水貂肺炎病原菌的检测及绿脓杆菌多价灭活疫苗的制备与免疫研究

发布时间：2018-01-20 10:33

  本文关键词： 水貂 肺炎病原菌 药敏试验 绿脓杆菌 多价灭活疫苗　出处：《东北林业大学》2015年硕士论文　论文类型：学位论文

【摘要】：水貂细菌性肺炎是困扰水貂养殖业的难题,为了调查引起该病的主要病原菌并探索安全有效的防治方法,2011年至2013年,对从山东、辽宁两省送检的水貂肺炎病例进行病原菌分离,共分离到38株细菌,结合生化鉴定和分子生物学鉴定方法,最终确定包括31株绿脓杆菌,4株肺炎克雷伯氏菌,1株臭鼻克雷伯氏菌,1株产酸克雷伯氏菌和1株与绿脓杆菌混合感染的鲍曼不动杆菌。绿脓杆菌包括三个血清型G型(22/31)、C型(6/31)、B型(3/31),G型为主要流行血清型。药敏试验结果显示所分离到的38株肺炎病原菌均对亚胺培南和美洛培南敏感,对氨苄青霉素和阿莫西林／克拉维酸耐药。所有分离到的病原菌均对小鼠有较强致病性。取绿脓杆菌G、C、B血清型各一株,做标准抗原,并免疫家兔,采集血清做标准抗体,通过方阵凝集试验确定标准抗原抗体最适反应浓度。将分离到的绿脓杆菌菌株免疫小鼠,采集血清,利用标准抗原检测免疫后小鼠血清效价,最终筛选了PA-L14(G型)、PA-L05(B型)、PA-L06(C型)三株免疫原性较好的菌株作为疫苗候选菌株。以终浓度为0.3%甲醛,经37℃、24h对菌液进行灭活处理。疫苗包含9×109CFU/mL的G型和B型绿脓杆菌,6×109CFU/mL的C型绿脓杆菌,配以20%铝胶佐剂,0.01%的硫柳汞,免疫健康无抗体的水貂进行抗体效价跟踪,结果显示免疫水貂在半月内机体可产生较高水平的抗体,该抗体水平可维持3个月,满足了在绿脓杆菌出血性肺炎流行季节对水貂的保护要求,大规模动物免疫回访结果显示,疫苗速发接种反应发生率低,预后良好,对预防接种和发病后紧急接种,均具有较好的保护性。
[Abstract]:Mink bacterial pneumonia is a difficult problem for mink breeding. In order to investigate the main pathogens causing the disease and explore safe and effective control methods, from 2011 to 2013, from Shandong Province. A total of 38 strains of bacteria were isolated from mink pneumonia cases in Liaoning Province. 31 strains of Pseudomonas aeruginosa were identified by biochemical identification and molecular biological identification. Four strains of Klebsiella pneumoniae, one strain of Klebsiella sinensis and one strain of Acinetobacter baumannii co-infected with Pseudomonas aeruginosa. Pseudomonas aeruginosa consists of three serotypes G (22 / 31). C / C / 31 / B was the main epidemic serotype. The results of drug sensitivity test showed that all the 38 strains of pathogenic bacteria were sensitive to imipenem and meropenem. Ampicillin and amoxicillin / clavulanic acid were resistant to ampicillin and amoxicillin / clavulanic acid. All the isolated pathogens were highly pathogenic to mice. The collected serum was used as standard antibody and the optimum reaction concentration of standard antigen antibody was determined by square array agglutination test. The isolated strains of Pseudomonas aeruginosa were immunized with mice and serum was collected. The serum titer of immunized mice was detected by standard antigen, and the PA-L14(G type PA-L05B was screened out. PA-L06(C type) three strains with good immunogenicity were used as vaccine candidates. The final concentration of formaldehyde was 0.3% at 37 鈩，

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