In three successive experiments, the immune functions of pigs persistently infected with the porcine reproductive and respiratory syndrome virus (PRRSV) have been evaluated. Non-specific immune responses were analyzed over a period of 12 weeks post-infection (PI). In addition, the capacity of PRRSV-infected pigs to develop an efficient immune response against pseudorabies virus (PRV) glycoproteins and to resist to a subsequent virulent challenge was investigated. Our results demonstrate that PRRSV produced minor effects on the immune system of pigs. The skin delayed type hypersensitivity (DTH) in response to phytohemagglutinine injection was slightly diminished one week after challenge, but was restored thereafter. However, three weeks after the infection, the total white blood cell count, and the number of CD2+, CD8+ and IgM+ cells were enhanced. The increase in numbers of CD8+ cells persisted for three consecutive weeks. Serum immunoglobulins in infected pigs also increased by week 3 PI and up to 8 weeks PI. These results show that PRRSV may have stimulating effects on the pig immune system during the phase of long-lasting infection. After immunization with PRV glycoproteins, the production of anti-PRV antibodies and skin DTH response against PRV glycoproteins were not affected. On the contrary, following a virulent PRV challenge, PRRSV-infected pigs developed a better secondary antibody response and their resistance to the infection was as effective as in control pigs. Taken together, our data do not support a systemic immunosuppressive effect of PRRSV, during the persistent phase of infection. Other mechanisms may therefore apply to explain the emergence of secondary infections in endemically infected herds.

Background The most common assay used to detect natural killer (NK) and cytotoxic T‐lymphocyte (CTL) activity is the 51Cr release assay. The numerous disadvantages of this method led us to evaluate cytotoxicity functions by flow cytometry. We described a flow cytometric assay to assess NK and CTL activity from different species. Methods This assay is based on a dual fluorescent staining of target cells. The dye, DIOC18(3) (3,3′‐dioctadecyloxacarbocyanine perchlorate), is used to stain the membrane of different target cells. Propidium iodide (PI) is used to label dead target and effector cells. This labeling allows a clear discrimination between both cell populations. Results A good correlation was observed between the percentage of target lysis and the effector‐to‐target cell (E/T) ratios with human and porcine peripheral blood mononuclear cells (PBMC) as effector cells. The flow cytometric assay was shown to be as sensitive and as reliable as the 51Cr release performed with human cells. The assay was also applied successfully to measure NK cell activity in other animal species (pig, rabbit, hen, and mouse) and to measure murine CTL activity against the influenza virus. Conclusions We provide evidence that the flow cytometric assay using DIOC18(3) is highly reproducible and is suitable to measure different types of cell cytotoxicity. Cytometry 41:289–297, 2000. © 2000 Wiley‐Liss, Inc.

-A better understanding of cell-mediated immune responses to classical swine fever virus (CSFV) is essential for the future development of improved vaccines. We analyzed the generation of cell-mediated and humoral immune responses in d/d histocompatible pigs following CSFV infection or vaccination. Viral infection induced high T cell responses with high primary and secondary CTL activity correlated with high IFN-g production, whereas vaccination with a live vaccine followed by infection mainly induced neutralizing antibody but low cell-mediated responses. Moreover, high IgG1 response was associated with high IFN-g response following infection whereas a weak IFN-g response was related to a good IgG2 response but a low IgG1 production. These data could reflect Th1/Th2-like balance of immune responses depending upon immunization protocols, which has not yet been described in the pig. T-cell responses to CSFV were evidenced by CSFV-specific CD25 upregulation on CD4 -CD8 + , but not on CD4 + CD8 -cells, which further illustrated the importance of CTL responses after infection. Our results indicated that generation of cell-mediated immune responses was much higher following intranasal/oral CSFV infection than after intramuscular vaccination, which implies that the capacity of new CSFV vaccines to induce higher T-cell responses should be considered. immunity / cytotoxicity / CSFV / d/d histocompatible pig