Construction Of Antigen Microarray For Detection Of6Strains Of Vibrio Bacteria In Cultured Fish

With the rapid development of aquaculture industry in China，increasingnumber of farmed aquatic animal species， intensive breeding and expandingdensity，lead to increasing frequency of disease outbreak. Bacterial disease is one ofthe most popular aquatic animal diseases，of which vibriosis breaks out frequentlyand spread widely. Vibrio is widely distributed in marine environment，it can infect avariety of aquacultured fish and cause high mortality，resulting in great economiclosses in quaculture industry.The basic principle of antigen microarray is spotting antigen on the support ofchip and detecting the corresponding antibody in serum. The antigen microarray hasbeen used in clinical medicine and epidemiological study in recent years. It showswide application prospects for its high sensitivity，specifidity，high-throughput andparallel analysis of multiple samples or biological indicators. However，there are fewreports of antigen microarray for the detection of bacterial disease of aquatic animal.In this paper，we combine antigen microarray technology and aquatic animalimmunology，and build the antigen microarray of6kinds of Vibrio bateria includingV. ichthyoenteri，V. anguillarum，V. parahaemolyticus，V. alginolyticus，V. fluvialisand V. harveyi in aquaculture fish. The resultes prove that the developed antigenmicroarray can be used in diagnosis and detection of the up-mentioned bacteria infish. The experimental methods and results are as follows：(1) After confirming the pathogenicity of Vibrio strains，we extracted theirantigenic components，including outer membrane protein (OMP)，extracellularproducts(ECP)，flagellum protein and ultrasonic liquid of the whole-bacteria. theconcentrations of the antigenic components were determined by Bradford，and thepathogenicity were confirmed by using flounder(Paralichthys olivaceus)anti-immunoglobulin monoclonal antibody labeled by HRP. The results showed thattha extracted antigenic components could react with tha antiserum of flounder， suggesting that they had good pathogenicity and could be used to build the antigenmicroarray.(2) Flounder antiserum was prepared by injecting healthy flounder withinactivated whole Vibrio bacteria. The antiserum levels of flounder after injectionwere detected by ELISA and the cross-reactions between antigenic components andflounder antiserum were analyzed. The results revealed that the levels of flounderantiserum were highest at4，5and6weeks after injection of inactivated wholeVibrio bacteria，therefore，this antiserum were then used in the detection of antigenmicroarray. Furthermore，the antiserum reacted strongly with the correspondingVibrio and there were various degrees of cross-reactions with other species ofVibrio， while the extracellular products and flagellum protein had nocross-reactions，which confirm the feasibility of constructing the antigen microarray.(3) Using the previously optimized antigen microarray supports，printingbuffer，immobilization time，blocking time，antibody incubation time and washbuffer，the antigen microarray of6strains Vibrio bacteria was built by spotting allantigenic components from the same Vibrio in one matrix，and then applied in thedetection of the Vibrio antiserum in flounder. The results demonstrated that all apotsin a matrix could reacted with the corresponding Vibrio antiserum，but no reaction orjust part reaction were seen with other Vibrio antiserum，suggesting that thedeveloped antigen microarray could be used for accurate detection of6strainsVibrio bacteria.(4) The developed antigen microarray was applied in the detection of Vibrio inartificially infected flounder and naturally infected turbot (Scophthalmusmaximus，Linnaeus). The results showed that the antiserum of flounder could react with allantigenic component spots of V. anguillarum in the antigen microarray，while theantiserum of turbot reacted with the antigenic components spots of V. alginolyticus，suggesting that the flounder and the turbot are infected with V. anguillarum and V.alginolyticus respectively. Meanwhile，the same result was got by ELISA. All theseresults revealed the the developed antigen microarray could be used in the detectionof Vibrio bacteria in the fish effectively. Antigen microarray is a kind of important immune chip and is the developmentof traditional immunological detection technique. The constructed antigenmicroarray in this paper can effectively detect6kinds of Vibriosis of aquaculturedfish，providing theoretical and practical basis for development of high-throughputdetecting technology of bacterial disease in aquaculture animals.