摘要
大菱鲆(Scophthalmus maximus)在分类学上属硬骨鱼纲(Osteichthyes)、鲽形目(Pleuronectiformes)、鲽亚目(Pleuronectoidei)、鲆科(Bothidae)、菱鲆属(Scophthalmus),为原产于欧洲的名贵经济鱼类,英文名为turbot,在我国俗称“多宝鱼”,是重要的海水经济增养殖鱼类之一。近年来,由于环境污染的日益加剧和养殖业的过度膨胀,各种传染性疾病尤其是病毒病开始广泛传播,病毒性疾病危害最大,并且极难防治。大菱鲆红体病是近年来对大菱鲆养殖业危害较大的一种病毒性疾病,其病原为大菱鲆红体病虹彩病毒(Turbot rddish body iridovirus,TRBIV)。大菱鲆死亡率逐年上升,给大菱鲆养殖业带来了巨大的经济损失。从根本上解决和预防大菱鲆病毒病的关键是要查清病毒的感染途径、感染机理以及研制病毒疫苗对大菱鲆进行免疫预防,而建立新的大菱鲆细胞系是进行病毒研究的理想体外研究体系和制备病毒疫苗的有效方法。
本文采用胰蛋白酶、透明质酸酶和Ⅱ型胶原酶消化法成功启动了大菱鲆肝组织的原代培养,原代培养启动一周后,组织块周围即有大量的细胞迁出,形成明显的生长晕,30天后便可长成细胞单层。通过比较DMEM/F-12和L-15两种培养液,发现DMEM/F-12更适合大菱鲆肝细胞的体外培养。确定了大菱鲆肝细胞的最适培养体系为:20℃下,pH值7.0,添加50μg/mL羧甲基壳多糖、50μg/mL N-乙酰葡萄糖盐酸盐、10ng/mL表皮生长因子(EGF)、10 ng/mL碱性成纤维细胞生长因子(bFGF)和20%胎牛血清的DMEM/F-12培养液。经继代培养后,已传至32代。
病毒感染体外培养细胞引起的细胞病变效应(cytopathic effects, CPE),是病毒在细胞内增殖的一个重要检测指标。本文利用TRBIV的敏感性细胞牙鲆腮细胞(Flounder gill,FG)为繁殖体系,以CPE为判断指标,对分离制备的TRBIV进行了鉴定,并测定其血凝效价作为病毒含量的标准,同时进行了体外最适繁殖条件如病毒接种量,吸附时间及细胞培养条件等的研究。结果表明,病毒接种量、病毒吸附时间、细胞培养条件均对TRBIV的体外繁殖产生明显的影响,如下繁殖条件可使病毒的繁殖速度最快:0.2 mL病毒液(血凝效价为1:64)接种于FG细胞,进行2h吸附后补加10%胎牛血清-MEM培养液,置22oC培养。另外还发现,病毒的感染力均受到病毒的收获时间和病毒液冻融次数的显著影响,在最佳繁殖条件下,收获在细胞中繁殖至第4天的病毒,且只冻融1次,可使其保持最强的感染活力,反复冻融会使病毒的感染活力显著降低。TRBIV的繁殖及其收获条件的确定,为后续大菱鲆病毒疫苗的研制奠定了基础。
在TRBIV的体外最适繁殖条件下,对TRBIV进行了大量增殖,利用传统的甲醛灭活法制备TRBIV灭活疫苗,加入终浓度为0.1%福尔马林,于37℃进行灭活处理48小时,即获得灭活TRBIV疫苗原液,置4℃保存,待使用时再行稀释并加入终浓度为5mg/mL的Al(OH)3佐剂,制成灭活TRBIV疫苗使用液。对TRBIV疫苗的安全性检测结果显示,灭活TRBIV疫苗液不会引起FG细胞病变,也不会造成健康正常大菱鲆染病,证明所得病毒疫苗已被有效灭活,具有高度安全性,可用于大菱鲆的预防免疫。TRBIV疫苗对大菱鲆的免疫保护力检测结果证明,实验室内注射灭活TRBIV疫苗对10尾大菱鲆的免疫保护率为90%,养殖场内浸泡灭活TRBIV疫苗对两个养殖场中50万尾和200万尾大菱鲆的免疫保护率分别为83%和85%,证明所得灭活TRBIV疫苗对大菱鲆具有极高的免疫保护率;血清抗体检测结果显示,TRBIV疫苗接种8天后,大菱鲆体内血清的血凝抑制效价为1∶20,表明TRBIV疫苗诱导大菱鲆产生了抗TRBIV的抗体,可显著增强抗TRBIV感染的免疫力。
Turbot, Scophthalmus maximus, has been one of important farmed fish species in coastal areas of northern China since it was introduced from Europe. However, epizootic disease of farmed turbots in China occurred recently because of intensive aquaculture with high density stocking and improper management. The pathogens were assumed to be parasite, bacteria, or virus. A recent study survey of farmed turbot diseases showed that“turbot reddish body syndrome”(RBS) was found in both juveniles and adults which was caused by a novel virus, turbot reddish body iridovirus (TRBIV). High mortalities of farmed turbot due to RBS diseases led to great economic loss. The best way for prevention is to best understand TRBIV infection, characterization and vaccination of TRBIV. The establishment of healthy and sensitive fish cell line is essential for isolation, identification and characterization of infectious viruses from fish.
To initiate the primary culture of liver cells successfully, turbot liver tissues were digested with trypsin (0.25%), hyaluronidase (0.5%) and Type II callagenase (0.2%). The experiment of using different mediums DMEM/F-12 and Leibovitz-15 for liver cell culture was concluded that the optimal medium was DMEM/F-12 which can supply liver cells better growth and division. Primary culture of this cell type was conducted at pH 7.0, 20℃using DMEM/F-12 medium supplemented with antibiotics, 20% filtered bovine serum (FBS), basic fibroblast growth factor (bFGF, 10 ng/mL), epidermal growth factors (EGF, 10ng/mL), N-Acetyl-D-glucosamine (50μg/mL), and carboxymethyl-chitosan (50μg/mL). The emergence of new cell growth began from the edges of seeded tissue explants and was observed one week later after tissue explanting. The cells grew quite fast, and appeared uniformly fibroblast-like morphology. Thirty days later, the cells grew into confluence in 25 cm2 tissue culture flasks. When the TF cell grew into monolayer, they were subcultured via routine trypsin-digestion method. The TF cells have been subcultured to passage 32.
In this study, TRBIV was separated from the diseased turbot with reddish body syndrome. By using cytopathic effect (CPE) in the form of cell rounding as a judgement index, monolayers cells of Flouder Gill (FG) cell line was used for TRBIV culture and the optimal propagation condition of TRBIV was studied. It was found that the amount of inoculated virus, adsorption time of virus and FG cell culture condition all had great effects on the in vitro propagation of TRBIV. And TRBIV propagated very fast when 0.2 mL virus solution was inoculated, adsorpted for 2 h, and virus inoculated FG cells was cultured in 10% bovine calf serum supplemented MEM medium at 22 oC. It was also found that the harvest time of propagated viruses and times of freeze-thaw of the harvested virus solution had enormous effects on the infective ability of TRBIV. The propagated TRBIV harvested on the 4th days after inoculation and freeze-thawed only once had highest infective ability. The determination of the optimal propagation condition of TRBIV will lay solid foundation for exploiture of turbot virus vaccine.
TRBIV greatly propagated under the optimal condition studied before was killed at 37℃for 48h with final concentration of 0.1% formalin to be used as inactivated vaccine for healthy turbot in this paper. The inactivated TRBIV vaccine was stored at 4℃, and used with Al(OH)3 of 5mg/mL after proper dilution. The detection of successful inactivation by formalin was performed to assure the TRBIV vaccine is safe for turbot and won’t cause fish infected. And the dectection results showed that inactivated TRBIV vaccine could’t infect FG cells and healthy turbot any more. Then healthy turbots were vaccinated with inactivated TRBIV vaccine via muscle injection in indoor culture and immersion in farmed fishpond culture, in the meanwhile, the control groups were inoculated with 0.9% salt water instead of TRBIV vaccine. The results showed that the turbot group vaccinated with TRBIV vaccine has higher survival, and the relative percentage survival is 90% in indoor culture of 10 turbots, while about 83% and 85% in two farmed fishpond culture respectively of 500,000 and 2,000,000 turbots. Moreover specific antibody titres could be detected of 1∶20 at 8 days after injecetion immunization. All of these results concluded that the inactivated TRBIV vaccine was effective for protecting turbots against infection of TRBIV.
引文
曾令兵,杨先乐,贺路等.左文功草鱼出血病细胞培养灭活疫苗旋转瓶法大规模生产工艺.中国水产科学, 1998, 5(2):62-67.
曾晓华.淋巴囊肿病毒的检测及其对牙虾鳃细胞系的感染研究[硕士论文].青岛:中国海洋大学,2004.
陈超然,陈萱,陈昌福等.酵母β-葡聚糖对受免异育银鲫免疫应答的增强作用.华中农业大学学报,2003, 22(4):380-384.
陈春娜,黄颖颖,李英文.大菱鲆常见病毒性疾病及其防治技术.水利渔业, 2006, 26(3):104-105.
陈怀青.鱼类免疫接种的理论与实践.国外水产, 1995, 3:7-10.
陈松林.鱼类胚胎干细胞研究进展.中国水产科学, 2001, 7(4):95-98.
陈秀男,郭光雄.疫苗在鱼病防治上的应用.中国水产(台湾), 1987, 411:32-39.
陈月英,钱冬,沈智华等.淡水鱼类细菌性败血症菌苗浸浴免疫研究.海洋与湖沼, 1998, 29(6): 597-602.
陈在贤,郑坚川,江育林.从患“红脖子病”甲鱼体分离到虹彩病毒.中国兽医学报, 1998, 18(2):135~139.
单晓枫,高云航,李影等.鱼用疫苗研究进展.中国兽药杂志,2005, 39(11): 19-22.
丁建民,赵守友,孙洪丽.鸡痘鹌鹁化弱毒细胞苗病毒释放工艺探索.辽宁畜牧兽医, 2004, (10): 30.
窦海鸽,刘彦,张悦等.大菱鲆病毒性疾病的综合防治对策.渔业现代化,2004, (6): 29-30.
杜平,范中善, Guo BY.病毒的细胞病变效应实验. //杜平.医用实验病毒学.北京:人民军医出版社, 1985: 2829-2833.
樊廷俊,耿晓芬,丛日山等.大菱鲆鳍细胞系的建立.中国海洋大学学报(自然科学版), 2007, 37(5):759-766 .
樊卫平,李敬云.不同血清浓度培养基对HIV产量的影响.军事医学科学院院刊, 2000, 24(1): 69-70.
范文辉,史成银,窦海鸽等.大菱鲆病毒性红体病及其综合防治的研究.科学养鱼, 2005, 1: 48-49.
方勤,肖调义,李旅等.四株草鱼呼肠孤病毒毒株的细胞感染特性比较研究.中国病毒学, 2002, 17(2): 182-184.
费小红,李楠,申红旗,等.鱼用疫苗的研究概况.水利渔业, 2007, 27(6):4-6.
弗雷什尼RI(Freshney RI). 1992.实用动物细胞培养技术(潘李珍,张海燕,高明庚等译. 1996). 北京:世界图书出版公司北京公司. 2-53.
郭玉娟,陈学年. A3α肽聚糖对受免彭泽鲫免疫应答影响的研究.中国预防兽医学报, 2006, 28(5):562-565.
韩志辉,赵永军,武德丽.犬传染性肝炎病毒在犬肾传代细胞中增殖过程的观察研究.青海畜牧兽医杂志, 1994(1): 16-17.
何福林,肖克宇,龚泽修.草鱼出血病病毒血凝作用及影响因素.湖南农业大学学报(自然科学版),2002, 28(1):48-50.
何建国,翁少萍.鱖鱼暴发性流行病病原的研究.中山大学学报(自然科学版), 1998, 37:74-77.
洪锡钧.南方鲶胚胎细胞培养及其细胞周期特性检测.水产学报, 1995, 21(3):240-245.
华鼎可.我国鱼病学研究的现状与进展.现代渔业信息, 1995, 10(3):9-15.
黄琪琰.中国水产动物疾病学研究进展.水产学报, 1996, 20(1):51-57.
姜泽东,周伯文,段晶晶.鱼类细胞培养的研究与应用.北京水产,2008,3(112):42-47.
金璐,金宇龙,王全民.动物疫苗的种类及其评价.吉林畜牧兽医, 2003, 7:32-33.
井上洁,池谷文夫,前野幸男等.养殖真鲷的虹彩病毒感染症.鱼病研究(日本), 1992, 27:19-27.
乐耕耘,吴娟,马雁冰等.戊型肝炎病毒在人胚肺二倍体细胞KMB17等传代细胞中的繁殖.中国医学科学院学报, 2001, 23(6): 590-593.
雷霁霖.大菱鲆的引进和新型养殖产业的发展.中国渔业经济, 2003, 5:16—22.
雷霁霖.海水养殖新品种介绍—大菱鲆.中国水产, 2000, 4:38.
李爱华,吴玉深,蔡桃珍等.嗜水气单胞菌和河弧菌二联疫苗对鲫的免疫效果.水生生物学报, 2002, 26(1): 52-56.
李爱玲,徐龙涛,臧鲁心等.犬瘟热病毒在Vero细胞上培养条件的优化研究.山东畜牧兽医, 2004, 1:8—9.
李霞,福田颖穗.金鱼疱疹病毒敏感细胞的体外培养.上海水产大学学报, 2003, 12(1): 12-18.
李向勇.大菱鲆(Scophthalmus maximus)病毒性出血病病毒基因克隆及序列分析[硕士学位论文].青岛:中国海洋大学, 2004.
李亚南,毛树坚.紫外线诱变建立草鱼抗出血病病原病毒AHZC8细胞株.水产学报, 1990.14(2):89-94.
梁文昌,陈美云,宋桂才等.鸡马立克氏病火鸡疱疹病毒冻干苗的生产条件初选.中国兽药杂志, 1998, 32(1): 30-32.
梁文昌,陈美云,宋桂才等.鸡马立克氏病火鸡疱疹病毒冻干苗的生产条件初选.中国兽药杂志,1998, 32(1):30-32.
林晓婷.大菱鲆红体病虹彩病毒的核酸探针检测及病毒感染牙鲆鳃细胞系的研究[硕士论文].青岛:中国海洋大学, 2006.
刘青,赵恒寿.鱼类常用免疫指标及其检测技术.渔业现代化, 2007, 34(3):28-30.
刘新建,李贵生.国内鱼类病毒病研究进展.生态科学, 2004, 23(3):282-285.
罗琳,蔡雪峰,谢碧文.我国养殖水生动物的病毒病.中国水产, 2001, 7:82-83.
牟建青,李贻刚,李峰,等.鹅副粘病毒冻融前后EID50比较.山东家禽,2003(10): 13.
潘杭君,孙红祥.免疫佐剂的研究进展.中国兽药杂志, 2004,38(1):32-37.
钱华金,张四明.鱼类细胞培养在渔业科学上的应用.淡水渔业,1991, (5):40-42.
史成银,王印庚,黄捷等.大菱鲆病毒性疾病研究进展.高技术通讯, 2003, 9:99-105.
史成银,王印庚,黄倢等.大菱鲆病毒性疾病研究进展.高技术通讯,2003, 13(9):99-105.
史成银,王印庚,秦蕾等.我国养殖大菱鲆病毒性红体病及其流行情况调查.海洋水产研究, 2 0 0 5, 26(1):1-6.
孙健和,严亚贤,陈怀青等.嗜水气单胞菌亚单位疫苗的研制.中国兽医学报, 1996,16(1):11-15.
孙建宏.免疫佐剂的研究进展.黑龙江畜牧兽医,1998, 2: 40-42.
孙修勤,洪旭光,张进兴等.牙鲆淋巴囊肿病毒在牙鲆鳃细胞系Fc-9307中的增殖.高技术通讯, 2002, 12:94-96.
帖金凤,张文福.病毒灭活检测技术的研究进展.中国消毒学杂志,2005, 22(4):450-452.
童裳亮.海洋动物的细胞培养与应用.生物工程进展, 1994, 6:46-48.
王广军.虹彩病毒生物学研究现状及展望.湛江海洋大学学报, 2004, 24(3):74-78.
王宏伟,王安利,王维娜等.水产动物细胞培养方法及前景.河北大学学报, 2001, 21(2):198-202.
王淼,胡本强.我国海洋环境污染的现状、成因和治理.中国海洋大学学报, 2006, 5: 1-6.
王世若,王兴龙,韩文瑜.现代动物免疫学(第二版).吉林:吉林科学技术出版社, 2001. 430-438.
王晓红,何建国.鱼类疫苗学研究.淡水渔业, 2001, 31(2): 36-38.
王晓红,何建国.鱼类疫苗学研究.淡水渔业, 2001, 31(2):36-38.
魏运亨.多宝鱼在声讨中奄奄一息30亿元产业链可能断掉中国经济网
吴金炉,曾志南.鱼类病毒病与鱼类病毒疫苗.海洋科学, 1999, 4 : 37-42.
吴淑勤,李新辉,潘厚军等.鲤鱼暴发性传染病病原的研究.水产学报, 1997, 21(增刊):50-56 .
吴志达,谢秀英.鸡传染性支气管炎病毒沪1株在鸡胚肾细胞上繁殖及电镜形态观察.中国兽医杂志, 1983, (7): 6-7.
夏春.鱼病诊断与防治手册.北京:中国农业大学出版社, 1999, 21-32.
夏春.鱼用疫苗的研究进展及评述田.中国兽医杂志, 2000, 26(4):40-41.
肖慧,李军,王祥红等.鲈鱼鳗弧菌病疫苗的制备及免疫防治效果.青岛海洋大学学报, 2003, 33(2):226-232.
肖琳琳,张庆华.蜂胶佐剂对异育银鲫嗜水气单胞菌灭活疫苗的免疫增强试验.海洋渔业, 2004, 26(4): 295-299.
肖显悦,田春雨,李凤华等. DNA疫苗及其在鱼类生产中的应用.生物技术通报, 2006(增刊):125-128.
徐洪涛,朴春爱,姜忠良等.养殖牙鲆淋巴囊肿病病原的研究.病毒学报, 2000, 16(3): 223-226.
许淑英.草鱼出血病细胞培养弱毒疫苗的制备及其免疫效果.水产学报,1994, 18(2):110-117.
杨净思,胡云章,蒋国润等.甲型肝炎减毒活疫苗生产中病毒释放的最佳方法探索.中国公共卫生,2001,17(8):687-688.
杨先乐,陈远新.鱼用疫苗的现状及其发展趋势.水产学报, 1996, 20(2):159-167.
杨先乐,吴明泉.草鱼出血病细胞培养灭活疫苗的研究:生产性免疫试验.水产科技情报, 1992, 19(1):10-14.
杨先乐,夏春,左文功.草鱼出血病细胞培养灭活疫苗的研究-细胞和病毒的扩大培养.淡水渔业, 1992, l:3-7.
杨先乐,夏春,左文功.草鱼出血病细胞培养灭活疫苗的研究—疫苗株的免疫原性及其有效免疫剂量的比较.水产学报, 1989, 13(2):138-143.
杨先乐.草鱼出血病细胞培养灭活疫苗的研究.水产学报, 1989, 13(2): 138-143.
姚泊,何建国.温度对白斑综合症杆状病毒致病力的影响.广州大学学报(自然科学版) , 2002, 1(4):17-19.
姚磊,盛鹏,朱德钟.肾综合征出血热病毒在体外培养的血管内皮细胞中增殖的研究.重庆医学, 2000,29(4): 294-295.
姚智慧,赵洪奎,董关木等.肾综合征出血热病毒感染正常人肝脏组织细胞培养研究.中华实验和临床病毒学杂志, 1995, (1): 19-21.
野中键.重要疾病预防与治疗—真鲷.养殖(日本), 1994, 31(增刊): 155-157. 殷震,刘景华.动物病毒学.科学出版社, 1985.
于大海,陈冠春,王笑梅.小牛血清对鸡传染性法氏囊病病毒增殖的抑制机制.病毒学报, 1988, 4(1): 33-38.
余俊红,沈继红,王祥红等.鳗弧菌口服微胶囊疫苗的制备及其对鲈鱼的免疫效.中国水产科学, 2001, 8(2):76-79.
于淼,管华诗,郭华荣等.鱼类细胞培养及其应用.海洋科学, 2002, 27(3): 4-8.
于秋涛,樊廷俊,汪小锋.宽纹虎鲨软骨细胞体外培养的启动.海洋科学, 2005, 29:33-37.
张立,张元兴,严春俞等.传染性法氏囊病病毒在鸡胚细胞上的优化繁殖.华东理工大学学报, 1996, 22(6): 707-711.
张铭,陈荪红等.淡水白鲳细胞系及其生长温度特性的研究.生物工程学报, 2001, 17(1):105-108.
张念慈,杨广智,等.草鱼吻端组织细胞株ZC-7901及其上皮样细胞亚株ZC-7901S1的建立和特性观察.水产学报, 1981, 6(2):111-120.
张奇亚,李正秋.中华鳖病毒(TSV)对鱼类细胞的感染.中国兽医学报, 2000, 20(1): 15-18.
张奇亚.我国水生动物病毒病研究概况.水生生物学报, 2002, 26(1):89-101.
张寿山,华鼎可译(美.D.P.安德森著).鱼类免疫学(第一版).北京:农业出版社. 1984: 82-113, 131-147.
张义兵.白鳍豚组织培养的初步研究.水生生物学报, 2002, 24(2):193-195.
张元兴,马悦,孙修勤.海洋鱼病疫苗开发与生物反应器大规模生产.高技术通讯, 2002, 2: 1-5.
张卓光,刘启良,王洪彬等. Vero细胞培养流行性感冒病毒的研究.微生物学免疫学进展, 2004, 32(3): 5-8.
赵锴.病毒疫苗的研究现状与展望.第二军医大学学报. 2003, 23:813-815.
郑春静,张铭.鱼类细胞体外培养及其应用.广西水产科技, 2003, 106(1): 15-18.
左颖,姜力,孙德君.不同收获方法对鸡马立克火鸡疱疹病活疫苗蚀斑数的影响.畜牧兽医科技信息, 2005(3): 35.
左颖,姜力,孙德君.不同收获方法对鸡马立克火鸡疱疹病活疫苗蚀斑数的影响.畜牧兽医科技信息,2005, 3: 35.
Ahne W, Bretnont M, Hedrick RP, et al. Special topic review iridoviruses associated with epizootic Haematoietic hecrosis virus (EHNV) in aquaculture. World Microbiology Biotechnology. 1997, 13: 367-373.
Alok D, Sampath KR, et al. Recombinant perciform GnRH-R activates different signaling pathways in fish and mammalian heterologous cell lines. Comparative Biochemistry and Physiology, 2001, 129: 375-380.
Amend, D.F. & D.C. Fender. Uptake of bovine serun albumin by rainbow trout from hyperosmotic solutions: a model for vaccinating fish. Science, 1976, 192:792-794.
Amend, D. F. & K. A. Johnson. status and future needs of Vibrio anguillanrum bacterins. Department of Biology. Standard , 1981, 49:403-417.
Anderson E D, Mourich D V. MolecularMarine Biology and Biotechnology, 2000, 5: 1l4-122.
Austin B. Bacterial fish pathogens:Disease in farmed and wild fish (3rded. ). Chichester: Praxis Publishing Ltd, 1999, 237-251
Awad M A, Nusbaum K E, Brady Y J. Preliminary studies of a newly developed subunit vaccine for channel catfish virus disease. Journal of Aquatic Animal Health, 1989, 1:233-237
Awad MA, et al.. Preliminary studies of a newly developed subunit vaccine for channel catfish virus disease. Journal of Aquaculture Animal Health, 1989, (1):233-237.
Balebona MC, et al. Neurotoxic effect on two fish species and a PC12 cell line of the supernate of Vibrio alginolyticus and Vibrio anguillarum. Veterinary Microbiology, 1998(63):61-6.
Bauer ON. Immunity of fish oocuring in infectious with Ichthyophtirius multifiliis. Dakl, Akad. Navk Ussr.,1953,93:377-379
Beat J,Bruschweiler, Friedrich E, et al. Cytotoxicity in vitro of organotin compounds to fish hepatoma cells PLHC-l(Poeciliopsis lucida). Aquatic Toxicology, 1995, 32: 143-160.
Bernard J. Viral haemorrhagic septicaemia in rainbow trout: Attempt to relate interferon production, antibody systesis and structure of the virus with the mechanism of virulence. Annals of Virology, 1985, 136(1):13-26.
Berthiaume L, AIain R, Robin J. Morphology and ultrastructure of lymphecystis disease virus, a fish iridovirus, grown this culture. Virology, 1984, 135(1):10-l9.
Beyenbach K W, Booz G, Kleinzeller. Transepithelial resistances across isolated perfused renal turbules of the winter flounder. Bulletin of Mount Desert Island Biological Laboratory, 1980, 20:78-82.
Biering E, Villoing S, Sommerset I, et al. Update on viral vaccines for fish. Developments in Biologicals, 2005, 121: 97-113.
Bloch B, Gravningen K, Larsen J L. Encephalomyelitis among turbot associated with a picornavirus-like agent. Diseases of Aquatic Organisms, 1991, 10:65-70.
Bloch B, Larsen J L. A case of severe general oedema in young farmed turbot associated with a herpes virus infection. Bulletin of European Association in Fish Pathology, 1994, 14:130-132.
Bloch B, Larsen J L. An iridovirus-like agent associated with systemic infection in cultured turbot, Scophthalmus maximus fry in denmmark. Disease of Aquatic Organisms, 1993, 15:235-240.
Boudino tP, Blanco M, Kinkelin PD. Virology, 1998, 249 (2):297-306.
Castric J, Baudin-Laurencin F, Coustans M F, et al. Isolation of infectious pancreatic necrosis virus, Ab serotype, from an epizootic in farmed turbot, Scophthalmus maximus. Aquaculture, 1987, 67:117-126.
Castric J, de Kinkelin P. Experimental study of the susceptibility of two marine fish species, sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus), to viral haemorrhagic septicaemia. Aquaculture, 1984, 41:203-212.
Chen SL, Sha ZX, Ren GC, et al. Development and characterization of a continuous embryonic cell line from turbot (Scophthalmus maximus). Aquaculture, 2005, 249(1-4):63-68.
Chinchar V G. Ranaviruses (family Iridoviridae): emerging cold-blooded killers. Archive of Virology, 2002, 147:447-470.
Chinchar, V.G., Essbauer, et al. Virus Taxonomy: VIII Report of the International Committee on the Taxonomy of Viruses, 2005, pp.163-175. Elsevier, London (ISBN: 0122499514)
Chou HY. Application of monoclonal antibody againstviralnucleo-protein to an aetiological study of infection heom atopoietic necrosis.Fish Disease, 1993, 16(2): 149-153.
Cipriano, R.& C.E. Stariiper. Immersion and injection vaccination of salmonids against furunculosis with an avirulent strain of Aeramomas salmonicida. Prog. Fish-culture., 1982, 44:167-169.
Clem L W, Moewus L, Sigel M M. Studies with cells from marine fish in tissue culture. Proceedings of the Society for Experimental Biology and Medicine, 1961, 108:762-766.
Croy, T.R.& D.F. Amend. Immunization of sockeye salmon(Oncorhynchus nerka) against vibriosis using the hyperosmotic infiltration technique. Aquaculture., 1977, 12:317-325.
Cuchens M A, Clem L W. Phylogeny of lymphocyte heterogeneity.Ⅱ.Differential effect of temperature on fish T-like and B-like cells.. Cell Immnology, 1977, 34:219-230.
Dannevig B H, Falk K, Namork E.Isolation of the causal virus infectious salmon anaemia (ISA) in a long-term cell line from Atlantic salmon heas kidney. General Virology, 1995, 76:1353-1359.
De Kinkelin P, Le Berre M. Nécrose bématopoiétique infect:euse de salmonidés: Production d’interféron circulant induite après L’infection expérimentale de la Truite Ar-en-ciel (Salmo gairdneri). Critical Reviews in Academy of Science in Paris, 1977, 284:101-104.
Dorson M.. Applied immunology of fish. In Immunologyical Memory, Edited by Manning M. J., 1984, PP.89-47, Elsevier/Amsterdam.
Duff D. C. B.. The oral immunization of trout against Bacterium salmonicida. Journal of Immunology, 1942, 44:87-94.
Duff, D. C. P..The oral immunization of trout againsr Bacterium salmonicida. Journal of Immunology , 1942,44:37-94.
Eggset G, Mikkelsen H, Killie J-E A. Fish & Shelfish Immunology ,1997 , 7 :247.
Emmart E W, Mossakowski. Primary cultures of tubular cells of the rectal gland of Squalus acanthial. Bulletin of Mount Desert Island Biology Laboratory, 1970, 23:83-84.
Engelmann K, Bohnke M, Friedl P. Isolation and long-term cultivation of human corneal endothelial cells. Investigative ophthalmology & visual science, 1988, 29(11): 1656-1662.
Engelmann K, Bohnke M. Growing human corneal endothelium in cell culture. Fortschritte of Ophthalmology, 1989, 86(1): 72-75.
Essbauer, S. and Ahne W.. Viruses of lower vertebrates. Journal of Veterinary Methods, 200l, 48:403-475.
Evelyn, T. P. T. & J. E. Ketchson. Laboratory and field observations of antivibrosis vaccines. In: Fish Diseases,Third COPRAQ-Session (Ed. by W. Ahne) .45-54. Springer-Verlag, Berlin, Heldelberg, New York. 1980
Fernandez-Alonso M, Alvarez F, Estepa A, et al. A model to study fish DNA immersion vaccination by using the green fluorescentprotein. Fish Diseases, 1999, 22(7): 237-241.
Fryer J L, Lannan C N. Three decades of fish cell culture: A current listing of cell lines derived from fish. Journal of Tissue Culture Methods, 1994(16): 87-94.
Fryer J L, Lannan C N. Three decades of fish cell culture: A current listing of cell lines derived from fish. Journal of Tissue Culture Methods, 1994, 16:87-94.
Fryer, J.L., et al.. Vaccination for control of infectious diseases in Pacific salmbn. Fish anthology, 1976,10(2):155-164.
Gilmore R D, Jr Engelking H M, Manning D S. Expression in Escherichia coli of an epitope of the glycoprotein of protects against viral challenge. Biological Technology, 1988, 6(3):295-300.
Gilmore,R.D.et Rl.. Expression in Escherichia cole of an epitope of the glycoprotein of infectious hematopoietic necrosis virus protects against viral challenge. Biological Technology., 1908, 6(3):295-300.
Gould , R.W. , et al.. Spray vaccination: A method for the immunization of fish. Fish Pathology, 1978,13:63-68.
Goven, B.A., et al.. Protection of channel catfish, Ictalarus punctatus Rafinesque, against Ichthyophtirius multifiliis Fouquet, by immunization . Journal of Fish Biology, 1980, 17:311-316.
Goven, B.A.. Protection of channel catfish( Ictalarus punctatus) against Ichthyophtirius multifiliis (Fouquet) by immunization with varying doses of Tetrohymena pyriformis (Lwoff) cillia. Aquaculture, 1981, 23:269-273.
Grizzl, J.M., Ltinok,et al. First isolation of largemouth bass virus. Diseases of Aquatic Organisms, 2002, 50(3):233-235.
Gudding R , Veterinary Immunology and Immuopathology , 1999 , 72:203.
Gupta RK. Alumimum compounds as vaccine adjuvants. Advanced Drug Delivery Reviews, 1998, 32(3):155-172.
Hart,S., et al.. Gut immnology in fist:a review. Development of Comparative Immunology, 1988, 12:453-480.
He J G, Weng S P, Zeng K, et al. Experimental transmission pathogenicity and physical-chemical properties of infections spleen and kidney necrosis virus (ISKNV). Journal of Fish Diseases, 2002, 23:1-4.
Hedrick R P, McDowell T S, Ahne W, et al. Properties of three iridovirus2 like agents associated with systemic infections of fish. Diseases of Aquatic Organisms, 1992, 13:203-209.
Hedrick, R.P. and McDowell, T.S.. Pr0Perties of Iridoviruses from ornamental fish.Veterinary Research, 1995, 26(5-6):423-427.
Hellberg H, Koppang E O, Torud B, et al. Subclinial herpesvirus inesction in farmed turbot Scophthalmus maximus. Diseases of Aquatic Organisms, 2002, 49:27-31.
Hengstberger S G, Hyatt A D, Speare R, et al. Comparison of epizootic haematopoietic necrosis and Bohle iridoviruses, recently isolated Australian iridoviruses. Diseases of Aquatic Organisms, 1993, 33:1-9.
Hoover K L(ed). Use of small fish species in carcinogenicity testing. National Cancer Institute Monograph, 1984, 65.
Húsgare S, Grotmol S, Hjeltnes B K, et al. Immune response to a recombinant capsid protein of striped jack nervous necrosis virus (SJNNV) in turbot Scophthalmus maximus and Atlantic halibutHippoglossus hippoglossus, and evaluation of a vaccine against SJNNV. Diseases of Aquatic Organisms, 2001, 45:33-44.
Iida J, Meijne A M, Oegema T R Jr, et al. A role of chondroitin sulfate glycosaminoglycan binding site in alpha4b et a1 integrin-mediated melanoma cell adhesion. Biological chemistry, 1998, 273(10):5955-5962.
Janet A W, Bayle J A. Detection of channel catfish virus in channel catfish, Ictalurus puncfatus(Rafinesque): use of a nucleic acid probe. Journal of Fish Diseases, 1985, 8:416-424.
Johnson K.A. &D.F. Amend. Efficacy of Vibrio anguillarum and Yersinia ruckeri bacterins applied by oral an anal intubation of salmonids .Journal of Fish Diseases., 1983, 6(5):473-476.
Joosten DHW, Tiemersma E, ThreelsA, et al. Oral vaccinationof fish againstVibrio anguillaramusing alginate microparticles. Fish and Shellfish Immunology, 1997, 7(7): 471-485.
King J A, Snow M, Skall H F, et al. Experimental susceptibility of Atlantic salmon Salmo salar and turbot Scophthalmus maximus to European freshwater and marine isolates of viral haemorrhagic septicaemia virus. Diseases of Aquatic Organisms, 2001, 47:25-31.
Kusov Y Y. Hepatitis A virus with membrane of infected cells . Soviet Program of Virology, 1986, 4: 89-93.
Kuzyk MA, Burian J, Machander D, et al. An efficacious recombinant subunitvaccine against the salmonid rickettsial pathogen Piscirickettsia salmonis. Vaccine, 2001, 19(17-19):2337-2344
Leong JC, Anderson E, Bootland LM. et al. Fish vaccine antigens produced or delivered by recombinant DNA technologies. Development of Biology Stand, 1997, 90(7): 267-277.
Leong JC, Crippen T, Drennan J, et al. Development of DNA vaccine for fish. Suisanzoshoku, 2000, 48(2): 285-290.
Li X, Trinh K Y, Hew C L. Expression and characterization of an active and thermally more stable recombinant antifreeze polypeptide from ocean pout,Macrozoarces americanus,in Escherichia coil:Improved expression by the modification of the secondary structure of the mRNA.Protein Engineering, 1991, 4(8):995-1002.
Lillehaug, A.. Oral immunization of rainbow trout, Salmo gairdneri Richaidson, against vibriosis with vaccines protected agaist digestive degradation. Journal of Fish Diseases, 1989, 12:579-581.
Lin JH, Yu CC, Lin CC, et al. An oral deliverysystem for recombinant subunit vaccine to fish. Development of Biology, 2005, 121: 175-180.
Lorenzen N, Lorenzen E, Einer-JensenK. Fish & Shellfish Immunology, 2002, l2: 439-453.
Lupiani B, Dopazo C P, Ledo A, et al. New syndrome of mixed bacterial and viral etiology iin cultured turbot Scophthalmus maximus. Journal of Aquatic Animal Health, 1989, 1:197-204.
Mao J H, Hedrick R P, Chinchar V G. Molecular characterization, sequence analysis, and taxonomic position of newly isolated fish iridoviruses. Virology, 1997, 229:212-220.
Mao J H, Tham T N, Gentry GA, et al. Cloning, sequence analysis, and expression of the major capsid protein of the iridovirus frog virus. Virology, 1996, 216:431-436.
Mark, E.H.&C.L. Leong. The glycoprotein of infectious hematopoietie necrosis virus elicits neutralizing antibody and protective responses. Virus Research, 1989, 13(3):213-230.
Marsden MJ, Vaughan LM. Alive(delta aroA)Aeromonas salmonicidavaccine for furunculosis preferentially stimulates T cell responses relative to B cell responses in rainbow trout(Oncorhynchusmykiss ), Infect Immune, 1996, 64(9): 3863-3869.
McGrogan D G, Ostland V E, Byrne P J et al. Systemic disease involving an iridovirus-like agent in cultured tilapia, Oreochromis niloticul L.-a case report. Journal of Fish Diseases, 1998, 21: 149-152.
Miller N, Sizemore R C, Clem L W. Phylogeny of lymphocyte hetèrogeneity. The cellular requirements for in vitro antibody response of channel cufish leukoeytes. Journal of Immunology, 1985, 134:2884-2888.
Moore J D , Ototake M, Nakanishi T. Fish & Shelfish Immunology ,1998 :393.
Nakanishi T, et a1. Cytotoxic T cell function in fish. Developmental and Comparative Immunology, 2001, 26:l31-139.
Newman S G. Bacterial vaccine for fish. Annual Review of Fish Diseases, 1993, 3:145-185.
Newman SG. Bacterial vaccine for fish. Annual Review of Fish Diseases, 1993, 32(3): 145-185.
Noonan B, Enzmann PJ, Trust TJ. Recominant infectious hematopoietic necrosisvirus and hemorrhagic septicemia virusglycoprotein epitopes expressed inAeromonas salmonicidainduce protective immunity in rainbow trout(Oncorhynchusmykiss). Applied and Environmental Micobiology, 1995, 61(10): 3586-3591.
Norman F, et a1.Production of amacrophage growth factors by a goldfish macrophag ecel line and macrophages derived from goldfish kidney leukocytes. Developmental and Comparative Immunology, 1998, 22(4):417-432.
Oberg LA, W irkkula J, Mourich D, et al. Bacterially expressed nucleoprotein of infectious hematopoietic necrosis virus augments protective immunity induced by the glycoprotein vaccine in fish. Virology, 1991, 65(8): 4486-4489.
P F Krnee, M K Patterson. Tissue Culture Method and Application. Acadenic Press, 1973, 21:133-146.
Paul P S, Mengehng W I. Vaccination of swine with an inactivated porcine parvovirus vaccine in the presence of passive immunity. American Veterinary Medical Association, 1986, 188(4): 410-413.
Paul PS,Mengehng WI.Vaccination of swine with an inactivated porcine parvovirus vaccine in the presence of passive immunity. Journal of the American Veterinary Medical Association, 1986, 188(4):410-413.
Qin Q.W, Lam TJ, SinYM, et a1. Electronmicroscopic observations of amarlnefishiridovimsisolatedfrom brown. Spotted grouper, Epinephelus tauvina. Journal of Virology methods, 2001, 98(1):l7-24.
Ribeiro L & Ahne W. Initiation of a line of pituitary cells from carp(Cyprinus carpio) to study the release of gonadotropin in vitro. Fish cell culture, 1982, 18:419-420.
Richards R H, Buchanan J S. Studies on Herpesvirus scophthalmi infection of turbot Scophthalmus maximus L.: histopathological observations. Journal of Fish Diseases, 1978, 1:251-258.
Rivas C, Noya M, Cepeda C, et al. Repliaction and morphogenesis of the turbot aquareovirus TRV in cell culture. Aquaculture, 1998, 160:47-62.
Robers R J. Fish pathology. 3rd ed. London: W B Saunders Pree. 2001.
Rodger H D, Kobs M, Macantney A, et al. Systemic iridovirus infection in fresh water angel fish, Pterophyllus scalare (Lichtenstein). Journal of Fish Diseases, 1997, 20: 69-72.
Ross, A.J.& G.W. Klontz. Oral immunization of rainbow trout (Salmo gairdneri) against the etiologic agent of "redmouth disease". Journal of the Fisheries Research Board of Canada, 1965, 22(3):713-719.
Rukyani, R.& Taukhid. The survival rate of Jumbo catfish( Clarias gariepinus) vaccinated against Ichthyophtirius multifiliis with Tetrahymena Pyriformis at various immersion time exposure. In: Asian Fish Health Bibligraphy and Abstracts I: Southeast Asia (Ed. by Arthur,J.R.), 22, Fish Health section. FS. Manila. 1990.
Samples J R, Binder P S, Nayak S K. Propagation of human corneal endothelium in vitro effect of growth factors. Experimental eye research, 1991, 52(2): 121—128.
Schaperclaus, W.. Fischkrankheiten.pp.708.Academic verlag, Berlin,1954.
Segner H, Behrens A, Joyce EM, et al. Transient induction of 7-ethoxyresorufin-O-deethylase (EROD) activity by medium change in the rainbow trout liver cell line, RTL-W1. Marine Environmental Research. 2000, 50(1-5):489-93.
Shang-Liang Tong, Hong-Zhi Miao, Hong Li. Three new continuous fish cell lines of SPH,SPS and RSBF derived from sea Pereh (Lateolabrax joponieus) and red bream. (Pagrosomus major). Aquaculture. 1998, 169:143-151.
Shen Linling, et a1. Channel catfish cytotoxic cells: a minireview. Developmental and Comparative Immunology, 2001, 26:141-149.
Shi Cheng-Yin, Yin-Geng Wang, Shao-Li Yang. The first report of an iridovirus-like age infection in farmed turbot, Scophthalmus maximus, in China. Aquaculture, 2004, 236:11–25.
Shi CY, Wang YG , Yang SL ,et al. The first report of an iridovirus-like agent infection in farmed turbot, Scophthalmus maximus, in China. Aquaculture, 2004, 236:11-25.
Shi CY, Wang YG, Yang SL , et al. The first report of an iridovirus-like agent infection in farmed turbot , Scophthalmus maximus , in China . Aquaculture , 2004 , 236 : 11-25.
Siegl G, deChastonay J, Kronauer G. Propagation and assay of hep-atitis a virus in vitro . Journal of Virology Methods, 1984, 9:53-67.
Sommerset I, Krossoy B, Biering E, et al. Vaccines for fish in aquaculture. Expert Review of Vaccines,2005 Feb, 4(1): 89-101.
Tamer, M.F.& M.T. Home. Factors infuencing the uptake of 14C-labelled Vibrio anguillarum vaccine in direct immersion experiments with rainbow trout, Salmo gairdneri Richardson. Journal of fish Biology, 1983, 22: 585-591.
Tidona C A, Darai G. The complete DNA sequence of lymphocystis disease virus. Virology, 1997, 230:207-216.
Tomonori S, et a1. Specific cell-mediated cytotoxicity against a virus-infected syngeneic cel fin in isogeneic ginbuma crucian carp. Developmental and Comparative Immunology, 2000, 24:633-640.
Traxler GS, Anderson E, Lapatra SE, et al. Naked DNA vaccination of Atlantic salmon Salmo salar against IHNV. Diseases of Aquatic Organisms, 1999, 38(3): 183-190.
Vanderheijden N, AlardD. The attenuatedV60 strain of channel catfish virus possesses a deletion inORF50 coding for a potentially secreted glycoprotein. Virology, 1996, 218(2): 422-426.
Vestergaard-Jorgensen, P.E.. Partial resistance of rainbow trout ( Salmo gairdneri) to viral haemorrhagic (VHS) following exposure to non-virulent Egtved virus. Nordic Veterinary Medicine, 1976, 28:570-571.
Walczak, E.M., et al. Properties of a vaccine for channel catfish virus disease and a method of administration. Development of Biology.Standard.,1981, 49:419-429.
Wang G, LaPatra S, Zeng L, et al. Establishment, growth, cryopreservation and species of origin identification of three cell lines from white sturgeon, Acipenser transmontanus. Methods in cell science, 2003, 25(3-4): 211-20.
Wassermann F E: In: Methods in virology, Vol: 4, (Naramorosch, K and Koprowski H eds), Academic Press, New York, 1968.
Witter R L, Sharma J M, Fadly A M. Pathogenicity of variantMarek’s disease virus isolants in vaccinated and unvaccinated chick-ens. Avian Diseases, 1980(24): 210-232.
Wolf K, Fish viruses and fish viral diseases . Cornell University Press, 1988.
Wolf K, Quimby M C. Established eurythemic line of fish cells in vitro. Science, 1962, 135:1065-1066.
Wolf K, Quimby MC. Established eurythemic line of fish cells in vitro. Science, 1962, 135:1065-1066.
Wolf K, Snieszko S F, Dunber C E. Infectious pancreatic necrosis, a virus-caused disease of fish . Excerpta Medica, 1959, 13(sect.1):228.
Wolf K. Fish virus and fish viral diseases [M]. Ithaca. NY: Cornell University Press, 1988, 459-472.
wolf K. Fish virus and fish viral diseases. Ithaca. NY: ComeU U niversity Press, 1988: 459-472.
Wolff JA, et al. Direct gene transfer into muscle in vivo. Science, 1990, 247:1456.
Wondergem R, Amsler K. Transmembrane protentials of cultured rectal gland cells of Raja erinacea. Bulletin of Mount Desert Island Biological Laboratory, 1986, 26:105-107.
Yu D H et al: Journal of Japapese Veterinary Medical Association, 1981, 37: 725, (in Japanese). YunboWei,Tingjun Fan, Guojian Jiang et al. Establishment of A Novel Fin Cell Line from Brown-marbledGrouper, Epinephelus fuscoguttatus (Forssk?l), and Evaluation of Its Viral Susceptibility. Aquaculture Research, 2009,1-9.