养殖鲆鲽类细菌性病原的鉴定及免疫的初步研究
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摘要
随着大菱鲆为主体的鲆鲽类养殖产业规模的扩大和集约化程度的不断提高，病害日益严重，成为其生产发展的主要制约因素。
2006年麦岛养殖牙鲆出现持续死亡，典型症状是体表或鳍基部溃烂，从病鱼体内分离到一株病原菌20060928-1，革兰氏阴性，直或弯短杆状，具极生单鞭毛，可运动，生理生化特征及16S rDNA序列分析菌鉴定为Vibrio harveyi。该菌感染牙鲆LD_(50)为2.02×10~6 CFU／尾，药敏试验表明菌必治、头孢唑肟对其有较强的抑制作用。
测定培养基初始pH、不同碳源对鳗弧菌生长的影响，利用均匀设计优化培养基配方，确定最佳培养条件初始pH为6.0，含NaCl 19‰，蛋白胨1.5％，蔗糖1.72％；选取4℃，0.40％福尔马林作用24 h作为其灭活条件，攻毒试验证实所制得疫苗是安全的。
为了从特异性免疫方面评价疫苗的效果，建立了一种双抗原夹心ELISA检测大菱鲆血清抗体效价的方法，该法采用抗原包被微孔板，待血清样品中抗体与包被抗原形成特异性结合物后，再加入HRP标记的抗原，形成抗原-抗体-酶标抗原复合物，利用底物(OPD)显色。与凝集法相比，该法灵敏度较高，且不需要制备二抗，操作步骤简单。
研究几种佐剂对大菱鲆的安全浓度范围，进行不同浓度梯度的浸浴免疫，四周后取其血清检测溶菌酶活力、蛋白含量及抗体效价，攻毒后计算免疫保护指数(PI)，注射组PI为33.6，****含量为39.3mg╱L的浸浴组也有一定效果，其PI值为6.61。
With the high stocking sensity and poor management of flatfish, diseases occurredseriously, and had becme the main factor restricting its development.
The cultured flounder died continually at Maidao in 2006, and the typicalsymptom was ulcer of body surface and fins. The pathogenic strain 20060928-1 wasisolated from the diseased fish. It was Gram-negative, right or curved rods shape,motile with a polar flagella, and it was shown from the conventional physiological,biochemical test and 16S rDNA sequence analysis that 20020928-1's characteristicswere very similar to those of Vibrio harveyi. The infection experiment showed LD_(50)value was 2.02×10~6 CFU/tail for flounder, only Ceftriaxone sodium and Ceftizoximewere effective to inhibit the growth of this pathogenic bacterium.
Effects of initial pH and different carbon sources on the growth of Vibrioanguillarum were studied. The optimized recipe of media contains sodium chloride19‰, peptone1.5%, sucrose 1.72%. The culture was inactivated for 24 h by 0.40%formalin under 4℃, and attack test was done to ensure the safety of vaccine.
To determine the titer of serum antibody, a new method was developed. Firstly,The microplates was coated with antigen, then labeled antigen was added whenantibody in serum samples had conjugated antigen. After antigen-antibody-antigencomplex had formed, Phenylenediamine (OPD) was used as substrate to color. Theprinciple of double antigen sandwich was used to detect serum antibody titers, whichwas simpler than microplate agglutination and had higher sensitivity.
Safety concentration of several adjuvants was studied, and then the immunizationexperiment of different gradients by bath immersion was done. Activity of lysozyme,protein content and antibody titer were detected. Two groups had better resultsaccording to Protection indices (PI) after attacking. One group is immunized byintraperitoneal injection (PI is 33.6), and another is immunized by bath immersion with33.9mg/L ****(PI is 6.61).
2006年麦岛养殖牙鲆出现持续死亡，典型症状是体表或鳍基部溃烂，从病鱼体内分离到一株病原菌20060928-1，革兰氏阴性，直或弯短杆状，具极生单鞭毛，可运动，生理生化特征及16S rDNA序列分析菌鉴定为Vibrio harveyi。该菌感染牙鲆LD_(50)为2.02×10~6 CFU／尾，药敏试验表明菌必治、头孢唑肟对其有较强的抑制作用。
测定培养基初始pH、不同碳源对鳗弧菌生长的影响，利用均匀设计优化培养基配方，确定最佳培养条件初始pH为6.0，含NaCl 19‰，蛋白胨1.5％，蔗糖1.72％；选取4℃，0.40％福尔马林作用24 h作为其灭活条件，攻毒试验证实所制得疫苗是安全的。
为了从特异性免疫方面评价疫苗的效果，建立了一种双抗原夹心ELISA检测大菱鲆血清抗体效价的方法，该法采用抗原包被微孔板，待血清样品中抗体与包被抗原形成特异性结合物后，再加入HRP标记的抗原，形成抗原-抗体-酶标抗原复合物，利用底物(OPD)显色。与凝集法相比，该法灵敏度较高，且不需要制备二抗，操作步骤简单。
研究几种佐剂对大菱鲆的安全浓度范围，进行不同浓度梯度的浸浴免疫，四周后取其血清检测溶菌酶活力、蛋白含量及抗体效价，攻毒后计算免疫保护指数(PI)，注射组PI为33.6，****含量为39.3mg╱L的浸浴组也有一定效果，其PI值为6.61。
With the high stocking sensity and poor management of flatfish, diseases occurredseriously, and had becme the main factor restricting its development.
The cultured flounder died continually at Maidao in 2006, and the typicalsymptom was ulcer of body surface and fins. The pathogenic strain 20060928-1 wasisolated from the diseased fish. It was Gram-negative, right or curved rods shape,motile with a polar flagella, and it was shown from the conventional physiological,biochemical test and 16S rDNA sequence analysis that 20020928-1's characteristicswere very similar to those of Vibrio harveyi. The infection experiment showed LD_(50)value was 2.02×10~6 CFU/tail for flounder, only Ceftriaxone sodium and Ceftizoximewere effective to inhibit the growth of this pathogenic bacterium.
Effects of initial pH and different carbon sources on the growth of Vibrioanguillarum were studied. The optimized recipe of media contains sodium chloride19‰, peptone1.5%, sucrose 1.72%. The culture was inactivated for 24 h by 0.40%formalin under 4℃, and attack test was done to ensure the safety of vaccine.
To determine the titer of serum antibody, a new method was developed. Firstly,The microplates was coated with antigen, then labeled antigen was added whenantibody in serum samples had conjugated antigen. After antigen-antibody-antigencomplex had formed, Phenylenediamine (OPD) was used as substrate to color. Theprinciple of double antigen sandwich was used to detect serum antibody titers, whichwas simpler than microplate agglutination and had higher sensitivity.
Safety concentration of several adjuvants was studied, and then the immunizationexperiment of different gradients by bath immersion was done. Activity of lysozyme,protein content and antibody titer were detected. Two groups had better resultsaccording to Protection indices (PI) after attacking. One group is immunized byintraperitoneal injection (PI is 33.6), and another is immunized by bath immersion with33.9mg/L ****(PI is 6.61).
引文
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