New insights in cellular immune response in colostrum-deprived pigs after immunization with subunit and commercial vaccines against Glässer’s disease

Cellular Immunology 277 (2012) 74–82

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New insights in cellular immune response in colostrum-deprived pigs after immunization with subunit and commercial vaccines against Glässer’s disease R. Frandoloso, S. Martínez-Martínez, S. Yubero, E.F. Rodríguez-Ferri, C.B. Gutiérrez-Martín ⇑ Microbiology and Immunology Section, Department of Animal Health, Faculty of Veterinary Medicine, University of León, 24007 León, Spain

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Article history: Received 18 November 2011 Accepted 15 May 2012 Available online 23 May 2012 Keywords: Haemophilus parasuis Glässer’s disease Vaccines Cell response

a b s t r a c t Four groups of colostrum-deprived pigs were immunized with Porcilis GlässerÒ (PG) or with subunit vaccines developed by us (rTbpA, NPAPTM or NPAPTCp) against Glässer’s disease, and they were challenged with 3  108 CFU of Haemophilus parasuis. A strong reduction in CD3+cdTCR+ cells was seen in non-immunized control and scarcely protected (rTbpA) groups, suggesting that these cells could represent a target of H. parasuis infection. A significant increase in CD172a+CD163+ cells was detected in all groups but PG, while a reduction in SLAIIDR+ molecules expression was observed after challenge in control animals. Significant increases in CD3e+CD8a+CD8b+ and B cells were detected respectively in control and NPAPT groups, and in scarcely (rTbpA) and well-protected (NPAPTM and NPAPTCp) groups. Finally, a greater response in CD4+CD8a cells was observed in NPAPTCp compared to NPAPTM and PG groups. These results state the potential of NPAPT antigen for developing effective vaccines against Glässer’s disease. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Fibrinous polyserositis, arthritis and meningitis (Glässer’s disease) caused by Haemophilus parasuis has become increasingly significant worldwide as a consequence of changes imposed in swine production methods [1]. This disease is also characterized by septicaemia or pneumonia [2]. To date, fifteen serovars of different virulence have been identified using immunodiffusion test [3], although several non-typable isolates are frequently recovered, reching up to 25% of them in some countries [4]. Control of Glässer’s disease has been relied on vaccination. Commercial or autogenous bacterins have usually afforded a strong protection against challenge with the homologous serovar [5,6], but more inconsistent results have been reported in crossprotection studies [1,7,8]. In addition, several subunit vaccines have been developed. A purified recombinant outer membrane protein (Omp) A showed a good antigenicity [9], and four other Omps (PalA, Omp2, D15 and HPS 06257) also yielded a strong potential to prevent Glässer’s disease [10]. More recently, some studies have proposed three ABC-type transporters (OppA, YfeA and PlpA) and one curli protein assembly (CsgG) [11], several recombinant virulent associated trimeric autotransporters [12], three Omps (SmpA, YgiW and FOG) [13], or a cell wall surface protein (6-phosphogluconate dehydrogenase) [14] as suitable candidates in future formulations against H. parasuis. ⇑ Corresponding author. Fax: +34 987 291 304. E-mail address: [email protected] (C.B. Gutiérrez-Martín). 0008-8749/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2012.05.010

However, few studies about the cell response developed after natural or experimental infection with H. parasuis have been reported. In this respect, only leukopenia [15], decrease in CD25+ lymphocytes [16], or increase in monocytes, granulocytes and aIgM+ cells, along with a concomitant decrease in CD3+ cells [17], have been described. To further understand the cellular immune response caused by this organism, the aim of this study was to investigate the changes in peripheral mononuclear cells (PBMC) in colostrum-deprived pigs immunized with subunit and commercial vaccines and then challenged with serovar 5, a highly virulent serovar of worldwide prevalence [2]. 2. Materials and methods 2.1. Vaccine formulations and immunization Four vaccines were compared. rTbpA and NPAPTM vaccines contained respectively rTbpA or NPAPT antigens adjuvanted with Montanide IMS 2215 VG PR (Seppic, Inc., Paris, France). NPAPTCp vaccine also contained NPAPT antigen but potentiated with neuraminidase from Clostridium perfringens (type VI). PG vaccine consisted of a commercially available vaccine (Porcilis GlässerÒ, Intervet, Spain), composed of inactivated H. parasuis cells. A total of 28 colostrum-deprived piglets were randomly assigned to one control (CTL) or one of four vaccinated groups. The rTbpA and NPAPTM groups (n = 6 each) received rTbpA or NPAPTM vaccines, respectively, by intramuscular injection; the NPAPTCp group (n = 6) received NPAPTCp vaccine intratracheally, and the PG group

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(n = 6) received PG vaccine intramuscularly at 28 and 42 days of age, as recommended by the manufacturer. All groups were challenged intratracheally with a lethal dose (3  108 CFU) of H. parasuis Nagasaki strain. The details have been already described [18]. 2.2. Blood collection and peripheral blood mononuclear cell (PBMC) isolation Blood samples were collected by aseptic venipuncture from the jugular vein into commercial tubes containing ethylenediamine tetra-acetic acid as anticoagulant, using a 21-g needle and a holder (VacutainerÒ, Becton Dickinson, USA) at the challenge day, and once a day up to 7th day post-challenge (dpc). Samples were processed immediately for PBMC isolation by means of a gradient centrifugation using lymphocyte separation medium (PAA Laboratories, Austria), as previously described [19]. Cell viability was evaluated by the trypan blue exclusion test. 2.3. Immunostaining For immunostaining, 50 ll of each sample (5  105 cells) diluted in FACS buffer (PBS containing 0.1% of BSA and 0.01% sodium azide) were used for each antibody labeling. Double or triple staining was performed using the monoclonal antibodies (mAbs) described in Table 1, which were combined for the recognition of the following surface molecules: CD3e, CD4 and CD8a; cdTCR and CD3e; aIgM and CD21; SLA-II and CD21; CD4 and CD4 naïve; CD172a and CD163; CD172a and SLA-II; CD8a, CD8b and CD3e. Most of these mAbs were generous gifts from Dr. Domínguez Juncal (INIA, Madrid, Spain). PBMCs were incubated in a V-shaped 96-well microplate (NUNC, USA) with primary mAbs for 20 min at 4 °C, washed in FACS buffer thrice. Non-specific epitopes were blocked using 5% swine normal serum for 10 min at 4 °C. This step was not carried out for the B cell staining (aIgM) because the 5C9 clone has affinity for the IgM a heavy-chain. After the blocking, the cells were incubated for 20 min at 4 °C with biotin or fluorescein isothiocyanateconjugated rat anti-mouse IgG2b (R12-3 clone), biotin-conjugated rat anti-mouse IgG2a (R19-15 clone), biotin-conjugated rat antimouse IgM (R6-60.2 clone), phycoerythrin-conjugated mouse anti-rat IgG2a (RG7/1.30 clone), or fluorescein isothiocyanate-conjugated rat anti-mouse IgG1 (A85-1 clone) (BD Pharmingen, USA). When the isotype of the primary mAb was the same as that used for labeling the secondary antibody, a simple blocking of Fab region with a 5% normal mouse serum diluted in FACS was carried out. After washing with FACS buffer, biotinilated antibodies were detected incubating cells with streptavidin-PerCp (peridinin–chlorophyll–protein complex) or APC (allophycocyanin) (BD Pharmingen, USA). Finally, the cells were resuspended in 400 ll of FACS buffer, transferred into a round-bottom tube (BD Falcon), and

Table 1 Monoclonal antibodies (mAbs) directed against porcine surface leukocyte antigens, used for double or triple staining (FACS analysis). Antibody

Clone

Isotype

Producer

Reference

CD3e CD4 CD8a TCRcd aIgM CD21 SLAII CD4 naïve CD172a CD163 CD8b

BB23-8E6-8C8 74-12-4 76-2-11 MAC320 5C9 Bly-4 1F12 2E3 BA1C11 2A10 PG164A

IgG2b IgG2b IgG2a IgG2a IgG1 IgG1 IgG2b IgM IgG1 IgG2a IgG2a

BD Pharmingen INIA BD Pharmingen BD Pharmingen INIA BD Pharmingen INIA INIA INIA INIA VMRD

[49] [50] [50] [51] [41] [42] [52] [53] [54] [52] [55]

75

propidium iodine (Sigma, USA) or 7-amino actinomycin D (7AAD) (BD Pharmingen, USA) were added for staining DNA of the death and damaged cells and their exclusion from analysis. A total of 20,000 events were acquired in a FACScan flow cytometer (Becton Dickinson, United Kingdom) operated by the CELLQuest™ software. The WinMDI 2.9 software was used for off-line data processing. The lymphocyte and monocyte populations were identified using the forward-scatter (FSC) versus side-scatter (SSC) dot plot, as it is represented in the Fig. 1a, and the results are shown as the total percent of each subpopulation inside their respective regions. Different lymphocyte subsets were analyzed using the SPSS statistical program, version 16.0 (Inc., Chicago, IL, USA). ANOVA and Tukey’s multiple comparison tests were used for comparing different groups at 1st and 2nd dpc in CTL group; at 1st, 2nd and 3rd dpc in rTbpA group, and until 7th dpc in surviving pigs. The GraphPad Prism statistical program, version 5.0 (San Diego, CA, USA) was used for drawing the figures. A value of P < 0.05 was considered significant.

3. Results 3.1. Characterization of surface molecule expression in PBMC subsets in control group Cell subsets implied in innate and acquired response were severely affected by challenge in the pigs belonging to CTL group (Table 2). Concerning the former, a significant decrease was observed for CD3+cdTCR+ cells at 1st and 2nd dpc compared to challenge, with a 31.8% (P = 0.02) and 74.3% (P = 0.0003) reduction in this subpopulation (Fig. 2). Contrarily, CD172a+CD163+ monocytes increased significantly at 1st (55%, P < 0.005) and 2nd dpc (168.4%, P < 0.0001) compared to the values obtained at challenge (Fig. 3). Surprisingly, contrary to the increase in CD172a+CD163+ monocytes, the expression of histocompatibility molecules (SLAIIDR+) fell progressively in CD172a+ monocytes, to more than 50% at 2nd dpc (P = 0.002) (Fig. 4). With regard to acquired immune response, a significant increase was seen in the number of CD4 CD8a+ lymphocytes at 1st dpc compared to the day before (P = 0.01). This rise was directly produced by a significant increase (P < 0.0001) in the rate of cytotoxic T cells, with CD3e+CD8a+CD8b+ phenotype at 1st dpc (Fig. 1i,ii and Table 2). The number of cells in both subpopulations decreased at 2nd dpc (when all animals in this group were found died), reaching values similar to those at challenge day. On the other hand, no significant differences were observed in CD4+CD8a+ and CD4+CD8a subsets; however, the rate of CD4+CD4naïve+ subpopulation decreased significantly at 1st and 2nd dpc (P = 0.02 and P = 0.01, respectively). The number of CD21+aIgM+ cells did not change significantly throughout the study, and no noticeable variations in the expression of SLAIIDR+ molecules in the surface of these lymphocytes were seen after challenge (Table 2).

3.2. Characterization of surface molecule expression in PBMC subsets in rTbpA group The values obtained are shown in Table 2. Similar results to those in CTL group were measured for CD3+cdTCR+ cells: their rates decreased significantly from 1st dpc until values about the half of those of challenge at 3rd dpc (P < 0.05). As for CTL group, CD172a+CD163+ monocytes rose significantly (more than four times) at 1st dpc (P < 0.0001), but from this time their number fell until the end of the study; even so, these values were twice higher than those found at challenge. A significant decrease (P < 0.001) of

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Fig. 1. Triple color analysis of swine peripheral cytotoxic lymphocytes (CD3e+CD8a+CD8b+) from CTL group (representative dot plots from swine nr 13). The lymphocytes were first stained with purified mAb PG164A, followed by biotinylated rat anti-mouse IgG2a, and then streptavidin–APC. Then, the cells were stained with FITC-conjugated CD3e and PE-conjugated mAb 76-2-11. Non-viable lymphocytes were excluded by staining with 7-AAD. (a) The viable PBMC were separated in lymphocyte (L) and monocyte (M) regions, using the forward-scatter (FSC) versus side-scatter (SSC) dot plot. (b) Histogram of the expression of CD3e+ cells from region L. Gate in CD3e+ includes the cytotoxic lymphocyte subset. (i and ii) Cells gated in CD3e+ lymphocytes. Percent of the CD3e+CD8a+CD8b+ subset (i) before challenge: 11.9%, (ii) 1st dpc: 19.7%.

Table 2 Mean ± standard deviation of peripheral blood mononuclear cell (PBMC) subsets in CTL and rTbpA groups. Experimental group and PBMC subset

CTL group cdTCR+ CD172a+CD163+ CD172a+SLAIIDR+ CD4 CD8a+ CD4+CD8a CD4+CD8+ CD4+CD4naïve+ CD8ab+ CD21+aIgM+ CD21+SLAIIDR+

Time Challenge

1st Day after challenge

2nd Day after challenge

39.3 ± 4.6 24.4 ± 3.2 32.5 ± 4.8 28.8 ± 5.3 5.5 ± 1.2 5.6 ± 1.7 4.3 ± 1.4 10.0 ± 1.9 12.0 ± 3.5 3.1 ± 2.2

26.8 ± 5.1a 37.8 ± 4.1a 18.6 ± 5.1a 40.0 ± 6.0a 5.3 ± 0.8 4.7 ± 0.8 1.5 ± 1.2a 18.8 ± 2.1c 8.8 ± 3.1 3.5 ± 1.7

10.1 ± 6.1b 65.5 ± 3.8c 15.9 ± 4.3a 34.2 ± 4.9 4.8 ± 1.4 4.3 ± 1.1 1.1 ± 0.9a 11.3 ± 1.3 11.3 ± 2.4 3.9 ± 1.3

23.1 ± 3.5 6.2 ± 1.2 35.0 ± 4.3 32.1 ± 4.3 12.3 ± 2.9 6.2 ± 1.3 5.1 ± 0.8 8.1 ± 4.9 11.2 ± 0.7 5.2 ± 0.8

13.8 ± 2.8a 26.1 ± 6.2c 41.5 ± 1.2 43.3 ± 3.5a 7.8 ± 1.8 5.8 ± 0.8 4.8 ± 1.1 17.3 ± 4.1 12.5 ± 1.3 5.8 ± 0.3

13.5 ± 2.3a 15.2 ± 1.4a 34.3 ± 3.8 23.7 ± 4.4 5.7 ± 2.1a 5.4 ± 1.1 5.3 ± 0.6 16.6 ± 4.9 39.5 ± 5.4c 15.4 ± 4.8a

3rd Day after challenge

rTbpA group

cdTCR+ CD172a+CD163+ CD172a+SLAIIDR+ CD4 CD8a+ CD4+CD8a CD4+CD8+ CD4+CD4naïve+ CD8ab+ CD21+aIgM+ CD21+SLAIIDR+ a b c

11.1 ± 1.4b 14.7 ± 0.4a 20.1 ± 4.3b 20.8 ± 2.8b 4.3 ± 2.7a 6.4 ± 0.7 4.1 ± 1.5 17.2 ± 5.4 41.1 ± 4.8c 19.3 ± 0.7b

Significant differences (P < 0.05) compared to challenge. Significant differences (P < 0.001) compared to challenge. Significant differences (P < 0.0001) compared to challenge.

the histocompatibility SLAIIDR+ molecules on the CD172a+ monocyte surface was also observed at 3rd dpc (Table 2). At challenge, CD4 CD8a+ lymphocytes reached 32.1% of the cells found in L gate, and increased until 43.3% at 1st dpc (P < 0.05); afterwards, these percentages decreased significantly

until 7th dpc (P < 0.05). On the other hand, CD4+CD8a cells fell significantly throughout the study (Table 2); however, no significant changes were detected for CD4+CD8a+, CD4+CD4naïve+ and CD8a+CD8b+ subsets. Even so, when the results of this latter subset were analyzed individually, a significant increase (P < 0.05) was

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Fig. 2. Two color analysis of swine peripheral cd T lymphocytes from CTL group. The expression of cd T cells was carried out on lymphocyte region. PBMC were first stained with purified mAb MAC320, followed by PE-conjugated mouse anti-rat IgG2a mAb RG7/1.30 and FITC-conjugated CD3e mAb BB23-8E6-8C8. Non-viable lymphocytes were excluded by staining with 7-AAD. (i) before challenge: 39.3% ± 4.6. (ii) 1st dpc: 26.8% ± 5.1. (iii) 2nd dpc: 10.1% ± 6.1.

Fig. 3. Two color analysis of swine peripheral monocytes from CTL group (representative dot plots from swine nr. 13). The expression of CD172a+CD163+ monocytes was carried out on monocyte region. PBMC were first stained with purified mAb BA1C11, followed by FITC-conjugated rat anti-mouse IgG1 mAb A85-1, purified mAb 2A10, biotinylated rat anti-mouse IgG2a, and streptavidin–PerCp. Non-viable monocytes were excluded by staining with propidium iodide. Percent of CD172a+CD163+ cells (i) before challenge: 22.9%, (ii) 1st dpc: 39.2%, and (iii) 2nd dpc: 63.1%.

Fig. 4. Two color analysis of swine peripheral monocyte from CTL group (representative dot plots from swine nr 13). The expression of CD172a+SLAIIDR+ monocytes was carried out on monocyte region. PBMC were first stained as indicated in Fig. 3, and then the cells were stained with purified mAb 1F12, biotinylated rat anti-mouse IgG2b, and streptavidin-PerCp. Non-viable monocytes were excluded by staining with propidium iodide. Percent of CD172a+SLAIIDR+ cells (i) before challenge: 28.1%, (ii) 1st dpc: 15.0%, and (iii) 2nd dpc: 13.3%.

found in four of the six pigs at 1st dpc. A strong response in CD21+aIgM+ cells was measured at 2nd dpc, being more than thrice higher than that seen the day before (P < 0.0001) (Fig. 5).

Similarly, a significant rise was observed in the expression of SLAIIDR+ molecules on the surface of CD21+ cells from 2nd dpc (P = 0.002 at 2nd dpc and P = 0.0005 at 3rd dpc).

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Fig. 5. Two color analysis of swine peripheral monocyte from rTbpA group (representative dot plots from swine nr 3). The expression of CD21+aIgM+ lymphocytes was carried out on lymphocyte region. PBMC were first stained with purified mAb 5C9, followed by FITC-conjugated rat anti-mouse IgG1 mAb A85-1 and PE-conjugated mAb Bly-4. Nonviable lymphocytes were excluded by staining with 7-AAD. Percent of CD21+aIgM+ cells (i) before challenge: 11.8%, (ii) 1st dpc: 12.5%, and (iii) 2nd dpc: 38.8%.

3.3. Characterization of surface molecule expression in PBMC subsets in NPAPTM, NPAPTCp and PG groups Quite different results compared to CTL and rTbpA groups were observed for these three vaccinated groups. CD3+cdTCR+ cells decreased significantly from 3rd dpc (P < 0.05, Fig. 6). On the other hand, CD172a+CD163+ monocytes increased significantly in NPAPTM and PG groups at 1st dpc (P < 0.0001) and only in NPAPTM group at 2nd dpc (P < 0.0001) compared to challenge. However, these values decreased in these three vaccinated groups from 2nd dpc until the end of the study, and this reduction was considered significant (P = 0.001) at 7th dpc compared to challenge in NPAPTCp and PG groups (Fig. 7). Concerning acquired immune response, PG and NPAPTM groups showed a significant rise in CD4 CD8a+ subset at 1st (P < 0.005) and 3rd dpc (P < 0.0001), respectively (Fig. 8A), which could be accounted for a rise in the rate of CD3e+CD8a+CD8b+ cytotoxic T cells, already seen from 2nd dpc in PG and NPAPTM groups (P < 0.05) and from 2nd dpc (P < 0.005) in NPAPTM pigs. In addition, the rise in this subset was especially noticeable in this latter group at 7th dpc (P < 0.0001) compared to the values obtained at challenge (Fig. 8D). Contrarily, no significant variations were achieved for

Fig. 6. Percents of the CD3+cdTCR+ cell subset in the pigs belonging to NPAPTM, NPAPTCp and PG groups. Significant differences between groups: ⁄P < 0.05, ⁄⁄ P < 0.005.

Fig. 7. Percents of the CD172a + CD163 + cell subset in the pigs belonging to NPAPTM, NPAPTCp and PG groups. Significant differences: ⁄P = 0.001, ⁄⁄P = 0.0001.

CD4 CD8a+, CD4+CD8a+, and CD3e+CD8a+CD8b+ subsets in the animals belonging to NPAPTCp group after challenge (Fig. 8A, C and D, respectively). A different tendency was observed for CD4+CD8a+ cells in NPAPTM group, with a significant increase after challenge, which was especially marked at 3rd dpc (P < 0.0005, Fig. 8C). No significant differences were noticed for this subset in PG group (Fig. 8C). On the other hand, the results observed for CD4+CD8a cells were different depending on the vaccination route (Fig. 8B). So, in the groups immunized intramuscularly (NPAPTM and PG), this subset reached about 5% of the lymphocytes measured in L gate throughout the study, in such a manner that no significant changes were detected, except for PG group at 2nd dpc (P < 0.05). However, numbers in CD4+CD8a cells in NPAPTCp group fell from almost 14% at 1st dpc to below 6% at 7th dpc (P < 0.005 compared to challenge, Fig. 8B). No significant differences were detected in CD4+CD4naïve+ subset in NPAPTM and PG groups, but a significant decrease was observed at 7th dpc in NPAPTCp group (about 6% at challenge vs about 2% at 7th dpc, P = 0.0001). A strong response of CD21+aIgM+ cells was measured in NPAPTCp, NPAPTM, and PG groups at 7th dpc (P < 0.0001), being 140%, 100% and 33% higher respectively than the percentages recorded at challenge (Fig. 9A). In addition, this response was

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Fig. 8. Percents of the T lymphocyte subsets being analyzed in L gate. Significant differences: ⁄P < 0.05,

⁄⁄

P < 0.005,

⁄⁄⁄

P < 0.0001.

Fig. 9. Percents of the B lymphocyte subsets being analyzed in L gate. Significant differences: ⁄P < 0.0001.

significantly higher in the NPAPTCp group (P = 0.003 compared to NPAPTM group, and P = 0.0001 compared to PG group). Similarly, a significant increase (P < 0.0001) in the expression of SLAIIDR+ molecules in the CD21+ lymphocytes was seen in these three vaccinated groups at 7th dpc, but it was also detected from 1st day after challenge in NPAPTM and PG groups (P < 0.0001) (Fig. 9B). 4. Discussion The cell immune response against H. parasuis has been poorly investigated to date. In this report, the changes of different blood

cellular subsets involved in both innate and adaptive immune response developed against Nagasaki strain (serovar 5), are described by comparing four vaccinated groups and a control non-vaccinated group. The clinical results showed that NPAPTM, NPAPTCp and PG vaccines were effective in preventing experimental Glässer’s disease, while rTbpA formulation was scarcely effective [18]. Our results evidenced the lympholytic activity caused by H. parasuis serovar 5 on the CD3+cdTCR+ cells of the piglets belonging to non-vaccinated group and in the vaccinated but non-protected animals (rTbpA group). A severe leukopenia measured from 20 to 48 h after infection by H. parasuis has been already

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observed [15]. Similarly, in colostrum-deprived pigs vaccinated with other formulations, a significant decrease in CD3+ subset after H. parasuis infection in non-protected animals was shown [17]. The fact that cd T cells are the most numerous lymphocyte subpopulation in the pheripheral blood of young pigs, along with the high proportion of NK cells [20], suggests that cell immune response of piglets is mainly dependent on the innate immunity mediated by cd T cells. On the basis of our results, it can be suggested that CD3+cdTCR+ cells are the main cellular immune target of H. parasuis infection, and this could be one of the mechanisms used by this pathogen for causing disease. Furthermore, the greatest susceptibility to Glässer’s disease is found in 4 to 6-week pigs, a time at which maternal antibody levels decrease considerably [21] and the proportion of cd T cells is especially high [20]. The mild decrease in the rate of CD3+cdTCR+ lymphocytes observed from 3rd dpc in the three vaccinated and protected groups was coincident with the increase of CD21+aIgM+ subset. This effect was due to a mere subpopulation change, but not to the lympholytic effect caused by H. parasuis infection. A strong response of CD172a+CD163+ cells was seen in the nonand scarcely protected groups and, in a lesser extent, in the wellprotected piglets during the first days after challenge. On the basis of the expression of CD163 antigen two porcine blood monocyte subsets have been proposed using 2A10 mAb, being CD163+ cells those that exhibit a most advanced maturation stage [22,23]. CD163+ monocytes produce high amounts of proinflammatory cytokines, such as tumor necrosis factor a [23,24], and interleukins 1b and 6 [25]. The increase in the proportion of mature monocytes seen in our study could be related to the pivotal role developed by these cells in the infectious inflammatory responses. In the infection target organs, a high transcription of CCL2 chemokine was detected (data not shown), that could induce increased levels of CD163+ monocytes in pheripheral blood, followed by their recruitment to the infection site. In a previous report [17], a significant increase in cells exhibiting SWC3 antigen (CD172a cells) was described after experimental infection with H. parasuis serovar 5, but not immediately after challenge, as it is seen in our study. Similar results were described [26] after experimental infection of pigs with Actinobacillus pleuropneumoniae. These authors observed a significant increase in blood CD172a+ cells and in the CD163+ monocytes located in the lung interstitial space. A striking observation was that the expression of SLAIIDR antigen in non- and scarcely protected piglets did not increase simultaneously along with CD172a subset, but decreased significantly. This result suggests that H. parasuis might modify the expression of this antigen in the surface of CD172a+ monocytes, thus becoming a serious problem in the presentation of processed antigens. A lot of changes were detected with regard to the adaptive response in peripheral blood after challenge. Conflicting results were observed regarding CD4+CD8a lymphocytes. This subset (T helper) plays a pivotal function in the regulation of B cell activity [27], providing help to the antibody production, and generation and maintance of CD8 T cell memory [28]. No significant changes in the CD4+CD8a cell rates were seen after infection in control group, which was in agreement to previous results [17,29]. However, the results found in the immunized groups were dependent on the protective effect of formulations. Interestingly, the piglets receiving NPAPTCp vaccine showed at challenge a rate of 50% of CD4+CD8a cells higher than those of the two other well-protected groups. This finding indicates that this vaccine itself, administration route or both factors simultaneously are capable of inducing a strong CD4+ cell response. Additionally, NPAPTCp and NPAPTM vaccines induced higher percentage of CD4+CD8a+ in pheripheral blood. This CD4+CD8a+ subset expresses in their surface high levels of CD29 (CD20high) antigen [30]. Previous studies have suggested that these cells could be in pig memory T cells because of their

capacity to proliferate in response to recall antigen and cooperate with B cells in antibody production [31,32]. CD4+2E3+ cells achieved approximately 5% of lymphocytes measured in the pigs belonging to immunized groups, without significant differences being observed after challenge. However, control animals exhibited a significant decrease after challenge, which could be explained as a result of the lympholytic activity performed by H. parasuis or due to the recruitment of these cells to secondary lymph organs for recognition of the antigens being presented by dendritic cells. Other striking result in our study was the response developed by cytotoxic T cells against H. parasuis. It is well known that CD3e+CD8a+CD8b+ cells play an important role against intracellular bacterial [33,34] or viral [35] infections. However, this is the first description of changes in the rates of this subset after H. parasuis infection. The rise of these cytotoxic cells could be explained on the basis of the studies indicating that H. parasuis is capable of internalizing into porcine endothelial [36–38] or epithelial cells [39,40], thus inducing a cell immune response against the cells exhibiting MHC class I molecules bound to H. parasuis antigens. B cells were studied using mAbs 5C9, which recognized IgM achain expressed in cytoplasmic membrane of most B cells [41], and Bly-4, which binds to CD21 of mature B cells [42,43]. As it could be expected, control animals did not develop a proliferative cell B response after challenge, thus indicating that these pigs had not been previously exposed to H. parasuis and, consequently, lacked memory B cells. A significant increase in mature B cells was seen in both scarcely and well protected groups. The rate of CD21+aIgM+ cells at challenge in the pigs showing an effective protection was about twice higher than that measured in rTbpA group. This finding states the efficacy of NPAPTM, NPAPTCp and PG vaccines in inducing a strong response of mature B cells in peripheral blood. Comparing these three groups, the highest rate of CD21+aIgM+ cells was detected in NPAPTCp group; therefore, this formulation resulted in not only the highest CD4+CD8 response but also in the best response of memory B cells. These results could be related to the immunization route and/or the adjuvant used in these pigs. The dispersible adjuvants based on TLR agonists (CpG and MPL) induced in mice CD4+ cell clonotypes exhibiting TCR with high affinity to antigen compared with depot-forming adjuvants [44]. Similarly, it could be hypothetically suggested that neuraminidase from C. perfringens, a dispersible molecule, could have stimulated the production of T helper cells with similar characteristics, due to the strong response of B cells detected in our study. A significant increase of aIgM+ lymphocytes a week after challenge was seen studying the effect of different vaccines against H. parasuis [17], which is in agreement with the results obtained by us in protected animals, and is coincident with the beginning of the clonal expansion of memory B cells [45]. This subpopulation increased at 2nd dpc in scarcely protected animals, which could be related to memory B cell response or the mitogenic activity of lipooligosaccharyde [46] or both together on B cells. Along with that of CD21+aIgM+ cells, an increase in the expression of SLAIIDR+ antigen was detected on the surface of these cells in all experimental groups except for control animals. It has been evidenced that the increase in the expression of this antigen had the same pattern as that of B cells after infection by A. pleuropneumoniae [47]. In addition, porcine B cells expressed constitutively SLAIIDR molecules on their surface [48]. This finding can explain the low expression of this antigen (about 4%) on the surface of CD21+ cells in control pigs, before and after challenge. On the other hand, the increase in class II molecules seen in immunized animals could suggest a higher capacity of mature B cells of responding to the antigens presented by antigen processing cells or independently. As conclusions, our study underlines the strong protection afforded by different vaccine formulations from the cell immune

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response point of view and provides new data on the pathogenesis of H. parasuis infection. The lympholytic effect of this pathogen on CD3+cdTCR+ cells and the response of cytotoxic T cells against it are reported for the first time. NPAPTM and NPAPTCp vaccines induced respectively a stronger response of CD21+aIgM+ B and helper T cells in comparison to commercial vaccine, thus revealing their potential for developing effective future formulations against Glässer’s disease. Acknowledgments Research in the laboratory of R.F. and S.M.M. was supported by long-predoctoral fellowships from the Spanish Ministry of Science and Innovation, as well as by grant AGL-2008-00110/GAN (Spanish Ministry of Science and Innovation) and grant AGL-2011-23195 (Spanish Ministry of Economy and Competitivity). We thank Dr. Domínguez Juncal (INIA, Madrid, Spain) for kindly providing most of the mAbs used in this study. We thank J.I. Rodríguez Barbosa (Biomedicine Institute, University of León) for help with FACS analysis, and Luiz Carlos Kreutz for reviewing this manuscript. References [1] V.J. Rapp-Gabrielson, G.J. Kocur, S.K. Muir, Haemophilus parasuis: immunity in swine after vaccination, Veterinary Medicine 92 (1997) 83–90. [2] S. Oliveira, C. Pijoan, Haemophilus parasuis: new trends on diagnosis, epidemiology and control, Veterinary Microbiology 99 (2004) 1–12. [3] P. Kielstein, V.J. Rapp-Gabrielson, Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts, Journal of Clinical Microbiology 15 (1992) 1019–1023. [4] C.E. Hill, D.S. Metcalf, J.I. MacInnes, A search for virulence genes of Haemophilus parasuis using differential display RT-PCR, Veterinary Microbiology 96 (2003) 189–202. [5] H. Bak, H.J. Riising, Protection of vaccinated pigs against experimental infections with homologous and heterologous Haemophilus parasuis, Veterinary Record 151 (2002) 502–505. [6] C.R. Hoffmann, G. Bilkei, The effect of a homologous bacterin given to sows prefarrowing on the development of Glässer’s disease in postweaning pigs after i.v. challenge with Haemophilus parasuis serotype 5, Deutsch Tierärztl Wochber 109 (2002) 271–276. [7] L. Takahaschi, S. Nagai, T. Yagihashi, T. Idehata, Y. Nakano, K. Senna, T. Maruyama, J. Murofushi, A cross protection experiment in pigs vaccinated with Haemophilus parasuis serovars 2 and 5 bacterins, and evaluation of a bivalent vaccine under laboratory and field conditions, Journal of Veterinary Medicine Science 63 (2001) 487–491. [8] A. Palzer, M. Ritzmann, K. Hienritzi, A field trial for early vaccination against Glässer’s disease using Porcilis Glässer, Schweiz Arch Tierheilkder 149 (2007) 389–394. [9] M. Zhang, Y. Guo, J. Zhao, Q. Hu, Y. Hu, A. Zhang, H. Chen, M. Jin, Molecular cloning, sequencing and expression of the outer membrane protein A gene from Haemophilus parasuis, Veterinary Microbiology 136 (2009) 408–410. [10] M. Zhou, Y. Gou, J. Zhao, Q. Hu, Y. Hu, A. Zhang, H. Chen, M. Jin, Identification and characterization of novel immunogenic outer membrane proteins of Haemophilus parasuis serovar 5, Vaccine 27 (2009) 5271–5277. [11] M. Hong, J. Ahn, S. Yoo, J. Hong, E. Lee, I. Yoon, J.K. Jung, H. Lee, Identification of novel immunogenic proteins in pathogenic Haemophilus parasuis based on genome sequence analysis, Veterinary Microbiology 148 (2011) 89–92. [12] A. Olvera, S. Pina, M. Pérez-Simó, V. Aragón, J. Segalés, A. Bensaid, Immunogenicity and protection against Haemophilus parasuis infection after vaccination with recombinant virulence associated trimeric autotransporters (VtaA), Vaccine 29 (2011) 2797–2802. [13] F. Yuan, S. Fu, J. Hu, J. Li, H. Chang, L. Hu, H. Chen, Y. Tian, W. Bei. Evaluation of recombinant proteins of Haemophilus parasuis strain SH0165 as vaccine candidates in a mouse model. Research in Veterinary Science 17 (2001) (Epub ahead of print). [14] S. Fu, F. Yuan, M. Zhang, C. Tan, H. Chen, W. Bei. Cloning, expression and characterization of a cell wall surface protein, 6-phosphogluconate dehydrogenase, of Haemophilus parasuis. Research in Veterinary Science Aug 11 (2011) (Epub ahead of print). [15] H. Amano, M. Shibata, K. Takahashi, Y. Sasaki, Effects of endotoxin pathogenicity in pigs with acute septicemia of Haemophilus parasuis infection, Journal of Veterinary Medicine Science 59 (1997) 451–455. [16] G. Müller, H. Kohler, R. Diller, A. Rassbach, A. Berndt, D. Schimmel, Influences of naturally occurring and experimentally induced porcine pneumonia on blood parameters, Research in Veterinary Science 74 (2003) 23–30. [17] A.J. Martín de la Fuente, C.B. Gutiérrez-Martín, J.I. Rodríguez-Barbosa, S. Martínez-Martínez, R. Frandoloso, F. Tejerina, E.F. Rodríguez-Ferri, Blood celular immune response in pigs immunized and challenged with Haemophilus parasuis, Research in Veterinary Science 86 (2009) 230–234.

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