Veterinary Microbiology 168 (2014) 357364

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Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Systemic and local immune response in pigs intradermally

and intramuscularly injected with inactivated Mycoplasma
hyopneumoniae vaccines
P. Martelli a,*, R. Saleri a, V. Cavalli a, E. De Angelis a, L. Ferrari a, M. Benetti a,
G. Ferrarini a, G. Merialdi b, P. Borghetti a
a

Department of Veterinary Science, University of Parma, Via del Taglio, 10 - 43126 Parma, Italy
Istituto Zooprolattico Sperimentale della Lombardia e dellEmilia Romagna B. Ubertini Sezione diagnostica di Bologna, Via Ficorini,
5 - 40127 Bologna, Italy

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 27 August 2013
Received in revised form 11 November 2013
Accepted 15 November 2013

The systemic and respiratory local immune response induced by the intradermal
administration of a commercial inactivated Mycoplasma hyopneumoniae whole-cell
vaccine (Porcilis1 MHYO ID ONCE MSD AH) in comparison with two commercial
vaccines administered via the intramuscular route and a negative control (adjuvant only)
was investigated. Forty conventional M. hyopneumoniae-free pigs were randomly assigned
to four groups (ten animals each): Group A = intradermal administration of the test vaccine
by using the needle-less IDAL1 vaccinator at a dose of 0.2 ml; Group B = intramuscular
administration of a commercially available vaccine (vaccine B); Group C = intramuscular
administration of the adjuvant only (2 ml of X-solve adjuvant); Group D = intramuscular
administration of a commercially available vaccine (vaccine D). Pigs were vaccinated at 28
days of age. Blood and bronchoalveolar lavage (BAL) uid samples were collected at
vaccination (blood only), 4 and 8 weeks post-vaccination. Serum and BAL uid were tested
for the presence of antibodies by ELISA test. Peripheral blood monomorphonuclear cells
(PBMC) were isolated to quantify the number of IFN-g secreting cells by ELISpot.
Moreover, cytokine gene expression from the BAL uid was performed. Total antibodies
against M. hyopneumoniae and specic IgG were detected in serum of intradermally and
intramuscularly (vaccine B only) vaccinated pigs at 4 and 8 weeks post-vaccination. M.
hyopneumoniae specic IgA were detected in BAL uid from vaccinated animals (Groups A
and B) but not from controls and animals vaccinated with the bacterin D (p < 0.05).
Signicantly higher gene expression of IL-10 was observed in the BAL uid at week 8 postvaccination in the intradermally vaccinated pigs (p < 0.05). The results support that the
intradermal administration of an adjuvanted bacterin induces both systemic and mucosal
immune responses. Moreover, the intramuscularly administered commercial vaccines
each had a different ability to stimulate the immune response both systemically and
locally.
2013 Elsevier B.V. All rights reserved.

Keywords:
Pig
M. hyopneumoniae
Vaccination
Intradermal

1. Introduction

* Corresponding author. Tel.: +39 0521 032698; fax: +39 0521 032692.
E-mail address: paolo.martelli@unipr.it (P. Martelli).
0378-1135/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetmic.2013.11.025

Mycoplasma hyopneumoniae (M. hyopneumoniae) is the

primary etiological agent of enzootic pneumonia (EP) in
pigs. The disease occurs worldwide and causes economic

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P. Martelli et al. / Veterinary Microbiology 168 (2014) 357364

losses to the pig industry (Maes et al., 2008). Vaccination,

improvement of housing conditions and management
procedures are currently applied for the control of EP
(Maes et al., 2008; Marchioro et al., 2013). It has been
estimated that approximately 70% of the industrial pig
population around the globe is vaccinated against M.
hyopneumoniae. This percentage is increasing in those
countries where pig production is switching rapidly from
the traditional backyard system to the intensive commercial herds. The currently available vaccines, consisting of
adjuvanted, inactivated, whole-cell preparations, have
been demonstrated to be effective in reducing the clinical
symptoms, the lung lesions, and the performance losses
associated with M. hyopneumoniae infections (Maes et al.,
2008).
The commercial vaccines induce specic antibodies in
serum, although no direct correlation has been found
between the induction of antibodies and protection
against M. hyopneumoniae (Djordjevic et al., 1997). Some
studies suggest that mucosal and cellular immune
responses might play an important role in the control
of this disease (Thacker et al., 2000), but the effects of
vaccination on mucosal and cell-mediated immunity
have been evaluated in only few studies. Thacker et al.
(2000) evaluated bronchoalveolar lavage (BAL) uid
and peripheral lymphocytes by ELISpot and ELISA, and
demonstrated that a bacterin-based vaccine induced
mucosal and systemic cell-mediated immune response.
Under the same experimental conditions, the vaccination
failed to induce changes in peripheral CD4+ and CD8+ cell.
In contrast, Kick et al. (2011) measured differences
between vaccinated and control pigs in the percentage of
these cell populations. The variability of the results
published on this topic support that the exact mechanism
of protection induced by vaccination is not completely
understood.
Furthermore, new routes of vaccine administration
have also been studied, not only to improve the
efciency of vaccination in terms of the immune
response, but also to increase the practical advantages
of control programs. Some previous studies considered
the importance of the route and the method of vaccine
administration for inducing optimum levels of protective
immunity. As well as the traditional intramuscular (IM)
route, a needle-less device has been proposed as a
relatively new tool for the intradermal administration of
vaccines and its application has been demonstrated to
induce an immune response similar or even better than
the intramuscular route, under eld conditions (Martelli
et al., 2007, 2009; Ferrari et al., 2011, 2013; Tassis et al.,
2012).
The study is aimed at evaluating the systemic and the
mucosal immune responses induced by a commercial
inactivated whole-cell M. hyopneumoniae vaccine administered via the intradermal route in comparison with the
more conventional intramuscular injection of two commercially available vaccines that have been shown to be
effective in reducing clinical signs and lung lesions
associated with M. hyopneumoniae infection. Consequently, the study is not intended to provide any
information about vaccination schemes.

2. Materials and methods

2.1. Experimental design
This blind, randomized, controlled study was performed in the isolation facilities of the Department of
Veterinary Science of Parma University, after the approval
of the Ethical Committee for Animal Experiments of Parma
University (approval number 21/12). Forty cross-bred
healthy piglets free of M. hyopneumoniae and PRRSV were
enrolled in this study at weaning (21 days of age), moved to
the isolation facilities and housed in the same room. All
animals were obtained from a herd that has been free from
M. hyopneumoniae and PRRSV based on repeated serological testing and the absence of EP lung lesions at
slaughterhouse. Moreover, before the enrollment, at 15
days of age, the animals were bled and serologically tested
for negativity to M. hyopneumoniae antibodies by using a
IDEXX M. hyo. commercial test kit (S/P ratio < 0.4). Upon
arrival, the animals were randomly allocated to four
different groups (10 piglets each) and individually
identied by a numbered ear tag. At 28 days of age, the
piglets were injected as follows: Group A = intradermal
(ID) administration of a commercial vaccine [Porcilis1 M.
hyo. ID ONCE MSD AH (formerly Intervet International),
Boxmeer, The Netherlands] by using the needle-less IDAL1
vaccinator at a dose of 0.2 ml (vaccine A); Group
B = intramuscular administration of a commercially available vaccine (vaccine B); Group C = intramuscular administration of the adjuvant only (2 ml of X-solve, the adjuvant
of vaccine A); Group D = intramuscular administration of a
commercially available vaccine (vaccine D). The dose of the
vaccines B and D is intentionally omitted in order to avoid
any possibility to identify the commercially available
products. All vaccines administered in this study were
licensed as a one dose.
Blood and bronchoalveolar lavage (BAL) uid were
collected at vaccination (blood only) and after 4 and 8
weeks. BAL uid was collected in restrained, standing
position by using a cannula via the mouth as commonly
performed for diagnostic purposes.
Blood was processed for serological investigations and
peripheral blood mononuclear cells (PBMC) isolation. BAL
uid was processed for antibody detection and centrifuged
for RNA extraction and subsequent cytokine gene expression quantication on the cellular pellet.
2.2. Nested and quantitative PCR (qPCR) for detection of M.
hyopneumoniae DNA
DNA was extracted from the BAL uid with the QIAGEN
protocol (Qiagen, DNeasy Blood & Tissue kit, Belgium). For
detection of M. hyopneumoniae DNA, both a nested and a
qPCR were performed as described by Stark et al. (1998)
and Marois et al. (2010) in order to avoid false negative
results.
2.3. Isolation of PBMC from blood
Porcine peripheral blood mononuclear cells (PBMC)
were isolated from 4 ml to 5 ml of pig blood samples

P. Martelli et al. / Veterinary Microbiology 168 (2014) 357364

collected in lithiumheparin as anticoagulant by density

gradient in Histopaque-10771solution (Sigma, St. Louis,
MO) according to the manufacturers instructions. Briey,
blood samples were taken and then stratied on Histopaque-10771 solution (1:1, v/v, Sigma) and centrifuged at
400  g for 30 min; puried PBMC were washed with
sterile PBS (Sigma) supplemented with 1% fetal bovine
serum (FBS) and resuspended in RPMI-1640 (Gibco,
Carlsbad, CA, USA) supplemented with 10% FBS, 2 mM Lglutamine, 100 mM non essential amino-acids, 50 mM 2bmercaptoethanol (Sigma) and 100 U/ml penicillin G,
100 mg/ml streptomycin and 0.25 mg/ml amphotericin B.
Such solution is referred as complete RPMI-1640 (cRPMI).
Cells were counted by inverted optical microscope and
concentration was assessed before being used for IFN-g
ELISpot assay. In all samples, PBMC were >98% viable as
conrmed by Trypan blue (Sigma) exclusion. After isolation, cell samples not processed immediately were frozen
at 80 8C using a Mr Frosty1 device (Sigma) gradient and
stored in liquid nitrogen for subsequent assays.
2.4. Total RNA extraction
Gene expression levels of relevant pro-inammatory
(TNF-a, IL-6) and adaptive immune (IFN-g, IL-10) cytokines were determined in swine PBMC. Total RNA
extraction and quantication was performed within 1
week after sample collection and storage. RNA extraction
was performed by using TRI-reagent (Ambion-Life Technologies, Grand Island, NY, USA) according to the
manufacturers instructions; purity and concentration
were assessed by UV-spectrophotometry at 260/280 and
260 nm respectively (GeneQuant Pro, Amersham Pharmacia Biotech-GE Healthcare Life Sciences, Little Chalfont,
UK). RNA integrity and quality were assessed by using an
Agilent Bioanalyzer 2100 and RNA 6000 Labchip kit
(Agilent Technologies, Santa Clara, CA, USA). RNA samples
were stored at 80 8C until reverse-transcription (RT).
2.5. Reverse-transcription (RT)

to

All RNA samples were DNAse-treated (Sigma) prior

cDNA synthesis. Total RNA (1 mg/20 mL) was

359

reverse-transcripted using a High-capacity cDNA Reverse

Transcription kit (Applied Biosystems, Foster City, CA). The
RT was performed by using a StepOne thermocycler
(Applied Biosystems, StepOne software v. 2.1) and, according to the manufacturers instructions, under the following
thermal conditions: 10 min at 25 8C; 120 min at 37 8C
followed by 5 min at 85 8C. All cDNA samples were stored at
20 8C until PCR was performed.
2.6. Quantication of cytokine gene expression by real-time
PCR (qPCR)
The cDNA samples (20 ng) were used as a template for
real-time quantitative PCR (qPCR) performed by using a
StepOne thermocycler (Applied Biosystems, StepOne
software v. 2.1). The cDNA (20 ng/20 mL) was amplied
in triplicate with Fast SYBR1 Green-1 Master Mix (Applied
Biosystems) and specic sets of primers optimized at
150 nM for IL-6 and 300 nM for the other cytokines. The
primers used are based on published sequences (Fisher
et al., 2006; Meissonnier et al., 2008) or were designed by
using Primer Express1 software package (Applied Biosystems) and purchased from Eurons MWG Operon
(Ebersberg, Germany). Details of each primer set for
detection of cytokine gene expression are reported in
Table 1.
The reference gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was selected among other tested
reference genes [e.g., b-actin (Meissonnier et al., 2008),
ribosome protein (RP)L-19 (Kiros et al., 2011), RPL-32
(Royaee et al., 2004)] as endogenous control according to
minimal intra-/inter-assay variation and to Fisher et al.
(2006), Facci et al. (2011) and Ferrari et al. (2011). Samples
were kept at 95 8C for 20 s (hold step) to allow DNApolymerase activation and then subjected to 40 cycles
consisting of a denaturation step at 95 8C for 3 s followed
by an annealing/extension step at 60 8C for 30 s. Fluorescence due to SYBR1 Green-1 incorporation was acquired at
the end of the extension step. No-template (NTC) and noRT controls were included in each experiment. A melting
curve analysis for specic amplication control was
performed (from 60 8C to 95 8C) at the end of the
amplication cycles. NTC controls were assumed as

Table 1
Details of the primer sequences of swine pro-inammatory (TNF-a, IL-6) and immune (IFN-g, IL-10) cytokines used for quantitative SYBR Green real-time
PCR amplication. The GAPDH gene was used as endogenous control gene.
Slope

r2

Amplied
product (bp)

98.5

3.36

0.99

118

for 50 -GGCAAAAGGGAAAGAATCCAG-30
rev 50 -CGTTCTGTGACTGCAGCTTATCC-30

95.9

3.43

0.99

87

NM_213948

for 50 -TGGTAGCTCTGGGAAACTGAATG-30
rev 50 -GGCTTTGCGCTGGATCTG-30

99.7

3.32

0.99

79

IL-10
(Royaee et al., 2004)

NM_214041

for 50 -TGAGAACAGCTGCATCCACTTC-30
rev 50 -TCTGGTCCTTCGTTTGAAAGAAA-30

106.7

3.17

0.98

114

GAPDH
(Primer Express)

NM_001206359

for 50 -GGTGAAGGTCGGAGTGAACG-30
rev 50 -GCCAGAGTTAAAAGCAGCCCT-30

100.3

3.32

0.99

70

Target gene

GenBank accession
number

Primer sequence

NM_214022

for 50 -ACTGCACTTCGAGGTTATCGG-30
rev 50 -GGCGACGGGCTTATCTGA-30

IL-6
(Meissonnier et al., 2008)

NM_214399

IFN-g
(Royaee et

TNF-a
(Meissonnier et

al., 2008)

al., 2004)

bp, base pairs; for, forward primer; rev, reverse primer.

qPCR
efciency (%)

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P. Martelli et al. / Veterinary Microbiology 168 (2014) 357364

negative and reliable if the quantication cycle (Cq) was

35.
DD
Data were analyzed according to the 2 Ct method
(Livak and Schmittgen, 2001) in which expression levels of
each cytokine, normalized to the GAPDH cDNA amount
and expressed as relative quantities (RQ).
2.7. Detection of M. hyopneumoniae-specic antibodies in
serum and BAL uid
Collected sera were analyzed for antibodies against M.
hyopneumoniae using a blocking ELISA (INGEZIM M. HYO
COMPAC, INGENASA, Madrid, Spain). To detect the specic
IgG in serum, peroxidase-labeled goat anti-porcine IgG
polyclonal antibodies (Bethyl Laboratories, Texas, TX, USA)
were used and M. hyopneumoniae antigen-coated microplates from IDEXX M. hyo. ELISA (IDEXX, Hoofddorp, The
Netherlands) commercial kit were used, as previously
described by Marchioro et al. (2013). In the BAL uid, IgG,
IgM and IgA antibodies against M. hyopneumoniae were
detected by using peroxidase-labeled goat anti-porcine
IgG, IgM and IgA polyclonal antibodies (Bethyl Laboratories), as previously reported by Marchioro et al. (2013).
The BAL uid was tested undiluted. The Optical Density
(OD) value was measured at 450 nm and the mean OD
value of the serum or BAL uid from the non-vaccinated
animals plus two folds the SD was used as cut-off value to
determine the number of positive samples in the
vaccinated groups for each immunoglobulin. Values equal
to or higher than the cut-off were considered as positive.
2.8. Enumeration of Mycoplasma-specic IFN-g secreting
cells
The number of Mycoplasma-specic IFN-g secreting cells
(SC) in the peripheral blood of pigs was determined by
ELISpot assay as previously described (Martelli et al., 2009;
Ferrari et al., 2011). Briey, PBMC isolated by Histopaque1.0771 gradient were plated at a density of 8  105 cells/
well in cRPMI supplemented with 10% FBS into 96-well
plates (MultiScreen1HTS-IP, Millipore, Billerica, MA, USA)
coated overnight at 4 8C with 10 mg/ml anti-pig IFN-g mAb
(clone P2G10, IgG1, BD Pharmingen, Franklin Lakes, NJ, USA)
and blocked with cRPMI for 2 h at 37 8C. For the in vitro
antigen recall, cells were stimulated with puried bacterins
(inactivated M. hyopneumoniae) at R = 100 bacterins/cells
(50 mg/ml) in cRPMI, for 20 h at 37 8C, 5% CO2. Afterwards,
plates were incubated for 1 h at 37 8C with 0.5 mg/ml antipig IFN-g biotin-labeled mAb (clone P2C11, BD Pharmingen)
and then with 1:750 AP-conjugated anti-biotin mAb in
PBS + 0.5% BSA (Vector Labs, Burlingame, CA, USA). Plates
were incubated for 7 min at room temperature with a BCIP/
NBT solution (BioRad, Hercules, CA, USA) and the reaction
was stopped with distilled water.
The number of Mycoplasma-specic IFN-g SC were
determined by using an AID1 ELISpot Reader and AID1
ELISpot software v. 6.0 (Autoimmun Diagnostika, Strassberg, Germany). As a positive control, 1  105 PBMC/well
were incubated with PHA (10 mg/ml); as a negative
control, 8  105 PBMC were incubated without antigen.
The background values were subtracted from the

respective counts of the stimulated cells and the immune

response was expressed as number of IFN-g SC/106 PBMC.
2.9. Statistical analysis
The statistical analysis was performed with SPSS 21.0
for Windows (IBM1 SPSS1 Statistics, IBM, NY, USA).
Cytokines and immunological parameters (immunoglobulins and number of IFN-g SC) were checked for normality
by analysis of variance (ANOVA). Group was used as xed
factor. For blood samples, data recorded at day 0 were used
as a covariate. When the criteria of normality were not
fullled, a non-parametric KruskalWallis ANOVA was
used. Results were considered signicantly different at
p < 0.05 (two-sided test).
3. Results
3.1. Nested and qPCR for detection of M. hyopneumoniae DNA
in BAL uid
All BAL uid samples from all experimental pigs were
negative for the presence of M. hyopneumoniae DNA.
3.2. Detection of M. hyopneumoniae-specic serum
antibodies
The serological changes of the animals are summarized
in Table 2. Ten piglets from each Groups A and B were
positive for M. hyopneumoniae at 4 and 8 weeks postvaccination, compared to none of the Groups C and D
piglets.
Results for IgG are summarized in Table 3. The OD value
was higher (p < 0.05) in Groups A and B than in Groups C
and D, both at 4 and 8 weeks post-vaccination. The OD
values for M. hyopneumoniae-specic IgG in Group D
vaccinated pigs did not differ over time in comparison to
the controls (Group C).
3.3. M. hyopneumoniae-specic IgG, IgM and IgA in BAL uid
IgG and IgM in BAL uid were not signicantly different
between groups at 4 and 8 weeks post-vaccination (data
not shown). Conversely, M. hyopneumoniae-specic IgA
antibodies were detected at each time point in Groups A
and B, as summarized in Table 4. Thus, signicantly higher
amounts of specic IgA antibodies were observed at 4
weeks in the vaccinated Group B (p < 0.05). At 8 weeks
post-vaccination, Groups A and B had signicantly higher
antibody response compared to control group (C) and
vaccinated D.
3.4. IFN-g, TNF-a, IL-10 and IL-6 gene expression in BAL
uid
There were no statistically signicant differences in
mRNA expression of TNF-a, IFN-g and IL-6 at any time.
During the post-vaccination period (+4 and +8 weeks),
statistically signicant changes of the investigated cytokines among groups were detected for IL-10 only. In fact, in
Group A, an increase of the IL-10 gene expression was

P. Martelli et al. / Veterinary Microbiology 168 (2014) 357364

361

Table 2
Results of ELISA serology against Mycoplasma hyopneumoniae at vaccination (0), 4 and 8 weeks post-vaccination (PV). Values are expressed as average
OD  SD at 450 nm (values with different letters within a column are signicantly different; p < 0.05).
Group

4 weeks PV

8 weeks PV

Number of seropositive overall pigs

Average OD  SD

0/10
1.47  0.47a

10/10
3.673  0.67a

10/10
3.88  0.88a

Number of seropositive overall pigs

Average OD  SD

0/10
1.3  0.3a

10/10
4.0  0.4a

10/10
4.53  0.53a

Number of seropositive overall pigs

Average OD  SD

0/10
1.58  0.58a

0/10
1.93  0.3b

0/10
1.61  0.61b

Number of seropositive overall pigs

Average OD  SD

0/10
1.41  0.41a

0/10
1.44  0.44b

0/10
1.27  0.27b

Table 3
Mycoplasma hyopneumoniae-specic IgG antibody response (mean  SD of the OD670nm) in the serum at vaccination (0), 4 and 8 weeks post-vaccination (PV)
(values with different letters within a column are signicantly different; p < 0.05).
0

4 weeks PV

8 weeks PV

Group
Number of seropositive overall pigs
Average OD  SD

0/10
0.34  0.04

6/10
0.97  0.09a

6/10
1.29  0.70a

Number of seropositive overall pigs

Average OD  SD

0/10
0.23  0.02

7/10
0.98  0.08a

6/10
1.40  0.03a

Number of seropositive overall pigs

Average OD  SD

0/10
0.33  0.03

0/10
0.44  0.09b

0/10
0.57  0.09b

Number of seropositive overall pigs

Average OD  SD

0/10
0.35  0.03

0/10
0.67  0.12b

0/10
0.65  0.08b

evident at 4 weeks post-vaccination without statistically

signicant differences (data not shown). Conversely, IL-10
mRNA expression at 8 weeks post-vaccination was
signicantly higher (p < 0.05) in Group A (intradermally
vaccinated animals) than in non-vaccinated pigs (Group C)
and in the intramuscularly vaccinated animals of Groups B
and D (Fig. 1).

week post-vaccination as compared to the controls (C) and

intramuscularly vaccinated Group D. In particular, at 4
weeks after treatment, vaccinated Groups A and B had the
highest mean values (Fig. 2). At both post-vaccination time
points (+4 and +8 weeks), the number of IFN-g SC in
Groups A and B were signicantly different as compared to
Groups C and D.

3.5. IFN-g secreting cells in peripheral blood

4. Discussion

The cell-mediated immune response evaluated as

number of M. hyopneumoniae-specic IFN-g secreting
cells (SC) in PBMC showed a signicant increase over time
in Groups A and B. In both groups, the course was
comparable. No IFN-g SC were detected at inclusion/
vaccination (time 0) in all groups. The IFN-g response was
evident in vaccinated pigs belonging to Groups A and B at 4

Vaccination with the intradermally administered

vaccine (Group A) induced the production of serum
antibodies in 100% of the animals 4 weeks after treatment,
comparable to the proportion of pigs that seroconverted
after the intramuscular administration of vaccine B.
Conversely, the pigs injected with vaccine D did not
seroconvert and were not different from the negative

Table 4
Mycoplasma hyopneumoniae IgA antibody (mean  SD of OD values at 450 nm) in BAL uid at 4 and 8 weeks post-vaccination (PV) (values with different letters
within a column are signicantly different; p < 0.05).
Group

4 weeks PV

8 weeks PV

Number of seropositive overall pigs

Average OD  SD

nt
nt

6/10
0.45  0.05a

6/10
0.63  0.03a

Number of seropositive overall pigs

Average OD  SD

nt
nt

6/10
0.53  0.03b

8/10
0.66  0.01a

Number of seropositive overall pigs

Average OD  SD

nt
nt

0/10
0.36  0.04a

0/10
0.34  0.05b

Number of seropositive overall pigs

Average OD  SD

nt
nt

0/10
0.36  0.04a

0/10
0.40  0.04b

nt, not tested.

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P. Martelli et al. / Veterinary Microbiology 168 (2014) 357364

Fig. 1. Post-vaccination gene expression levels of TNF-a, IL-6, IFN-g and IL-10 in the BAL uid of vaccinated (A, B and D groups) and non-vaccinated control
(C) pigs. Data are reported as relative (RQ: relative quantication) values normalized to GAPDH gene expression levels (mean values  standard deviation).
The asterisk refers to statistically signicant differences between groups; p < 0.05.

control (Group C). These are supported by the levels of IgG

specic to Mycoplasma hyopneumoniae detected in sera.
The trend of IgG levels increased over time from 4 to 8
weeks post-vaccination with 6070% of positive animals
at 4 and 8 weeks.
Although these results are in agreement with other
studies, (Sibila et al., 2007; Villarreal et al., 2011; Marchioro
et al., 2013), it is well known that the concentration of

Fig. 2. Post-vaccination frequencies of Mycoplasma hyopneumoniaespecic IFN-g secreting cells in PBMC of vaccinated (A, B and D
Groups) and non-vaccinated control (C) pigs. Data are expressed as
number of IFN-g secreting cells (SC) per million of PBMC (IFN-g SC/106
PBMC) and each value represents the mean response  standard deviation
(values with different letters are signicantly different between groups;
p < 0.05).

serum antibodies does not correlate with clinical protection

against Mycoplasma hyopneumoniae (Djordjevic et al., 1997;
Thacker et al., 1998).
Quantication of IFN-g SC via ELISpot in PBMC was
performed to evaluate the systemic M. hyopneumoniaespecic cell-mediated immune response after vaccination.
In fact, cell-mediated immunity seems to be important in
the protection from the effects of M. hyopneumoniae
(Thacker et al., 2000).
These data are in agreement with the previously
discussed results on the humoral compartment of immunity. In particular, 4 weeks after vaccination and at the end
of the study, the intradermally (Group A) and intramuscularly (Group B) vaccinated animals showed signicant
higher numbers of M. hyopneumoniae-specic IFN-g SC
compared to the other groups. Additionally, a positive
correlation between IgG levels in sera and IFN-g SC
number (q = 0.65; p < 0.05) was also detected in the A and
B treated pigs compared to the others.
This demonstrates that vaccines A and B stimulate the
induction of a specic Th1 immune response that is
involved in the protection against the microorganisms.
Another recent study (Marchioro et al., 2013) obtained
comparable results in terms of production of IL-12 and IFNg in vaccinated piglets conrming that the immune
response induced by vaccination involves both humoral
and cellular immunity.
Regarding the local immune response, IgA specic
antibodies from BAL uid were detected at all experimental time points in intradermally vaccinated animals

P. Martelli et al. / Veterinary Microbiology 168 (2014) 357364

and in intramuscularly vaccine B treated pigs. Specic

locally secreted IgA may play a pivotal role in preventing
the adhesion of the microorganism to the epithelium,
sustaining a protective effect during the infection. The
evaluation of the mucosal immune response was further
addressed by the analysis of cytokine gene expression in
BAL uid. In particular, the higher gene expression level of
IL-10 in BAL uid in intradermally vaccinated animals
could explain two biological effects: this may be associated
with a positive regulation of a Th2-mediated immune
response inuencing the IgA local secretion as observed in
this study or, as reported by other authors (Conti et al.,
2003; Vranckx et al., 2012; Villarreal et al., 2011;
Marchioro et al., 2013), with the effect of vaccination on
the prevention of tissue damage caused by the excess of
inammatory cytokines upon M. hyopneumoniae infection.
Taken together, under the conditions of this study,
intradermal vaccination with a bacterin induced a
systemic humoral and cell-mediated immune response
as well as local humoral immunity, which was comparable
to that obtained by the intramuscular administration of an
inactivated, whole cell, adjuvanted vaccine. Even if we
cannot speculate about the efcacy in terms of prevention
of clinical signs and reduction of EP-like lung lesions of the
vaccine showing a poor priming of the cell-mediated and
local (IgA secretion in BAL uid) immune response, this
unexpected result highlights the variability of the efcacy
of commercial vaccines and some cases of failure in
clinical protection under eld conditions (Simionatto
et al., 2013).
All these results and conclusions are worth investigating to further characterize the role of the local and
systemic immunity in relation to clinical protection and
the application of these immune parameters for the
evaluation of M. hyopneumoniae vaccine efcacy under
eld conditions.
Conict of interest
The authors state they have no conict of interest. MSD
Animal Health, manufacturer of the intradermal vaccine,
funded the study.
Acknowledgements
The Ph.D. studies of Dr. Giulia Ferrarini and Dr. Michele
Benetti are funded by a Pre-doctoral grant of the
Department of Veterinary Science, University of Parma
(Italy) in Veterinary Science. The Post-doctoral Research
Fellowship of Dr. Luca Ferrari is funded by a Post-doctoral
grant of the Department of Veterinary Science, University
of Parma (Italy) on Immunity in swine: study of the
efciency of the immune and neuroendocrine response.
References
Conti, P., Kempuraj, D., Kandere, K., Di Giocchino, M., Barbace, R.C.,
Castellani, M.L., et al., 2003. IL-10, an inammatory/inhibitory cytokine, but not always. Immunol. Lett. 86 (April (2)) 123129.
Djordjevic, S.P., Eamens, G.J., Romalis, L.F., Nicholls, P.J., Taylor, V., Chin, J.,
1997. Serum and mucosal antibody responses and protection in
pigs vaccinated against Mycoplasma hyopneumoniae with vaccines

363

containing a denatured membrane antigen pool and adjuvant. Aust.

Vet. J. 75 (July (7)) 504511.
Facci, M.R., Auray, G., Meurens, F., Buchanan, R., van Kessel, J., Gerdts, V.,
2011. Stability of expression of reference genes in porcine peripheral
blood mononuclear and dendritic cells. Vet. Immunol. Immunopathol. 141, 1115.
Ferrari, L., Borghetti, P., Gozio, S., De Angelis, E., Ballotta, L., Smeets, J.,
Blanchaert, A., Martelli, P., 2011. Evaluation of the immune response
induced by intradermal vaccination by using a needle-less system in
comparison with the intramuscular route in conventional pigs. Res.
Vet. Sci. 90, 6471.
Ferrari, L., Martelli, P., Saleri, R., De Angelis, E., Cavalli, V., Bresaola, M.,
Benetti, M., Borghetti, P., 2013. Lymphocyte activation as cytokine
gene expression and secretion is related to the porcine reproductive
and respiratory syndrome virus (PRRSV) isolate after in vitro homologous and heterologous recall of peripheral blood mononuclear cells
(PBMC) from pigs vaccinated and exposed to natural infection. Vet.
Immunol. Immunopathol. 151 (February (34)) 193206.
Fisher, T., Buttner, M., Rziha, H.J., 2006. T helper 1-type cytokine transcription in peripheral blood mononuclear cells of pseudorabies virus
(Suid herpesvirus 1)-primed swine indicates efcient immunization.
Immunology 101, 378387.
Kick, A.R., Tompkins, M.B., Mark, H.J., Routh, P.A., Almond, G.W., 2011.
Evaluation of peripheral lymphocytes after weaning and vaccination
for Mycoplasma hyopneumoniae. Res. Vet. Sci. 91 (December) 6872.
Kiros, T.G., van Kessel, J., Babiuk, L.A., Gerdts, V., 2011. Induction, regulation and physiological role of IL-17 secreting helper T-cells isolated
from PBMC, thymus, and lung lymphocytes of young pigs. Vet.
Immunol. Immunopathol. 144, 448454.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression
data using real-time quantitative PCR and the 2(Delta Delta C(T))
method. Methods 25, 402408.
Maes, D., Segales, J., Meyns, T., Sibila, M., Pieters, M., Haesebrouck, F.,
2008. Control of Mycoplasma hyopneumoniae infections in pigs. Vet.
Microbiol. 126 (January) 297309.
Marchioro, S.B., Maes, D., Flahou, B., Pasmans, F., Del Pozo Sacristan, R.,
Vranckx, K., Melkebeek, V., Cox, E., Wuyts, N., Haesebrouck, F., 2013.
Local and systemic immune responses in pigs intramuscularly
injected with an inactivated Mycoplasma hyopneumoniae vaccine.
Vaccine 31 (February (9)) 13051311.
Marois, C., Dory, D., Fablet, C., Madec, F., Kobisch, M., 2010. Development
of a quantitative real-time TaqMan PCR assay for determination of the
minimal dose of Mycoplasma hyopneumoniae strain 116 required
to induce pneumonia in SPF pigs. J. Appl. Microbiol. 108 (May)
15231533.
Martelli, P., Cordioli, P., Alborali, L.G., Gozio, S., De Angelis, E., Ferrari, L.,
Lombardi, G., Borghetti, P., 2007. Protection and immune response in
pigs intradermally vaccinated against porcine reproductive and
respiratory syndrome (PRRS) and subsequently exposed to a heterologous European (Italian cluster) eld strain. Vaccine 25, 34003408.
Martelli, P., Gozio, S., Ferrari, L., Rosina, S., De Angelis, E., Quintavalla, C.,
Bottarelli, E., Borghetti, P., 2009. Efcacy of a modied live porcine
reproductive and respiratory syndrome virus (PRRSV) vaccine in pigs
naturally exposed to a heterologous European (Italian cluster) eld
strain: clinical protection and cell-mediated immunity. Vaccine 27,
37883799.
Meissonnier, G.M., Pinton, P., Laftte, J., Cossalter, A.M., Gong, Y.Y., Wild,
C.P., Bertin, G., Galtier, P., Oswald, I.P., 2008. Immunotoxicity of
aatoxin B1: impairment of the cell-mediated response to vaccine
antigen and modulation of cytokine expression. Toxicol. Appl. Pharmacol. 231, 142149.
Royaee, A.R., Husmann, R.J., Dawson, H.D., Calzada-Nova, G., Schnitzlein,
W.M., Zuckermann, F.A., Lunney, J.K., 2004. Deciphering the involvement of innate immune factors in the development of the host
response to PRRSV vaccination. Vet. Immunol. Immunopathol. 102,
199216.
Sibila, M., Nofrarias, M., Lopez-Soria, S., Segales, J., Valero, O., Espinal, A.,
et al., 2007. Chronological study of Mycoplasma hyopneumoniae infection, seroconversion and associated lung lesions in vaccinated and
non-vaccinated pigs. Vet. Microbiol. 122 (May (12)) 97107.
Simionatto, S., Marchioro, S.B., Maes, D., Dellagostin, O.A., 2013. Mycoplasma hyopneumoniae: from disease to vaccine development. Vet.
Microbiol. 165 (August (34)) 234242.
Stark, K.D., Nicolet, J., Frey, J., 1998. Detection of Mycoplasma hyopneumoniae by air sampling with a nested PCR assay. Appl. Environ.
Microbiol. 64 (February (2)) 543548.
Tassis, P.D., Papatsiros, V.G., Nell, T., Maes, D., Alexopoulos, C., Kyriakis,
S.C., Tzika, E.D., 2012. Clinical evaluation of intradermal vaccination
against porcine enzootic pneumonia (Mycoplasma hyopneumoniae).
Vet. Rec. 170, 261.

364

P. Martelli et al. / Veterinary Microbiology 168 (2014) 357364

Thacker, E.L., Thacker, B.J., Kuhn, M., Hawkins, P.A., Waters, W.R., 2000.
Evaluation of local and systemic immune responses induced by
intramuscular injection of a Mycoplasma hyopneumoniae bacterin
to pigs. Am. J. Vet. Res. 61 (November (11)) 13841389.
Thacker, E.L., Thacker, B.J., Boettcher, T.B., Jayappa, H., 1998. Comparison
of antibody production, lymphocyte stimulation, and protection
induced by four commercial Mycoplasma hyopneumoniae bacterins.
Swine Health Prod. 6 (May (3)) 107112.

Villarreal, I., Maes, D., Vranckx, K., Calus, D., Pasmans, F., Haesebrouck, F.,
2011. Effect of vaccination of pigs against experimental infection with
high and low virulence Mycoplasma hyopneumoniae strains. Vaccine
29 (February (9)) 17311735.
Vranckx, K., Maes, D., Marchioro, S.B., Villarreal, I., Chiers, K., Pasmans, F.,
et al., 2012. Vaccination reduces macrophage inltration in bronchusassociated lymphoid tissue in pigs infected with a highly virulent
Mycoplasma hyopneumoniae strain. BMC Vet. Res. March, 8.