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Journal of General Virology (2012), 93, 1556–1562 DOI 10.1099/vir.0.041749-0 Protective effect of the maternally derived porcine circovirus type 2 (PCV2)-specific cellular immune response in piglets by dam vaccination against PCV2 challenge Yeonsu Oh,3 Hwi Won Seo,3 Kiwon Han, Changhoon Park and Chanhee Chae Correspondence Chanhee Chae Seoul National University, College of Veterinary Medicine, Department of Veterinary Pathology, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea [email protected] Received 2 February 2012 Accepted 10 April 2012 The objective of the present study was to evaluate (i) the passive transfer of maternally derived functional porcine circovirus type 2 (PCV2)-specific lymphocytes of seronegative sows immunized with the PCV2 vaccine to newborn piglets and (ii) the functional role of the maternally derived PCV2-specific cellular immune response in protecting newborn piglets from challenge with PCV2. After ingesting colostrums, piglets from vaccinated sows (PT01 and PT02) have significantly higher numbers of PCV2-specific gamma interferon-secreting cells, an increased PCV2-specific delayed type hypersensitivity response, and a stronger proliferative response of peripheral blood mononuclear cells compared with piglets from non-vaccinated seronegative sows (PT03 and PT04). In the PCV2 challenge study, the number of serum genomic PCV2 copies was significantly less in piglets from vaccinated sows (PT02) compared with piglets from non-vaccinated sows (PT04) at 7–28 days post-inoculation (P,0.05 and P,0.001). The histopathological lesions and immunohistochemical scores were significantly lower in piglets of vaccinated sows compared with those of non-vaccinated sows. To our knowledge, this is the first report of transferring a maternally derived PCV2-specific cellular immune response from vaccinated dams to their offspring. Maternally derived adaptive cellular immune responses play a critical role in protecting newborn piglets challenged with PCV2 at 3 weeks of age. INTRODUCTION Porcine circovirus type 2 (PCV2) is associated with a number of diseases and syndromes collectively known as porcine circovirus-associated disease (PCVAD), including post-weaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), reproductive failure, porcine respiratory disease complex and exudative epidermitis (Chae, 2004, 2005). PMWS, the most common and the most severe form of PCVAD, is clinically characterized by wasting, progressive weight loss, enlarged lymph nodes and dyspnoea (Chae, 2004, 2005). Since PCV2 vaccines were introduced onto the world market in 2006, vaccination has become an important tool for controlling PMWS. Several commercial PCV2 vaccines are available in the global market, including an inactivated PCV2 vaccine that has been administered to sows (Pejsak et al., 2010). Vaccination of sows provides their offspring with maternal antibody protection against PMWS and subclinical infection of PCV2 and may control PCV2 infections (Kurmann et al., 2011; Pejsak et al., 2010). However, the efficacy of the piglet 3These authors contributed equally to this work. 1556 vaccine which is different from sow vaccine used in this study is unrelated to the serum antibody response. Instead, it is due to the cellular immune response induced by the PCV2 vaccine (Fort et al., 2009b; Kim et al., 2011; Opriessnig et al., 2009). These results suggest that the passive transfer of the maternally derived cellular, rather than the humoral, immune response may play a critical role in controlling PCV2 infection in newborn piglets later. The passive transfer of the maternally derived PCV2-specific cellular immune response to piglets has not been studied. Hence, the objective of the present study was to evaluate the (i) passive transfer of maternally derived functional PCV2-specific lymphocytes to newborn piglets from sows immunized with the PCV2 vaccine and (ii) the functional role of the maternally derived PCV2-specific cellular immune response in protecting newborn piglets from challenge with PCV2. RESULTS Body weight Significant differences in mean body weight were not detected among the four groups until 14 days post-inoculation (p.i.). Downloaded from www.microbiologyresearch.org by 041749 G 2012 SGM IP: 88.99.165.207 On: Fri, 12 May 2017 11:18:19 Printed in Great Britain Protective effect of maternal PCV2 cellular immunity Body weight in challenged piglets from non-vaccinated sows (PT04) was significantly lower at 21 (8.4±4.6 kg, P,0.05) and 28 (10.1±4.3 kg, P,0.001) days p.i. compared with piglets in groups PT01 (12.9±2.1 kg at 21 days p.i. and 18.3±1.2 kg at 28 days p.i.), PT02 (12.1±1.2 kg at 21 days p.i. and 15.8±1.4 kg at 28 days p.i.) and PT03 (12.5±0.8 kg at 21 days p.i. and 15.5±1.3 kg at 28 days p.i.). Anti-PCV2 ELISA and serum virus neutralization titre Anti-PCV2 titres were determined by ELISA and serum virus neutralization (SVN) titre in vaccinated sows between 14 and 0 days antepartum, with a mean titre of 6.5±0.4 log2 at 0 days antepartum in vaccinated sows. No serum passively transferred anti-PCV2 antibodies were detected in piglets through either ELISA or SVN from non-vaccinated sows at any point of the experiment. No serum passively transferred anti-PCV2 antibodies were detected through either ELISA or SVN prior to colostrums ingestion, regardless of the sows’ vaccination status (at 221 days p.i.). After colostrums ingestion, piglets (PT01 and PT02) were passively transferred high anti-PCV2 antibody titres (mean ELISA EU titre: 5389±402 for PT01 and 5427±309 for PT02 and mean SVN titre: 6.50±1.80 log2 for PT01 and 6.40±0.42 log2 for PT02). From 221 to 28 days p.i., the anti-PCV2 titres in the non-challenged piglets (PT01) decreased gradually from 5389±402 EU to 2532±115 EU and from 6.50±1.80 log2 to 4.56±1.70 log2, in ELISA and SVN, respectively. After challenge, seroconversion of SVN titre started at 0–7 days p.i. in the challenged piglets from vaccinated (PT02) and from non-vaccinated 10 (PT04) sows. No SVN titres were detected in serum from non-challenged piglets (PT03) (Fig. 1). Quantification of PCV2 DNA No genomic copies of PCV2 were detected in any piglets’ serum samples at 221, 220 or 0 days p.i. No genomic copies of PCV2 were detected in any of the serum samples from non-challenged piglets (PT01 and PT03). Among challenged pigs from vaccinated sows (PT02), ten were viraemic at 7 days p.i., nine were viraemic at 14 days p.i. and eight were viraemic at 28 days p.i. All challenged pigs from non-vaccinated sows (PT04) were viraemic throughout the experiment. The number of genomic copies of PCV2 in serum was significantly lower in challenged piglets from vaccinated sows (PT02) than in challenged piglets from non-vaccinated sows (PT04), from 7 to 28 days p.i. (P,0.05 or P,0.001) (Fig. 2). Identifying lymphocyte subsets In the flow-cytometry analyses, most changes in the relative proportions of lymphocyte subsets could be attributed to the treatments received. At 1 day post-partum, vaccinated sows showed an increase in the relative proportions of CD4+CD8+ cells (59.19±3.75 % vs 38.36±7.59 %, P, 0.0001), CD3+CD4+ cells (44.95±0.86 % vs 41.53± 6.32 %, P50.044), CD3+CD8+ cells (73.23±4.00 % vs 60.33±11.68 %, P50.029) and CD25+ cells (13.79± 1.46 % vs 10.18±1.76 %, P50.017) compared with nonvaccinated sows. Colostrums of vaccinated sows increased proportions of CD4+CD8+ cells (30.33±2.52 % vs 25.74± 1.48 %, P50.003), CD3+CD4+ cells (19.60±0.86 % vs 17.15±1.52 %, P50.040), CD3+CD8+ cells (49.58±0.73 % vs 40.68±1.78 %, P,0.0001) and CD25+ cells (8.83± 1.58 % vs 6.16±0.88 %, P50.049) compared with those from non-vaccinated sows. 9 7 † † *,† † † 6 † 5 † † * † 4 3 2 1 _21 _20 0 7 14 21 28 3.5 log10 PCV2 genomic copies ml–1 serum Serum neutralization titre (log2) 8 3.0 † 2.5 † 2.0 † 1.5 1.0 0.5 _21 Time p.i. (days) * _20 0 7 14 21 28 Time p.i. (days) Fig. 1. Mean values of the serum neutralization antibody titre in the different groups. Non-challenged piglets (PT01; m) and challenged piglets (PT02; &) from vaccinated sows, and nonchallenged piglets (PT03; g) and challenged piglets (PT04; h) from non-vaccinated sows. Variation is expressed as SD. Significant difference is indicated at P-value ,0.05* or ,0.0013. http://vir.sgmjournals.org Fig. 2. Mean values of the genomic copy number of PCV2 DNA in blood samples from challenged piglets (PT02; &) from vaccinated sows and challenged piglets (PT04; h) from non-vaccinated sows. Variation is expressed as SD. Significant difference is indicated at P-value ,0.05* or ,0.0013. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 11:18:19 1557 Y. Oh and others After ingesting colostrums, the relative proportions of CD3+CD4+ cells were significantly different at day 220 (P,0.001) and day 0 (P,0.05), and the proportions of CD25 cells were significantly different at day 0 (P,0.001) in the peripheral blood mononuclear cells (PBMCs) from piglets from vaccinated sows (PT02) and in those from piglets from non-vaccinated sows (PT04). After challenge with PCV2, piglets from vaccinated sows (PT02) had significantly different relative proportions of all tested lymphocytes subsets except CD4+CD8+ and CD25 cells at 14 and 21 days p.i. in the PBMCs compared with challenged piglets from non-vaccinated sows (PT04) (Fig. 3). After ingesting colostrums, the relative proportions of CD3+CD4+ cells were significantly different at day 220 (28.42±5.50 % for PT01 vs 19.08±1.44 % for PT03, P,0.001) and day 0 (20.44±5.18 % for PT01 vs 14.44± 4.1 % for PT03, P,0.05), and the proportions of CD25 cells were significantly different at day 0 (5.01±1.21 % for PT01 vs 1.21±0.3 % for PT03, P,0.05) in the PBMCs from piglets from vaccinated sows (PT01) and in those from piglets from non-vaccinated sows (PT03). PCV2-specific lymphocyte stimulation assay The stimulation index (SI) value for colostrums from vaccinated sows was significantly greater compared with that of non-vaccinated sows. There was no difference in the proliferation response of PBMCs stimulated by phytohaemagglutinin (PHA) in piglets from vaccinated and nonvaccinated sows. However, stimulation with PCV2 antigen resulted in a higher proliferative response of PBMCs in piglets from vaccinated sows (PT01 and PT02) compared with piglets from non-vaccinated sows (PT03 and PT04). The SI value for PT01 and PT02 piglets was significantly higher at 220 days p.i. (1 day of age), than for PT03 and PT04 piglets (P,0.001) (Fig. 4). PCV2-specific gamma interferon (IFN-c)-secreting cells No PCV2-specific IFN-c-secreting cells (IFN-c-SCs) were detected in the PBMCs from piglets of either vaccinated or non-vaccinated sows before colostrums ingestion (at 221 days p.i.). After colostrums ingestion, PCV2-specific IFN-c-SCs were detected in the PBMCs of piglets from vaccinated sows (PT01 and PT02). PCV2-specific IFN-cSCs gradually decreased in challenged piglets (PT02) until 0 day p.i. and gradually increased after PCV2 challenge. PCV2-specific IFN-c-SCs developed in challenged piglets from non-vaccinated sows (PT04) between 14 and 21 days p.i. No PCV2-specific IFN-c-SCs were detected in the PBMCs from non-challenged piglets (PT03) (Fig. 5). Delayed type hypersensitivity (DTH) At 36 h after intradermal injection of PCV2 antigen, piglets from vaccinated sows (PT01 and PT02) showed skin reactions characterized by circumscribed and often erythematous nodules. The nodules regressed slowly after 48 h and left no scar tissue. No erythematous nodules were observed in piglets from non-vaccinated sows (PT03 and PT04). Piglets from vaccinated and non-vaccinated sows showed DTH responses to the non-specific mitogen PHA. No difference in the magnitude of PHA-induced DTH response size was observed between piglets from the vaccinated and non-vaccinated sows (10.82±0.78 mm vs 10.99±0.41 mm, P50.892). Piglets from vaccinated sows had significantly greater PCV2-specific DTH responses (14.07±2.73 mm) than piglets from non-vaccinated sows (0.80±0.30 mm, P,0.0001). No erythematous nodules were observed at saline injection sites for any piglets. 2.5 35 30 † † † † † 25 * 20 * † 15 * † 10 * 5 _21 _20 0 7 14 21 28 2.0 * 1.5 * 1.0 0.5 0 Colostrum Time p.i. (days) Fig. 3. Lymphocyte subsets analysis in the different groups; CD4+CD8+ (m), CD3+CD4+ (&) and CD25 ($) from challenged piglets (PT02) from vaccinated sows and CD4+CD8+ (g), CD3+CD4+ (h) and CD25 (#) from challenged piglets (PT04) from non-vaccinated sows. Variation is expressed as SD. Significant difference is indicated at P-value ,0.05* or ,0.0013. 1558 PCV2 Ag-specific cell SI Per cent among million PBMCs ml–1 40 0 h after birth 24 h after birth Fig. 4. PCV2-specific lymphocyte SI. Lymphocytes were isolated from colostrums of vaccinated (&) and non-vaccinated (h) sows and from piglets from vaccinated and non-vaccinated sows before (BC) and 24 h after (AC) colostrums ingestion. Variation is expressed as SD. Significant difference is indicated at P-value ,0.001*. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 11:18:19 Journal of General Virology 93 Protective effect of maternal PCV2 cellular immunity PCV2 IFN- -SCs per million PBMCs 120 100 † 80 † *,† *,† 60 40 * * 20 _21 _20 0 7 14 21 28 Time p.i. (days) Fig. 5. Mean number of PCV2-specific IFN-c-SCs in nonchallenged piglets (PT01; m) and challenged piglets (PT02; &) from vaccinated sows, and non-challenged piglets (PT03; g) and challenged piglets (PT04; h) from non-vaccinated sows. Variation is expressed as SD. Significant difference is indicated at P-value ,0.05* or ,0.0013. Lymph node lesions Mild to moderate lymphoid depletion and histiocytic replacement of lymphoid follicles associated with PCV2 infection was observed in the lymph nodes from challenged piglets from non-vaccinated sows (PT04). No histopathological lymph node lesions were observed in piglets from any other group. The morphometric analysis of histopathological changes in the lymph nodes showed significantly reduced scores in challenged piglets from vaccinated sows (PT02) (lesion score 1.45±0.69) compared with challenged piglets from non-vaccinated sows (PT04) (lesion score 3.17±0.75, P50.001). The mean number of PCV2-positive cells per unit area of the lymph node in challenged piglets from non-vaccinated sows (PT04) (immunohistochemical score 88.12±29.15) was significantly greater than that of challenged piglets from vaccinated sows (PT02) (immunohistochemical score 29.94±10.69, P,0.001). Statistical analysis In challenged piglets from vaccinated (PT02) and nonvaccinated sows (PT04), the number of genomic copies of PCV2 in the blood was correlated with SVN titre (PT02: r250.480, P50.032 and PT04: r250.545, P50.013), PCV2specific IFN-c-SCs (PT02: r250.532, P50.007 and PT04: r250.799, P50.001), histopathological lesion score (PT02: r250.826, P50.001 and PT04: r250.469, P50.048) and immunohistochemical score (PT02: r250.789, P50.001 and PT04: r250.597, P50.003). DISCUSSION The present study has demonstrated that maternally derived PCV2-specific cellular immunity can be passively http://vir.sgmjournals.org transferred to newborn piglets by dam vaccination against PCV2. Antigen-specific DTH responses and lymphocyte proliferation indicate an anti-PCV2-specific adaptive cellular response. Furthermore, piglets that received maternal PCV2-specific memory T lymphocytes mount DTH reactions in response to intradermal injection of PCV2 antigen. The DTH response was PCV2-specific because skin lesions characteristic of the DTH response were only detected upon intradermal injection with PCV2 antigens and not with mock preparations. The PCV2specific proliferation was also detected by stimulating the PBMCs with PCV2 antigen, whereas colostral lymphocytes from non-vaccinated sows did not proliferate following stimulation. Lymphocytes isolated from piglets before suckling did not respond to PCV2 stimulation (data not shown), which indicates that newborn piglets are naı̈ve to PCV2 at birth. Therefore, the DTH and lymphocyte proliferation responses observed in newborn piglets from vaccinated sows were due to the action of PCV2 antigenspecific maternal colostral lymphocytes. Serum PCV2 contributes to the development of PCVAD, such as PMWS. Viraemia contributes to viral distribution throughout the lymphoid tissues (Kim et al., 2003). High PCV2 viraemia has been associated with the development of PCVAD (Larochelle et al., 2003; Meerts et al., 2006). Hence, reduction of viraemia may protect piglets from PCV2 infection. Reducing the PCV2 load in the blood coincided with the appearance of both serum virus neutralizing antibodies and PCV2-specific IFN-c-SCs, as reported previously (Fort et al., 2009a). Moreover, a good correlation was observed between serum neutralizing antibodies and decreased PCV2 replication with a lack of clinical PCVAD under experimental (Meerts et al., 2005, 2006) and field (Fort et al., 2007) conditions when PCVAD pigs were compared with pigs subclinically infected with PCV2. The development of IFN-c-SCs is another key component of the developing anti-PCV2 adaptive cellular response. In the present study, passively transferred PCV2specific IFN-c-SCs were observed in newborn piglets from vaccinated, but not non-vaccinated, sows. The level of immune protection against PCV2 infection depends on the development of mechanisms that reduce PCV2 spread and develop memory T lymphocytes. We confirmed that maternally derived PCV2-specific T lymphocytes mount an immune response, which highlights the clinical importance of protective T lymphocyte-mediated cellular responses to PCV2. In the present study, the numbers of CD4+CD8+ and CD3+CD4+ lymphocytes were decreased, while the number of CD3+CD8+ was unchanged in the challenged piglets from non-vaccinated sows (PT04) compared with challenged piglets from vaccinated sows (PT02). This observation indicated that CD3+ and CD4+ lymphocytes were reduced by PCV2 infection. These results agree with previous findings of reduced CD3+ and CD4+ cells in pigs with PMWS, while absolute and relative CD8+ cell numbers are similar in both normal and diseased pigs Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 11:18:19 1559 Y. Oh and others (Segalés et al., 2001). A selective loss of CD3+ and CD4+ cells during PCV2 infection may impair the immune system in pigs and result in co-infections with other viral and bacterial pathogens. In field conditions, PMWS in pigs is frequently observed in combination with other viral and bacterial pathogens (Kim et al., 2002; Pallarés et al., 2002). In conclusion, our results suggest that maternally derived colostral lymphocytes from sows immunized with PCV2 vaccine transferred into the neonates’ circulation and participated in the cellular immune response in a PCV2 antigen-specific manner. Our findings are supported by in vivo DTH responses and in vitro lymphocyte proliferation and the presence of IFN-c-SCs. To our knowledge, this is the first confirmation of the transfer of a maternally derived PCV2-specific cellular immune response from vaccinated dams to their offspring. These maternally derived adaptive cellular immune responses play a critical role in protecting newborn piglets from PCV2 infection even if PCV2 infection should occur as late as 8–14 weeks of age. Further studies are needed to confirm the duration of maternally derived adaptive cellular immunity to protect newborn piglets against PCV2 infection. for histopathological examination and immunohistochemistry as described previously (Kim et al., 2011). All methods were approved by the Seoul National University Institutional Animal Care and Use Committee. Sample collection. Blood samples from each sow were collected by jugular venipuncture at 35, 14 and 0 days antepartum. Ten millilitres of colostrum was manually collected from three udders in 50 ml conical tubes from all sows after disinfection with alcohol. Eighty newborn piglets (four piglets per sow) were randomly selected on the day of birth and subjected to blood collection at 221 (0 h before colostrums ingestion), 220 (24 h after colostrums ingestion; 1 day old), 0 (21 days old), 7 (28 days old), 14 (35 days old), 21 (42 days old) and 28 (49 days old) days p.i. Serology. Serum samples were stored at 220 uC and tested using a commercially available ELISA (Serelisa PCV2 Ab Mono Blocking ELISA; Synbiotics). SVN tests were performed as described previously (Allan et al., 1994; Pogranichnyy et al., 2000). The air-dried cells were stained for PCV2 antigens using an immunoperoxidase assay (Rodrı́guez-Arrioja et al., 2000). Quantification of PCV2 DNA. DNA extraction from collected serum samples was performed using the QIAamp DNA mini kit (Qiagen). DNA extracts were used to quantify PCV2 genomic DNA copy numbers by real-time PCR as described previously (Gagnon et al., 2008). Flow cytometry. PBMCs were prepared as described previously METHODS Sow experiment design. Twenty conventional crossbred pregnant sows with a second parity were obtained from a porcine reproductive and respiratory syndrome virus (PRRSV)-free herd. All sows were serologically negative for PCV2, porcine parvovirus, PRRSV and swine influenza virus. Sows were moved to a research facility, housed individually in separate rooms and randomly allocated into one of two groups. Group 1 (ST01, n510) sows were vaccinated intramuscularly twice at 5 and 2 weeks antepartum with a 2 ml dose of a commercial inactivated PCV2 vaccine (Circovac) according to the manufacturer’s recommendation. Group 2 (ST02, n510) sows served as non-vaccinated controls. At parturition, sows farrowed naturally and no cross-fostering was applied to ensure maternal lymphocyte transfer into blood circulation of piglets. The ten vaccinated sows delivered 105 live and three stillborn piglets. The ten non-vaccinated sows delivered 107 live and two stillborn piglets. A total of 80 newborn piglets (four piglets per sow) were randomly selected on the day of birth and notched in their ear with a unique identification number. After selection, selected piglets stayed with their sows until weaning at 3 weeks of age in a separate room within the facility and unselected piglets were removed from the sow. Piglet experimental design. At weaning, 40 piglets randomly selected once from the litters of each of the vaccinated sows (ST01) were allocated into group 1 (PT01, n520) or 2 (PT02, n520). Another 40 piglets (four piglets per sow) randomly selected from the litters of non-vaccinated sows (ST02) were allocated into group 3 (PT03, n520) or 4 (PT04, n520). Piglets in groups 2 (PT02) and 4 (PT04) were intranasally administered 2 ml PCV2b (strain SNUVR000463; 5th passage) pool containing 1.06105 TCID50 ml21 (Kim et al., 2003). The same amount of sterile PBS was intranasally administrated in nonchallenged pigs in groups 1 (PT01) and 3 (PT03). The live weight of each pig was measured 221, 214, 27, 0, 7, 14, 21 and 28 days p.i. All piglets were tranquilized by an intravenous injection of azaperon (Stresnil; Janssen Pharmaceuticals) and euthanized by electrocution at 28 days p.i. Tissue samples were collected at necropsy and processed 1560 (Williams, 1993) and were incubated with R-PE- or FITC-conjugated mouse mAbs [anti-swine CD3 (R-PE), CD4a (R-PE and FITC) and CD8a (FITC); SouthernBiotech] or non-conjugated anti-swine CD25 (VMRD) for 30 min at 4 uC in the dark and washed twice. For CD3, CD4a and CD8a staining, cells stained with conjugated antibodies were resuspended immediately in supplemented RPMI 1640 medium. For CD25 staining, cells stained with primary non-conjugated antibodies were incubated with FITC-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) for 30 min at 4 uC in the dark, washed twice and resuspended in medium before evaluation. Cells were analysed using a FACSCalibur flow cytometer (Becton Dickinson) as described previously (Sosa et al., 2009). PCV2-specific lymphocyte stimulation assay. The 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay for PCV2-specific lymphocyte stimulation was performed as described previously (Lee & Ge, 2010; Wang et al., 2011) with slight modifications. PBMCs were stimulated with PCV2 antigen. Cells were adjusted to a density of 26106 cells per well in 180 ml, placed in 96-well tissue culture plates (Corning Laboratories Science Products; Corning) and incubated with 20 ml PCV2 antigen, PHA (Roche Diagnostics GmbH) as a positive control, or PBS as a negative control for 60 h at 37 uC in a 5 % humidified CO2 atmosphere. Following this incubation, 100 ml MTT (0.5 mg ml21; aMReSCO) was added to each well and the cells were incubated for another 4 h. The formazan was dissolved with 100 ml DMSO and the plates were shaken lightly for 30 min. The reduction of MTT was quantified by absorbance at a wavelength of 550 nm using a microplate reader (model-550; Bio-Rad). The results were expressed as the mean differential SI (SI5optical density with PCV2 antigen/optical density without the antigen) of each sample. Enzyme-linked immunospot assay (ELISPOT). The numbers of PCV2-specific IFN-c-SCs in PBMCs were determined by a commer- cial linked ELISPOT assay kit (MABTECH) using a commercial mAb (swine IFN-c Cytosets kits; Biosource) following a previously described protocol (Dı́az & Mateu, 2005). Preparation of PCV2 antigen. PCV2 antigen was prepared as described previously (Bautista & Molitor, 1997). The same PCV2 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 11:18:19 Journal of General Virology 93 Protective effect of maternal PCV2 cellular immunity strain, as for challenge of the pigs, was propagated in PCV-free PK15 cells to a titre of 104 TCID50 ml21 and treated with two freeze–thaw cycles. Inactivation was confirmed by the absence of virus antigen in PK15 cells as determined by immunoperoxidase assay (Rodrı́guezArrioja et al., 2000). DTH. DTH test was performed on 80 piglets (four piglets per sow) at 4 days of age. Piglets were injected intradermally on the left inguinal area with 250 mg partially purified PCV2 antigen from infected PK15 cells. PHA (20 mg ml21 in 0.1 ml) and saline (0.1 ml) were used as positive and negative controls, respectively. Induration was measured with a micrometer after 36 h. Orthogonal diameters of the induration were measured for each reaction and the sums of these diameters were recorded. Histopathology. For the morphometric analysis of histopathological changes in lymph nodes, three superficial inguinal lymph node sections were examined blindly. The scores ranged from 0 (normal, i.e. no lymphoid depletion or granulomatous replacement) to 5 (severe lymphoid depletion and granulomatous replacement) as described previously (Kim et al., 2011). Immunohistochemistry. Immunohistochemistry was performed using a polyclonal anti-PCV2 antibody as previously described (Kim et al., 2011). Single sections (10 randomly selected fields) from each formalin-fixed lymph node sample were used for the virus-infected morphometric analysis, as described previously (Kim et al., 2003). Chae, C. (2004). Postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology. Vet J 168, 41–49. Chae, C. (2005). A review of porcine circovirus 2-associated syndromes and diseases. Vet J 169, 326–336. Dı́az, I. & Mateu, E. (2005). Use of ELISPOT and ELISA to evaluate IFN-c, IL-10 and IL-4 responses in conventional pigs. Vet Immunol Immunopathol 106, 107–112. Fort, M., Olvera, A., Sibila, M., Segalés, J. & Mateu, E. (2007). Detection of neutralizing antibodies in postweaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs. Vet Microbiol 125, 244–255. Fort, M., Fernandes, L. T., Nofrarias, M., Dı́az, I., Sibila, M., Pujols, J., Mateu, E. & Segalés, J. (2009a). Development of cell-mediated immunity to porcine circovirus type 2 (PCV2) in caesarean-derived, colostrum-deprived piglets. Vet Immunol Immunopathol 129, 101–107. Fort, M., Sibila, M., Pérez-Martı́n, E., Nofrarı́as, M., Mateu, E. & Segalés, J. (2009b). One dose of a porcine circovirus 2 (PCV2) sub- unit vaccine administered to 3-week-old conventional piglets elicits cell-mediated immunity and significantly reduces PCV2 viremia in an experimental model. Vaccine 27, 4031–4037. Gagnon, C. A., del Castillo, J. R. E., Music, N., Fontaine, G., Harel, J. & Tremblay, D. (2008). Development and use of a multiplex real-time quantitative polymerase chain reaction assay for detection and differentiation of Porcine circovirus-2 genotypes 2a and 2b in an epidemiological survey. J Vet Diagn Invest 20, 545–558. Kim, J., Chung, H.-K., Jung, T., Cho, W.-S., Choi, C. & Chae, C. (2002). Statistical analysis. Summary statistics were calculated for all groups to assess the overall quality of the data, including normality. For single comparisons, ANOVA with a post-hoc Tukey’s test was used to compare the primary variables (sow and colostrum lymphocyte subsets, DTH response, PCV2-specific lymphocyte stimulation assay, histopathological lesion score and immunohistochemical score) among groups. The continuous data for PCV2 serology, PCV2 DNA quantification, PCV2-specific IFN-c-SCs and lymphocyte subsets of the piglets were analysed using an ANOVA for each time point. When a one-way ANOVA test had a significance of P,0.05, Tukey’s Honestly Significant Difference test was used to determine the significance of individuals between group differences. The Pearson’s correlation coefficient was used to assess the relationship among viraemia, serum virus neutralization titre, PCV2-specific IFN-c-SCs and the Spearman’s rank correlation coefficient was used to assess histopathological lesion score and immunohistochemical score. A value of P,0.05 was considered significant. Postweaning multisystemic wasting syndrome of pigs in Korea: prevalence, microscopic lesions and coexisting microorganisms. J Vet Med Sci 64, 57–62. Kim, J., Choi, C. & Chae, C. (2003). Pathogenesis of postweaning multisystemic wasting syndrome reproduced by co-infection with Korean isolates of porcine circovirus 2 and porcine parvovirus. J Comp Pathol 128, 52–59. Kim, D., Kim, C. H., Han, K., Seo, H. W., Oh, Y., Park, C., Kang, I. & Chae, C. (2011). Comparative efficacy of commercial Mycoplasma hyopneu- moniae and porcine circovirus 2 (PCV2) vaccines in pigs experimentally infected with M. hyopneumoniae and PCV2. Vaccine 29, 3206–3212. Kurmann, J., Sydler, T., Brugnera, E., Buergi, E., Haessig, M., Suter, M. & Sidler, X. (2011). Vaccination of dams increases antibody titer and improves growth parameters in finisher pigs subclinically infected with porcine circovirus type 2. Clin Vaccine Immunol 18, 1644–1649. Larochelle, R., Magar, R. & D’Allaire, S. (2003). Comparative serologic and virologic study of commercial swine herds with and without postweaning multisystemic wasting syndrome. Can J Vet Res 67, 114–120. ACKNOWLEDGEMENTS Lee, G. & Ge, B. (2010). Inhibition of in vitro tumor cell growth by This research was supported by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries; contract research funds of the Research Institute for Veterinary Science (RIVS) from the College of Veterinary Medicine; and the Brain Korea 21 Program for Veterinary Science in the Republic of Korea. RP215 monoclonal antibody and antibodies raised against its antiidiotype antibodies. Cancer Immunol Immunother 59, 1347–1356. Meerts, P., Van Gucht, S., Cox, E., Vandebosch, A. & Nauwynck, H. J. (2005). Correlation between type of adaptive immune response against porcine circovirus type 2 and level of virus replication. Viral Immunol 18, 333–341. Meerts, P., Misinzo, G., Lefebvre, D., Nielsen, J., Bøtner, A., Kristensen, C. S. & Nauwynck, H. J. (2006). Correlation between REFERENCES Allan, G. M., Mackie, D. P., McNair, J., Adair, B. M. & McNulty, M. S. (1994). Production, preliminary characterisation and applications of monoclonal antibodies to porcine circovirus. Vet Immunol Immunopathol 43, 357–371. the presence of neutralizing antibodies against porcine circovirus 2 (PCV2) and protection against replication of the virus and development of PCV2-associated disease. BMC Vet Res 2, 6–16. Opriessnig, T., Patterson, A. R., Madson, D. M., Pal, N. & Halbur, P. G. (2009). Comparison of efficacy of commercial one dose and two dose Bautista, E. M. & Molitor, T. W. (1997). Cell-mediated immunity to PCV2 vaccines using a mixed PRRSV-PCV2-SIV clinical infection model 2-3-months post vaccination. Vaccine 27, 1002–1007. porcine reproductive and respiratory syndrome virus in swine. Viral Immunol 10, 83–94. Pallarés, F. J., Halbur, P. G., Opriessnig, T., Sorden, S. D., Villar, D., Janke, B. H., Yaeger, M. J., Larson, D. J., Schwartz, K. J. & other http://vir.sgmjournals.org Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 11:18:19 1561 Y. Oh and others authors (2002). Porcine circovirus type 2 (PCV-2) coinfections in US field cases of postweaning multisystemic wasting syndrome (PMWS). J Vet Diagn Invest 14, 515–519. Segalés, J., Alonso, F., Rosell, C., Pastor, J., Chianini, F., Campos, E., López-Fuertes, L., Quintana, J., Rodrı́guez-Arrioja, G. & other authors (2001). Changes in peripheral blood leukocyte populations Pejsak, Z., Podgórska, K., Truszczyński, M., Karbowiak, P. & Stadejek, T. (2010). Efficacy of different protocols of vaccination in pigs with natural postweaning multisystemic wasting syndrome (PMWS). Vet Immunol Immunopathol 81, 37–44. against porcine circovirus type 2 (PCV2) in a farm affected by postweaning multisystemic wasting syndrome (PMWS). Comp Immunol Microbiol Infect Dis 33, e1–e5. Sosa, G. A., Quiroga, M. F. & Roux, M. E. (2009). Flow cytometric Pogranichnyy, R. M., Yoon, K. J., Harms, P. A., Swenson, S. L., Zimmerman, J. J. & Sorden, S. D. (2000). Characterization of immune response of young pigs to porcine circovirus type 2 infection. Viral Immunol 13, 143–153. Rodrı́guez-Arrioja, G. M., Segalés, J., Balasch, M., Rosell, C., Quintanta, J., Folch, J. M., Plana-Durán, J., Mankertz, A. & Domingo, M. (2000). Serum antibodies to porcine circovirus type 1 and type 2 in pigs with and without PMWS. Vet Rec 146, 762–764. 1562 analysis of T-lymphocytes from nasopharynx-associated lymphoid tissue (NALT) in a model of secondary immunodeficiency in Wistar rats. Immunobiology 214, 384–391. Wang, J., Chen, B., Jin, N., Xia, G., Chen, Y., Zhou, Y., Cai, X., Ding, J., Li, X. & Wang, X. (2011). The changes of T lymphocytes and cytokines in ICR mice fed with Fe3O4 magnetic nanoparticles. Int J Nanomedicine 6, 605–610. Williams, P. P. (1993). Immunomodulating effects of intestinal absorbed maternal colostral leukocytes by neonatal pigs. Can J Vet Res 57, 1–8. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 11:18:19 Journal of General Virology 93