Download Protective effect of the maternally derived porcine circovirus type 2

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.).
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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
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6
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5
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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
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2.5
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2.0
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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.
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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.
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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
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25
*
20
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10
*
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_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*.
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Journal of General Virology 93
Protective effect of maternal PCV2 cellular immunity
PCV2 IFN- -SCs per million PBMCs
120
100
†
80
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*,†
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
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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
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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
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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
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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
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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
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