【作者】 佟延南；

【导师】 关瑞章；

【作者基本信息】 集美大学 ， 水产养殖学， 2009， 硕士

【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。更多还原

【Abstract】 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.更多还原