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Veterinary Microbiology 168 (2014) 357–364

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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 Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna ‘‘B. Ubertini’’ – Sezione diagnostica di Bologna, Via Ficorini,
5 - 40127 Bologna, Italy

b

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) fluid samples were collected at
vaccination (blood only), 4 and 8 weeks post-vaccination. Serum and BAL fluid 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 fluid was performed. Total antibodies
against M. hyopneumoniae and specific IgG were detected in serum of intradermally and
intramuscularly (vaccine B only) vaccinated pigs at 4 and 8 weeks post-vaccination. M.
hyopneumoniae specific IgA were detected in BAL fluid from vaccinated animals (Groups A
and B) but not from controls and animals vaccinated with the bacterin D (p < 0.05).
Significantly higher gene expression of IL-10 was observed in the BAL fluid 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.
ß 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 ß 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

Furthermore. under field conditions (Martelli et al. Kick et al. 2013.1. (2000) evaluated bronchoalveolar lavage (BAL) fluid and peripheral lymphocytes by ELISpot and ELISA. Under the same experimental conditions. Consequently. ID ONCE – MSD AH (formerly Intervet International). randomized. hyopneumoniae and PRRSV were enrolled in this study at weaning (21 days of age). The Netherlands] by using the needle-less IDAL1 vaccinator at a dose of 0. 2008. All animals were obtained from a herd that has been free from M. 2. Group B = intramuscular administration of a commercially available vaccine (vaccine B). hyopneumoniae (Djordjevic et al. As well as the traditional intramuscular (IM) route. inactivated. DNeasy Blood & Tissue kit. It has been estimated that approximately 70% of the industrial pig population around the globe is vaccinated against M. (2011) measured differences between vaccinated and control pigs in the percentage of these cell populations. whole-cell preparations. Belgium). Ferrari et al. Group D = intramuscular administration of a commercially available vaccine (vaccine D). after the approval of the Ethical Committee for Animal Experiments of Parma University (approval number 21/12). Blood and bronchoalveolar lavage (BAL) fluid were collected at vaccination (blood only) and after 4 and 8 weeks. hyopneumoniae antibodies by using a IDEXX M.. For detection of M. Thacker et al.. Blood was processed for serological investigations and peripheral blood mononuclear cells (PBMC) isolation. commercial test kit (S/P ratio < 0. standing position by using a cannula via the mouth as commonly performed for diagnostic purposes. Marchioro et al. consisting of adjuvanted. the piglets were injected as follows: Group A = intradermal (ID) administration of a commercial vaccine [Porcilis1 M. before the enrollment. although no direct correlation has been found between the induction of antibodies and protection against M.3. hyo. but also to increase the practical advantages of control programs. 2. Forty cross-bred healthy piglets free of M. not only to improve the efficiency of vaccination in terms of the immune response. Tassis et al... hyopneumoniae infection. 2008). the lung lesions.2 ml (vaccine A). 2013). Martelli et al. / Veterinary Microbiology 168 (2014) 357–364 losses to the pig industry (Maes et al. 2011. the animals were bled and serologically tested for negativity to M. (2010) in order to avoid false negative results. This percentage is increasing in those countries where pig production is switching rapidly from the traditional backyard system to the intensive commercial herds. The commercial vaccines induce specific antibodies in serum. 1997). the animals were randomly allocated to four different groups (10 piglets each) and individually identified by a numbered ear tag. Experimental design This blind. 2009. Some previous studies considered the importance of the route and the method of vaccine administration for inducing optimum levels of protective immunity. both a nested and a qPCR were performed as described by Stark et al. at 15 days of age.358 P. but the effects of vaccination on mucosal and cell-mediated immunity have been evaluated in only few studies. In contrast. Isolation of PBMC from blood Porcine peripheral blood mononuclear cells (PBMC) were isolated from 4 ml to 5 ml of pig blood samples .4). controlled study was performed in the isolation facilities of the Department of Veterinary Science of Parma University. and demonstrated that a bacterin-based vaccine induced mucosal and systemic cell-mediated immune response. hyopneumoniae infections (Maes et al. All vaccines administered in this study were licensed as a one dose. (1998) and Marois et al. The currently available vaccines. Some studies suggest that mucosal and cellular immune responses might play an important role in the control of this disease (Thacker et al. Vaccination. improvement of housing conditions and management procedures are currently applied for the control of EP (Maes et al. hyo. 2000). Moreover. 2008)..2. The dose of the vaccines B and D is intentionally omitted in order to avoid any possibility to identify the commercially available products. 2012). 2007. The study is aimed at evaluating the systemic and the mucosal immune responses induced by a commercial inactivated whole-cell M. Materials and methods 2. hyopneumoniae. The variability of the results published on this topic support that the exact mechanism of protection induced by vaccination is not completely understood. Nested and quantitative PCR (qPCR) for detection of M. new routes of vaccine administration have also been studied. moved to the isolation facilities and housed in the same room. 2. BAL fluid was processed for antibody detection and centrifuged for RNA extraction and subsequent cytokine gene expression quantification on the cellular pellet. hyopneumoniae DNA DNA was extracted from the BAL fluid with the QIAGEN protocol (Qiagen. hyopneumoniae and PRRSV based on repeated serological testing and the absence of EP lung lesions at slaughterhouse.. have been demonstrated to be effective in reducing the clinical symptoms.. At 28 days of age. Upon arrival. and the performance losses associated with M. 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. BAL fluid was collected in restrained. the study is not intended to provide any information about vaccination schemes. 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. Group C = intramuscular administration of the adjuvant only (2 ml of X-solve. the adjuvant of vaccine A). Boxmeer.. hyopneumoniae DNA.. the vaccination failed to induce changes in peripheral CD4+ and CD8+ cell.

99 79 IL-10 (Royaee et al. 50 mM 2bmercaptoethanol (Sigma) and 100 U/ml penicillin G. 2. The GAPDH gene was used as endogenous control gene. RNA integrity and quality were assessed by using an Agilent Bioanalyzer 2100 and RNA 6000 Labchip kit (Agilent Technologies.. 100 mM non essential amino-acids. USA) supplemented with 10% FBS. 2. MO) according to the manufacturer’s instructions..9 3.7 3. 2008) NM_214399 IFN-g (Royaee et TNF-a (Meissonnier et al. Total RNA extraction and quantification was performed within 1 week after sample collection and storage. 2. A melting curve analysis for specific amplification control was performed (from 60 8C to 95 8C) at the end of the amplification cycles. v/v. Quantification 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. rev. The cDNA (20 ng/20 mL) was amplified in triplicate with Fast SYBR1 Green-1 Master Mix (Applied Biosystems) and specific sets of primers optimized at 150 nM for IL-6 and 300 nM for the other cytokines. RNA extraction was performed by using TRI-reagent (Ambion-Life Technologies. Fluorescence due to SYBR1 Green-1 incorporation was acquired at the end of the extension step. 2. Facci et al. 2004)] as endogenous control according to minimal intra-/inter-assay variation and to Fisher et al.5.32 0. Total RNA (1 mg/20 mL) was 359 reverse-transcripted using a High-capacity cDNA Reverse Transcription kit (Applied Biosystems. IL-6) and adaptive immune (IFN-g. Meissonnier et al.. Carlsbad. The reference gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was selected among other tested reference genes [e.. The RT was performed by using a StepOne thermocycler (Applied Biosystems.. 2008) or were designed by using Primer Express1 software package (Applied Biosystems) and purchased from Eurofins MWG Operon (Ebersberg.1) and. 2004) NM_214041 for 50 -TGAGAACAGCTGCATCCACTTC-30 rev 50 -TCTGGTCCTTCGTTTGAAAGAAA-30 106. according to the manufacturer’s instructions. 2011). 2 mM Lglutamine. Cells were counted by inverted optical microscope and concentration was assessed before being used for IFN-g ELISpot assay. USA) according to the manufacturer’s instructions. blood samples were taken and then stratified on Histopaque-10771 solution (1:1. (2011) and Ferrari et al. NTC controls were assumed as Table 1 Details of the primer sequences of swine pro-inflammatory (TNF-a. CA. After isolation. 2008) al. for. ribosome protein (RP)L-19 (Kiros et al. StepOne software v. purity and concentration were assessed by UV-spectrophotometry at 260/280 and 260 nm respectively (GeneQuant Pro.99 87 NM_213948 for 50 -TGGTAGCTCTGGGAAACTGAATG-30 rev 50 -GGCTTTGCGCTGGATCTG-30 99. All cDNA samples were stored at 20 8C until PCR was performed. CA). 100 mg/ml streptomycin and 0. Slope r2 Amplified product (bp) 98. Germany). Total RNA extraction Gene expression levels of relevant pro-inflammatory (TNF-a.3 3. UK). under the following thermal conditions: 10 min at 25 8C. CA. Reverse-transcription (RT) to All RNA samples were DNAse-treated (Sigma) prior cDNA synthesis.32 0.6. Louis.. NY. Foster City. 2. Martelli et al. b-actin (Meissonnier et al. 2004) bp. Amersham Pharmacia Biotech-GE Healthcare Life Sciences.4. USA)..1).43 0. No-template (NTC) and noRT controls were included in each experiment. Grand Island..7 3. The primers used are based on published sequences (Fisher et al.99 70 Target gene GenBank accession number Primer sequence NM_214022 for 50 -ACTGCACTTCGAGGTTATCGG-30 rev 50 -GGCGACGGGCTTATCTGA-30 IL-6 (Meissonnier et al. RNA samples were stored at 80 8C until reverse-transcription (RT). 120 min at 37 8C followed by 5 min at 85 8C. 2006.. 2008).98 114 GAPDH (Primer Express) NM_001206359 for 50 -GGTGAAGGTCGGAGTGAACG-30 rev 50 -GCCAGAGTTAAAAGCAGCCCT-30 100. In all samples. Such solution is referred as complete RPMI-1640 (cRPMI).17 0. 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. Briefly.g.P. IL-10) cytokines were determined in swine PBMC. Details of each primer set for detection of cytokine gene expression are reported in Table 1. Little Chalfont. purified PBMC were washed with sterile PBS (Sigma) supplemented with 1% fetal bovine serum (FBS) and resuspended in RPMI-1640 (Gibco. Santa Clara. qPCR efficiency (%) .99 118 for 50 -GGCAAAAGGGAAAGAATCCAG-30 rev 50 -CGTTCTGTGACTGCAGCTTATCC-30 95.. base pairs.36 0. PBMC were >98% viable as confirmed by Trypan blue (Sigma) exclusion. / Veterinary Microbiology 168 (2014) 357–364 collected in lithium–heparin as anticoagulant by density gradient in Histopaque-10771solution (Sigma. St. 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. (2006). IL-6) and immune (IFN-g. forward primer.25 mg/ml amphotericin B.5 3. reverse primer. RPL-32 (Royaee et al. (2011). Sigma) and centrifuged at 400  g for 30 min. StepOne software v. IL-10) cytokines used for quantitative SYBR Green real-time PCR amplification.

Billerica. Germany). 3. For blood samples. as a negative control. as summarized in Table 4. Millipore. Madrid. hyopneumoniae were detected by using peroxidase-labeled goat anti-porcine IgG. Conversely. hyopneumoniae using a blocking ELISA (INGEZIM M. CA. Burlingame. USA) were used and M. hyopneumoniae-specific serum antibodies The serological changes of the animals are summarized in Table 2. significantly higher amounts of specific IgA antibodies were observed at 4 weeks in the vaccinated Group B (p < 0. hyopneumoniae-specific antibodies in serum and BAL fluid Collected sera were analyzed for antibodies against M. statistically significant changes of the investigated cytokines among groups were detected for IL-10 only. IBM.. At 8 weeks post-vaccination. DD Data were analyzed according to the 2 Ct method (Livak and Schmittgen. Martelli et al. Strassberg.5% BSA (Vector Labs. HYO COMPAC. IL-10 and IL-6 gene expression in BAL fluid There were no statistically significant differences in mRNA expression of TNF-a. 2001) in which expression levels of each cytokine. both at 4 and 8 weeks post-vaccination. hyopneumoniae-specific IgA antibodies were detected at each time point in Groups A and B. MA.05). Texas. 2. Detection of M. 6. Plates were incubated for 7 min at room temperature with a BCIP/ NBT solution (BioRad. Ferrari et al. As a positive control. INGENASA. cells were stimulated with purified bacterins (inactivated M. Detection of M.9. data recorded at day 0 were used as a covariate.8. Statistical analysis The statistical analysis was performed with SPSS 21. 2.0 for Windows (IBM1 SPSS1 Statistics. NY. Cytokines and immunological parameters (immunoglobulins and number of IFN-g SC) were checked for normality by analysis of variance (ANOVA). Hercules. USA) coated overnight at 4 8C with 10 mg/ml anti-pig IFN-g mAb (clone P2G10. 2009. hyopneumoniae at 4 and 8 weeks postvaccination. 3. (2013).4. normalized to the GAPDH cDNA amount and expressed as relative quantities (RQ). Groups A and B had significantly higher antibody response compared to control group (C) and vaccinated D.05 (two-sided test).0 (Autoimmun Diagnostika. IgG. During the post-vaccination period (+4 and +8 weeks). in Group A. When the criteria of normality were not fulfilled. The OD values for M. IFN-g and IL-6 at any time. hyo. 3.2. an increase of the IL-10 gene expression was . 3. / Veterinary Microbiology 168 (2014) 357–364 negative and reliable if the quantification cycle (Cq) was 35. IgM and IgA polyclonal antibodies (Bethyl Laboratories). Briefly. TX. as previously reported by Marchioro et al. Results 3. 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. IgG1. The Optical Density (OD) value was measured at 450 nm and the mean OD value of the serum or BAL fluid 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. NJ. hyopneumoniae) at R = 100 bacterins/cells (50 mg/ml) in cRPMI. For the in vitro antigen recall. CA. as previously described by Marchioro et al. Group was used as fixed factor. USA). ELISA (IDEXX. To detect the specific IgG in serum.. hyopneumoniae antigen-coated microplates from IDEXX M. In fact. M. Values equal to or higher than the cut-off were considered as positive. 1  105 PBMC/well were incubated with PHA (10 mg/ml). TNF-a. 8  105 PBMC were incubated without antigen. 5% CO2. 2011). Enumeration of Mycoplasma-specific IFN-g secreting cells The number of Mycoplasma-specific IFN-g secreting cells (SC) in the peripheral blood of pigs was determined by ELISpot assay as previously described (Martelli et al. 2. hyopneumoniae-specific IgG in Group D vaccinated pigs did not differ over time in comparison to the controls (Group C). 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. Spain). hyopneumoniae-specific IgG. BD Pharmingen) and then with 1:750 AP-conjugated anti-biotin mAb in PBS + 0. USA) and the reaction was stopped with distilled water.7. plates were incubated for 1 h at 37 8C with 0.05) in Groups A and B than in Groups C and D. The number of Mycoplasma-specific IFN-g SC were determined by using an AID1 ELISpot Reader and AID1 ELISpot software v. M. Nested and qPCR for detection of M. USA) and blocked with cRPMI for 2 h at 37 8C. The Netherlands) commercial kit were used. a non-parametric Kruskal–Wallis ANOVA was used. Results for IgG are summarized in Table 3. for 20 h at 37 8C. Thus. (2013). Results were considered significantly different at p < 0. USA).3. compared to none of the Groups C and D piglets.1. hyopneumoniae DNA. IgM and IgA antibodies against M. BD Pharmingen. Hoofddorp. peroxidase-labeled goat anti-porcine IgG polyclonal antibodies (Bethyl Laboratories. Ten piglets from each Groups A and B were positive for M. IFN-g.360 P. Franklin Lakes. hyopneumoniae DNA in BAL fluid All BAL fluid samples from all experimental pigs were negative for the presence of M. In the BAL fluid.5 mg/ml antipig IFN-g biotin-labeled mAb (clone P2C11. Afterwards. The BAL fluid was tested undiluted. IgM and IgA in BAL fluid IgG and IgM in BAL fluid were not significantly different between groups at 4 and 8 weeks post-vaccination (data not shown). The OD value was higher (p < 0.

44b 0/10 1.05).05b D Number of seropositive overall pigs Average OD  SD nt nt 0/10 0.47  0.93  0. Group 0 4 weeks PV 8 weeks PV A Number of seropositive overall pigs Average OD  SD 0/10 1.03 0/10 0.09b 0/10 0.45  0. p < 0.34  0.08b evident at 4 weeks post-vaccination without statistically significant differences (data not shown).34  0.44  0.47a 10/10 3.65  0.04 6/10 0.33  0.01a C Number of seropositive overall pigs Average OD  SD nt nt 0/10 0. the number of IFN-g SC in Groups A and B were significantly different as compared to Groups C and D.61b D Number of seropositive overall pigs Average OD  SD 0/10 1.58  0.67a 10/10 3.97  0.35  0.02 7/10 0.3  0. comparable to the proportion of pigs that seroconverted after the intramuscular administration of vaccine B. IL-10 mRNA expression at 8 weeks post-vaccination was significantly higher (p < 0.05a 6/10 0. 3. Group 0 4 weeks PV 8 weeks PV A Number of seropositive overall pigs Average OD  SD nt nt 6/10 0.66  0.98  0. 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 fluid at 4 and 8 weeks post-vaccination (PV) (values with different letters within a column are significantly different. the course was comparable.70a B Number of seropositive overall pigs Average OD  SD 0/10 0.40  0. Discussion The cell-mediated immune response evaluated as number of M.03a C Number of seropositive overall pigs Average OD  SD 0/10 0.41a 0/10 1.44  0.09b D Number of seropositive overall pigs Average OD  SD 0/10 0.53  0.41  0.04b nt.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.05). vaccinated Groups A and B had the highest mean values (Fig.29  0.12b 0/10 0.04a 0/10 0. at 4 weeks after treatment.63  0.03b 8/10 0.88a B Number of seropositive overall pigs Average OD  SD 0/10 1.04a 0/10 0. week post-vaccination as compared to the controls (C) and intramuscularly vaccinated Group D.4a 10/10 4.57  0. 0 4 weeks PV 8 weeks PV A Group Number of seropositive overall pigs Average OD  SD 0/10 0.3a 10/10 4.27b Table 3 Mycoplasma hyopneumoniae-specific IgG antibody response (mean  SD of the OD670nm) in the serum at vaccination (0).3b 0/10 1. 4 and 8 weeks post-vaccination (PV).23  0. Conversely.61  0. Values are expressed as average OD  SD at 450 nm (values with different letters within a column are significantly different.27  0.53a C Number of seropositive overall pigs Average OD  SD 0/10 1.53  0.05). hyopneumoniae-specific IFN-g secreting cells (SC) in PBMC showed a significant increase over time in Groups A and B. . / Veterinary Microbiology 168 (2014) 357–364 361 Table 2 Results of ELISA serology against Mycoplasma hyopneumoniae at vaccination (0). In both groups. 1). At both post-vaccination time points (+4 and +8 weeks).P.36  0. not tested.88  0.36  0. 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.0  0. Martelli et al. p < 0. No IFN-g SC were detected at inclusion/ vaccination (time 0) in all groups.03a B Number of seropositive overall pigs Average OD  SD nt nt 6/10 0.673  0.40  0.67  0. In particular. p < 0.5.09a 6/10 1. IFN-g secreting cells in peripheral blood 4.08a 6/10 1. 4 and 8 weeks post-vaccination (PV) (values with different letters within a column are significantly different.58a 0/10 1. 2).03 0/10 0. Conversely.

a positive correlation between IgG levels in sera and IFN-g SC number (q = 0.05. control (Group C).05).. This demonstrates that vaccines A and B stimulate the induction of a specific Th1 immune response that is involved in the protection against the microorganisms. These are supported by the levels of IgG specific to Mycoplasma hyopneumoniae detected in sera.362 P. it is well known that the concentration of Fig. B and D Groups) and non-vaccinated control (C) pigs.65.. 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 significantly different between groups. 2013). 4 weeks after vaccination and at the end of the study. hyopneumoniae (Thacker et al. Another recent study (Marchioro et al. 2013) obtained comparable results in terms of production of IL-12 and IFNg in vaccinated piglets confirming that the immune response induced by vaccination involves both humoral and cellular immunity. Villarreal et al. These data are in agreement with the previously discussed results on the humoral compartment of immunity. Post-vaccination frequencies of Mycoplasma hyopneumoniaespecific IFN-g secreting cells in PBMC of vaccinated (A. Although these results are in agreement with other studies. IFN-g and IL-10 in the BAL fluid of vaccinated (A.. the intradermally (Group A) and intramuscularly (Group B) vaccinated animals showed significant higher numbers of M. Marchioro et al. In particular. hyopneumoniaespecific cell-mediated immune response after vaccination. In fact.05) was also detected in the A and B treated pigs compared to the others. Data are reported as relative (RQ: relative quantification) values normalized to GAPDH gene expression levels (mean values  standard deviation). Quantification of IFN-g SC via ELISpot in PBMC was performed to evaluate the systemic M. Martelli et al. Post-vaccination gene expression levels of TNF-a. Thacker et al.. hyopneumoniae-specific IFN-g SC compared to the other groups.. 2000). 1997.. p < 0. / Veterinary Microbiology 168 (2014) 357–364 Fig. 2007.. (Sibila et al. B and D groups) and non-vaccinated control (C) pigs. p < 0. 2011. The trend of IgG levels increased over time from 4 to 8 weeks post-vaccination with 60–70% of positive animals at 4 and 8 weeks. The asterisk refers to statistically significant differences between groups. IL-6. IgA specific antibodies from BAL fluid were detected at all experimental time points in intradermally vaccinated animals . p < 0. 1998). cell-mediated immunity seems to be important in the protection from the effects of M. 2. serum antibodies does not correlate with clinical protection against Mycoplasma hyopneumoniae (Djordjevic et al. 1. Additionally. Regarding the local immune response.

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Stability of expression of reference genes in porcine peripheral blood mononuclear and dendritic cells. K. V. studies of Dr. Bottarelli.. Evaluation of the immune response induced by intradermal vaccination by using a needle-less system in comparison with the intramuscular route in conventional pigs. but not always. this unexpected result highlights the variability of the efficacy of commercial vaccines and some cases of failure in clinical protection under field conditions (Simionatto et al. B. Flahou. Luca Ferrari is funded by a Post-doctoral grant of the Department of Veterinary Science. P. Vet. H.. F. Microbiol.J. Meyns. G. Taken together.. Pasmans. 2007. intradermal vaccination with a bacterin induced a systemic humoral and cell-mediated immune response as well as local humoral immunity.G. Eamens.. 142–149. 1997. Schnitzlein. J. Maes. F. Laffitte. 90. 2009.Y.. P.. Even if we cannot speculate about the efficacy 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 fluid) immune response. S. Rec. Zuckermann.... Melkebeek.. S.. Nofrarias. Maes.D.. Michele Benetti are funded by a Pre-doctoral grant of the Department of Veterinary Science. Sci.. Kick. D.. Borghetti. Papatsiros. Immunopathol. Specific locally secreted IgA may play a pivotal role in preventing the adhesion of the microorganism to the epithelium. Clinical evaluation of intradermal vaccination against porcine enzootic pneumonia (Mycoplasma hyopneumoniae). E. R. Martelli et al. Bresaola. Analysis of relative gene expression data using real-time quantitative PCR and the 2(Delta Delta C(T)) method. E.. Borghetti. Microbiol. 141.. J. Immunology 101. 2013. E. Valero.M. 75 (July (7)) 504–511. 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Vet. A. / Veterinary Microbiology 168 (2014) 357–364 and in intramuscularly vaccine B treated pigs. P.. Pharmacol. Lunney..... Maes. Res. Vet. Microbiol... M. O. G. F. C. Schmittgen..P.. Aust.. 378–387. sustaining a protective effect during the infection. 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. 170. as reported by other authors (Conti et al.. Sci. 64–71. De Angelis.. van Kessel.. Benetti. Meissonnier. R. Almond.. Nicholls. S. Gozio.J. 2013. the higher gene expression level of IL-10 in BAL fluid in intradermally vaccinated animals could explain two biological effects: this may be associated with a positive regulation of a Th2-mediated immune response influencing the IgA local secretion as observed in this study or. Simionatto. 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