养殖刺参腐皮综合征2种致病菌间接荧光抗体快速检测方法

Rapid detection of 2 pathogenses of skin ulcer syndrome in cultured Apostichopus japonicus by indirect fluorescence antibody technique

以养殖刺参(Apostichopus japonicus)腐皮综合征的2种致病菌:灿烂弧菌(Vibrio splendidus)和假交替单胞菌(Pseudoalteromonas nigrifaciens)为抗原,分别制备兔抗血清。以载玻片为介质,建立了2种病原菌的间接荧光抗体检测技术(IFAT)。交叉反应、阻断试验和吸收试验结果均表明本方法特异性强。对人工感染实验中的养殖水体及发病刺参溃烂组织检测,可以检出水体和患病刺参溃烂组织中的相应病原菌,病原菌被染成明亮的黄绿色,检测灵敏度2.4×104cell/mL。冰冻切片检测结果显示,在刺参肿胀嘴部与溃烂肌肉处有大量染成黄绿色的细菌颗粒。结果表明:采用IFAT可以对刺参腐皮综合征2种致病菌进行准确快速的检测。该方法的建立对刺参腐皮综合征的流行病学调查及快速诊断具有重要意义。

An indirect fluorescence antibody technique for the rapid detection of Vibrio splendidus and Pseudoalteromonas nigrifaciens-the two pathogens of the skin ulcer syndrome of sea cucumber Apostichopusjaponicus, has been developed on glass slides. The antiserum of V. splendidus （KH 1 ） and P. nigrifaciens （KH2） were prepared with rabbits.Cross-reactions of antisera with other 11 main marine pathogens were detected by agglutination, and the results showed low cross-reaction which was eliminated by heightening the dilution of antisera. The cross reaction assay and absorb assay indicated the method was successful in distinguishing those pathogens. Working concentration of two antisera and FITC-goat-anti-rabbit IgG （FITC-IgG）were assayed by grads dilutions： dilution titers of KH1,KH2 and FITC-IgG were 400,200 and 80 respectively for detecting bacteria, while dilution titers of KH1, KH2 and FITC-IgG were 200,100 and 80 respectively for detecting frozen section of tissues. The sensitivity of the method was 2.4 × 10^4 cells/mL. An artificial infection experiment on sea cucumber by 2 pathogens were carried out, and bacteria cultured from tissues （muscle ulceration, mouth tumidity, muscle） of diseased A. japonicus, water, and frzoen sections of tissues were detected by IFAT. Results showed the established method could detect the pathogens from diseased sea cucumber, water and even healthy sea cucumber in infection test. V. splendidus and P. rtigrifaciens were stained with bright yellow-green. Positive cells in frozen sections indicated that the mouth tumidity and skin ulceration symptoms of diseased sea cucumber were caused by the invading of bacteria. The overall detecting results suggest that applying this technique properly in clinical diagnosis of skin ulcer syndrome of A. japonicus would be more efficient and accurate. [Journal of Fishery Sciences of China,2010, 17 （ 2 ） ： 329-336}