养殖鳗鲡病原菌快速诊断试剂盒研制及其应用
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摘要
鳗鲡俗称鳗鱼,是名贵经济鱼类之一,目前中国鳗鲡养殖产量居世界首位,主要养殖种类为欧洲鳗鲡(Anguilla anguilla(Linnaeus))和日本鳗鲡(Anguilla japonica(Temminck et Schlegel)),美洲鳗鲡(Anguilla rostrata(Lesueur))也在某些地区开始试养。在养殖过程中,各种疾病不断发生,其中细菌性疾病造成的危害最大。但是,目前对鳗鲡病原菌的现场快速检测的试剂盒研究很少,尚无行之有效的方法。本研究利用新制备的10株病原菌的免疫血清和实验室以前研制的23种免疫血清组成快速诊断试剂盒,并用此试剂盒对鳗鱼主养区福建省和广东省的不同地区、不同时间、不同规格的鳗鲡所患病原菌进行现场快速检测,结果如下:
1.应用本实验室分离得到的10株病原菌免疫注射新西兰大白兔,获得了免疫血清效价达1024及以上有8种;1种血清效价为512;1种血清效价为256。
2.将10种免疫血清与本实验室分离得到29株病原菌(包括制备免疫血清的10株病原菌)进行玻片凝集交叉反应,结果显示:没有交叉反应的免疫血清7种,有交叉反应的免疫血清3种,交叉菌株的数量分别为1、2和4株,交叉菌株的凝集效价与制备血清菌株的凝集效价比值最高为1/2,最低为1/8。
3.用HiTrap Protein A HP亲和层析柱在AKTApurifier-100蛋白纯化仪器上纯化了有交叉反应的3种血清,纯化后,用纯化前后血清蛋白含量的比值计算出回收率,同时用纯化前后的比活力的比值计算出纯化倍数,结果显示:样品纯化后,最高回收率为30%,最低回收率为7.5%,最高纯化倍数为85,最低纯化倍数3.1。并且通过纯化,3种血清与29株病原菌的玻片凝集试验无出现明显的交叉反应。
4.利用可检测33种鳗鲡病原菌的快速诊断试剂盒在福建省四个地区(闽东、闽南、闽西、闽北)及广东省台山市对不同时间、不同地区、不同种类的鳗鲡病原菌进行现场快速检测,结果显示:福建省欧洲鳗鲡在4～6月和8～10月这两个时间段过检测到12株病原菌,其中4～6月检测到9株,8～10月检测到10株,有6株病原菌不能同时存在于两个时间段,说明不同时间鳗鲡所感染的病原菌不同。对于欧洲鳗鲡,在4～6月份,共检测了三个地区,检测到9株病原菌,仅仅有一株病原菌可以在三个地区同时出现;在8～10月,共检测了四个地区,检测到9株病原菌,有两株病原菌在四个地区同时出现;说明在同一时间内不同地区的病原菌是有差别的。8～10月对广东省台山市日本鳗鲡进行检测,检测到5株病原菌,其中有3株在福建省日本鳗鲡养殖场也检测到,说明两地的日本鳗鲡所感染的病原菌没有明显差异。另外两地的欧洲鳗鲡共检测到12株病原菌,日本鳗鲡共检测到13株,仅仅有3株同时存在于两种鳗鲡,说明不同种类的鳗鲡病原菌差别较大。
Eels (Anguilla spp.), are one of the most popular and valuable economic farmed fish. Eel aquaculture production in China ranks first in the world. European eel (Anguilla anguilla) and Japanese eel (A. japonica) are the main cultivated eels. Now American eel (A. rostrata) has also been cultivated in some areas. In intensive eel cultivation, the diseases occured are inevitable, and bacterial diseases are the main problem and often lead to great economic loss. The different bacteria are very different in the sensitiveness to the medicine. Therefore, to prevent or cure the bacterial diseases it is very important to develop diagnostic kits for rapid and accurate diagnose the species of pathogenic bacterium. In this study, we developed rapid diagnostic kits, including the 10 new made pathogens immune serums and 23 others made before in our laboratory. They have been used to detect pathogenic bacteria of eel on-site at different times and size of eels in different regions of Fujian Province and Guangdong Province. The results are as follows:
1.Ten different pathogenic bacteria strains were used to immune the New Zealand rabbits to produce immune serums. Titer of each immune serum was detected using the method of slide agglutination. The results showed the titres of eight serums were above 1024, the titres of other two were 512 and 256, respectively.
2. The cross-reaction was assayed by the method of slide agglutination between ten kinds of immune serums and 29 known strains of bacteria, including ten strains of bacteria used for producing the above immune serums. The results show that there are seven serums with no cross-agglutinate while there are three serums with cross-agglutinate. The highest agglutinating titer ratio between cross-strains and the strains for producing serums was up to 1/2, and the lowest was 1/8.
3. The three cross-reactive serums were purified with HiTrap Protein-A HP column through the AKTApurifier-100 purification system. After the purification, no obvious agglutination is observed among 29 tested bacterial strains when tested by slide agglutination; the maximum recovery of sample is 30%, the minimum recovery of 7.5%; and the highest multiples of purification is 85-fold, the lowest multiples of 3.1.
4.The rapid diagnostic kits have been applied to investigate different pathogenic bacteria in two species of cultivated eels from four regions of Fujian Province (Eastern, Southern, Western, Northern) and Taishan in Guangdong Province during different period of time (from April to June and from August to October). The results are as follows: 1) There are totally 12 different pathogenic bacteria detected in Anguilla anguilla from Fujian Province during the period of time. Although there are nine strains and ten strains of pathogens detected from April to June and from August to October respectively, there are only six pathogens detected at both the period of time. The results indicated that eels could be infected by different pathogenic bacteria during different period of time.2) From April to June, there are nine pathogenic bacteria detected in European eel from three regions of Fujian, in which only one pathogen detected simultaneously. And from August to October, a total of nine pathogens were detected in European eel from all of the four regions of Fujian, in which two pathogens were detected simultaneously. The results show different pathogenic bacteria could be found in different regions at the same time. 3) There are five pathogens detected at Taishan in Guangdong province from August to October, in which only three pathogenic bacteria are the same as detected in Japanese eel fromFujian province. The detection result show it was no significant difference of pathogen infection in Japanese eel at two areas. In addition, there were 12 strains of pathogenic bacteria detected from European eel, and 13 strains detected in Japanese eel, among which only three pathogens could be detected in both the specices of eel at the same time. These results show that pathogenic bacteria are significant different, because of each different species of eels.
1.应用本实验室分离得到的10株病原菌免疫注射新西兰大白兔,获得了免疫血清效价达1024及以上有8种;1种血清效价为512;1种血清效价为256。
2.将10种免疫血清与本实验室分离得到29株病原菌(包括制备免疫血清的10株病原菌)进行玻片凝集交叉反应,结果显示:没有交叉反应的免疫血清7种,有交叉反应的免疫血清3种,交叉菌株的数量分别为1、2和4株,交叉菌株的凝集效价与制备血清菌株的凝集效价比值最高为1/2,最低为1/8。
3.用HiTrap Protein A HP亲和层析柱在AKTApurifier-100蛋白纯化仪器上纯化了有交叉反应的3种血清,纯化后,用纯化前后血清蛋白含量的比值计算出回收率,同时用纯化前后的比活力的比值计算出纯化倍数,结果显示:样品纯化后,最高回收率为30%,最低回收率为7.5%,最高纯化倍数为85,最低纯化倍数3.1。并且通过纯化,3种血清与29株病原菌的玻片凝集试验无出现明显的交叉反应。
4.利用可检测33种鳗鲡病原菌的快速诊断试剂盒在福建省四个地区(闽东、闽南、闽西、闽北)及广东省台山市对不同时间、不同地区、不同种类的鳗鲡病原菌进行现场快速检测,结果显示:福建省欧洲鳗鲡在4～6月和8～10月这两个时间段过检测到12株病原菌,其中4～6月检测到9株,8～10月检测到10株,有6株病原菌不能同时存在于两个时间段,说明不同时间鳗鲡所感染的病原菌不同。对于欧洲鳗鲡,在4～6月份,共检测了三个地区,检测到9株病原菌,仅仅有一株病原菌可以在三个地区同时出现;在8～10月,共检测了四个地区,检测到9株病原菌,有两株病原菌在四个地区同时出现;说明在同一时间内不同地区的病原菌是有差别的。8～10月对广东省台山市日本鳗鲡进行检测,检测到5株病原菌,其中有3株在福建省日本鳗鲡养殖场也检测到,说明两地的日本鳗鲡所感染的病原菌没有明显差异。另外两地的欧洲鳗鲡共检测到12株病原菌,日本鳗鲡共检测到13株,仅仅有3株同时存在于两种鳗鲡,说明不同种类的鳗鲡病原菌差别较大。
Eels (Anguilla spp.), are one of the most popular and valuable economic farmed fish. Eel aquaculture production in China ranks first in the world. European eel (Anguilla anguilla) and Japanese eel (A. japonica) are the main cultivated eels. Now American eel (A. rostrata) has also been cultivated in some areas. In intensive eel cultivation, the diseases occured are inevitable, and bacterial diseases are the main problem and often lead to great economic loss. The different bacteria are very different in the sensitiveness to the medicine. Therefore, to prevent or cure the bacterial diseases it is very important to develop diagnostic kits for rapid and accurate diagnose the species of pathogenic bacterium. In this study, we developed rapid diagnostic kits, including the 10 new made pathogens immune serums and 23 others made before in our laboratory. They have been used to detect pathogenic bacteria of eel on-site at different times and size of eels in different regions of Fujian Province and Guangdong Province. The results are as follows:
1.Ten different pathogenic bacteria strains were used to immune the New Zealand rabbits to produce immune serums. Titer of each immune serum was detected using the method of slide agglutination. The results showed the titres of eight serums were above 1024, the titres of other two were 512 and 256, respectively.
2. The cross-reaction was assayed by the method of slide agglutination between ten kinds of immune serums and 29 known strains of bacteria, including ten strains of bacteria used for producing the above immune serums. The results show that there are seven serums with no cross-agglutinate while there are three serums with cross-agglutinate. The highest agglutinating titer ratio between cross-strains and the strains for producing serums was up to 1/2, and the lowest was 1/8.
3. The three cross-reactive serums were purified with HiTrap Protein-A HP column through the AKTApurifier-100 purification system. After the purification, no obvious agglutination is observed among 29 tested bacterial strains when tested by slide agglutination; the maximum recovery of sample is 30%, the minimum recovery of 7.5%; and the highest multiples of purification is 85-fold, the lowest multiples of 3.1.
4.The rapid diagnostic kits have been applied to investigate different pathogenic bacteria in two species of cultivated eels from four regions of Fujian Province (Eastern, Southern, Western, Northern) and Taishan in Guangdong Province during different period of time (from April to June and from August to October). The results are as follows: 1) There are totally 12 different pathogenic bacteria detected in Anguilla anguilla from Fujian Province during the period of time. Although there are nine strains and ten strains of pathogens detected from April to June and from August to October respectively, there are only six pathogens detected at both the period of time. The results indicated that eels could be infected by different pathogenic bacteria during different period of time.2) From April to June, there are nine pathogenic bacteria detected in European eel from three regions of Fujian, in which only one pathogen detected simultaneously. And from August to October, a total of nine pathogens were detected in European eel from all of the four regions of Fujian, in which two pathogens were detected simultaneously. The results show different pathogenic bacteria could be found in different regions at the same time. 3) There are five pathogens detected at Taishan in Guangdong province from August to October, in which only three pathogenic bacteria are the same as detected in Japanese eel fromFujian province. The detection result show it was no significant difference of pathogen infection in Japanese eel at two areas. In addition, there were 12 strains of pathogenic bacteria detected from European eel, and 13 strains detected in Japanese eel, among which only three pathogens could be detected in both the specices of eel at the same time. These results show that pathogenic bacteria are significant different, because of each different species of eels.
引文
[1]孙颖杰,陈学群.鳗鲡养殖加工与出口[M].福建科学技术出版社,2005.
[2]樊海平.我国鳗鲡养殖业的现状与发展对策(上)[J].科学养鱼,2006,2:1-2.
[3]樊海平.中国鳗鲡养殖业的现状与发展对策[N].中国渔业报,2007.
[4]松井魁,角佳英明,青江弘.鳗鲡生物学和人工养殖[M].刘海金,张世义译.北京:科学出版社,1978,1-11.
[5]王广军,谢骏,余德光.鳗鲡繁殖生物学研究进展[J].南方水产,2005,1:71-75.
[6]薛祥朝.土池养殖欧洲鳗鲡技术探讨[J].中国水产,1999,2:26-27.
[7]肖九兰.欧洲鳗鲡山区土池养殖技术[J].中国水产,2004,4:48-49.
[8]陈爱平,朱泽闻,王立新等.2006年中国水产养殖病害检测报告(二)[J].科学养鱼,2007,8:48-49.
[9]陈爱平.鳗鲡养殖病害、用药情况及对策[J].科学养鱼,2003,8:38.
[10]黄志斌.鳗鲡病害及其防治技术(上)[J].科学养鱼,2008,10:71-72.
[11]黄志斌,刘志军.鳗鲡病害及其防治技术(下)[J].科学养鱼,2008,11:71-73.
[12]缪庞捷.鳗鲡常见疾病及其防治[J].科学养鱼,2008,11:83.
[13]林明辉,黄志斌,邓国成.鳗鲡爱德华氏菌的防治[J].江西水产科技,2008,2:25.
[14]卢迈新.鳗鲡常见病害的综合防治技术[J].科学养鱼,2008,6:71.
[15]池宝兴.鳗鲡水霉病综合防治技术[J].齐鲁渔业,2008,4(25):10.
[16]陈三木.鳗鲡鱼玻豆虫病的防治方法[J].渔业致富指南,2008,4:60.
[17]刘庆营.鳗鱼养殖常见疾病及其控制方法[J].渔业致富指南,2008,20:51-52.
[18]苏文钦,苏少龙.浅谈鳗鲡疾病无公害的防治[J].中国水产,2007,2:62-63.
[19]钟全福.欧洲鳗鲡肠炎病的防治技术[J].中国水产,2003,10:50-51.
[20]艾红,李永振.养殖鳗鲡的主要疾病与防治研究现状[J].湛江海洋大学学报,1998,18:71-76.
[21]赵文,孙兆和.精养条件下欧洲鳗的病害及防治[J].国外水产,1995,4:20-24.
[22]袁定清.我国养殖鳗鲡病害防治技术(上)[J].科学养鱼,2005,8:14-15.
[23]颜青,颜江华,方莹,等.欧鳗“狂游病”病毒的分离及观察[J].厦门大学学报(自然科学版),2001,40(5):1175-1178.
[24]宫崎照熊.ニホンウナギのヘルべスウイルス性鰓病[J].養殖,1995,32(6):9.
[25]宫崎照熊.ニホンウナギのてブトウイルス病[J].養殖.1995,32(11):31.
[26]黄琪琰.水产动物疾病学[M].上海:上海科学技术出版社,1993:123-124.
[27]林明辉,黄志斌,江小燕.鳗鲡爱德华氏菌病的防治[J].内陆水产,2007,10:35.
[28]朱清旭.鳗鲡的主要病害及防治[J].科学养鱼,2007,11:76.
[29] Hoshina T. Studies on red-fin disease of eel [J]. Special Research Report of Tokyo University of Fisheries, 1962, 6: 105.
[30]陈会波,林阳东.鳗鲡赤鳍病病原菌的分离鉴定和耐药性的研究[J].水生生物学报,1992,16(1):40-46.
[31] Liu CI. The pathological studied on gill diseases in eels [J]. Jcrr Fish, 1978: 45-57.
[32]曾华.养殖欧洲鳗鲡(Anguilla anguilla)狂游病的研究[D].广州:中山大学,2000.
[33] Sakata T, Hattori M. Characteristics of Vibrio vulnificus isolated from diseased tilapia [J]. Fish Pathology, 1998, 23(1): 33-40.
[34] Amaro C, Biosca E, Esteve C, et al. Comparative study of phenotypic and virulence properties in Vibrio vulnificus biotype 1 and 2 obtained from a european eel farm experiencing mortalities[J]. Dis Aquat Org, 1992, 13: 29-35.
[35]郑亚平.鳗鲡常见细菌性疾病的防治[J].渔业致富指南,2004,13:47-48.
[36]樊海平,曾占壮,余培建,等.由豚鼠气单胞菌引起的欧洲鳗鲡败血症[J].水产学报,1999,23(3):313-318.
[37]董传甫,林天龙,陈日升,等.日本鳗鲡败血腹水病病原研究[J].水产科学,2002,21(1):5-8.
[38]宋学宏,李蒙英.欧洲鳗鲡红头病病原及组织病理研究[J].苏州大学学报(自然科学版),2001,17(3):85-91.
[39] Fouz B, Amaro C. Isolation of a new serovar of Vibrio vulnificus pathogenic for eels cultured in freshwater farms [J]. Aquaculture, 2003, 217: 677-682.
[40]陈会波,翁燕玲.鳗鲡肝肾病病原菌的研究[J].汕头大学学报(自科版),1991,6(1):65-72.
[41]王国良,徐兴林.鳗鲡爱德华氏病病原菌及一新种[J].水产学报,1993,17(3):224-229.
[42]何义进,包颖.微生态学在鱼病防治中的应用[J].水产科技情报,1997,24(1):17-19.
[43]储卫华.鱼类细菌性疾病快速诊断技术进展[J].水利渔业,2000,20:29-30.
[44]高汉娇.水库暴发性鱼病及其防治技术研究[J].水利渔业,1995,2:11-13.
[45]杨振国,李学勤.淡水养殖鱼类主要细菌性传染病快速诊断的研究[J].黑龙江畜牧兽医,1997,8:5-8.
[46]樊景凤,李光,王斌,等.间接免疫荧光抗体技术检测凡纳滨对虾红体病病原——副溶血弧菌[J].海洋环境科学,2007,26(6):501-503.
[47]吴淑勤,石存斌,黄志斌,等.鳗鲡混合感染迟缓爱德华氏菌和嗜水气单胞菌的快速诊断[J].鱼类病害研究,1993,3-4:14-17.
[48] Ryoko Ushikoshi, Takaya Inui, Nobuhiro Mano, et al. A method for specific antibody detection from Japanese Eel by indirect ELISA using cuticular antigen of Anguillicola crassus.[J] Fish Pathology, 1999, 34(2): 81-83.
[49] Guitang W, Jeong-Ho K, Mamoru S, et al. Detection of antibody against the monogenean heterobothrium okamotoi in tiger puffer by ELISA[J].Fish Pathology, 1997, 32(3): 1231-1234.
[50]马家好.Dot-ELISA快速检测鱼类运动性气单胞菌的研究[J].大连水产学院学报,1997,12(3):72-78.
[51]钱冬.应用酶联免疫吸附法检测暴发病病原—嗜水气单胞菌的研究[J].水产养殖,1993,4:14-17.
[52]杨鸢劼，陈辉.酶联免疫吸附法快速检测鳗弧菌[J].上海交通大学学报(农业科学版),2005,23:69-76.
[53]沈素芳,储卫华,陆承平.鱼类致病性气单胞菌诊断试剂盒的研制[J].中国兽医学报,2000,20(5):462-464.
[54]张明琴,窦远,张建华,等.核酸探针的原理及应用[J].生物学通报,2006(41):11-12.
[55]余俊红,陈吉祥,历云,等.聚合酶链反应(PCR)检测花鲈鳗弧菌感染[J].黄渤海海洋,2002,20(2):60-64.
[56]陆承平,陈怀青.PCR检测嗜水气单胞荫毒素基因[J].中国动物检疫,1995,12(5):5-7.
[57]孔杰,刘萍,张岩.PCR技术在对虾病原检测中的应用[J].生物工程进展,1994,14(6):43-46.
[58]夏春,马志宏,陈慧英,等.聚合酶链反应(PCR)法检测产β-溶血素嗜水气单胞菌[J].水生生物学报,1999,23(3):288-289.
[59] Carlos R.16S rRNA gene sequence analysis of photobacterium damselae and nested PCR methed for nested pmethod for rapid detection of the causative agent of fish pasteruellosis [J]. Appl environ Microbiol, 1999, 65(7): 2942-2946.
[60] Gonzalez I.Rapid enumeration of Eseheriehia coli in oysters by a quantitafive PCR -ELISA [J]. Appl Microbiol, 1999, 86(2): 231-236.
[61] Kakizaki E, Yoshida T, Kawakami H, et al. Detection of bacterial antigens using immuno-PCR [J]. Lett Appl Microbiol, 1996, 23(2): 101-103.
[62]林端,董燕红,袁茵,等.鱼类虹彩病毒PCR快速检测试剂盒的准确度评价及其应用[J].海洋环境科学,2007(26):190-193.
[63]樊海平,徐娟儿,黄晓沨.福建养殖鳗鲡细菌性疾病的调查与防治[J].海洋湖沼通报,1996(2):66-70.
[64]朱立平,陈学清.免疫学常用试验方法[M].人民军医出版社,2000:18-22,99-102.
[65]凌红丽,陆承平,陈怀青,等.嗜水气单胞菌检验程序的研究[J].中国动物免疫, 1998,3(15):1-3.
[66]黄士懿,陈俊荣.鳗鱼的营养价值(一)[J].台湾鳗讯,2003:228.
[67]杨堂保.关于鳗鲡病害问题的一些看法[J].江西水产科技,1998,2:2-4.
[68]樊海平.鳗鲡病害发生的现状及防治关键[J].科学养鱼,2000,5:5-6.
[69] Esteve-Gassent MD, Nielsen ME, Amaro C.The kinetics of antibody production in mucus and serum of European eel (Anguilla anguilla)after vaccination against Vibrio vulnificus:development of a new method for antibody quantification in skin mucus [J] . Fish and Shellfish Immunology, 1999, 15: 51-61.
[70]朱振久,张冉,杨慧军等.短程免疫法制备高效价抗血清研究[J].湖南师范大学学报(医学版),2006,3(1):42-44.
[71]王钦富.免疫学及免疫学检验试验技术[M].中国医药科技出版社,1995:1-9.
[72]林清华.免疫学试验[M].武汉大学出版社,1999:78-88.
[73] Karunasagar.I, Rosalind.G. Immunological response of the Indian major carps to Aeromonas hydrophila aceine [J]. Journal of diseases , 1991, 14: 413-417.
[74]张俊杰,吴元明,刘慧萍.兔抗人ERA抗血清的制备[J].第四军医大学学报,2001,17(1):73-75.
[75]鄢庆枇,覃映雪,陈强等.大黄鱼血清IgM纯化及其兔抗血清的制备[J].中国水产科学,2006,13(3):475-479.
[76]邢小红,吴元明,黄高升.兔抗人Bit1抗血清的制备及其纯化[J].细胞与分子免疫学杂志,2004,20(6):764-768.
[77]王娅,王全勇,王淑兰.伤寒、副伤寒免疫血清的制备[J].数理医药学志,2005,18(2):178-179.
[78]罗映辉,文质,陈利玉,刘水平,夏忠弟.制备高效价细菌免疫血清的试验研究[J].实用预防医学,2003,2(10):23-24.
[79]蔡玲斐.不同免疫途径制备伤寒抗血清效价的比较[J].杭州医学高等专科学校校报,2002,23(3):106-107.
[80] Bouvet JP. A modified gel filtration technmique predicting an unusual exclusion volume of IgM, a simple way of preparation monoclonal IgM [J]. Immunol Meth, 1984, 66: 229.
[81] Clegradin P. Trandem purification of mouse IgM momoclonal antibodies produced in vitro using anion exchange and gel fast protein liquid chromatography [J]. Chromatograph, 1986, 358: 209.
[82] Lamoyi E. Preparation of F (ab) ' fragment from mouse IgG of various subclasses [J]. Immunol Meth, 1983, 56: 235.
[83] Ogden JR. Purification of murine monoclonal antibodies by cap rylic acid [J]. Immunol Meth, 1982, 53: 133.
[84] Roberts UJ, Barth SL. Expression of messenger ribonacleic acid encloding the inhibin lactivin system during mid and late gestation rat embrogogenesis [J]. Endocrinol, 1994, 134(2): 914.
[85] Barton DE, Yang Feng TL, Mason AJ, et al. Mapping of genes for inhibin subunits alpha, teta A, and beta B on human and mouse chromosomes and studies of jsd mice [J]. Genomics, 1989, 5 (1): 91.
[86]徐颖华.抗百日咳单克隆抗体的纯化及应用进展[J].药品评价,2005,2(2):113-116.
[87]汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2001:38-59.
[88]黄文树.锯缘青蟹一种新的阴离子抗菌肽scygonadin及其基因的分离与鉴定[D].福建:厦门大学,2006:35-36.
[89]萨姆布鲁克J,弗里奇EF,曼尼阿蒂斯T.分子克隆试验指南第二版[M].北京:科学出版社,1998:883-884.
[90] Goding JW. Affinity chromatography monoclonal antibodies; principles and practice [J].San Diego: Acdemic Press Inc, 1993: 327.
[91]张晓楠,黄勇,陈苏民,陈南春,等.兔抗Red蛋白抗血清的纯化及MC3T3细胞中Red蛋白表达时相的分析[J].科学技术与工程,2006(6):681-684.
[92]邓瑞春,明伟,白云秀,汪劲松,等.A蛋白亲和层析纯化动物血清抗体的效果比较[J].免疫学杂志,1999,15(2):128-130.
[93]韩先朴.鳗鲡养殖[M].农业出版社,1990:142-184.
[94]韩先朴,陈光辉.鳗鲡爱德华氏菌病的研究[J].水生生物学报,1989,13(3):259-263.
[95]樊海平,钟全福.今年上半年养殖鳗鲡病害发生概况与流行趋势[J].科学养鱼,2006,8:50-51.
[96]樊海平,钟全福.当前鳗鲡养殖病害流行趋势预测及应对措施[J].农家之友,2006,12:51.
[97]胡成钰,洪一江,饶伟贤,等.欧洲鳗鲡(Anguilla anguilla)病害调查[J].南昌大学学报,2002,26(1):88-91.
[98]黄孝湘.欧洲鳗的病害防治[J].孝感学院学报,2003,23(3):18-21.
[99]林岗,汪彦音,金莺.日本鳗鲡春鳃病的流行病学调查及防治研究[J].福建畜牧兽医,2002,3:7-8.
[100]余金梅.鳗鱼肝肾病、烂尾病的防治[J].北京水产,2003,1:16.
[101]袁定清.养殖欧鳗脱粘败血综合症及防治[J].科学养鱼,2000,7:29.
[102]顺美.鳗鱼红点病的防治[J].南方科技报,2003,3(22):8.
[2]樊海平.我国鳗鲡养殖业的现状与发展对策(上)[J].科学养鱼,2006,2:1-2.
[3]樊海平.中国鳗鲡养殖业的现状与发展对策[N].中国渔业报,2007.
[4]松井魁,角佳英明,青江弘.鳗鲡生物学和人工养殖[M].刘海金,张世义译.北京:科学出版社,1978,1-11.
[5]王广军,谢骏,余德光.鳗鲡繁殖生物学研究进展[J].南方水产,2005,1:71-75.
[6]薛祥朝.土池养殖欧洲鳗鲡技术探讨[J].中国水产,1999,2:26-27.
[7]肖九兰.欧洲鳗鲡山区土池养殖技术[J].中国水产,2004,4:48-49.
[8]陈爱平,朱泽闻,王立新等.2006年中国水产养殖病害检测报告(二)[J].科学养鱼,2007,8:48-49.
[9]陈爱平.鳗鲡养殖病害、用药情况及对策[J].科学养鱼,2003,8:38.
[10]黄志斌.鳗鲡病害及其防治技术(上)[J].科学养鱼,2008,10:71-72.
[11]黄志斌,刘志军.鳗鲡病害及其防治技术(下)[J].科学养鱼,2008,11:71-73.
[12]缪庞捷.鳗鲡常见疾病及其防治[J].科学养鱼,2008,11:83.
[13]林明辉,黄志斌,邓国成.鳗鲡爱德华氏菌的防治[J].江西水产科技,2008,2:25.
[14]卢迈新.鳗鲡常见病害的综合防治技术[J].科学养鱼,2008,6:71.
[15]池宝兴.鳗鲡水霉病综合防治技术[J].齐鲁渔业,2008,4(25):10.
[16]陈三木.鳗鲡鱼玻豆虫病的防治方法[J].渔业致富指南,2008,4:60.
[17]刘庆营.鳗鱼养殖常见疾病及其控制方法[J].渔业致富指南,2008,20:51-52.
[18]苏文钦,苏少龙.浅谈鳗鲡疾病无公害的防治[J].中国水产,2007,2:62-63.
[19]钟全福.欧洲鳗鲡肠炎病的防治技术[J].中国水产,2003,10:50-51.
[20]艾红,李永振.养殖鳗鲡的主要疾病与防治研究现状[J].湛江海洋大学学报,1998,18:71-76.
[21]赵文,孙兆和.精养条件下欧洲鳗的病害及防治[J].国外水产,1995,4:20-24.
[22]袁定清.我国养殖鳗鲡病害防治技术(上)[J].科学养鱼,2005,8:14-15.
[23]颜青,颜江华,方莹,等.欧鳗“狂游病”病毒的分离及观察[J].厦门大学学报(自然科学版),2001,40(5):1175-1178.
[24]宫崎照熊.ニホンウナギのヘルべスウイルス性鰓病[J].養殖,1995,32(6):9.
[25]宫崎照熊.ニホンウナギのてブトウイルス病[J].養殖.1995,32(11):31.
[26]黄琪琰.水产动物疾病学[M].上海:上海科学技术出版社,1993:123-124.
[27]林明辉,黄志斌,江小燕.鳗鲡爱德华氏菌病的防治[J].内陆水产,2007,10:35.
[28]朱清旭.鳗鲡的主要病害及防治[J].科学养鱼,2007,11:76.
[29] Hoshina T. Studies on red-fin disease of eel [J]. Special Research Report of Tokyo University of Fisheries, 1962, 6: 105.
[30]陈会波,林阳东.鳗鲡赤鳍病病原菌的分离鉴定和耐药性的研究[J].水生生物学报,1992,16(1):40-46.
[31] Liu CI. The pathological studied on gill diseases in eels [J]. Jcrr Fish, 1978: 45-57.
[32]曾华.养殖欧洲鳗鲡(Anguilla anguilla)狂游病的研究[D].广州:中山大学,2000.
[33] Sakata T, Hattori M. Characteristics of Vibrio vulnificus isolated from diseased tilapia [J]. Fish Pathology, 1998, 23(1): 33-40.
[34] Amaro C, Biosca E, Esteve C, et al. Comparative study of phenotypic and virulence properties in Vibrio vulnificus biotype 1 and 2 obtained from a european eel farm experiencing mortalities[J]. Dis Aquat Org, 1992, 13: 29-35.
[35]郑亚平.鳗鲡常见细菌性疾病的防治[J].渔业致富指南,2004,13:47-48.
[36]樊海平,曾占壮,余培建,等.由豚鼠气单胞菌引起的欧洲鳗鲡败血症[J].水产学报,1999,23(3):313-318.
[37]董传甫,林天龙,陈日升,等.日本鳗鲡败血腹水病病原研究[J].水产科学,2002,21(1):5-8.
[38]宋学宏,李蒙英.欧洲鳗鲡红头病病原及组织病理研究[J].苏州大学学报(自然科学版),2001,17(3):85-91.
[39] Fouz B, Amaro C. Isolation of a new serovar of Vibrio vulnificus pathogenic for eels cultured in freshwater farms [J]. Aquaculture, 2003, 217: 677-682.
[40]陈会波,翁燕玲.鳗鲡肝肾病病原菌的研究[J].汕头大学学报(自科版),1991,6(1):65-72.
[41]王国良,徐兴林.鳗鲡爱德华氏病病原菌及一新种[J].水产学报,1993,17(3):224-229.
[42]何义进,包颖.微生态学在鱼病防治中的应用[J].水产科技情报,1997,24(1):17-19.
[43]储卫华.鱼类细菌性疾病快速诊断技术进展[J].水利渔业,2000,20:29-30.
[44]高汉娇.水库暴发性鱼病及其防治技术研究[J].水利渔业,1995,2:11-13.
[45]杨振国,李学勤.淡水养殖鱼类主要细菌性传染病快速诊断的研究[J].黑龙江畜牧兽医,1997,8:5-8.
[46]樊景凤,李光,王斌,等.间接免疫荧光抗体技术检测凡纳滨对虾红体病病原——副溶血弧菌[J].海洋环境科学,2007,26(6):501-503.
[47]吴淑勤,石存斌,黄志斌,等.鳗鲡混合感染迟缓爱德华氏菌和嗜水气单胞菌的快速诊断[J].鱼类病害研究,1993,3-4:14-17.
[48] Ryoko Ushikoshi, Takaya Inui, Nobuhiro Mano, et al. A method for specific antibody detection from Japanese Eel by indirect ELISA using cuticular antigen of Anguillicola crassus.[J] Fish Pathology, 1999, 34(2): 81-83.
[49] Guitang W, Jeong-Ho K, Mamoru S, et al. Detection of antibody against the monogenean heterobothrium okamotoi in tiger puffer by ELISA[J].Fish Pathology, 1997, 32(3): 1231-1234.
[50]马家好.Dot-ELISA快速检测鱼类运动性气单胞菌的研究[J].大连水产学院学报,1997,12(3):72-78.
[51]钱冬.应用酶联免疫吸附法检测暴发病病原—嗜水气单胞菌的研究[J].水产养殖,1993,4:14-17.
[52]杨鸢劼，陈辉.酶联免疫吸附法快速检测鳗弧菌[J].上海交通大学学报(农业科学版),2005,23:69-76.
[53]沈素芳,储卫华,陆承平.鱼类致病性气单胞菌诊断试剂盒的研制[J].中国兽医学报,2000,20(5):462-464.
[54]张明琴,窦远,张建华,等.核酸探针的原理及应用[J].生物学通报,2006(41):11-12.
[55]余俊红,陈吉祥,历云,等.聚合酶链反应(PCR)检测花鲈鳗弧菌感染[J].黄渤海海洋,2002,20(2):60-64.
[56]陆承平,陈怀青.PCR检测嗜水气单胞荫毒素基因[J].中国动物检疫,1995,12(5):5-7.
[57]孔杰,刘萍,张岩.PCR技术在对虾病原检测中的应用[J].生物工程进展,1994,14(6):43-46.
[58]夏春,马志宏,陈慧英,等.聚合酶链反应(PCR)法检测产β-溶血素嗜水气单胞菌[J].水生生物学报,1999,23(3):288-289.
[59] Carlos R.16S rRNA gene sequence analysis of photobacterium damselae and nested PCR methed for nested pmethod for rapid detection of the causative agent of fish pasteruellosis [J]. Appl environ Microbiol, 1999, 65(7): 2942-2946.
[60] Gonzalez I.Rapid enumeration of Eseheriehia coli in oysters by a quantitafive PCR -ELISA [J]. Appl Microbiol, 1999, 86(2): 231-236.
[61] Kakizaki E, Yoshida T, Kawakami H, et al. Detection of bacterial antigens using immuno-PCR [J]. Lett Appl Microbiol, 1996, 23(2): 101-103.
[62]林端,董燕红,袁茵,等.鱼类虹彩病毒PCR快速检测试剂盒的准确度评价及其应用[J].海洋环境科学,2007(26):190-193.
[63]樊海平,徐娟儿,黄晓沨.福建养殖鳗鲡细菌性疾病的调查与防治[J].海洋湖沼通报,1996(2):66-70.
[64]朱立平,陈学清.免疫学常用试验方法[M].人民军医出版社,2000:18-22,99-102.
[65]凌红丽,陆承平,陈怀青,等.嗜水气单胞菌检验程序的研究[J].中国动物免疫, 1998,3(15):1-3.
[66]黄士懿,陈俊荣.鳗鱼的营养价值(一)[J].台湾鳗讯,2003:228.
[67]杨堂保.关于鳗鲡病害问题的一些看法[J].江西水产科技,1998,2:2-4.
[68]樊海平.鳗鲡病害发生的现状及防治关键[J].科学养鱼,2000,5:5-6.
[69] Esteve-Gassent MD, Nielsen ME, Amaro C.The kinetics of antibody production in mucus and serum of European eel (Anguilla anguilla)after vaccination against Vibrio vulnificus:development of a new method for antibody quantification in skin mucus [J] . Fish and Shellfish Immunology, 1999, 15: 51-61.
[70]朱振久,张冉,杨慧军等.短程免疫法制备高效价抗血清研究[J].湖南师范大学学报(医学版),2006,3(1):42-44.
[71]王钦富.免疫学及免疫学检验试验技术[M].中国医药科技出版社,1995:1-9.
[72]林清华.免疫学试验[M].武汉大学出版社,1999:78-88.
[73] Karunasagar.I, Rosalind.G. Immunological response of the Indian major carps to Aeromonas hydrophila aceine [J]. Journal of diseases , 1991, 14: 413-417.
[74]张俊杰,吴元明,刘慧萍.兔抗人ERA抗血清的制备[J].第四军医大学学报,2001,17(1):73-75.
[75]鄢庆枇,覃映雪,陈强等.大黄鱼血清IgM纯化及其兔抗血清的制备[J].中国水产科学,2006,13(3):475-479.
[76]邢小红,吴元明,黄高升.兔抗人Bit1抗血清的制备及其纯化[J].细胞与分子免疫学杂志,2004,20(6):764-768.
[77]王娅,王全勇,王淑兰.伤寒、副伤寒免疫血清的制备[J].数理医药学志,2005,18(2):178-179.
[78]罗映辉,文质,陈利玉,刘水平,夏忠弟.制备高效价细菌免疫血清的试验研究[J].实用预防医学,2003,2(10):23-24.
[79]蔡玲斐.不同免疫途径制备伤寒抗血清效价的比较[J].杭州医学高等专科学校校报,2002,23(3):106-107.
[80] Bouvet JP. A modified gel filtration technmique predicting an unusual exclusion volume of IgM, a simple way of preparation monoclonal IgM [J]. Immunol Meth, 1984, 66: 229.
[81] Clegradin P. Trandem purification of mouse IgM momoclonal antibodies produced in vitro using anion exchange and gel fast protein liquid chromatography [J]. Chromatograph, 1986, 358: 209.
[82] Lamoyi E. Preparation of F (ab) ' fragment from mouse IgG of various subclasses [J]. Immunol Meth, 1983, 56: 235.
[83] Ogden JR. Purification of murine monoclonal antibodies by cap rylic acid [J]. Immunol Meth, 1982, 53: 133.
[84] Roberts UJ, Barth SL. Expression of messenger ribonacleic acid encloding the inhibin lactivin system during mid and late gestation rat embrogogenesis [J]. Endocrinol, 1994, 134(2): 914.
[85] Barton DE, Yang Feng TL, Mason AJ, et al. Mapping of genes for inhibin subunits alpha, teta A, and beta B on human and mouse chromosomes and studies of jsd mice [J]. Genomics, 1989, 5 (1): 91.
[86]徐颖华.抗百日咳单克隆抗体的纯化及应用进展[J].药品评价,2005,2(2):113-116.
[87]汪家政,范明.蛋白质技术手册[M].北京:科学出版社,2001:38-59.
[88]黄文树.锯缘青蟹一种新的阴离子抗菌肽scygonadin及其基因的分离与鉴定[D].福建:厦门大学,2006:35-36.
[89]萨姆布鲁克J,弗里奇EF,曼尼阿蒂斯T.分子克隆试验指南第二版[M].北京:科学出版社,1998:883-884.
[90] Goding JW. Affinity chromatography monoclonal antibodies; principles and practice [J].San Diego: Acdemic Press Inc, 1993: 327.
[91]张晓楠,黄勇,陈苏民,陈南春,等.兔抗Red蛋白抗血清的纯化及MC3T3细胞中Red蛋白表达时相的分析[J].科学技术与工程,2006(6):681-684.
[92]邓瑞春,明伟,白云秀,汪劲松,等.A蛋白亲和层析纯化动物血清抗体的效果比较[J].免疫学杂志,1999,15(2):128-130.
[93]韩先朴.鳗鲡养殖[M].农业出版社,1990:142-184.
[94]韩先朴,陈光辉.鳗鲡爱德华氏菌病的研究[J].水生生物学报,1989,13(3):259-263.
[95]樊海平,钟全福.今年上半年养殖鳗鲡病害发生概况与流行趋势[J].科学养鱼,2006,8:50-51.
[96]樊海平,钟全福.当前鳗鲡养殖病害流行趋势预测及应对措施[J].农家之友,2006,12:51.
[97]胡成钰,洪一江,饶伟贤,等.欧洲鳗鲡(Anguilla anguilla)病害调查[J].南昌大学学报,2002,26(1):88-91.
[98]黄孝湘.欧洲鳗的病害防治[J].孝感学院学报,2003,23(3):18-21.
[99]林岗,汪彦音,金莺.日本鳗鲡春鳃病的流行病学调查及防治研究[J].福建畜牧兽医,2002,3:7-8.
[100]余金梅.鳗鱼肝肾病、烂尾病的防治[J].北京水产,2003,1:16.
[101]袁定清.养殖欧鳗脱粘败血综合症及防治[J].科学养鱼,2000,7:29.
[102]顺美.鳗鱼红点病的防治[J].南方科技报,2003,3(22):8.