Cellular adaptive immune response against porcine circovirus type 2 in subclinically infected pigs

Abstract

Background: Porcine circovirus type 2 (PCV2) is a dominant causative agent of postweaning multisystemic wasting syndrome (PMWS), a multifactorial disease complex with putative immunosuppressive characteristics. Little is known about adaptive PCV2-specific immune responses in infected pigs. Therefore, the T and B cell responses following PCV2 infection in 3-week old SPF piglets infected with PCV2 or PCV2 plus porcine parvovirus (PPV) were studied.

Results: All animals were asymptomatically infected. At 7 days post infection (d p.i.), B lymphocyte and T lymphocyte numbers decreased in the dual infected, but not in the single infected piglets. At this time point a transient PCV2 viraemia was noted in the PCV2 infected groups. Antibodies against the infecting virus were detectable at day 24-28 p.i. for anti-PCV2 antibodies and at day 10 p.i. for anti-PPV antibodies, with no apparent influence of PCV2 on the early PPV antibody development. In the animals infected with PPV alone, IFN-gamma secreting cells (SC) that were not specific for PCV2 were detected by ELISPOT assay at day 7 p.i. Interestingly, this response was absent in the PCV2/PPV dual infected animals. PCV2-specific IFN-gamma SC were observed in the PCV2/PPV infected group at 7 d p.i. and in the PCV2 single infected group at 21 d p.i. A reduction in the numbers of IFN-gamma SC was observed following anti-CD4 and anti-CD8 antibody treatment, suggesting roles for both CD4+ and CD8+ T cells in the response against PCV2 infection. This was supported by an observed increase in the percentage of IFN-gamma positive CD8hi cytotoxic T cells as well as IFN-gamma positive CD8-/low helper T cells after PCV2 in vitro re-stimulation.

Conclusions: Infection of weaned SPF piglets with PCV2 alone or combined with PPV does not induce disease and in both cases a relatively slow anti-PCV2 antibody response and weak T lymphocyte responses were found. Knowledge on such immunological characteristics is important for both PCV2 pathogenesis and vaccination.

Figure 1

Body temperature and average daily weight gain of animals infected with PCV2 and/or PPV. (A) Body temperature. The rectal body temperature was measured for all animals at daily intervals. Mean values of the groups are shown. (B) Weight gain. The average daily weight gain (kg) is shown for each animals within a group (filled and open symbols); group mean values are represented by the bar symbol.

Figure 2

Anti-PPV and anti-PCV2 Ab titres. (A) Anti-PPV specific antibodies were measured with a commercial competitive Ab ELISA, at time points (days p.i.) indicated on the x-axis. The values are given as percent inhibition (PI), calculated as described in Materials and Methods. Means of the groups, +/- SD, are shown.(B) Anti-PCV2 IgG Ab titres were measured by an indirect ELISA. The log₁₀ of the reciprocal end-point dilution is given for the time points (days p.i.) indicated on the x-axis. Mean values for the groups are shown by the histograms, +/- standard deviation (SD).

Figure 3

B- and T-lymphocyte counts. (A) B lymphocyte counts. PBMC were isolated at the time points (days post-infection (p.i.)) indicated on the x-axis, and stained with anti-IgG H+L Ab to analyse the B cell numbers. The absolute B cell counts were calculated and expressed as ×10³ cells/ml. Individual animals are represented by the open and filled symbols, with the mean values for the groups shown by the continuous line. (B) T lymphocyte counts. The above PBMC were stained for CD3a expression, to analyse the T lymphocyte counts. These counts are given as × 10³ cells/ml for the individual animals (open and filled symbols), with the group mean values shown as the continuous line. *P < 0.05.

Figure 4

IFN-γ secreting cells after ex vivo PCV2 re-stimulation. (A) PBMC from two adult PCV2-immune animals were used as positive controls to evaluate the ability of the test to detect PCV2 specific IFN-γSC. The PBMC were re-stimulated for 24 h with PCV2-infected cell lysate, or mock cell lysate as negative control, at increasing MOI based on the titre of the PCV2 (TCID₅₀/cell, x-axis). The IFN-γSC were measured by ELISPOT assay as described in Materials and Methods, and expressed per 10⁶ cells. (B-D) PBMC were isolated from the piglets which had received PBS (control), PCV2 single infection, PCV2/PPV double infection and PPV single infection. The cells were re-stimulated with PCV2 or mock antigen at 1 TCID₅₀/cell (or equivalents dilution for the mock) for 24 h, and the IFN-γSC detected by ELISPOT assay. The mean values of the groups, +/- SD, are shown for PBMC isolated before infection (0 days) (B), 7 d p.i. (C) and 21 d p.i. (D). The IFN-γSC are calculated per 10⁶ cells.

Figure 5

Comparison of the PCV2 re-stimulation profile for freshly isolated compared to frozen and then in vitro expanded PBMC. PBMC were re-stimulated with PCV2, mock antigen, or medium alone directly after isolation ("PBMC fresh; 24 h"). Aliquots of the PBMC were frozen under liquid nitrogen, before thawing and expanding by culture in the presence of rpoIL-2 together with PCV2, mock antigen, or medium alone. This expansion was for 24 h ("PBMC thawed; 24 h"), 3 days ("PBMC thawed; 3 days") or 5 days ("PBMC thawed; 5 days") prior to analysis for IFN-γ SC by the ELISPOT assay. Means of triplicates +/- SD of two experiments are shown.

Figure 6

Characterization of anti-PCV2 specific T lymphocytes. (A) Anti-CD4 and -CD8 mAbs reduce IFN-γ SC. PBMC from PCV2-immune animals were treated with mAbs against the CD4 and CD8 T cell receptors, or anti-CD1 as control for 1 h prior to PCV2 or mock antigen re-stimulation for 5 days (as in Fig. 5). The IFN-γ SC were measured by ELISPOT assay, and calculated per 10⁶ cells. Mean values of triplicates of one representative experiment +/- SD are shown. (B and C) IFN-γ SC detected by flow cytometry. PBMC from PCV2-infected piglets (3 months after infection) were re-stimulated with PCV2 or mock antigen for 5 days prior to staining for the presence of intracellular IFN-γ. The cells were also labelled for the surface markers CD8 to differentiate MHC class I restricted CTLs as CD8^hi cells from Th cells located within the CD8^-/lo cell population. The latter includes also NK cells and γ/δ T cells. Values shown on the y-axis represent the percentage of IFN-γ positive cells. The symbols represent the individual animals. In C the percentage of the IFN-γ positive cells within the blasting cells defined by gating on cells with high forward scatter is shown. For B and C, monocytic cells were excluded based on their CD172a expression.