| Oligodeoxynucleotides containing unmethylated CpG motifs (CpG-ODN) have been proved to be a potent and safe vaccine adjuvant. However, the application of CpG-ODN in poultry vaccines was limited because of its high cost. The objective of this study was to sceen CpG-ODN with efficient adjuvant activity and low cost in chickens and to compare the immunoadjuvant effect of bacteria CpG-DNA and CpG-ODN in chickens. Firstly, four sequences of CpG-ODN were designed based on the CpG-ODN2006which was template and positive sequences of chicken. Then the effects of different CpG-ODN on inactivated avian H5N1influenza virus (IAIV) vaccine were investigated in chicken in vivo and in vitro test. In vitro test:the immunostimulatory activity of different CpG-ODN on chicken spleen lymphocytes and chicken bone marrow-derived dendritic cells (BM-DCs) were investigated. In vivo test,330seven-day-old chickens were randomly divided into eleven groups. All the chickens were immunized twice (at9days and16days old). Chickens from group1were intranasally immunized with PBS liquol. Chickens from group2were intranasally immunized with IAIV only. Chickens from group3and group4were intranasally immunized with IAIV respective together with CpG-ODN F1and CpG-ODN F2(30μg each chicken). The chickens from group5to group7were intranasally immunized with IAIV respective together with different dosages of CpG-ODN F3(10μg,30μg,80μg each chicken). Chickens from group8to group11were intranasally immunized with IAIV respective together with CpG-ODN F4, CpG-ODN F5, CpG-ODN F6and bacterial CpG-DNA (30μg each chicken). Firstly the expression levels of IL-6mRNA, IL-12mRNA and IFN-ymRNA in nasal cavity and trachea were detected to investigate the effects of different CpG-ODN on local cell immunity. The levels of AIV-specific IgA in the lavage fluid of nasal cavity and trachea were detected to investigate the effects of different CpG-ODN on the local humoral immunity. The levels of AIV-specific IgG and HI antibody in serum were detected to investigate the effects of different CpG-ODN on systemic immunity. Finally the expression levels of TLR21mRNA in nasal cavity and trachea were detected to investigate immunostimulatory activity of dfifferent CpG-ODN on chicken.1The immunostimulatory activity of different CpG-ODN on chicken spleen lymphocytesAccording to the relationship between function and structure features of CpG-ODN, four sequences of CpG-ODN were designed. Six sequences of CpG-ODN in the study were numbered F1to F6. The immunostimulatory activities of different CpG-ODN on the chicken spleen lymphocytes were analyzed using a WST-8Cell Counting Kit-8(CCK-8). The results showed that the stimulation indices (SI) of all the CpG-ODN (except for CpG-ODN F2) and bacteria CpG-DNA on the spleen lymphocytes were significantly higher than that of PBS (P<0.01), and no significant differences were detected among these sequences, whereas the SI of CpG-ODN F1, CpG-ODN F3and CpG-ODN F6were significantly higher than that of bacteria CpG-DNA (P<0.05). These results demonstrated that the designed CpG-ODN had much better immunostimulatory effects on chicken spleen lymphocytes.2The immunostimulatory activity of different CpG-ODN on chicken BM-DCsThe chicken BM-DCs were stimulated respectively by different CpG-ODNs or bacteria CpG-DNA for24hours. Then the expression levels of the CD40and CD86on the surface of cells were detected by the flow cytometry. The results showed that among all the groups, the designed CpG-ODN F3showed the strongest ability to up-regulate the expression of CD40and CD86molecules. In addition, the BM-DCs stimulated by CpG-ODN F3showed the strongest ablity to induce the T-cell proliferative response.The results demonstrated that the designed CpG-ODN could induce activation and maturation of chicken BM-DCs efficiently, and CpG-ODN F3had the strongest ability to stimulate chicken BM-DC maturation.3The effect of different CpG-ODN on local mucosal immunity of chickensThe real time PCR was applied to detect the expression levels of IL-6mRNA, IL-12mRNA and IFN-ymRNA in nasal cavity and trachea. The results showed that the expression levels of IL-6mRNA, IL-12mRNA and IFN-ymRNA in nasal cavity and trachea from chikens immunized with IAIV plus CpG-ODN (except for CpG-ODN F2) or CpG-DNA were significantly increased compared with that immunized with IAIV only (P<0.01). In addition, the expression levels of these cytokines in nasal cavity and trachea were much higer after immunization with IAIV respective plus CpG-ODN F1, CpG-ODN F3, CpG-ODN F6and CpG-DNA.The level of AIV-specific IgA antibody in the lavage fluid of nasal cavity and trachea was detected by indirect ELISA. The results showed that the levels of AIV-specific IgA antibody in nasal lavage fluid of all groups reached peak at the5th week after the first immunization, and that was the highest in the chickens receiving IAIV and CpG-DNA. The levels of AIV-specific IgA antibody in the tracheal lavage fluid of all groups reached peak at the3rd week after the first immunization, and that was the highest in the chickens receiving IAIV and CpG-ODN F3or CpG-DNA. In addition, after immunization with IAIV respective plus different dosages of CpG-ODN F3, the level of AIV-specific IgA antibody was the highest at the moderate dose,.The results of two essays above prompted that intranasal immunization with IAIV and designed CpG-ODN (especially CpG-ODN F3) could significantly increase the local mucosal immunity in respiratory tract.4The effect of different CpG-ODN on systemic immunity of chickensThe level of AIV-specific HI antibody in serum was detected by HI test. The results showed that the HI degree in the chickens receiving IAIV and CpG-ODN (except for CpG-ODN F4) or CpG-DNA reached peak at the4th week after the first immunization, whereas that in the chickens receiving IAIV alone was always quite low after the first immunization.The level of AIV-specific IgG antibody in the serum was detected by indirect ELISA. The results showed that the levels of IgG antibody in serum from chickens receiving IAIV and CpG ODN (except for CpG-ODN F2) or CpG-DNA were significantly higher than that from chickens receiving IAIV alone (P<0.01). The levels of IgG antibody in serum were much higher in the chickens receiving IAIV and CpG-DNA, CpG-ODN F1, CpG-ODN F3or CpG-ODN F6.Besides, after immunization with IAIV respective plus different dosages of CpG-ODN F3, the HI degree and IgG antibody levels in serum were the highest at the moderate dose.The results of two essays above prompted that intranasal immunization with IAIV and designed CpG-ODN (especially CpG-ODN F3) could improve the systemic immunity level at a certain extent.5The effect of different CpG-ODN on the expression level of chicken TLR21mRNAThe real time PCR was applied to detect the expression level of TLR21mRNA in nasal cavity and trachea of chicken. The results showed that the expression levels of TLR21mRNA in nasal cavity and trachea from chickens receiving IAIV and CpG-ODN (except for CpG-ODN F2) or CpG-DNA were significantly higher than that immunized with IAIV only (P<0.01). In nasal cavity, the expression level of TLR21mRNA in the chickens immunized with IAIV and CpG-ODN F3was the highest. In trachea, the expression levels of TLR21mRNA in the chickens immunized with IAIV respective plus CpG-ODN F3and bacterial CpG-DNA were the highest but no significant changes were deteced between them (P>0.05). The study suggested that the designed CpG-ODN could activate the TLR21and up-regulate the expression of TLR21mRNA, and CpG-ODN F3had the strongest ability to up-regulate the expression of TLR21mRNA. |
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