中华鳖细菌疫苗免疫佐剂的筛选及其初步应用

Selection and application of immunization adjuvant in four-strain inactivated bacterial vaccines in soft shelled turtle

将分离自中华鳖Trionyxsinensis主要细菌性疾病——肠道出血性败血症、鳖穿孔病(疖疮病)、红脖子红底板病、鳖腐皮病的4株病原菌,在体积分数为3.5%的福尔马林溶液中灭活24 h。在灭活的4株菌中添加不同佐剂,充分混匀后分别制备成总含菌量为6×109个/mL的4价菌苗,分别免疫8只ICR小鼠,并设不加佐剂免疫组及空白组作为对照。经3次免疫后,随机选3只小鼠抽血制备血清,用间接ELISA方法测定抗体效价,除空白对照组外,其它4组小鼠的免疫血清效价为1∶1.6×104～1∶2.56×105,各免疫组的效价依次为弗氏佐剂>白油佐剂>蜂胶佐剂>不加佐剂。另外5只小鼠用于免疫保护性试验。结果表明:3种佐剂的免疫保护率均为100%,而不加佐剂组免疫保护率为80%,空白对照组死亡率为100%。考虑到弗氏佐剂的成本较高且白油佐剂有副作用,故选择蜂胶作为免疫佐剂,制备了中华鳖4价菌苗。用该菌苗免疫接种50只体重为100～150 g/只的健康中华鳖,注射剂量为0.2 mL/只,并设置对照组(注射等量的生理盐水),共接种20只健康中华鳖。分别于免疫后第20、30、40、50天随机采集3只受免疫的中华鳖和对照组的1只中华鳖血液并制备血清,用浙江省淡水水产研究所制备的针对中华鳖IgM的单抗3H3介导的ELISA方法,测定血清中的抗体效价。结果表明:当中华鳖免疫后40 d,血清中抗体效价达到最高,为1∶6.4×104;免疫后50 d将受免中华鳖分成4组,分别用4种菌株以5LD50(9×106个/mL)进行攻毒试验(0.5 mL/只),结果免疫保护率均达到100%,而对照组死亡率均为100%。

Four strains of Aeromonas sobria isolated from the cultivated soft - shelled turtle with bacterial diseases were inactivated with 3.5% formalin for 24 hours, and three different substances A, B and C were chosen to be adjuvants. The A as a common adjuvant was made by white oil and Tween- 80, B as propolis adjuvant and C Freund＇s adjuvant buyable including complete and incomplete. The proper four - strain inactivated bacteria vaccines （6 × 109cfu/mL）mixed with A, B and C to immunize 8 ICR mouse separately, and the adjuvant- free group and blank group were designed as a control. The immune sera of three mice were collected after three time immunization in the four groups. The multi -clone antibodies in the groups sera were proved to have high ELISA titers between 1：1.6 × 10^4 and 1：2.56 × 10^5 except that of the blank control. The titers of immune sera of the immunized groups with fourstrain inactivated bacterial vaccines were appending C, A, B and adjuvant -free groups in turn from high to low as C, A, B in turn. Considering the expensive price of the adjuvant C and the side effect of adjuvant A, the adjuvant B was the best. Propolis adjuvant was chosen as immune adjuvant of the four inactivated bacterial vaccines above with concentration 6 × 109cfu/mL. Fifty healthy turtles were immunized with the vaccines above by intramuscular injection in the hind leg at a dose of 0.2 mL individually. The turtles in the blank group were injected with the same dose with saline water. The immune sera of the three turtles were collected from 10 days to 100 days after the injection. The ELISA titers of the multi - clone antibody against the vaccines were evaluated with TAS - ELISA, and showed that the ELISA titer was 1 ： 1.28 × 104 reaching a peak on the 40^th day after the administration. The Relative Survival Percent （ RSP, % ） of the turtles were found 100% on the 50^th day after immunization, indicating that the vaccine had a good protection and a practical prospect.