Study On Immune Enhancement Of Bacterial CpG DNA In Chicken Inoculated With Inactivated Avian Influenza Virus H9

Bacterial DNA has immunostimulatory effects on both innate and adaptive immune responses in numerous species including human and animals. It may directly activate B cells, monocytes macrophages, and dendritic cells in vitro or in vivo by upregulating the expression of costimulatory molecules that drive immune responses and secrete a variety of cytokines, including high levels of interleukin(IL)-12, IL-1, and tumor necrosis factor (TNF)- . Bacterial DNA can also activate natural killer (NK) cells and T cells indirectly. The activity was attributed to CpG motifs including unmethylated CpG nucleotides flanked by specific bases which are common in bacterial DNA and considerably less common in vertebrate DNA. Our Lab once confirmed that bacterial DNA from a E Coli Yak strain has a strong immunostimulatory activity. This thesis was aimed to further study the immune enhancement in chickens as a novel vaccine adjuvant. Furthermore, the production technique was studied.1. E Coli Yak strain was used as the reference strain to study the best production technique. Total DNA was respectively extracted from the bacteria with 3 kinds of DNA extraction methods including STET, SDS-CTAB and STET-CTAB. The result showed the extracted DNA using 3 kinds of methods accorded with essential demand of the quality standards of the product. All of the products induced a higher level of T lymphocyte proliferation than did the blank control group. The recovery rate of DNA was the higest for STET-CTAB (3.0mg/100 ml bacterial culture) with the most value of A260/A280 (1.95).2. 15-day-old chickens were immunized by the combination of the inactivated virus with bacterial CpG DNA. The proliferation of both T and B lymphocytes was detected by XTT colorimetric assays, and anti-ATV H9 antibodies were tested by hemagglutination inhibition (HI) assays. The results showed that the chickens inoculated with the inactivated virus combined with CpG DNA (or CpG-DNA/AIV H9) showed a higher level of antibody toAIV H9 and stronger proliferation of T and B lymphocytes than those of the groups innoculated the inactivated virus alone. The survival rate was 88.9% for the group with bacterial CpG-DNA/AIV H9 and 30.0% for the group with AIV H9 alone and 0.00% for the blank control group. Therefore the bacterial CpG DNA induced a stronger immune response as an adjuvant of ATV H9 in chicken.3. The immunostimulatory effects of bacterial DNA of E.coli Yak strain and two plasmid pUC18, pcDNA3.1 were compared by acting as adjuvants of inactivated Avian Influenza virus H9 vaccine. The results showed that proliferation of both T and Bcells,antibody responses to inactivated AIV H9 vaccine, and survival rate in groups with bacterial CpG DNA or plasmids as adjuvants were much higher than those in the group injected with the virus alone. These results obviously indicated that bacterial CpG DNA was a strong adjuvant that can significantly enchance the immunogenicity of inactivated AIV H9 vaccine in chicken.4. Different doses of bacterial CpG DNA were applied to investigate dose-dependent effects for chicken immunization. 15-day-old chickens were immunized by combination of inactivated virus with bacterial CpG DNA from 25 g, 50 g, 75 g, and 100 g per chicken. The proliferation of both T and B lymphocytes was detected by XTT colorimetric assays, and anti-AIV H9 antibodies were tested by hemagglutination inhibition (HI) assays. The results showed that all of the immunized groups were detected apparently improved humoral and cellular immune response. All the chickens inoculated with inactivated virus and CpG DNA showed a higher level of antibody to AIV H9 and proliferation of T and B lymphocytes than those of blank group. Although bigger dosage of bacterial CpG DNA enhanced stronger immune response, there were no significant difference among groups with various doses and 25p.g per chicken of bacterial CpG DNA could stimulate apparently stronger immune responses to ATV H9 than those in blank control group.5. Bacterial DNA and mineral oil adjuvant were separately...