Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 143–155
Contents lists available at ScienceDirect
Comparative Immunology, Microbiology
and Infectious Diseases
journal homepage: www.elsevier.com/locate/cimid
Cytokine expression, glucocorticoid and growth hormone changes
after porcine reproductive and respiratory syndrome virus (PRRSV-1)
infection in vaccinated and unvaccinated naturally exposed pigs
Paolo Borghetti a,∗,1 , Roberta Saleri b , Luca Ferrari a , Marina Morganti a , Elena De Angelis a ,
Valentina Franceschi a , Ezio Bottarelli a , Paolo Martelli a,1
a
b
Department of Animal Health, University of Parma, Italy
Department of Animal Production, Veterinary Biotechnology and Food Safety, University of Parma, Italy
a r t i c l e
i n f o
Article history:
Received 7 May 2010
Accepted 20 June 2010
Keywords:
PRRSV infection
Vaccination
Cytokines
Growth hormone
Cortisol
Immunity
a b s t r a c t
The objective of this paper was to study the changes of some cytokines and neuroendocrine
hormones in vaccinated and unvaccinated pigs that were naturally infected by a PRRSV1 (porcine reproductive and respiratory syndrome virus) heterologous field strain. We
analyzed gene expression of pro-inflammatory (TNF-␣, IL-1␤, MCP-1, IL-6), pro-immune
(IFN-␥) and anti-inflammatory cytokines (IL-10) in PBMC, as well as hormonal (GH and cortisol) levels in blood samples of pigs obtained in a field trial previously reported [Martelli
P, Gozio S, Ferrari L, Rosina S, De Angelis E, Quintavalla C, et al. Efficacy of a modifiedlive porcine reproductive and respiratory syndrome virus (PRRSV) vaccine in pigs naturally
exposed to a heterologous European (Italian cluster) field strain: clinical protection and cellmediated immunity. Vaccine 2009;27:3788–99]. All vaccinated pigs showed an increase
in pro-inflammatory and pro-immune cytokine gene expression with respect to controls
and a prompt increase in GH that could be consistently associated with pro-inflammatory
cytokines in sustaining innate immunity; moreover, the higher levels of cortisol indicates
the activation of the hypothalamus–pituitary–adrenal (HPA) axis response. In contrast,
unvaccinated pigs showed down-regulation of the cortisol and GH responses, and the proinflammatory and pro-immune cytokines remained at a basal or low level, with an increase
of TNF-␣ and IL-6 in association with a higher level of IL-10 in the late phase of natural infection. The associated trends of pro-inflammatory and anti-inflammatory cytokines together
with the cortisol level demonstrate that a previous vaccination promotes an early immune
responsiveness in pigs and a more efficient control of inflammation in the late phase of
infection with a heterologous PRRSV isolate; both events could sustain clinical protection.
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
∗ Corresponding author at: Department of Animal Health, Faculty of
Veterinary Medicine, University of Parma, Via del Taglio, 10, 43126 Parma,
Italy. Tel.: +39 0521 032725; fax: +39 0521 032732.
E-mail address: paolo.borghetti@unipr.it (P. Borghetti).
1
Equally contributed.
0147-9571/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.cimid.2010.06.004
Porcine reproductive and respiratory syndrome (PRRS)
is one of the most important diseases in the world because
of its high prevalence and clinical impact on swine herds,
manifested as reproductive failure and respiratory disease.
PRRS virus has a complex interaction with the immune
system by replication in and negative modulation of innate
tissue cells such as macrophages, monocytes and dendritic cells. In contrast to other viral infections, PRRSV
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P. Borghetti et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 143–155
causes an acute infection, with viremia lasting approximately 1 month, followed by a phase of chronic persistence
in lymphoid tissues up to several months. The mechanisms by which PRRSV escapes the immune system
and by which the immune response is not triggered to
give effective clearance remain partially unknown. During
PRRSV infection, the humoral and cell-mediated immune
responses develop but their specific role in protection and
in eliminating the virus is still conflicting [1,2]. A supported hypothesis is that infection of permissive immune
cells, monocytes/macrophages and dendritic cells, does not
prime efficient pathogen recognition, with a consequent
down-regulation of inflammatory cytokines and a weak
innate immune response that compromises the following
onset and development of the antigen-specific adaptive
immune response [3–7].
Moreover, despite the observation that exposure to the
virus induces protective immunity against re-exposure to
an homologous virus [8–10], infected pigs can become
re-infected and clinically affected when exposed to a heterologous PRRSV isolate. At present, it is not possible to
predict the degree of protection provided by individual
PRRSV strains that have genetic similarity; it has been
shown that variable levels of protection may be achieved
in modified live vaccine (MLV)-vaccinated pigs after infection with unrelated PRRSV strains [10–16], but the role
played by the innate and/or adaptive immune response in
this protection is still unclear [17].
Furthermore, although important information about
the immune response of pigs to experimental infection
by American and European genotype strains have been
obtained [18–21], few studies have been carried out on
natural infection and the related respiratory disease. In
the present study, pigs were vaccinated with an attenuated European-type vaccine (DV strain, Lelystad cluster)
and naturally exposed, under farm conditions, to an Italian wild-type strain of PRRSV-1. As previously described
[15], the conditions of this infection model following natural exposure to a heterologous PRRSV-1 isolate allowed
the assessment of vaccination efficacy in terms of clinical
protection because the PRRS-related disease occurred with
overt clinical abnormalities.
For a successful resolution of infection, efficient activation of innate/inflammatory and acquired immunity is
required to block pathogen replication and invasion, as well
as to promote tissue clearance of the pathogens and/or
infected cells. The most prominent pro-inflammatory
cytokines [interleukin [(IL)-1 IL]-1, tumour necrosis factor␣ (TNF-␣), IL-6, IL-8, monocyte chemoattractant protein-1
(MCP-1)] are produced early after pathogen recognition
and play a pivotal role in eliciting the innate response as
well as in priming and coordinating the adaptive immune
response. However, if production is impaired, the innate
response will be delayed and inefficient in clearing the
pathogen. On the other hand, if production is not dampened
and controlled by feed-back mechanisms, the persistence
of cytokines will increase tissue damage and worsen the
severity of the disease. Thus, qualitative and quantitative,
time-related changes in cytokine production during infection can be studied as functional markers of protective
immunity and/or of the outcome of the disease [3,6,22–25].
In fact, during infection, the host’s defensive response
is modulated by an integrated response, involving a bidirectional communication between the immune and the
neuroendocrine systems; pro-inflammatory cytokines and
hormones are the effectors of this coordinated and controlled cross-talk, which potentiates innate immunity,
controls potential harmful effects due to uncontrolled
inflammation and permits the return to a homeostatic
state, therefore playing a pivotal role in the efficiency of
the immune response against infectious agents. Recently,
a lot of knowledge has been obtained on the molecular signals orchestrating this integrated adaptive response; this
information has permitted investigators to focus on the
systemic mediators that drive the efficiency of the response
and also the signalling alterations and control pathway dysfunctions that may be involved in the persistence and/or
over-expression of inflammation and consequent tissue
damage, thereby influencing the clinical outcome of the
disease [26–28]. Indeed, neuroendocrine regulatory factors, such as glucocorticoids and somatotropic hormones,
are known to play a major role in the development of
the immune system, in the modulation of the innate
immune response during infection and in the return to
the homeostatic state after the clearance of the pathogen
[29,30]. In a previous study we analyzed the immunological
response and the clinical evolution of a naturally occurring infection by a PRRSV-1 field strain in vaccinated and
unvaccinated pigs. The obtained results showed that vaccination with a MLV conferred clinical protection and that
the overall vaccine efficacy was 0.72. The present study
aims at investigating, in the same blood samples, some
additional parameters (cytokines, cortisol and GH) as indicators of the integrated adaptive neuroendocrine-immune
response. These associated parameters could provide new
insight to monitor the development and the role of the
innate immune response in PRRSV infected animals in combination with the outcomes of the disease.
2. Materials and methods
2.1. Animals and experimental design
The protocol was described in details in a previous
paper [15]. Briefly, a total of 30, 4-week-old, conventional pigs were purchased from a PRRSV-free, Mycoplasma
hyopneumoniae-free farm. Pigs were randomly divided into
three groups (designated IM, ID and C groups) and conventionally housed in an isolation barn located away from the
farm of origin (site 2 unit). Different groups were housed
in different rooms.
After 1 week of acclimatization (5 weeks of age), the pigs
of IM (n = 10 pigs) and ID (n = 10 pigs) groups were vaccinated with Porcilis® PRRS at a dose of 104.5 TCID50 per pig
via the intramuscular (2 ml) or intradermal route (0.2 ml),
respectively. Intradermal vaccination was performed by
using a needle-less vaccinator (I.D.A.L.® Intervet BV,
Boxmeer, The Netherlands). Pigs from group C (n = 10 pigs)
were not vaccinated and served as controls. The PRRSV
vaccine used was the attenuated European virus strain DV
(Porcilis® PRRS, Intervet BV, Boxmeer, The Netherlands).
The vaccine was suspended in a tocopheryl acetate-
P. Borghetti et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 143–155
containing aqueous adjuvant (Diluvac Forte® , Intervet BV).
A strict control group with no vaccination and challenge is
missing because this is impossible to be achieved in a field
setting.
Forty-five days post-vaccination (PV), IM, ID and C
groups (30 pigs) were moved to the site 3 unit, which
housed a conventional, continuous-flow herd, to be naturally exposed to field pathogens. The experimental pigs
were commingled with 30 resident animals and housed in
the same pen for the duration of the experiment. During
the post-exposure period (PE), blood samples were collected on day 0 (45 days PV), 7, 14, 21, 28 and 34 PE for the
quantification of pro-inflammatory and anti-inflammatory
cytokine gene expression and hormone (GH, cortisol) plasmatic levels.
Pigs were monitored daily for general and respiratory
clinical signs throughout the post-exposure period (35
days). Pigs were evaluated in a blind manner by visual
inspection for a period of at least 15 min and scores
were given for general clinical signs (rectal temperatures,
appetite and level of consciousness/lethargy) and respiratory signs as previously described in details [15].
The “recipient-site 3” herd was positive for porcine
circovirus type 2 (PCV2) and free from post-weaning
multisystemic wasting syndrome and porcine circovirus
associated disease (PCVD). There was a history of respiratory problems caused by PRRSV, along with the most
common bacteria. In the 2 weeks preceding the exposure
of experimental pigs, in animals of the recipient herds
(resident pigs) that were affected by acute respiratory disease and subsequently died, microbiological, pathological
and serological investigations were carried out. PRRSV was
identified by polymerase chain reaction (PCR) in the pneumonic lungs and blood of dead pigs, and the virus was
isolated from all samples. Pasteurella multocida and Streptococcus spp. were also isolated from pneumonic lungs.
Moreover, blood samples were taken from 20 pigs that
had recovered from respiratory disease (convalescent samples) to evaluate seroconversion to the most common
agents of respiratory disease. Seroconversion was shown
for PRRSV (ELISA kit, IDEXX Laboratories Inc., Westbrook,
ME, USA) and M. hyopneumoniae but not for Actinobacillus pleuropneumoniae and Aujeszky’s disease. Low titers of
antibodies to swine influenza virus (SIV) at hemagglutination inhibition were obtained from some samples and were
inconclusive. No lesions referable to PCVD were detected
from pathological investigations.
The isolated PRRSV (05R1421) from the resident pigs
and from the naturally exposed experimental animals
belongs to the Italian cluster of the PRRSV-1. The nucleotide
sequence of the open reading frame 5 from strain 05R1421
is 84% identical to that of the DV strain (vaccine strain) [15].
2.2. Cytokine gene expression
Cytokine (TNF-␣, IL-6, IFN-␥, IL-1␤, IL-10, MCP-1) gene
expression was evaluated in PBMC as follows. PBMC
were separated by density gradient centrifugation by
using Histopaque-1077® solution (Sigma–Aldrich® , St.
Luis, MO, USA) and washed twice with phosphate-buffered
145
saline (PBS). RNA was extracted by TRI Reagent® solution (Applied Biosystems-Ambion® , Foster City, CA, USA).
Briefly, 1 ml of TRI Reagent® solution was added to 1 × 107
PBMC and RNA was extracted according to the manufacturer’s instructions.
RNA quantification was carried out by using a spectrophotometer (GeneQuant pro® , Amersham Pharmacia
Biotech-GE Healthcare Life Sciences, Little Chalfont, UK).
Reverse transcription (RT) was carried out with the
Ready-To-GoTM You-Prime First-Strand Beads kit (Amersham Pharmacia Biotech-GE Healthcare Life Sciences, Little
Chalfont, UK) as described by the manufacturer. Two
micrograms of total RNA were used in the RT. Aliquots
(5 ␮l) from the generated cDNA were used for the subsequent PCR amplification in the reaction buffer containing
1.5 ␮l of MgCl2 (50 mM), 1 ␮l of dNTPs (12.5 mM) and 1 ␮l
of Taq DNA polymerase (1 ␮g/␮l), to a final volume of
50 ␮l. Amplification was carried out for 27 cycles, when
the reaction was in the middle of the linear range (before
reaching the amplification plateau). Each cycle consisted
of denaturation at 94 ◦ C for 1 min, annealing at a specific
temperature for each primer set for 1 min, extension at
72 ◦ C for 1 min; at the end of the 27th cycle, an additional
extension was carried out for 5 min. The primer sequences,
annealing temperatures and amplification product weights
(base pairs, bp) are summarized in Table 1. The amplified PCR products were visualized after electrophoresis
on a 2% agarose gel containing SYBR® Safe DNA gel stain
(Invitrogen Corp., Carlsbad, CA, USA). The density of each
band was quantified by densitometric analysis by using
Scion Image software (Scion Capture Driver 1.2 for ImagePro Plus; Scion, MA). Ribosomal 18S RNA was used as an
internal standard according to manufacturer’s instructions
(QuantumRNATM 18S Internal Standards Kit, Ambion Inc.,
Austin, TX, USA). The values are shown as ratio of the band
intensity of each cytokine normalized with the corresponding ribosomal 18S and expressed as arbitrary units (A.U.).
2.3. Hormonal analysis
2.3.1. GH assay
Plasma samples were assayed for GH by ELISA as previously described [31] and modified for porcine GH (pGH).
Briefly, 40 ␮l of unknown or standard samples (ranging from 39 to 5000 pg of pGH) were incubated with
100 ␮l of anti-pGH antiserum (1:300,000). After incubation for 48 h at 4 ◦ C, 10 ng/well of biotinyl-pGH were
added, followed by incubation for 2 h at 4 ◦ C. Extravidin conjugated with peroxidase (1:2000) was then
added in 100 ␮l of assay buffer, followed by incubation
for 1 h; subsequently, 100 ␮l of ABTS (2,2 -azinobis(3ethylbenzothiazolinesulphonic acid)/H2 O2 were added,
and the reaction was stopped after 25 min. The absorbance
was measured at 405 nm using a Spectra Shell Microplate
Reader. ED90 , ED50 and ED10 were 0.039, 0.166 and
5 ng/well, respectively. The intra- and inter-assay coefficients of variation were 5.3% and 7.8%, respectively.
pGH
(AFP10864B)
and
anti-pGH
antiserum
(AFP5672099) were provided by Dr. A.F. Parlow (National
Hormone and Pituitary Program, Harbor-University of
California-Los Angeles Medical Center, La Jolla, CA).
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P. Borghetti et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 143–155
Table 1
Oligonucleotide sequences designed for the detection of different porcine cytokines, with the respective annealing temperatures and amplified products
(bp).
Cytokine
Primer set
Annealing temperature (◦ C)
Amplified product (bp)
IL-1␤ [14]
F: 5 -ACA GGG GAC TTG AAG AGA G-3
R: 5 -CTG CTT GAG AGG TG CTG ATG T-3
54.5
285
IL-6
F: 5 -ATG AAC TCC CTC TCC ACA AGC-3
R: 5 -TGG CTT TGT CTG GAT TCT TTC-3
56.0
493
IL-10
F: 5 -AGC CAG CAT TAA GTC TGA GAA-3
R: 5 -CCT CTC TTG GAG CTT GCT AA-3
56.0
394
TNF-␣ [56]
F: 5 -CCA CCA ACG TTT TCC TCA CT-3
R: 5 -AAT AAA GGG ATG GAC AGG GG-3
57.3
351
IFN-␥
F: 5 -CTC TCC GAA ACA ATG AGT TAT ACA-3
R: 5 -GCT CTC TGG CCT TGG AA-3
55.0
503
MCP-1 [21]
F: 5 -ATC CTC CAG CAT GAA GGT-3
R: 5 -GGA AAT GAA TTG TCT TCA GAT-3
53.7
354
2.3.2. Cortisol assay
The concentration of plasma cortisol was determined
by a validated radioimmunoassay, as previously described
[32]. Cortisol, H3 -labeled cortisol and anti-cortisol antiserum were purchased from Sigma–Aldrich (St. Luis, MO,
USA). The intra- and inter-assay coefficients of variation
were 7.4% and 9.5%, respectively.
2.4. Statistics
Group comparison for hormonal and cytokine data at
each time point was performed by Analysis of Variance
and Dunnett’s Two-Sided Multiple-Comparison Test With
Control. Comparison of data at different time points within
the same group was performed by Repeated Measures
Analysis of Variance (full model) and Dunnett’s Two-Sided
Multiple-Comparison Test. p-Values less than an alpha of
0.05 (probability of Type I Error) were considered significant throughout this study. Statistical analysis was
carried out using the SPSS System for Windows, Version
14.0.
The Pearson’s correlation coefficient was used to determine the degree of association between pairs of variables.
The statistical significance of the correlation was determined by the F test, using the statistical package NCSS
2007.
3. Results
3.1. Cytokine gene expression
The results of cytokine gene expression are shown
in Table 2 where the statistically significant differences
at the comparison between time-points within the same
group are reported. The statistically significant differences
between groups are shown in Figs. 1–6. The comparison between the vaccinated groups (IM and ID) did not
show any statistically significant difference for any of the
cytokines at any of the considered times. On the contrary,
significant differences were recorded when vaccinated
groups were compared with controls (unvaccinated pigs).
It is worth remembering that, as briefly reported above and
detailed in a previous paper [15], experimental pigs became
naturally infected by the PRRSV field isolate, starting from
day 4 PE. From day 11 to day 14 all pigs were viremic at
PCR.
IFN-␥ gene expression in PBMC (Table 2) showed a slight
increase during the first week PE in all groups. Then, IFN-␥
rose significantly (p < 0.05) only in the vaccinated groups,
reaching the highest levels at 14 and 21 days PE; after
the third week, IFN-␥ in vaccinated pigs fell to basal levels on day 28 PE. In contrast, in unvaccinated pigs, IFN-␥
gene expression did not show any significant changes during the whole period, however, an increasing trend was
observed from the third week PE. IFN-␥ gene expression
was significantly higher (p < 0.05) at 14 and 21 days PE in
the vaccinated pigs and at 28 and 34 days in the controls
(Fig. 1).
IL-6 gene expression (Table 2) increased significantly
(p < 0.05) in all groups during the first week. In both vaccinated groups, IL-6 gene expression showed a further
significant increase at 14 days PE, and thereafter a decrease
to basal levels at 21 up to 34 days PE. At 14 days PE the levels
of this cytokine in both vaccinated pigs were significantly
higher compared with controls (Fig. 2). In unvaccinated animals, IL-6 showed a significant transient increase in the first
week PE, then it decreased up to day 21. After 21 days PE,
IL-6 gene expression began to significantly rise and continued up to day 34 PE. In this time period, the higher level of
IL-6 in controls was statistically significant compared with
vaccinated pigs (Fig. 2).
In the vaccinated groups, both IL-1␤ and MCP-1 gene
expression (Table 2) showed a significant increase at 7 days
PE, when the pigs started to become infected by the field
PRRSV, with a decline to a basal level at 14 and 21 days PE
and 1–2 weeks post-infection, respectively. In control pigs,
no changes in IL-1␤ and MCP-1 expression were observed
over time, with the exception of a significant increase in
MCP-1 on day 34 (3 weeks post-infection) compared with
vaccinated pigs. At 7 days PE, IL-1␤ gene expression in both
vaccinated groups was significantly higher as compared to
controls (Fig. 3). MCP-1 gene expression was significantly
higher (p < 0.05) at 7 and 14 days PE in vaccinated pigs and
at 34 days in controls (Fig. 4).
TNF-␣ gene expression (Table 2) in PBMC from vaccinated animals showed a significant increase at 14 days PE,
P. Borghetti et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 143–155
147
Table 2
Levels of pro-inflammatory and immunomodulatory cytokine gene expression in PBMC of IM-vaccinated and ID-vaccinated pigs compared with unvaccinated control (C) pigs in the post-exposure (PE) period.
Group
Days PE
0
“Cytokines”
TNF-␣
IM
ID
C
+7
62.0 ± 12.1a
55.6 ± 16.2a
72.5 ± 7.2a
83.55 ± 5.6a
86.3 ± 2.6a
87.7 ± 7.5a
+14
+21
+28
+34
71.8 ± 8.3a
80.0 ± 7.2b
73.5 ± 15.1a
95.3 ± 5.5b
94.2 ± 4.4c
71.5 ± 15.7a
86.3 ± 2.8b
86.7 ± 4.6c
94.0 ± 2.5b
52.8 ± 6.2c
54.8 ± 9.5a
93.2 ± 13.1b
45.2 ± 4.4c
49.3 ± 4.2a
96.8 ± 17.8b
139.8 ± 24.3b
125.3 ± 15.8b
90.2 ± 6.8a
90.2 ± 13.4a
87.3 ± 6.7a
81.3 ± 12.8a
84.8 ± 23.1a
86.2 ± 5.9a
90.7 ± 11.4a
82.0 ± 16.6a
78.2 ± 11.2a
88.3 ± 10.9a
72.0 ± 9.9a
77.2 ± 11.8a
88.7 ± 11.0a
87.6 ± 15.6b
83.6 ± 23.8b
49.8 ± 14.1a,b
61.8 ± 25.1c
54.0 ± 4.6c
50.7 ± 14.1a,b
36.2 ± 9.2a
35.2 ± 4.7a
65.3 ± 12.1b
35.2 ± 6.3a
35.3 ± 7.1a
70.5 ± 12.7b
91.2 ± 5.7a
89.0 ± 5.5a
86.7 ± 8.1a
90.7 ± 5.9a
80.2 ± 15.1a
83.5 ± 10.0a
81.3 ± 9.2a
85.0 ± 12.1a
107.5 ± 14.4b
IL-1␤
IM
ID
C
IL-6
IM
ID
C
34.2 ± 27.2a
30.8 ± 15.5a
40.7 ± 22.5a
78.0 ± 8.2b
75.0 ± 7.6b
66.0 ± 18.5b
MCP-1
IM
ID
C
78.8 ± 14.7a
83.2 ± 15.2a
88.5 ± 7.4a
133.2 ± 30.4b
122.8 ± 5.3b
87.0 ± 6.9a
103.2 ± 16.1b
101.0 ± 24.3b
85.5 ± 10.5a
IFN-␥
IM
ID
C
49.3 ± 14.6a
55.2 ± 19.6a
46.0 ± 26.2a
73.2 ± 3.5b
73.7 ± 5.1b
61.5 ± 14.5a
91.0 ± 8.1c
90.4 ± 12.7b
45.0 ± 21.1a
89.0 ± 3.1c
90.7 ± 1.5b
61.8 ± 20.2a
48.2 ± 7.5a
53.8 ± 5.8a
69.7 ± 11.4a
51.8 ± 8.3a
49.3 ± 6.7a
75.0 ± 12.8a
IL-10
IM
ID
C
45.3 ± 8.6a
64.8 ± 19.1a
58.5 ± 23.1a
72.7 ± 7.6b
65.5 ± 11.6a
68.8 ± 13.1a
77.0 ± 12.1b
86.5 ± 5.1b
79.8 ± 13.2a
58.0 ± 7.6a
55.8 ± 11.2a
86.5 ± 5.7b
23.3 ± 7.2c
28.0 ± 5.4c
50.4 ± 10.8c
18.8 ± 5.5c
21.8 ± 6.1c
48.8 ± 18.9c
IM
ID
C
10.1 ± 1.2a
9.4 ± 0.9a
6.8 ± 0.5a
18.6 ± 1.6b
17.2 ± 3.1b
7.6 ± 0.81a
14.4 ± 6.3b
15.1 ± 4.9b
12.2 ± 5.0a
8.5 ± 0.5a
10.1 ± 3.2a
9.1 ± 2.6a
8.3 ± 1.6a
8.8 ± 0.9a
9.7 ± 1.8a
7.9 ± 1.6a
8.7 ± 0.8a
10.1 ± 1.8a
IM
ID
C
23.6 ± 4.9a
24.1 ± 4.5a
22.9 ± 5.1a
23.3 ± 3.5a
26.4 ± 2.9a
10.1 ± 3.5b
9.5 ± 5.0b
16.3 ± 6.4b
10.1 ± 1.9b
8.3 ± 2.2b
7.1 ± 4.6b
6.7 ± 3.2b
7.1 ± 1.4b
8.8 ± 3.0b
9.8 ± 2.5b
8.8 ± 1.1b
8.2 ± 2.0b
10.5 ± 2.2b
Hormones
GH
Cortisol
Values are expressed as mean ± S.D. of the cytokine/18S gene expression ratio (arbitrary units).
Different superscript letters indicate a statistical difference (p < 0.05) among time points within the same group.
and a declining trend 21 days PE, reaching basal levels at
28 and 34 days PE. In unvaccinated pigs, TNF-␣ expression significantly increased on day 21 PE and remained at
significantly higher levels until the end of the experiment,
compared with vaccinated animals (Fig. 5).
IL-10 levels (Table 2) slightly increased up to 14
days PE in all groups; in vaccinated animals, IL-10 gene
expression showed a prompt and marked decrease from
14 days PE to the end of the observations. Conversely,
unvaccinated pigs maintained a significantly higher level
with respect to vaccinated pigs from 21 to 34 days PE
(Fig. 6).
The study of the statistical correlations showed that in
unvaccinated pigs IL-6 and TNF-␣ are positively correlated
with clinical signs as reported by Martelli et al. [15] with a
p value of 0.01 and 0.03, respectively. The r values are 0.42
Fig. 1. Course of IFN-␥ gene expression in PBMC of PRRSV-vaccinated (ID and IM groups) and unvaccinated (C group) pigs in the post-exposure period. Data
are shown as ratios of IFN-␥/18S rRNA gene expression, arbitrary units (A.U.). Asterisk (*) indicates a statistically significant difference (p < 0.05) between
vaccinated (groups IM and ID) and unvaccinated pigs.
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P. Borghetti et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 143–155
Fig. 2. Course of IL-6 gene expression in PBMC of PRRSV-vaccinated (ID and IM groups) and unvaccinated (C group) pigs in the post-exposure period. Data
are shown as ratios of IL-6/18S rRNA gene expression, arbitrary units (A.U.). Asterisk (*) indicates a statistically significant difference (p < 0.05) between
vaccinated groups IM and ID) and unvaccinated pigs.
Fig. 3. Course of IL-1␤ gene expression in PBMC of PRRSV-vaccinated (ID and IM groups) and unvaccinated (C group) pigs in the post-exposure period. Data
are shown as ratios of IL-1␤/18S rRNA gene expression, arbitrary units (A.U.). Asterisk (*) indicates a statistically significant difference (p < 0.05) between
vaccinated (groups IM and ID) and unvaccinated pigs.
Fig. 4. Course of MCP-1 gene expression in PBMC of PRRSV-vaccinated (ID and IM groups) and unvaccinated (C group) pigs in the post-exposure period.
Data are shown as ratios of MCP-1/18S rRNA gene expression, arbitrary units (A.U.). Asterisk (*) indicates a statistically significant difference (p < 0.05)
between vaccinated (groups IM and ID) and unvaccinated pigs.
P. Borghetti et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 143–155
149
Fig. 5. Course of TNF-␣ gene expression in PBMC of PRRSV-vaccinated (ID and IM groups) and unvaccinated (C group) pigs in the post-exposure period.
Data are shown as ratios of TNF-␣/18S rRNA gene expression, arbitrary units (A.U.). Asterisk (*) indicates a statistically significant difference (p < 0.05)
between vaccinated (groups IM and ID) and unvaccinated pigs.
Fig. 6. Course of IL-10 gene expression in PBMC of PRRSV-vaccinated (ID and IM groups) and unvaccinated (C group) pigs in the post-exposure period. Data
are shown as ratios of IL-10/18S rRNA gene expression, arbitrary units (A.U.). Asterisk (*) indicates a statistically significant difference (p < 0.05) between
vaccinated (groups IM and ID) and unvaccinated pigs.
and 0.37 for IL-6 and TNF-␣, respectively. No statistically
significant correlations were detected in vaccinated pigs.
3.2. Hormonal changes
The results regarding hormone plasma levels are shown
in Table 2 where the statistically significant differences
at the comparison between time-points within the same
group are reported. The statistically significant differences
between groups are shown in Figs. 7 and 8.
In the first week of exposure, the plasmatic level of
cortisol fell in unvaccinated pigs whereas remained at significantly higher levels in vaccinated animals (Fig. 7). On
day 14 PE, plasmatic cortisol levels reached basal levels in
all considered animals and maintained those levels up to
the end of the experimental period.
The GH plasmatic trend showed significant differences
between vaccinated and unvaccinated groups in the PE
period (Fig. 8). On day 7 PE, plasmatic GH significantly
increased increased significantly (p < 0.05) in vaccinated
pigs (both groups, IM and ID), with statistically significant
differences observed at the comparison with unvaccinated
pigs. Thereafter, GH levels decreased to the basal level at 21
days PE. In unvaccinated pigs, GH showed a slight transient
increase at 14 days PE.
In vaccinated pigs the levels of GH are statistically correlated (p < 0.01) with the levels of gene expression of IFN-␥
(r = IM: 0.33; ID: 0.38), IL-6 (r = IM: 0.46; ID: 0.63), TNF-␣
(r = IM: 0.36; ID: 0.52), IL-1␤ (r = IM: 0.49; ID: 0.54), and
MCP-1 (r = IM: 0.52; ID: 0.65).
Moreover, the levels of IL-10 in vaccinated pigs, belonging to both groups, were positively correlated with the
cortisol course (p < 0.001) (r = IM: 0.31; ID: 0.53). No statistical correlations between cytokines and hormones were
detected in control animals. Conversely, the levels of cortisol is negatively correlated (p < 0.001; r = −0.56) with
clinical signs measured as reported by Martelli et al. [15].
4. Discussion
As a previous paper [15] is the underlying study for
this follow-up research, to make reading easier the major
results are briefly summarized. After exposure, accord-
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P. Borghetti et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 143–155
Fig. 7. Course of cortisol plasma levels in PRRSV-vaccinated (ID and IM groups) and control (C group) pigs in the post-exposure period. Data shown as
mean values ± S.D. Asterisk (*) indicates a statistically significant difference (p < 0.05) between vaccinated (groups IM and ID) and unvaccinated pigs.
ing to PCR results, PRRSV infection first occurred on day
4 PE, and between days 7 and 14, all of the experimental pigs (vaccinated by both routes of administration and
unvaccinated) were infected. Moreover, gross pathology
and microbiological investigations were performed on two
experimental pigs that died, and P. multocida was isolated
from pneumonic lungs. Nested-PCR and immunofluorescence assay for M. hyopneumoniae gave consistently
negative results. SIV was not isolated from pneumonic
lungs. No seroconversion to A. pleuropneumoniae, M. hyopneumoniae, Aujeszky’s disease virus and SIV was detected
at any considered time. At vaccination and on days 0 and 21
PE, all serum samples were negative for the PCV2 genome
by quantitative PCR. At the end of the study (day 34 PE),
1 pig (IM group) out of 27 had a viral load in serum of
3.45 log10 [15]. These results confirm that PRRSV and P.
multocida were involved in the outcome of the observed
disease and do not allow to completely exclude the effect
of other significant concurrent/secondary sub-lethal bacterial infections in a proportion of these pigs that may
have altered some of the later responses. It is well known
that in field the existence of whatever other pathological
conditions could affect the results presented in this study.
Therefore, whatever other potential infection under the
conditions of this field experiment should have affected
all treatment groups (vaccinated and unvaccinated animals) so that, if significant differences were obtained,
these must be mainly attributed to vaccination. The clinical evaluation recorded statistically significant differences
between vaccinated and controls, clearly supporting that
vaccination conferred clinical protection (vaccine efficacy
of approximately 70%) as detailed in a previous report [15].
Furthermore, in accordance with previous reports [14,15],
the results of this study show that the immune parameters are not affected by the route of administration of the
vaccine, as intradermally vaccinated pigs did not show any
differences in terms of cytokine and hormonal trends compared to intramuscularly injected animals.
The study investigated the induction of some proinflammatory (IL-1␤, TNF-␣, IL-6, MCP-1), pro-immune
(IFN-␥) and anti-inflammatory (IL-10) cytokines in blood
leucocytes from MLV-vaccinated pigs and unvaccinated
pigs naturally infected with a PRRSV-1 field strain. Several studies have analyzed cytokine gene expression in
vivo and in vitro and/or cytokine secretion in the blood
or bronchoalveolar lavage (BAL) fluid during experimental
infection by PRRSV [5,33–35]. However, the results were
not conclusive and were occasionally contrasting.
Fig. 8. Course of growth hormone (GH) plasma levels in PRRSV-vaccinated (ID and IM groups) and control (C group) pigs in the post-exposure period. Data
shown as mean values ± S.D. Asterisk (*) indicates a statistically significant difference (p < 0.05) between vaccinated (groups IM and ID) and unvaccinated
pigs.
P. Borghetti et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 143–155
The present study evaluated gene expression of systemic cytokines in PBMC with the objective of assessing
cytokine temporal patterns. Many immune cell subpopulations are represented in PBMC (i.e. monocytes, dendritic
cells, innate and acquired cytotoxic cells, regulatory cells)
and their proportion and functional status are expression of
immune cell recruitment, migration and activation against
pathogens. We therefore used PBMC as a biological sample and gene expression as a parameter to functionally
measure inflammatory and immune cell reactivity during PRRSV infection. The Authors considered that gene
expression in PBMC could also be a suitable parameter
for achieving a more stable measurement of cytokine production for the evaluation of immune cell activation over
time. Indeed, the determination of cytokine protein levels in serum could be erratic and less informative under
the conditions of this experiment, namely natural infection.
Moreover, cytokine levels in serum are not strictly related
to the levels produced locally in the target organs (e.g. BAL
fluid) [3,33].
Furthermore, to our knowledge, this is the first study
evaluating cytokine profiles in relation to the time-related
changes of some immune-modulating hormones, namely
cortisol and GH.
The time-related changes of pro-inflammatory and proimmune cytokine expression in vivo demonstrate that
natural infection by PRRSV does not activate an early
and efficient inflammatory and innate immune response,
which agrees with other laboratory trials that showed
how PRRSV infection alters the immune response, mainly
inducing down-regulation of pro-inflammatory and proimmune cytokines [3,4,20,33].
IFN-␥ production is a fundamental event for the effectiveness of innate and acquired immunity against viruses;
innate [natural killer (NK) cells and ␥/␦ T lymphocytes] and
acquired (helper and cytotoxic T lymphocytes) immune
cells produce IFN-␥ as the pivotal paracrine and autocrine
signal for their activation [25,36]. IFN-␥ gene expression did not show any significant increase after field
PRRSV exposure in unvaccinated pigs. This impaired cellmediated immune response was confirmed by the late
changes in IFN-␥ secreting cells (SC), natural and MHCspecific cytotoxic cells during PRRSV infection occurring at
21 days PE [15].
It is well-known that PRRSV infection can induce a
systemic production of IFN-␥, characterized by interindividual variability in terms of intensity and occurrence,
coupled with extremely variable efficiency of the innate
and adaptive immune response that sustains viral clearance [1,2,37].
Under the conditions of this experiment, in unvaccinated pigs infected by PRRSV-1, the levels of proinflammatory and pro-immune cytokines significantly
increased only when infection was established by 2 weeks;
conversely, the expression of these cytokines in PBMC
of vaccinated pigs was higher as soon as the first week
post-infection. These different trends sustain the prompt
immune reactivity of systemic inflammatory and immune
cells in vaccinated animals, and the impaired, delayed reaction in controls coupled with the occurrence of more severe
clinical signs, namely a higher proportion of feverish pigs
151
associated with lethargy and anorexia, as reported in the
previous paper [15].
We observed that PRRSV natural infection does not
induce variations in systemic TNF-␣ gene expression up
to 1–2 weeks post-infection in unvaccinated pigs. It is
interesting to note that TNF-␣ gene expression was low
in the first 2 weeks of infection when the health status
of the infected control pigs was impaired by fever and
general clinical abnormalities [15]. This result is in accordance with that by Thanawongnuwech et al. [5] whose
study assessed cytokine gene expression in pulmonary
alveolar macrophages; moreover, Van Reeth et al. [33]
detected minimal TNF-␣ levels at 10 days post-infection
when the maximal pathology associated with PRRSV infection occurs. Some mechanisms have been hypothesized to
explain this feature: PRRSV strongly inhibits TNF-␣ production and conversely it is well-known that increased TNF-␣
levels can play a pivotal role in viral clearance by inflammatory cells in the respiratory tract [5]. It is known that TNF-␣
has a pivotal role in innate immune response efficiency,
showing antiviral effect, activation and differentiation of
the monocyte/macrophage lineage, and activation of NK
cells to produce IFN-␥ [25].
Taken together, we can speculate that the lack of TNF-␣
gene expression in PBMC indicates a reduced reactivity of systemic inflammatory and innate cells, which can
also negatively affect their efficiency when recruited in
the respiratory tract. The evidence of higher TNF-␣ gene
expression in PBMC of vaccinated infected pigs demonstrates that the reactivity of these cells to produce this
cytokine has been primed and, at the time of infection, they
are able to play a more efficient functional role in clinical
protection.
The results of this trial show a significant, prompt
increase of IL-1␤ soon after infection in vaccinated piglets
and no modification in its gene expression in unvaccinated
pigs. In the unvaccinated pigs, IL-1␤ gene expression in
PBMC did not show any changes during the whole postexposure period. Van Reeth and Nauwynck [3] reported an
early increase of IL-1␤ after experimental challenge with
PRRSV in the BAL fluid; the authors hypothesized that this
increase can be related to systemic clinical signs (i.e. fever).
Contrarily, our results obtained from a different biological
sample (systemic PBMC vs. BAL fluid) lead to a different
interpretation in terms of functional meaning: in our case,
cytokine gene expression in PBMC has to be evaluated as a
marker of efficiency of innate cell reactivity rather than as
an indicator of severity of local and systemic abnormalities.
Our data confirm that clinical signs in unvaccinated
pigs occur without early signals of inflammatory and
innate reactivity (i.e. IL-1␤, TNF-␣ and MCP-1), even in
the severely affected animals. Conversely, the patterns of
the mentioned cytokines in vaccinated pigs indicate that,
although infection occurs with the same timing as that of
unvaccinated pigs, the vaccinated pigs are able to show and
sustain more efficient reactivity of innate immunity within
the first week post-infection, even in the case of infection
by a genetically different, heterologous field isolate (85%
of homology). This difference in immune reactivity in vaccinated pigs parallels with a less severe outcome of the
disease in comparison with controls.
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P. Borghetti et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 143–155
The results of IL-6 gene expression are more difficult to
interpret. It is likely that the changes of IL-6 activity should
be functionally evaluated in terms of their occurrence in the
early (acute) or late (chronic) phase of inflammation, taking into account the complex and modulating role exerted
during the immune response and IL-6’s interplay with
other pro-immune factors, namely neuro-immune hormones. At the beginning of the acute inflammatory phase,
IL-6 mediates the acute phase reaction (APR) and modulates the over activity of inflammation; it is well-known
that IL-6 is also critical in controlling the extent of local
and systemic acute inflammation, particularly in decreasing the levels of pro-inflammatory cytokines by exerting
a protective effect against potential damage and promoting anti-inflammatory activity [38]. However, if its levels
persist and chronic inflammation occurs, IL-6 can have a
detrimental effect by favouring the recruitment, proliferation and survival of mononuclear cells at the site of injury
[39]. We detected low IL-6 levels of gene expression in
unvaccinated pigs exposed to the PRRSV field isolate in
the first 2 weeks post-infection in accordance with the
results by Thanawongnuwech et al. [5], who demonstrated
increased levels of IL-6 only after 28 dpi, with no increased
levels of IL-1␤. Conversely, in vaccinated pigs, higher levels
of IL-6 were observed only during the first week postinfection, followed by a prompt and long-lasting significant
decrease. Moreover, the observed early increase of IL-1␤
preceding the increase of IL-6 in vaccinated pigs is expected
because the former cytokine is a strong inducer of IL-6 production during viral infection [40]. Thus, in this paracrine
interaction, an increase in both IL-1␤ and IL-6 can sustain,
along with TNF-␣, a more efficient innate response. In contrast, in unvaccinated pigs considered in this trial, the late
occurrence and persistence of IL-6 gene expression in PBMC
could impair activation of the adaptive immune response
by further inhibition of Th1 differentiation [41] and could
drive chronicization of the inflammation [38], with a strong
influence on the outcome of the observed disease in unvaccinated pigs. Moreover, the positive statistically significant
correlation between IL-6 and TNF-␣ with clinical signs in
controls [15] corroborate this last assumption.
MCP-1 is a chemokine involved in the onset of inflammation; it mediates mainly the chemotaxis of monocytes
and also of memory T lymphocytes and NK cells [42].
MCP-1 and its receptor play an important role in the
T cell-mediated immune response against intracellular
pathogens by controlling leukocyte recruitment and clearance of pathogens [43–45]. Its production is induced by
IL-6, TNF-␣ and IFN-␥ [38,42].
The pattern of MCP-1 in the present study was characterized by an increase and subsequent decrease in
vaccinated pigs during the first week of infection, which
indicates, similarly to the other pro-inflammatory cytokine
pattern, namely IL-1␤ and TNF-␣, that an early danger signal was produced and mediated prompt activation of an
inflammatory/innate response. On the contrary, in unvaccinated pigs, the increase of MCP-1 was observed later
with respect to infection (4 weeks); the difference in its
level, compared with those of vaccinated animals, differs
significantly and coincides with a higher level of IL-6, a
strong inducer of MCP-1 production. The combination of
these two cytokines in PBMC is consistent with a delayed
recruitment of monocytes and with a chronicization of
inflammation [38]. It is well-known that under several conditions, high and persistent levels of MCP-1 are involved
in many autoimmune- or infection-mediated inflammatory diseases associated with monocyte recruitment with
un-controlled inflammatory damage [42].
During infection, activation of the HPA axis is one
of the major neuroendocrine features of APR. Indeed,
pro-inflammatory cytokine (IL-1, TNF-␣, IL-6) production,
which sustains the APR, is tightly controlled by intrinsic
anti-inflammatory mechanisms and also by the stimulation of the HPA and the release of cortisol by adrenal glands
[26,46]. It is well known that the increase of glucocorticoid
(GC) plasmatic levels suppresses the further release of proinflammatory cytokines, preventing long-term and/or over
activation of inflammation.
Interestingly, PRRSV infection down-regulates the cortisol response; our results on plasmatic levels of cortisol
are in accordance with those by Sutherland et al. [47],
who observed that plasma cortisol, considered an indicator of HPA axis activation, was suppressed during PRRSV
infection. Our results confirm and further characterize the
above feature: we demonstrated that, in unvaccinated pigs,
cortisol down-regulation is associated with no increase
of pro-inflammatory cytokines which are the major systemic signals for HPA activation during the acute phase
reaction [48]. Interestingly, in vaccinated animals cortisol maintained statistically significant higher levels than
in unvaccinated animals in the first week post-infection.
At present, a potential direct or indirect mechanism by
which PRRSV infection can alter HPA activation and GC
production is unknown and warrants further study.
If GC have a central role in the control of local and systemic inflammatory responses during and after pathogen
challenge and in the prevention of excessive inflammation, it is not surprising that a failure of GC production or
tissue resistance to their inhibitory effects may result in
uncontrolled inflammation and tissue damage, and thus
increased susceptibility to and severity of the related
disease, influencing survival [26,27,30,49,50]. We can
hypothesize that in unvaccinated pigs the down-regulation
of cortisol production in the early phase of infection may
have negatively influenced HPA responsiveness so that
in the subsequent late phase cortisol did not increase to
counterbalance the rise of pro-inflammatory cytokines (i.e.
TNF-␣, IL-6 and MCP-1). In unvaccinated animals, the longterm maintenance of higher levels of TNF-␣ and IL-6 after
infection in association with low levels of cortisol could
be consistent with a long-lasting inflammatory status and
lack of control of the disease, thereby influencing the overt
clinical signs observed in the unvaccinated animals.
IL-6 is also a key cytokine in the neuro-immune communication during infection. It has important effects on
the production of corticotrophin-releasing hormone by the
hypothalamus and the secretion of adrenocorticotropic
hormone, prolactin and GH from the pituitary gland
[51,52]. In vaccinated pigs, we detected increased levels of
IL-6 gene expression statistically correlated with increases
of GH and IFN-␥; in this context, IL-6 could act positively on
innate immunity. High and early IL-6 levels are consistent
P. Borghetti et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 143–155
with an APR, whereas prolonged production of IL-6 and the
related deregulation of the HPA axis have been considered
an important pathway affecting health during inflammation, chronic stress, aging and metabolic disease [38,53];
therefore, the persistence of this cytokine can be a valuable
marker for ongoing infections [54].
Furthermore, this is the first study considering the GH
course during PRRSV natural infection. It is well known
that pro-inflammatory cytokines (i.e. IL-1 and TNF-␣) have
a direct effect on pituitary GH secretion during the acute
phase response and several studies have suggested that
this hormone plays an important role in the improvement of innate immune responses during infection [30].
The infection in unvaccinated pigs did not significantly
increase plasmatic GH, indicating inefficient pituitary activation. Contrarily, both after vaccination with an MLV (data
not shown) and, in vaccinated animals, after infection with
PRRSV under field conditions, an early and prompt rise of
GH was clearly detected. In a previous study, we demonstrated that vaccination against PRRSV increases plasmatic
GH regardless of a weaning-dependent increase in plasmatic cortisol [55].
The rise of plasmatic GH during infection could be a
reliable indicator of immune responsiveness in vaccinated
pigs; GH has a positive effect on the development and
maturation of lymphoid cells and on cytokine secretion in
primary lymphoid organs such as thymus, and promotes
the efflux of mature lymphoid cells in the blood and the
migration in secondary lymphoid organs and target tissues.
Moreover, GH improves the efficiency of innate immune
cell activity, mainly NK cells [30,56,57]. Recently in pig,
Thacker et al. [58] showed that plasmid-mediated supplementation of growth hormone-releasing hormone before
vaccination may enhance protection against M. hyopneumoniae pneumonia and reduces the clinical outcome of the
disease.
Indeed, there is evidence that IL-6 may regulate GH
secretion [59] and, in turn, GH increases IL-6 secretion
[60] and directly affects the innate immune response [61].
Since a paracrine influence exists between GH and IL6, under a stress condition such as immune challenge,
GH appears fundamental to increasing the levels of these
cytokines for prompt imprinting of an innate response to
pathogens.
IL-10, mainly produced by Th2 and regulatory T lymphocytes, but also by activated macrophages, is a crucial
cytokine in controlling the extent of the immune response
and preventing immune-mediated tissue damage. Conversely, early over-expression and/or persistence of IL-10
might prevent or delay the clearance of pathogens, thereby
resulting in persistent infection [62]. In the present study,
high levels of IL-10 occurred in vaccinated and unvaccinated pigs from the beginning of PRRSV infection; this is
consistent with the delay of the acquired response in the
first 2–3 weeks post-infection and consequently of the inefficient viral clearance during the period of observation [15].
Some studies indicate that enhanced in vivo and in vitro
IL-10 expression in different permissive or unpermissive
immune cells might be an important mechanism for modulating the host’s immune response during experimental
infection by PRRSV and for playing an essential role in a
153
virus evading the immune response, which prevents the
development of protective immunity [7,12,63,64].
Similar to Thanawongnuwech et al. [5], we found
high levels of IL-10 gene expression during the whole
period of exposure, but compared with vaccinated
pigs, unvaccinated pigs showed significantly higher levels of IL-10 gene expression during the last 2 weeks
post-exposure.
In the model proposed in this field study, the evidence
for differences of IL-10 gene expression in PBMC between
vaccinated and unvaccinated pigs, with higher levels of this
cytokine in the latter animals, can parallel with the severity
of the clinical scores we observed, and conversely with the
protection induced by vaccination. In contrast, the statistically correlated trends between IL-10 and cortisol sustain a
more balanced control of inflammation in the early phase of
the disease. It can be hypothesized that the difference in the
immune response and, likely, in the severity of the disease
between vaccinated and control groups depends mainly on
the quality of the innate immune response, which was evaluated as a time-related, pro-inflammatory cytokine and
anti-inflammatory balance. In our study, vaccinated animals, in contrast to unvaccinated pigs, showed a prompt
increase of TNF-␣ gene expression balanced by high levels of IL-10 and a rapid decrease of both cytokines after
infection with a heterologous PRRSV-1 field isolate. Interestingly, other Authors, although using other approaches,
have suggested that the quality of the cytokine pattern
induced by a given strain is important for modulating the
entire immune response against PRRSV. In agreement with
this, the present study and a previous paper [15], Diaz et
al. [65] found that strains inducing TNF-␣ and IL-10 simultaneously yielded a more sustained cell-mediated immune
response, measured as IFN-␥ SC.
The early and prompt increase after PRRSV infection,
and the subsequent decrease to basal levels within 1–2
weeks, of IL-1␤, IL-6, TNF-␣, MCP-1 and IFN-␥ in vaccinated animals is a further, clear indication of both a more
efficient responsiveness of peripheral innate immune cells
(i.e. monocytes, NK cells) in the early phase of infection and
a more efficient control of inflammation in a later phase.
Both aspects coincides with the clinical protection (70%)
induced by vaccination as previously reported by Martelli
et al. [15] in the same experimental animals.
On the contrary, unvaccinated animals subsequently
infected with a heterologous PRRSV-1 strain show an
inefficient and delayed immune response; furthermore,
long-term maintenance of TNF-␣, IL-6 and IL-10 levels and
deregulation of cortisol production could be markers of
uncontrolled inflammatory damage and could functionally
explain the severity of clinical signs.
Thus, the associated trends of pro-inflammatory and
anti-inflammatory cytokines together with the cortisol
level could provide indicators to study the progression of
the persistent PRRSV infection and its severity.
Conflict of interest statement
None of the authors of this paper has a financial or personal relationship with other people or organisations that
154
P. Borghetti et al. / Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 143–155
could inappropriately influence or bias the content of the
paper.
Acknowledgements
[17]
[18]
This work was supported by a grant from the University
of Parma (Italy)-FIL 2007 (FIL0797239).
Ph.D. studies of Dr. Luca Ferrari and Marina Morganti
were funded by a pre-doctoral grant of the University of
Parma (Italy) in “Experimental and Comparative Immunology and Immunopathology”.
[19]
[20]
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