Identification Of Bacterial Pathogens Of Flatfish And Primary Study On The Immunoprotect Against Pathogens

With the high stocking sensity and poor management of flatfish, diseases occurredseriously, and had becme the main factor restricting its development.The cultured flounder died continually at Maidao in 2006, and the typicalsymptom was ulcer of body surface and fins. The pathogenic strain 20060928-1 wasisolated from the diseased fish. It was Gram-negative, right or curved rods shape,motile with a polar flagella, and it was shown from the conventional physiological,biochemical test and 16S rDNA sequence analysis that 20020928-1's characteristicswere very similar to those of Vibrio harveyi. The infection experiment showed LD₅₀value was 2.02×10⁶ CFU/tail for flounder, only Ceftriaxone sodium and Ceftizoximewere effective to inhibit the growth of this pathogenic bacterium.Effects of initial pH and different carbon sources on the growth of Vibrioanguillarum were studied. The optimized recipe of media contains sodium chloride19‰, peptone1.5%, sucrose 1.72%. The culture was inactivated for 24 h by 0.40%formalin under 4℃, and attack test was done to ensure the safety of vaccine.To determine the titer of serum antibody, a new method was developed. Firstly,The microplates was coated with antigen, then labeled antigen was added whenantibody in serum samples had conjugated antigen. After antigen-antibody-antigencomplex had formed, Phenylenediamine (OPD) was used as substrate to color. Theprinciple of double antigen sandwich was used to detect serum antibody titers, whichwas simpler than microplate agglutination and had higher sensitivity.Safety concentration of several adjuvants was studied, and then the immunizationexperiment of different gradients by bath immersion was done. Activity of lysozyme,protein content and antibody titer were detected. Two groups had better resultsaccording to Protection indices (PI) after attacking. One group is immunized byintraperitoneal injection (PI is 33.6), and another is immunized by bath immersion with33.9mg/L ****(PI is 6.61).