Development And Application Of Detection Methods For Chick Cellular Immune Responses

Cellular immune responses plays a key role in the defense of bacteria,which has been confirmed in animal models such as mice.Due to lack of related biological reagents and methods,and complexity of avian immune system,the study of avian cellular immunity is greatly restricted,which seriously limits the development and application of novel vaccines against poultry diseases.Therefore,the establishment of detection techniques for avian cellular immune responses will promote the development of related avian biological products and the effective control of diseases.In this study,a set of analytical techniques including detection of T cell proliferation,analysis of chick T cell subsets,and analysis of cytotoxic T cell killing ability were established for evaluating chick cellular immune responses.Herein,the attenuated vaccine against Salmonella Pullorum and the Newcastle Disease-Infectious Bronchitis Vaccine were used to immunize the chicks.The cellular immune technology platform was used to analyze and evaluate the cellular immune response induced by the vaccines,which provides new insights into the development and improvement of poultry vaccines.1.Development of detection methods for chick cellular immune responsesChick spleen was aseptically collected and single cell suspension was prepared to isolate the lymphocytes.The T cell proliferation was detected by BrdU ELISA within the stimulation of ConA.The T cell subsets and cell apoptosis were analysed by flow cytometry.CD8+T cells were separated by using flow cytometry and magnetic activated cell sorting(MACS).BrdU ELISA results showed that ConA stimulation could significantly induce chick T cell proliferation compared with unstimulated group.CD4+and CD8+T cell subsets were distinct population detected by flow cytometry and the purity of sorted CD8+T cells was over 97%.The purity of CD8 T cells separated by MACS was 94%,which determing high sorting speed and efficiency.Besides,Annexin V-FITC/PI was effective method to detect apoptosis and death of chick cells.The above techniques will be helpful to evaluate chick cell-mediated immune responses.2.Analysis and evaluation of cellular immune responses induced by Salmonella Pullorum vaccine candidate strain3-days-old chicks were immunized intramuscularly with the Salmonella Pullorum attenuated vaccine candidate strain S06004?spiC,and the PBS injection group was used as a control.The established chick cellular immune response analysis technology platform was used to evaluate the immune response induced by S06004?spiC strain.The cell proliferation assay showed that,the lymphocytes were significantly stimulated by Salmonella Pullorum specific antigen in vitro at 14 d(P<0.001)and 21 d(P<0.01)after immunization with S06004?spiC strain compared with the PBS control group.The analysis of T cell subsets showed that the population of CD8 T cell subsets increased significantly at 14 d,28 d and 35 d after immunization.In the CTL killing experiment,the killing ability of CTL cells in the immunized group was significantly higher than that in the non-immunized group(P<0.05).It was determined that IFN-y content in serum of immunized group in ELISA was higher than that of control group at 5 d after immunization.The IgG antibodies cound be detected at 7 d after immunization with S06004?spiC,and acquiring the highest value at 21 d.Taken together,our chick cellular immune response analysis technology platform could be used to evaluate the cellular immune response induced by Salmonella Pullorum attenuated vaccine candidate S06004?spiC.And the immunization of S06004?spiC strain can induce a strong cellular immune responses,which providing crucial data to support the exploitation and development of Salmonella Pullorum vaccine.3.Analysis and evaluation of cellular immune responses induced by Newcastle Disease-Infectious Bronchitis live vaccineThe Newcastle Disease-Infectious Bronchitis live vaccine was used to immunize 3-days-old chicks by nasal and eye drops,and the PBS immunized group was used as a control to detect the immune response induced by the vaccine.At 28 d after immunization,T cell subsets showed significant changes.Targeting the kidney cells,the killing ability of CTL cells in the immunized group was higher than that in the non-immunized group.The results of transcriptional level test showed that the expression levels of cytokines in chick spleen were higher than those in the control group,and the expression levels of IL-2,IL-4 and IL-10 were significantly different at 14 d.The level of IgA antibodies in tracheal lavage cound be detected at 7 d after immunization,and peaked at 28 d.The level of IgG antibodies in serum was significantly higher than that of the control group at 21 d after immunization,and the difference was most significant at 28 d.In conclusion,chick Newcastle Disease-Infectious Bronchitis combined live vaccine can induce strong chick cellular immunity,humoral immunity and mucosal immune response in immunized animal.Meanwhile the established techniques demonstrated to be a powerful tool to analyze chick cellular immune responses,and to evaluate chick cellular immunity induced by Newcastle Disease-Infectious Bronchitis live vaccine.These results will provid new theoretical basis for the application and improvement of the vaccine.