| 摘要: |
| 于1994年8月—1995年5月建立中国对虾病原菌-副溶血弧菌的间接荧光抗体检测技术，其中副溶血弧菌的特异抗血清由家兔制备，羊抗兔免疫球蛋白用异硫氰酸荧光素标记，并以罗丹明标记的牛血清白蛋白为背景染色。应用该技术在山东省莱州市大华水产实业公司养殖场检测中国对虾样品中的副溶血弧菌。结果表明，养成期人工感染对虾中，副溶血弧菌主要集中于中国对虾的注射点附近的肌肉及血淋巴中，说明由肌肉注射而进入体内的病原菌主要转移部位是血淋巴；菌浴感染苗期中国对虾中，尽管虾苗外观未见异常，但可检测到大量副溶血弧菌；苗期对虾中的副溶血弧菌现场检测，3组发病虾苗样品中均检测到副溶血弧菌，3组外观健康的虾苗样品中，其中1组样品中检测到副溶血弧菌，另外2组样品未检测到副溶血弧菌，表明间接荧光抗体技术不仅可用于诊断发病的感染对虾，也可用于检测带菌状态或未发病的感染对虾。 |
| 关键词: 间接荧光抗体诊断技术 中国对虾 副溶血弧菌 |
| DOI： |
| 分类号: |
| 基金项目:欧洲共同体“发展中国家生命科学和技术计划”资助!STD3（TS3-CT94-0269） |
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| STUDY ON DIAGNOSIS OF VIBROSIS IN PENAEUS CHINENSIS BY INDIRECT FLUORESCENT ANTIBODY STAINING PROCEDURE |
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Zhang Xiaohua1, Xu Huaishu1, Xu Bing1, Ji Weishang1, Han Yin2, Yang Xuesong3, Ma Jiankang3
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1.College of Marine ha Sciences. Ocean University of Qingdao, Qingdao 266003;2.Ningbo Fisheries Research Institute, Zhejiang 315010;3.Dahua Fishery Production Group Company of Laizhou, Shandong 261413
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| Abstract: |
| An indirect fluorescent antibody technique (iFAT) incorporating fluoresce in isothiocyanate conjugated anti-rabbit globulin goat serum, and rhodamine isothiocyanate conjugated bovine serum albumin (RITC-BSA, BBL, Cockeysville, MD) as background stain has been developed for the detection of Vibrio parahaemolyticus during August 1994-May 1995. The iFAT was based on Xu et al. (1984). V. parahaemolyticas, strains 4-7, 1.1614, 1.1615, J and QM1 were used as antigens. Rabbits were immunized five times in 2 weeks intervals by injecting 0.1, 0.2, 0.5, 1.0 and 2.0 ml bacterial suspension intravenously, with three rabbits for each strain (Tab.1). On the 7th day after the last injection, antibody titers were determined by slide agglutination. If the titers were higher than 1280, the blood was withdrawn by venipuncture and antiserum was prepared. 52 strains of Vibrio (non-V. parahaemolyricus spp.) and related organisms were used for cross reactivity studies (Tab. 2). Each strain proven to have cross reactivity was grown on five Marine Agar slopes at 28°C for 24 hours, washed three times (3000 r / min, 20 min) in sterile 0.85% saline, and added to the pooled antiserum. The mixture was stirred at room temperature for 30 min, then overnight at 4°C. Cells were removed by centrifugation, followed by filtration through 0.2μm Nuclepore membrane filters. After being absorbed, the cross reactivity of the antiserum was tested again. For the case of no agglutination, the polyvalent antiserum for V. parahaemolyricus was used. Before absorption, with 52 Vibrio strains and other bacteria, 38 strains had cross-reactions, 6 strains did not have cross-reactions, and 8 strains were not tested. Cross-reactivity titers of most strains were not more than 1196, but one strain of V. anguillarium was 1 : 256 (Tab.2). After absorption, there were no cross reactivities between antiserum and 52 strains of bacteria. The specificity of antiserum was suitable for the demands of immunological examinations of shrimp pathogens.
Levels of bacteria in adult Penaeus chinensis challenged with V. parahaemolyticus and in larval shrimps were detected using this method in Dahua Hatchery, Laizhou, Shandong, P. R. China. The results of V. parahaemolyticus in adult challenged shrimp show that V. parahaemolyricus mainly exist in the muscle tissues near the injection site (Plate I: 1) and in haemolymph (Plate I: 2). There were only a few bacteria in other tissues, such as muscle far from injection site, limbs, hepatopancreas and heart. There were no bacteria in the eyeball fluid. This indicates that the major infection site of pathogens entering shrimps by intramuscular injection is haemolymph. The results of V. paralpaewrolyticus in challenged larval shrimps show that a lot of V. parahaemolyticus can be detected in the samples collected from the challenged shrimp larvae, though none of the shrimps died during challenge. None of the bacteria could be detected in control samples. A field-scale test of V. parahaemolyticus in shrimp hatchery show that V. parahaemolyticus can be detected in all the three groups of diseased shrimp larvae samples and one group of health shrimp larvae sample. Therefore, iFAT can be used not only to diagnose the clinically diseased shrimp, but also to recognize subclinically diseased or carrier shrimps. |
| Key words: Indirect fluorescent antibody staining, Penaeus chinensis, Vibrio paraljzaemolyticus |
