Study On White-sports Disease In Internal Organs Of Pseudosciaena Crocea

In the recent years, a new epidemic disease occurred in net-cage cultured large yellow croaker (Pseudosciaena crocea) in Xiangshan bay, Zhejiang Province resulting in severe losses in spring and winter. Diseased fish showed no obvious symptoms on the outside, but many white spots of0.5-1.0mm in diameter in the spleen and kidney were observed. The isolation and identification of the causative agent in combination with16S rRNA sequencing showed the bacterium Pseudomonas putida to be responsible for that disease. The tissues of diseased fish were pathologically analyzed by paraffin histological section techniques and the isolated strain PT01was determined as the causative agent by artificial infection. Affected fish showed obvious inflammation and cell deformation or disintegration of the liver, kidney and spleen.This paper reports the development of a visual loop-mediated isothermal amplification (LAMP) assay for rapid detection of the pathogen. Four primers, F3, B3, FIP and BIP, were designed on the basis of DNA sequence of the rpoN gene of P.putida. After optimization of the reaction conditions, the detection limit of LAMP assay was4.8cfu per reaction,10-fold higher than that of the conventional PCR. The assay showed high specificity to discriminate all isolates from ten different Gram-negative bacterias. The assay also successfully detected the pathogen DNA in the tissues of infected fish. For visual LAMP without cross-contamination, SYBR green Ⅰ was embedded in a microcrystalline wax capsule and preseted in the reaction tubes; after the reaction the wax was melted at85℃to release the dye and allow intercalation with the DNA amplicons. The simple, highly sensitive, highly specific and cost-effective characteristics of visual LAMP may encourage its application in the rapid diagnosis of this pathogen.Besides we detected the P.putida extracellular enzyme, the detected strains do not produce casein enzyme, amylase, phospholipase, and do not have hemolytic. We observed the biofilm formation of P.putida by silver staining, and then prepared lipopolysaccharide (LPS), outer membrane protein (OMP) and whole-bacterial antigen(FKC), we did the artificial immunity of large yellow croaker using the all immune components. After21days we set out the serum agglutination antibody titer, lysozyme activity, phagocytic activity and immune protection rates to explore the impact of these protective antigens on the immune function of large yellow croaker. We can find the blood leukocyte phagocytic activity and serum lysozyme activity of the FKC immune group has increased to a certain extent, but compared with the control group, they did not have significantly difference (P>0.05). However, The serum antibody titer of it was significantly higher (P<0.05). The antibody titer, lysozyme activity and phagocytic activity of the LPS and OMP immune group was not significantly different (P>0.05) compared with the control group. Following we did the intraperitoneal challenge with P.putida PT01, the result also verified that the FKC immune group can provide some protection (RPS of40%).