring was done in a blinded fashion and scores ranged from 0 (no signal) to 3 (> 50 % of the lymphoid follicles containing cells with PCV2 antigen staining) (Opriessnig et al., 2004b).
Overall lymphoid lesion score.
The overall microscopic lymphoid lesions scores, which account for lymphoid depletion, histiocytic inflammation and PCV2 antigen present in lymphoid tissues, were calculated for each pig as described previously (Opriessnig et al., 2004b) and ranged from 0 (normal) to 9 (severe).
Sequencing.
PCR products amplified from virus recovered from one randomly selected pig from each inoculation group at 49 days p.i were sequenced and compared with the respective inoculum. Nested PCR was used to amplify a fragment of the PCV2 genome including the entire ORF2 gene for sequencing and sequence comparison (Opriessnig et al., 2006c). For the PCR, Invitrogen Platinum PCR SuperMix High Fidelity was used. PCR products were run on a 1 % agarose gel and the expected 820 bp products were excised, purified and sequenced at the Virginia Bioinformatics Institute at Virginia Tech using an Automated DNA Sequencer (Applied Biosystems). Sequences were analysed with the MacVector computer program and compared with the sequences of the original virus inocula.
Differential PCR.
Serum samples obtained from singly inoculated and reinoculated pigs obtained at 42 and 49 days p.i. were tested for PCV2a and PCV2b by differential quantitative real-time PCR (Veterinary Diagnostic Laboratory at Kansas State University). The PCR was performed according to laboratory-specific protocols and was able to detect and differentiate between the PCV2a and PCV2b cluster isolates used in this study.
Statistical analysis.
Summary statistics were calculated for all groups to assess the overall quality of the data, including normality. Analysis of variance (ANOVA) was used for cross-sectional assessment of continuous measures. The rejection level for the null hypothesis was 0.05 followed by pairwise testing using the Tukey Kramer adjustment. In order to summarize and simplify the clinical observations, response feature analysis and a chi-square test was used. Non-repeated measures of necropsy and histopathology data were assessed using non-parametric Kruskal–Wallis one-way ANOVA. If this was significant (P<0.05), pairwise Wilcoxon tests were used to assess differences between groups. All data were analysed for clusters (PCV2a versus PCV2b) followed by analysis for isolates. The data were summarized for pigs inoculated at 4 and at 9 weeks of age if the effect of age was non-significant in an initial ANOVA testing.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Phylogenetic analysis confirmed that isolates Can-17639 and NC-16845 grouped closely with other PCV2b isolates (Fig. 1).
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Effect of age on PCV2 infection.
Analysis of the data indicated no effect of age of inoculation on the number of PCV2 DNA copies in serum (P=0.238) or the severity of microscopic lymphoid tissue lesions (P=0.215). Therefore, the microscopic lesion and PCR data for the pigs inoculated at 4 and 9 weeks of age were combined for further evaluation.
There was a significant difference between age groups in anti-IgG antibody response: at 14 days p.i., the mean PCV2 IgG S : P ratio in pigs challenged at 9 weeks of age (0.242±0.017; mean±SEM) was significantly higher (P<0.001) than that in pigs challenged at 4 weeks of age (0.121±0.016). No significant differences between groups inoculated with PCV2a and PCV2b (P=0.074 and 0.925 for challenge at 4 and 9 weeks of age, respectively) or individual isolates were noted.
Anti-PCV2 IgG antibody levels.
Pigs in all inoculated groups seroconverted to PCV2 between 14 and 35 days p.i. Pigs in the PCV2a group had a significantly higher antibody response in comparison with PCV2b pigs at 14 and 21 days p.i. (P=0.019 and 0.016, respectively) (Fig. 2a). When analysed by individual isolates, ISU-40895 had significantly (P<0.05) lower S : P ratios at 14 days p.i. in comparison with ISU-4838. From 28 to 49 days p.i., this difference was reversed but remained significant (P<0.05). Similarly, the levels of anti-PCV2 IgG antibodies in Can-17639-inoculated pigs were lower than those observed in the NC-16845-inoculated pigs at 35 and 49 days p.i. (P=0.020 and 0.028, respectively) (Fig. 2b).
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). PCV2 DNA was detected on all days and in all pigs following inoculation with ISU-4838, ISU-40895 and NC-16845; however, the incidence of PCR-positive serum samples was lower in Can-17639-inoculated pigs (5/12 pigs at 7 days p.i., 9/12 pigs at 14 days p.i., 5/6 pigs at 21 days p.i., 6/6 pigs at 28, 35 and 42 days p.i. and 5/6 pigs at 49 days p.i.). The amount of Can-17639 PCV2 DNA was reduced significantly (P<0.001) at 7 (1.7±0.6) and 14 days p.i. (3.6±0.7) compared with all other groups (Fig. 3b).
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). Additionally, the pigs inoculated with ISU-40895 and NC-16845 had significantly (P<0.05) more severe lesions compared with the negative control pigs and Can-17639-inoculated pigs but were not different (P>0.05) from each other. In pigs that were necropsied at 51 days p.i., there was mild-to-severe lymphohistiocytic interstitial nephritis present in individual PCV2a- and PCV2b-inoculated pigs. The overall PCV2-associated lymphoid lesions appeared to have resolved in all groups and were not different (P=0.533) between clusters and isolates (Table 1).
Prior exposure to PCV2 induces protective immunity against heterologous cluster challenge
Anti-PCV2 IgG antibody levels.
After reinoculation, the PCV2-antibody levels in PCV2a–b and PCV2b–a inoculated groups were not different (P>0.05) compared with those in singly infected pigs (Fig. 4). A significant decrease or increase in anti-PCV2 IgG antibodies was not observed in dual-infected groups.
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Incidence and amount of PCV2 DNA in serum samples.
PCV2 DNA levels in pigs inoculated with PCV2a and then PCV2b or PCV2b and then PCV2a were not different (P>0.05) compared with those in single-infected pigs (data not shown).
Sequencing and PCR typing of the PCV2 DNA present in reinoculated (challenged) pigs.
ORF2 sequencing of the DNA obtained from one randomly selected pig from each group at 49 days p.i. revealed the recovery of the PCV2 used for initial inoculation 49 days previously in all groups including reinoculated groups. We were not able to detect the isolate used for reinoculation/challenge in any of the serum samples analysed. This finding was further confirmed by PCV2a-/PCV2b-specific quantitative real-time PCR. A total of 48 serum samples (24 on day 42 and 24 on day 49) were analysed and the PCV2 cluster present in 47/48 serum samples was identified as the initial cluster that was used to inoculate the pigs 42 and 49 days previously, respectively. A mixed PCV2a and PCV2b population was detected in one out of 48 samples on day 42. One week later, on day 49, the cluster used for reinoculation was no longer detectable in serum from this pig.
Microscopic lesions and incidence of PCV2 antigen in tissues.
Pigs inoculated at 4 weeks of age with PCV2a or PCV2b and reinoculated at 9 weeks of age with the heterologous isolate had normal lymphoid tissues or mild PCV2-associated lesions similar to what is expected in the resolving stages of infection (Table 1). There was a reduction in the PCV2-associated lesions in pigs inoculated at 35 days p.i. with either PCV2a or PCV2b if they had been exposed to the heterologous isolate 35 days previously (Fig. 5), and the reduction was significant (P=0.023) for PCV2a–b versus PCV2b.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Gagnon et al. (2007) investigated 83 Canadian PCV2 cases submitted in 2005 and 2006 and found that 79.5 % of the sequences were PCV2b. Twenty-four PCV2 sequences from isolates collected from six herds located in Iowa, Kansas and North Carolina in 2005 were analysed and 21 of the sequences were determined to be PCV2b (Cheung et al., 2007). Similarly, Horlen et al. (2007) further characterized the PCV2 isolates involved in an outbreak of PCVAD in Kansas and found that all PCV2 genomic sequences recovered from affected animals belonged to the PCV2b cluster. In the current study, cloned isolates from the PCV2a and PCV2b clusters were compared side by side in the conventional SPF pig model. Clones were used instead of cell-culture-propagated virus(es) to reduce the risk of inoculation of pigs with an unknown second virus in the cell culture. Since the clone utilized may not necessarily be representative of the virulence of the original viral isolate, two PCV2 clones from each cluster were used.
As in our previous study (Opriessnig et al., 2006c), these results confirm differences in virulence among PCV2 isolates. However, significant differences in virulence between isolates from the ‘old’ PCV2a and the ‘new’ PCV2b clusters were not found in our model. Neither the PCV2a isolates nor the recent PCV2b isolates recovered from field cases with high mortality induced clinical disease in singly inoculated conventional pigs. This outcome is different from what is usually seen under field conditions typical of PCVAD. This could be explained in part by the limited stress under experimental conditions (unrestricted access to feed, optimized housing, reduced numbers of pen-mates) and the exposure to PCV2 only as opposed to repeated exposure to multiple pathogens under field conditions.
A previous study using the germ-free pig model and cell culture-derived PCV2a and PCV2b clones found that both the PCV2a and PCV2b isolates induced severe disease in the gnotobiotic pig model (Lager et al., 2007). In that study, five out of eight gnotobiotic pigs inoculated with PCV2 developed clinical PCVAD, which is in contrast to our study, in which clinical disease was not observed. This further highlights the increased disease susceptibility of gnotobiotic pigs compared with conventional pigs; however, due to the small numbers of pigs used in the study by Lager et al. (2007) and the limited data presented on microscopic parameters, it could not be concluded definitively that differences in virulence existed between PCV2 clusters.
The anti-PCV2 IgG response was reduced in pigs inoculated with ISU-4838 past 21 days p.i. and isolate ISU-4838 induced less severe PCV2-associated microscopic lesions compared with isolate ISU-40895. Similar observations were found with the two PCV2b isolates used for the first time in the present study. Pigs inoculated with Can-17639 had significantly reduced numbers of PCV2 DNA copies in serum and significantly less severe microscopic lesions compared with NC-16845. These findings are quite interesting, as the difference between the two PCV2b isolates was limited to one amino acid change located in the replicase gene, which is not used routinely for sequencing in epidemiological investigations. However, when the data were combined for analysis of all PCV2a- and PCV2b-inoculated pigs, apart from the anti-PCV2 IgG antibody response at 14 and 21 days p.i., no significant differences were observed for any of the other parameters evaluated (amount of anti-PCV2 neutralizing antibodies, amount of PCV2 DNA in serum and overall lymphoid lesion scores) between the PCV2a and PCV2b groups. The initial higher anti-PCV2 IgG levels in the PCV2a group could be explained by isolate variation (Fig. 2a) rather than a truly stronger humoral response against cluster PCV2a. We therefore cannot confirm or rule out that there are significant differences in virulence between PCV2a cluster isolates and PCV2b cluster isolates in the conventional pig model.
In the second part of this study, we evaluated cross-protection between heterologous PCV2 clusters. Pigs were initially inoculated with PCV2a or PCV2b isolates and 5 weeks later inoculated with the heterologous isolate. Under the conditions of this study, the isolate used for the second challenge was not able to induce active infection or lesions in the pigs, as determined by comparison with pigs without prior exposure (Fig. 5). It needs to be pointed out that, although Fig. 5 shows a significant (P=0.046) difference in mean group lymphoid lesions between PCV2a- and PCV2b-inoculated pigs, cluster PCV2a included a total of 12 pigs inoculated with ISU-40895 and ISU 4838, whereas cluster PCV2b was represented by only six pigs inoculated with NC-16845. Pigs inoculated with Can-17639 at 9 weeks of age were not included in this part of the experiment.
Based on the results of sequencing and differential PCR, we showed that replication of the second challenge isolate was blocked by almost 100 % in reinoculated pigs, probably through generation of an effective immune response after the first challenge. Neutralizing antibodies appear to be crucial in decreasing PCV2 replication and preventing clinical disease (Meerts et al., 2005, 2006), and the amounts of anti-PCV2a and anti-PCV2b neutralizing antibody were similar across all groups at the time of second challenge. The ability of PCV2a isolates to cross-protect against infection with PCV2b isolates is further supported by the success of the recently introduced commercial PCV2 vaccines used in the field (Opriessnig et al., 2007; Fort et al., 2008). All commercial vaccines available today are based on the PCV2a cluster and yet they are very successful in reducing morbidity and mortality in herds with PCVAD (Opriessnig et al., 2007), from which PCV2b is now most commonly detected.
It still remains unclear why PCV2b has apparently emerged as the predominant cluster in the field since 2005. Based on serological investigations, the majority of herds in North America were seropositive prior to the onset of the PCVAD outbreaks in 2005 (Larochelle et al., 2003; Magar et al., 2000; Opriessnig et al., 2004c) and yet the herds were apparently not protected and subsequently went through severe PCVAD. One possible explanation for this is that protection induced by maternal antibodies versus active inoculation might be different, since there is no cellular immunity afforded through passively acquired antibodies. However, cellular immunity appears to play a major role in reducing PCV2 viraemia and lesions, as indicated by early work with PCV2 vaccines, where pigs did not develop a detectable humoral immune response to PCV2, yet were protected against subsequent challenge (Fenaux et al., 2004a). Another explanation for the recent devastating PCVAD outbreaks in North America would be the presence of an unknown triggering agent that enhances replication of PCV2b specifically and thereby increases disease severity.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 5 February 2008;
accepted 15 June 2008.
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) and PCV2b (Can-17639, NC-16845) (
) clusters. (b) Individual groups ISU-40895 (
), ISU-4838 (
), NC-16845 (
) and Can-17639 (
). An S : P ratio equal or greater than 0.2 is considered to be positive. Error bars represent SEM. *, Significant (P<0.05) differences between clusters.
