【作者】 冯守明；

【导师】 战文斌；

【作者基本信息】 中国海洋大学 ， 水产养殖， 2008， 博士

【摘要】 大菱鲆(Scophthalmus maximus)是珍贵的海洋经济鱼类,目前已经在我国大规模养殖。随着高密度工厂化养殖的迅速发展,诸多疾病相继出现,造成了重大的经济损失,其中,腹水病是危害最大的疾病之一,其病原是迟缓爱德华氏菌(Edwardsiella tarda)。目前,对鱼类细菌病的防治方法主要采取频繁使用抗生素及化学药品的方法,其所带来的耐药性、食品及环境安全问题愈加突出。因此,免疫成为防治鱼类细菌性疾病的安全有效方法。本文采用组织制片观察、单克隆抗体免疫组化标记、间接荧光法和流式细胞术等手段,首先,对不同年龄的养殖大菱鲆粘膜及淋巴细胞进行研究,描述并比较了不同年龄大菱鲆的粘膜及免疫组织的细胞学基础;然后,以置备的迟缓爱德华氏菌灭活全菌和主要OPM为抗原,对大菱鲆进行腹腔注射、直接浸泡和肛门灌注免疫接种,以Ig+细胞相对数量及血清特异抗体凝集效价为指标,对大菱鲆粘膜免疫与系统免疫应答进行评价和比较,阐述了大菱鲆粘膜组织具有相对独立的特异性免疫应答功能,以及不同抗原的免疫效果。具体研究内容和结果如下:(1)大菱鲆粘膜及免疫组织的细胞学基础研究。组织切片显微观察发现,大菱鲆皮肤、鳃、后肠、头肾和脾中存在淋巴细胞。利用大菱鲆IgM单抗免疫组化标记发现,除皮肤外,鳃、后肠、头肾和脾中均原位存在Ig+细胞,其中,鳃组织中Ig+细胞存在于鳃小片的上皮与支持细胞之间的间隙中,后肠Ig+细胞存在肠道的固有层和粘膜下层中,外周血Ig+细胞主要存在于淋巴细胞群中,脾Ig+细胞分布在实质区,且更倾向于分布在脾窦周围,头肾Ig+细胞无规律地散布于肾组织中。采用间接荧光标记和流式细胞仪对受精后2.5和10月龄大菱鲆的皮肤、鳃、后肠、外周血和头肾中Ig+细胞进行检测、分析后发现,同一年龄大菱鲆的不同组织中Ig+细胞的数量不同,同时,不同年龄鱼的同一组织的Ig+细胞数量也有差异。2.5月龄大菱鲆Ig+细胞分布为:皮肤0.00±0.00%、鳃1.58±0.40%、后肠2.53±0.70%、外周血17.0±0.39%和头肾21.0±1.79%;10月龄鱼Ig+细胞分布为:皮肤0.94±0.74%,鳃1.60±0.71%、后肠3.91±1.06%、外周血28.95±0.39%和头肾21.54±0.90%。(2)迟缓爱德华氏菌的分离、鉴定及抗原的制备。从患腹水病大菱鲆体内分离纯化出主要病原菌株,用生化鉴定及16S rRNA基因序列测定和分析方法鉴定为迟缓爱德华氏菌;用0.5%福尔马林灭活法,制备了安全的迟缓爱德华氏菌灭活全菌抗原;用Sarkosyl法,制备了安全的迟缓爱德华氏菌主要OPM抗原。(3)大菱鲆粘膜及淋巴细胞对迟缓爱德华氏菌灭活全菌抗原的免疫应答。直接浸泡后,鳃组织中的Ig+细胞数量的出现峰值时间比外周血、头肾组织提前7天,鳃中的Ig+细胞数量极显著高于未免疫组,而外周血和头肾的Ig+细胞数量与未免疫组大菱鲆差异不显著;腹腔注射后,鳃组织Ig+细胞数量的出峰值时间与外周血、头肾相同,鳃中Ig+细胞数量与对照组差异不显著,且极显著低于浸泡免疫组,外周血和头肾的Ig+细胞数量则极显著高于对照组及浸泡免疫组。另外,浸泡免疫后,免疫鱼体针对全菌抗原的血清抗体效价显著高于对照组,且其达到峰值的时间与浸泡免疫后鳃组织中Ig+细胞的峰值时间相同,表明浸泡免疫后大菱鲆的血清特异抗体主要来源于鳃组织中Ig+细胞的特异性免疫应答。(4)大菱鲆粘膜及淋巴细胞对不同抗原的免疫应答。用迟缓爱德华氏菌灭活全菌及主要OPM分别对大菱鲆进行腹腔注射、直接浸泡和肛门灌注免疫后发现,注射OPM的大菱鲆外周血中Ig+细胞数量和血清抗体效价均不显著低于注射迟缓爱德华氏菌灭活全菌大菱鲆;浸泡OPM大菱鲆鳃中Ig+细胞数量和血清抗体效价均不显著高于浸泡灭活全菌的大菱鲆,肛门灌注OPM大菱鲆后肠、外周血中Ig+细胞相对数量和血清抗体效价均不显著高于注射灭活全菌大菱鲆。本文研究结果表明,粘膜组织具有特异性免疫应答的组织细胞学基础,对抗原可以产生显著的相对独立的特异性免疫应答;大菱鲆体内存在粘膜免疫和系统免疫,不同免疫途径对不同免疫组织的诱导作用不同,浸泡免疫主要诱导体表粘膜组织的免疫应答,而注射免疫则主要诱导系统组织的免疫应答,肛门灌注则可能对肠粘膜和系统免疫组织的诱导作用相近;另外,大菱鲆的粘膜和系统免疫组织对不同抗原的特异性免疫应答水平亦不同。本研究结果对鱼类免疫途径的选择及鱼用疫苗的研制具有重要参考价值。更多还原

【Abstract】 Turbot (Scophthalmus maximus) is an economically important fish and recently culturing has become popular in China. However, with the development of intensive culture, infectious diseases have become a major obstacle to the cultivation of it and caused a big loss. The swollen abdomen is the most important disease of turbot, and its agent is the Edwardsiella tarda. Problems, such as drug resistance, food and environmental security, have emerged due to over using of aquacultural drug to prevent and cure infectious diseases. Therefore, immunization has become the most important method in preventing and curing fish diseases. In this study, with methods of micro observation, immunohistochemistry, indirect fluorescence analyze technology, flow cytometry and agglutination tires analyze, the lymphoid tissues and cells were observed and compared in mucosal and systemic lymphoid organ in turbot, Ig+ cells and plasma antibody level were measured and compared in unvaccinated turbots and vaccinated ones by inactivated E. tarda and OPM via direct immersion (d.i.), intraperitoneal injection (i.p.) and anal incubation (a.i.). The immune responses were evaluated in mucosal and systemic lymphoid tissues in turbot vaccinated by different antigen via different route. The independent immunological role of mucosal tissues was discussed. The following are the details.(1) The tissue observation of mucosal and systemic immune organs of turbot. Lymphocytes presented in skin, gill, hindgut, head kidney, spleen and peripheral blood (PBL) after micro observation to tissue sections. The immunoglobulin-positive cell (Ig+ cell) could be locally observed in these tissues above except in the skin by immunohistochemistry with a monoclonal antibody (mouse-anti-turbot IgM). Ig+ cells of gill were located in filament sinusoid between the epithelial cells and the support cells. Ig+ cells of hindgut presented within the lamina propria or the lower layers of lamina epithelialis. Ig+ cells in PBL often appeared among lymphocytes. Ig+ cells were often distributed throughout the parenchyma in spleen, but tended to concentrate around melano-macrophage centres (MMCs). Ig+ cells scattered in the interstitial tissue in head kidney. The Ig+ cells isolated from skin, gills, hindgut kidney and PBL were measured in healthy turbots (aged 2.5 and 10 months post-fertilization, mpf) by flow cytometry. Results were that the percentage of Ig+ cells respectively was 0±0.00% of skin, 1.58±0.40% of gills, 2.53±0.70% of hindgut, 17.05±0.39% of PBL, 21.06±1.79% of kidney in turbot aged 2.5 mpf and was 0.94±0.74% of skin, 1.60±0.71% of gills, 3.91±1.06% of hindgut, 28.95±0.39% of PBL, 21.54±0.90% of kidney in turbot aged 10 mpf.(2) Isolation and identification of E. tarda and preparation of antigens. The main pathogenic bacteria was isolated from turbot suffered swollen abdomen. Biochemical identification and 16S rRNA gene sequence analysis were used to identify the main pathogenic bacteria. The results showed that it was identified as E.tarda. Then the killed cell antigen was prepared with method of 0.5% formalin-killing. The major OPM antigen was also prepared with the extraction method of Sarkosyl. (3) The immune response of mucosal and systemic lymphoid tissues in turbot after immunization with inactivated E. tarda. After d.i., a 7-day advanced phase in peaking time of number of Ig+ cells was observed in gill compared with in PBL or kidney from immunized turbot. A high elevation of number of Ig+ cells was observed in the gill, and this was significantly different from the control group’s. However, a slight elevation in the number of Ig+ cells was observed in the kidney and PBL in vaccinated turbots via bath, and this was insignificantly different from their respective control group’s. After i.p., the peaks of number of Ig+ cells in gills, PBL and kidney reached on the same time point from immunized turbot. A slight elevation of Ig+ cells was observed in the gills from turbot vaccinated by i.p., and the increase was not significant compared with its control’s and was significantly lower than vaccinated turbot via bath. But, a clear increase of the number of Ig+ cells was observed in kidney and PBL in turbot vaccinated by i.p., and a significant difference was observed in the kidney and PBL compared with their respective controls. In addition, the peak of agglutination tire in plasma was on the same time point as the peak of the number of Ig+ cells in gill in turbot vaccinated by d.i, and it was higher significantly than its control’s. This showed that immune response to E .tarda was significant and mainly from gill in turbot vaccinated by d.i..(4) The immune response to different antigen in turbot. After immunization with inactivated E. tarda and major OPM via d.i., i.p. and a.i., the followings were obtained: The number of Ig+ cells in PBL and the plasma agglutination tire was respectively insignificantly lower in turbot vaccinated by OPM than in one vaccinated by inactivated E. tarda via i.p.. The number of Ig+ cell in PBL and the plasma agglutination tire was respectively insignificantly higher in turbot vaccinated by OPM than in one vaccinated by inactivated E. tarda via d.i.. The number of Ig+ cell in hindgut and PBL as well as the plasma agglutination tire was respectively insignificantly lower in turbot vaccinated by inactivated E. tarda than in one vaccinated by major OPM via a.i..Therefore, our results clearly supported the existence of mucosal and systemic compartments in immune system, and the mucosal compartment had tissue and cell conditions to conduct a independent special immune response to an antigen in turbot. The administration route could play an important role in inducing immune response of the two immune compartments, the d.i mainly elicited mucosal immune, the i.p. could induce a more intensive systemic immune, and the a.i. almost had a same role to induce responses in gut mucosal and systemic tissue. Additionally, the different antigen had different immunological role upon mucosal and systemic compartment in fish. These are essential for designing vaccination strategies in fish.更多还原