【作者】 张生；

【导师】 曾忠良； 李安兴；

【作者基本信息】 西南大学 ， 基础兽医学， 2007， 硕士

【摘要】 近年来我国水产养殖业发展迅猛，随着高密度、集约化、大规模水产养殖业的出现，鱼类疾病频繁爆发。特别是细菌性疾病种类多、发病率和死亡率高，给水产养殖业造成了巨大的经济损失。由于药物的频繁使用，导致了大量耐药性病原菌的出现，同时也造成大量的药物残留，对人类食品安全及人类健康提出巨大挑战。现代水产养殖业，接种疫苗已被作为评价标准渔业养殖计划的一个重要组成部分，开发环保高效的鱼病疫苗成为当今水产养殖产业的当务之急。本研究主要针对给淡、海水鱼类养殖业带来巨大危害的海豚链球菌（Streptococcus iniae S．iniae）病，以制备的海豚链球菌灭活疫苗为研究对象，主要进行了海豚链球菌灭活疫苗对淡水罗非鱼和海水花鲈鱼的保护性试验。通过本试验筛选了最佳疫苗免疫原用量和最佳免疫佐剂，同时也对药物免疫调节剂在鱼类中应用进行了初步探讨。为海豚链球菌灭活疫苗的推广应用打下了基础。将S．iniae海水株HD-1、HD-4，S．iniae淡水株TBY-1，TBY-5的培养液灭活后，制备弗氏佐剂灭活疫苗，腹腔注射接种罗非鱼(1×10¹⁰CFU×mL^-10.1 mL×尾^-1)，经过两次免疫，初次免疫后35天，分别用对应的菌株攻毒，统计14天内各组鱼的死亡情况，计算各免疫组的相对保护率（RPS），并测定各组鱼血清抗体效价的变化。结果表明：HD-1株弗氏佐剂灭活疫苗保护率达到93.6％，最高血清抗体凝集效价达到1：64；TBY-1株弗氏佐剂灭活疫苗保护率达到100％，最高血清抗体凝集效价达到1：64。海水株HD-1和淡水株TBY-1免疫效果均较好，可作为海水鱼和淡水鱼制苗菌株的候选。利用S．iniae海水株HD-1对大亚湾地区主要的海水养殖经济鱼类石斑鱼、红鳍笛鲷、卵圆鲳鲹、花鲈及美国红鱼进行攻毒试验。结果显示花鲈鱼对S．iniae海水株HD-1最为敏感，可以作为S．iniae海水株HD-1灭活疫苗试验用鱼进行进一步的相关试验。将S．iniae海水株HD-1制备成10¹⁰CFU×mL^-1、10⁹CFU×mL^-1及10⁸CFU×mL^-1三种菌体抗原浓度的白油佐剂灭活疫苗对花鲈鱼进行了免疫保护性试验，攻毒结果显示：10¹⁰CFU×mL^-1组相对保护率为100％、10⁹ CFU×mL^-1组相对保护率为92.5％、10⁸CFU×mL^-1组相对保护率为85％、对照组的死亡率是100％；最高抗体滴度分别为1：16、1：64、1：32。10¹⁰CFU×mL^-1组相对保护率与10⁹ CFU×mL^-1组相对保护率比较，无显著性差异（p＞0.05）；与10⁸CFU×mL^-1组相对保护率为85％比较，10¹⁰CFU×mL^-1组保护率较高，具有显著性差异（p＜0.05）。综合考虑生产实际等各项指标，本试验采用10⁹CFU×mL^-1菌体抗原浓度为制苗用菌体抗原量的浓度。将含10⁹CFU×mL^-1菌体抗原浓度的S．iniae海水株HD-1灭活并制备成白油佐剂疫苗和铝胶佐剂疫苗，对花鲈鱼进行免疫保护性试验，攻毒结果显示：白油佐剂疫苗获得92.5％的相对保护率，铝胶佐剂疫苗组获得82.5％的相对保护率；两种疫苗的最高抗体滴度分别为1：64、1：32。两者比较具显著性差异（p＜0.05）。白油佐剂疫苗获得较好的保护效果，可以有效防制花鲈鱼海豚链球菌病。将左旋咪唑和中药芦荟多糖分别以一定的量加入到10⁹CFU×mL^-1菌体抗原浓度的S．iniae海水株HD-1白油佐剂灭活疫苗中制备成复合佐剂疫苗（compound adjuvant vaccine），同时设白油佐剂疫苗基础上单独注射芦荟多糖组、白油佐剂组及PBS组，对花鲈鱼进行免疫保护性试验，攻毒结果显示：各试验组相对保护率差异性不显著（p＞0.05）；但白油佐剂灭活疫苗加单独注射芦荟多糖组鱼血清抗体滴度比其他各组要高（p＜0.05），而左旋咪唑复合佐剂疫苗组鱼血清抗体效价显著低于白油佐剂灭活疫苗组（p＜0.05），另外各组血清抗体滴度之间比较没有显著性差异。测定各组血清中溶菌酶变化显示，单独注射免疫增强剂芦荟多糖组血清溶菌酶含量明显比其他各组都要高（p＜0.05），试验结果表明：中药免疫增强剂芦荟多糖能刺激鱼类产生非特异性免疫应答反应并提高其血清抗体效价。更多还原

【Abstract】 In decades, with rapid development of aquaculture industry with the high-density and the production on a large scale, the diseases of cultured fish took place frequently, especially the bacteriosis, with high variety, morbidity and mortality, caused huge economic loss to aquaculture. A frequent use of chemical and antibiotic resulted in occurrence of drug- resistant pathogenic strains, simultaneously brought about severe public health and sanitation problems, such as drug residue and food security, to which our human being have to faced and challenged with. In modern aquaculture industry, vaccination will offer a good alternative to strategy of fish disease provention in the highly qualitative fishery cultivation. So developing highly effective vaccine for cultured fish have been an urgent matter in aquaculture industry. In present studies, we mainly focused on Streptococcus iniae, as one of important pathogens in cultured freshwater fish and seawater fish, which caused severe economic losses to the aquaculture industries, developing a vaacine to prevent streptococcosis of fish. The efficacy of S. iniae inactivated vaccine in the freshwater fish tilapia （Oreochromis niloticus） and the sea water Japanese sea perch （Lateolabrax japonicus） were evaluated, the best inoculated doses of antigen were balanced and the best immune adjuvant were choosed. Simultaneously the use of immunostimulants in fish was discussed preliminarily. Our experimental results formed a good scientific basic for the preparation and application of Streptococcus iniae inactivated vaccine in cultured fish.Formalin killed whole-cell vaccines were prepared with sea water strain HD-1, HD-4 and freshwater strain TBY-1, TBY-5 of S. iniae respectively. Tilapia were vaccinated with vaccine (1×10¹⁰ colony-forming units of bacterial per fish) mixed with equal doses in volume of Freund’s ajuvant by intraperitoneal （i.p.） injection, through twice immunization to fish, the protective efficacies of four kinds of vaccines were compared by challenge of fish with the corresponding strain on the 35th day after the first vaccination. The number of fish died in each group was recorded in 14 days, the relative percent survivl （RPS） of each immune group was statisticsed and the titer of serum antibody in each group fish was detected by bacterial agglutination assays to be determined the efficacy of vaccine on fish. The results showed that the Freund’s ajuvant vaccine of S. iniae sea water strain HD-1 could present 93.6% RPS for vaccinated fish and the serum antibody titer of immunized fish reached 1:64; TBY-1 strain vaccine had a 100% RPS with high serum antibody titer by 1:64. As a result, we suggested that HD-1 and TBY-1 strain could be used as the candidate strains to prepare vaccine of cultured fish.To detect the sensitivity of mariculture fish to S. iniae, Epinephelus coioides, Sciaenops ocellatus, Lutjanus erythopterus, Trachinotus ovatus and Lateolabrax japonicus, as main mariculture species in Guangdong, were infected with HD-1 strain. The result showed that Lateolabrax japonicus were most sensitive to S. iniae seawater HD-1 strain, which would be as animal model for inactivated S. iniae vaccine experiment.Three types of inactivated S.iniae HD-1 strain vaccine in bacterial concentration of 1×10¹⁰ CFU×rnL^-1,1×10⁹ CFU×mL^-1 and 1×10⁸ CFU×mL^-1 mixed with equal volume of white oil adjuvant were prepared. After immunized Japanese sea perch （L. japonicus） had been challenged with corresponding virulent strain, the fish vaccinated with 10¹⁰ CFU×mL^-1, 10⁹ CFU×mL^-1, 10⁸ CFU×mL^-1 doses showed high immune protection with RPS of 100%, 92.5%, 85% and high antibody titer with 1:16, 1:64, and 1:32 respectively, while all fish in non-immnized control group died. Antigen dose of 10⁹ CFU×mL^-1 concentrations was most suitable for S.iniae HD-1 strain vacine to immunize: maricultured fish.To detect immunoprotection of S.iniae HD-1 strain vacine to Japanese seap erch （L. japonicus）, two type of vacine with a dose of 1×10⁹ CFUxmL^-1 killed HD-1 strain mixed with equal volume of white oil and alhydrogel adjuvant respectively were prepared and used to vacinate 40 tail fish. After experimenal fish were challenged with corresponding virulent stains, the group immunized with white oil and alhydrogel adjuvant vaccine had high immuoprotection with RPS of 92.5% and 82.5%, and high antibody titers with 1:64 and 1:32 respectively, while all fish in non-immnized control group died. So we thought that white oil adjuvant vaccine could elicit good immune effect and protect Japanese sea perch from streptococcicosis.Respectively added the Levamisole and the aloe polysaccharide of traditional Chinese medicine by a certain quantity into the S.iniae HD-1 strain vacine with a dose of 1×10⁹ CFU×mL^-1 killed HD-1 strain mixed with equal volume of white oil, simultaneously the aloe polysaccharide alone injection group in white oil adjuvant vaccine foundation, the vaccine with white oil adjuvant group and one non-immunized control group. After experimenal fish were challenged with corresponding virulent stains, it was not significant （p＞0.05） difference with RPS in experimental groups. The antibody titer of the aloe polysaccharide alone injection group were higher than the other groups （p＜0.05）; the antibody titer of Levamisole group was lower than the white oil adjuvant group （p＜0.05）, the other gorups were not significant differently. Measurese the changing tendency of antalzyme in each group’s serum showed that the immunoenhancer of aloe polysaccharides alone injection group obviously higher than the other groups （p＜0.05）. our experiments proved that the immunoenhancer of aloe polysaccharides can stimulate fish to produce nonspecific immune response.更多还原