| The present study aims was to investigate mechanism of pCI-chIL-4-chIL-2-EGFP gene adjuvant on chicken coccidiosis vaccine immue effect.The changes in the number of activated T lymphocytes,B lymphocytes,antigen presenting cells in the immune organs(thymus,spleen,bursa of Fabricius)and intestines(duodenum,jejunum,cecal tonsils)of chickens and interleukin 2(IL-2),interleukin 4(IL-4),interferon-γ(IFN-γ)and α-tumor necrosis factor(TNF-α)m RNA and protein content in the blood and tissues,antibody titer of Newcastle disease(ND)and avian influenza(AI)vaccine were detected to comparison of the immune effect of chicken coccidiosis trivalent four strain live vaccine with and without pCI-chIL-4-chIL-2-EGFP on chickens.The test results showed that:(1)The number of activated T cells in thymus medulla,cecal tonsil propria and spleen summary in adjuvant group(T1 group)was significantly or extremely significantly higher than that in vaccine group(C1 group)and blank group(C0 group)(P < 0.05 or P < 0.01)after 7 day inoculation.The number of activated T cells in lamina propria of the intestine(duodenum,jejunum and cecal tonsils),thymus medulla,spleen summary,periarterial lymphatic sheath and splenic cord in the T1 group was significantly or extremely significantly higher than that of the C1 and C0 groups(P < 0.05 or P < 0.01)after 14 day inoculation.The number of activated T cells in lamina propria of the intestine,thymus medulla and splenic cord in T1 group was significantly or extremely significantly higher than that in C1 group and C0 group(P< 0.05 or P < 0.01)after 28 day inoculation.(2)The number of activated B cells in the jejunum and cecum tonsil propria,thymus cortex,splenic cord and medulla of bursa of fabric in T1 group was significantly(P < 0.05)or extremely significantly(P <0.01)higher than that in C1 and C0 groups after 7 day inoculation.The number of activated B cells in lamina propria of three intestinal,thymus cortex,spleen summary and bursa of fabricius(cortex and medulla)of chickens in T1 group was significantly or extremely significantly higher than that in C1 and C0groups(P < 0.05 or P < 0.01)after 14 and 28 day inoculation..(3)The number of antigen-presenting cells in the villous lamina propria of cecum tonsil in T1 group was significantly higher than that in C1 group(P < 0.05),and extremely significantly higher than that in C0group(P < 0.01)after 7 day inoculation.The number of antigen-presenting cells in the villous lamina propria of duodenum and cecal tonsil and spleen summary in T1 group was significantly(P < 0.05)or extremely significantly(P < 0.01)higher than that in C1 and C0 groups after 14 day inoculation.The number of antigen-presenting cells in the villous lamina propria of three intestinal segments,thymus medulla,spleen summary,bursa cortex and medulla in T1 group was significantly or extremely significantly higher than that in C1 and C0 groups(P < 0.05 or P < 0.01)after 28 day inoculation.(4)There were no significant differences in the titers of Newcastle disease and avian influenza antibodies between T1 group and C1 group(P > 0.05)after 7 day inoculation.The antibody titer of Newcastle disease in the T1 group was significantly(P < 0.05)greater than that of the C1 group,and extremely significantly(P < 0.01)greater than that of the C0 group,but there was no significant difference in the antibody titer of avian influenza after 14 day inoculation.The antibody titers of Newcastle disease and avian influenza in T1 group were significantly or extremely significantly higher than those in C1 and C0 groups(P < 0.05 or P < 0.01)after 28 day inoculation.(5)The results of enzyme-linked immunosorbent assay(ELISA)showed that the level of IL-4 protein in T1 group was significantly or extremely significantly higher than that in C1 group and C0 group after 7days inoculation(P < 0.05 or P < 0.01).The level of γ-IFN protein in group C1 was extremely significantly higher than that in group T1 and group C0(P < 0.01)after 14 day inoculation.The protein levels of IL-2,IL-4,γ-IFN and α-TNF in T1 group were significantly or extremely significantly higher than those in C1 and C0 groups(P < 0.05 or P < 0.01)after 28 day inoculation.(6)Fluorescence quantitative PCR detection found that: 7d after vaccination,the expression of IL-2m RNA in muscle and spleen of chickens in T1 group was significantly higher than that in C1 group(P <0.05),IL-4 m RNA in muscle and cecal tonsils was significantly higher than that in C1 and C0 groups(P <0.05),the m RNA levels of IFN-γ in cecal tonsils were significantly(P < 0.05)and extremely significantly(P < 0.01)higher than those in C1 and C0 groups,respectively.The m RNA levels of IL-2 in muscle,spleen,jejunum and caecal tonsil in T1 group were significantly or extremely significantly higher than those in C1 group and C0 group(P < 0.05 or P < 0.01),IL-4 m RNA in muscle,spleen and jejunum was significantly or extremely significantly higher than that in group C1(P < 0.05 or P < 0.01),IFN-γ m RNA in the jejunum and cecal tonsils were significantly or extremely significantly(P < 0.05 or P < 0.01)greater than that in the C1 and C0 groups,TNF-α m RNA in liver,spleen,jejunum and caecal tonsil were significantly or extremely significantly(P < 0.05 or P < 0.01)higher than those in C1 and C0 groups after 14 day inoculation.The m RNA level of IL-2 in jejunum,liver and caecal tonsil in T1 group was significantly or extremely significantly higher than that in C1 group(P < 0.05 or P < 0.01),IL-4 and IFN-γ in liver,spleen,jejunum and cecal tonsils were significantly or extremely significantly higher than(P < 0.05 or P < 0.01)those in C1 and C0 groups.TNF-α m RNA in the liver,spleen and cecal tonsils was significantly or extremely significantly higher than that in C1 and C0 groups(P < 0.05 or P < 0.01)after 28 day inoculation.In conclusion,the pCI-CHIL-4-CHIL-2-EGFP gene adjuvant can promote the proliferation and activation of T cells and B cells in duodenum,jejunum,caecal tonsil,spleen,thymus and bursal of fabricius,and promote the expression of MHC-II molecules in antigen-presenting cells.Gene adjuvant can increase the expression of IL-2,IL-4,IFN-γ and TNF-α genes in chicken body,and can also increase the antibody titer of Newcastle disease and avian influenza vaccine. |
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