Responsiveness Of Lymphocytes From CSF-immunized Porcine Lymphoid Tissues To PRRSV Peptides Stimulation

Porcine reproductive and respiratory syndrome is caused by PRRSV, commonly known as “blue ear disease of porcine” in China. PRRSV whose origin was a mystery began to appear in swine industry all around the world in the late 1980 s, causing severe economical losses. PRRS often accompanied with CSF(classical swine fever) increases the incidence rate, mortality rate, and makes pathogenic mechanism more complex, so preventive treatment is not perfect enough so far.In the course of PRRS infection, cytokines like IFN-γ and IL-12 are mainly involved in immune prevention. However, IL-10 plays an important role in immune suppression. This experiment was designed to detect the changes of IFN-γ, IL-12 and IL-10 secreted by T lymphocytes which were isolated from the immune organs of healthy piglets after the cell stimulation of PRRSV-specific peptides. The experiment selected 3 piglets with no antigens and antibodies of CSFV, PRRSV, PCV, PRV, inoculating the CSFV vaccine when they were up to the age of 4 weeks. And after another 4 weeks’ isolation rearing, blood were taken through anterior vena cava for serum to detect if there were nucleic acid and how their immune status was. What we did above is just to exclude the possibility of wild virus’ interference to test results. Bone marrow of the limbs(4 samples), lymph nodes(inguinal, mesenteric, jaw, shoulders and hilar lymph node, totally of 5 samples), thymus and spleen(three samples including the head, the middle and tail part) were operated out of bodies lively and antiseptically, and then T lymphocytes marked correctly were separated from each kind of sample in these organs correspondingly. However we must try to identify if they were T lymphocytes by flow cytometry. Next, these cells should be divided into two groups in the 96 pore plate, one group of T lymphocytes in culturing 24 h, the other in culturing 72 h. They were both added four kinds of PRRSV-specific peptides to stimulate, leaving a hole as blank control. The four PRRSV-specific peptides were PRRSV-GP5(aa 100-108), PRRSV-GP5(aa96-104), PRRSV-N(aa 106-114), PRRSV-N(aa 49-57), also for short G1, G2, N1 and N2. All of these samples were held in 37℃ for 12 h. After centrifugation, the secretion of IFN-γ, IL-12 and IL-10 in the supernatant were detected by ELISA. Finally, using the statistical software called SPSS to analyse the test result.The result of T lymphocytes for culturing 24 h shows that cells in marrow, gave a reduction of IFN-γ significantly after stimulation by G1、G2 and N1 peptides compared with the blank control group reduced significant(P<0.05); The content of IL-12 after stimulation by G1、G2、N1 peptides compared with the blank control group reduced significantly(P<0.05); The content of IL-10 after stimulation by four specific peptides compared with the blank control group reduced significantly(P<0.05). T lymphocytes in lymph nodes stimulated by G1、G2、N1 peptides,the content of IFN-γ reduced but not obviously; The content of IL-12 after stimulation by G2、N1、N2 peptides compared with the blank control group increased significantly(P<0.05); The content of IL-10 after stimulation by the four specific peptides reduced significantly(P<0.05), compared with the blank control group; T lymphocytes in spleen stimulated by the four PRRSV-specific peptides, the secretion of IFN-γ reduced significantly(P<0.05), compared with the blank control group; The content of IL-12 after stimulation by the four specific peptides didn’t change obviously; The content of IL-10 after stimulation by the four specific peptides reduced significantly compared with the blank control group,especially stimulated by the N1 and N2 peptides, which is significant(P<0.05). T lymphocytes in thymus stimulated by the four PRRSV-specific peptides, the secretion of IFN-γ increased significantly(P<0.05), compared with the blank control group; The content of IL-12 after stimulation by the G1、N1 and N2 peptides reduced significantly(P<0.05); The content of IL-10 after stimulation by the four specific peptides reduced significantly(P<0.05), compared with the blank control group.The result of T lymphocytes for 72 h shows that cells in bone marrow could increase the secretion of IFN-γ significantly(P<0.05), stimulated by the G1、G2 and N2 peptides. And the content of IL-12 reduced after stimulation by the N1 and N2 peptides, compared with the blank control group; The content of IL-10 after stimulation by the four specific peptides, didn’t change obviously. T lymphocytes in lymph nodes stimulated by the G1、G2 and N1 peptides,gave a reduction of IFN-γ, especially after the stimulation of the G2 peptide significantly(P<0.05); The content of IL-12, stimulated by the four PRRSV-specific peptides, reduced significantly(P<0.05). The content of IL-10 decreased after stimulation by the G1、N1 and N2 peptides, compared with the blank control group; T lymphocytes in spleen could increase the secretion of IFN-γ, stimulated by the G1、N1 and N2 peptides, especially under the stimulation of the N2 peptide, the difference was significant(P<0.05); The secretion of IL-12 after stimulation by the N2 peptide, increased significantly(P<0.05); The content of IL-10 after stimulation by the four specific peptides, compared with the blank control group, reduced significantly(P<0.05). T lymphocytes in thymus could increase the secretion of IFN-γ significantly(P<0.05), stimulated by the four PRRSV-specific peptides; The content of IL-12 after stimulation by the G2、N1 and N2 peptides, increased significantly(P<0.05), compared with the blank control group; The content of IL-10 after stimulation by G2 and N2 peptides, increased significantly(P<0.05).As the result shows, when swines are immuned by CSFV vaccine successfully, their immune systems will have a strong response to the stimulation of PRRSV-specific peptides. Also, the four specific peptides have a significant inhibitory effect on the secretion of IL-10, inhibiting the occurrence of immune suppression, which is conducive to the production of immune stimulating cytokines. At the same time, the effects of G1, G2 and N1 peptides on the secretion of IFN-γ and IL-12 are not obvious, while the N2 peptide, which affects the secretion of IFN-γ and IL-12, has a tendency to promote the secretion of the two, but should be verified further. In addition, these cytokines of immune organs were detected under vitro-culturing conditions. As far as it can be seen, when stimulation occurred at the point of cell-culturing 24 h, the secretion of cytokines is more regular than that at 72 h, which is conducive to the evaluation of PRRSV-specific peptides’ effect and provides more accurate reference data for synthetic peptide vaccine of PRRSV.