[鱼師]诺卡氏菌口服疫苗的制备与免疫效果研究

Preparation and Immunoefficacy of Oral Vaccine Against Nocardia seriolae

以海藻酸钠为包被体,[鱼師]诺卡氏菌(Nocardia seriolae)全菌灭活疫苗为内芯,制备[鱼師]诺卡氏菌口服微球疫苗,口灌体质量(45±5)g的珍珠龙胆石斑鱼(Epinephelus fuscoguttatus♀×E.lanceolatus♂)。一周灌3 d停4 d,4周后每尾腹腔注射100μL[鱼師]诺卡氏菌液,攻毒浓度为1×10~8CFU/mL,对照组口服生理盐水微球,然后测定保护率及肝中碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、超氧化物歧化酶(SOD)、溶菌酶(LSZ)以及过氧化氢酶(CAT)活性,用RT-PCR研究相关免疫基因的表达。结果显示,口服疫苗免疫保护率(RPS)为67%,疫苗组ACP活力在免疫1周时达到峰值;CAT活力在免疫后先升高再降低,在免疫1周时达峰值,第3周时仍与对照组差异显著;LSZ活力第2 d、21 d、28 d显著低于对照组;SOD活力整体显著低于对照组;免疫组与对照组相比,口服疫苗免疫后的珍珠龙胆石斑鱼激发TLR信号通路,促进TLR2、TLR3、MyD88基因表达量上调。细胞因子类相关基因IL-12p40、TNF-α转化生长因子TGF-β、免疫球蛋白IgM,及适应性免疫相关基因MHC表达量均有上调,但不同组织上调程度不同。

Oral microsphere vaccine against Nocardia seriolae was prepared using sodium alginate as the coating body and the whole bacterium inactivated as the core and orally gavaged into hybrid grouper(Epinephelus fuscoguttatus♀×E.lanceolatus♂) with body weight of(45 ±5) g 3 times a week for 4 weeks.At the end of the experiment,the hybrid grouper was challenged with Nocardia seriolae at a dose of 100 μL of Nocardia seriola solution at concentration of 1×10^(8) CFU/mL by intraperitoneal injection,the fish in the control group was orally administered by normal saline microspheres and then the protection rate and activities of alkaline phosphatase(AKP),acid phosphatase(ACP),superoxide dismutase(SOD),lysozyme(LSZ) and catalase(CAT) in the liver and the expression of related immune genes was detected by RT-PCR.The results showed that there was protective rate of 67% in the oral vaccine groups.The peak ACP activity was observed in the vaccine group at the first week of immunization,and the CAT activity was increased first and then decreased after immunization.The was significantly lower activity of LSZ in the vaccine group than that in the control group on the 2nd,21st and 28th day,and the activity of SOD was significantly lower in the vaccine group than that in the control group,the oral vaccine induced the up-regulation of TLR signaling pathway in grouper,including the up-regulation of TLR2,TLR3 and Myd88.Cytokine-related genes IL-12p40,TNF-α,transforming growth factor TGF-β,immunoglobulin IgM,and adaptive immune-related genes MHC were shown to be up-regulated,with the different extent of up-regulation in various tissues.