锦鲤腐皮病灭活疫苗制备及使用.docVIP

锦鲤腐皮病灭活疫苗制备及使用 　　摘要：从患有腐皮病的锦鲤（Cryprinus carpio）肝、肾、脾、血液里分离到1株优势菌，对该菌进行纯化培养并经人工感染试验，确定该菌为病原菌。经16S rRNA序列分析，鉴定该病原菌为蜡样芽孢杆菌（Bacillus cereus）。用不同浓度福尔马林和不同温度灭活蜡样芽孢杆菌，筛选其灭活效果并制得灭活疫苗。将灭活疫苗免疫健康锦鲤后测定其抗体效价变化及免疫保护率。结果表明，0.2%的福尔马林溶液在28 ℃下灭活24 h及4 ℃灭活36 h为蜡样芽胞杆菌的适宜灭活条件。首次免疫后第20天，免疫Ⅰ组、Ⅱ组、Ⅲ组、Ⅳ组血清抗蜡样芽孢杆菌的抗体效价达到高峰，分别为1∶128、1∶64、1∶64、1∶32；第30天同样剂量加强免疫后，各免疫组血清抗体效价达到更高水平，分别为1∶256、1∶256、1∶128、1∶64；免疫后第50天时攻毒，Ⅰ组、Ⅱ组、Ⅲ组、Ⅳ组免疫保护率分别为66%、66%、55%、44%。 　　关键词：蜡样芽孢杆菌（Bacillus cereus）；锦鲤（Cryprinus carpio）；灭活疫苗；抗体效价；免疫保护力 　　中图分类号：S942.5 文献标识码：A 文章编号：0439-8114（2016）20-5320-04 　　DOI：10.14088/j.cnki.issn0439-8114.2016.20.038 　　Abstract： A dominant bacteria strain was isolated from the liver，kidney，spleen and blood of diseased Cryprinus carpio and confirmed as pathogenic bacteria strain by purified culture and artificial infection. It was identified as Bacillus cereusr by 16SrRNA sequence analysis. The optimal inactivated condition for preparing the inactivated vaccine of Bacillus cereusr was determined through treated with different concentrations of formalin and temperature on bacteria. The antibody titers of serum and relative percentage of survival（RPS） were determined after immune healthy Cryprinus carpi. The results showed that the optimal inactivation conditions of Bacillus cereusr were 28 ℃ for 24 h or 4 ℃ for 36 h with the concentration of 0.2% formalin. On the 20th day after the first immunization the serum antibody titer of Cryprinus carpi against Bacillus cereus reached the peak and that of immunized groupⅠ，Ⅱ，Ⅲ and Ⅳ were 1∶128，1∶64，1∶64 and 1∶32，respectively. The antibody titers of all immunized groups reached a higher level when got second immunization with the same dose as the first immunization and that of groupⅠ，Ⅱ，Ⅲ and Ⅳwere 1∶256，1∶256，1∶128 and 1∶64 on the 30th day after the first immunization，respectively. The RPS of immunized groupⅠ，Ⅱ，Ⅲ and Ⅳ were 66%，66%，55% and 44% on the 50th day after the first immunization，respectively. 　　Key words：Bacillus cereus； Cryprinus carpio； inact