Detection Of Vibrio Anguillarum And Its Virulent Factor And Study Of Attenuated Mutagenesis And Inactivated Vaccine

A pathogenic Vibrio anguillarum strain M3 was isolated from diseased left-eyed flounder (Paralichthys olivaceus).The extracellular products (ECP) of M3 was virulent to flounders,goldfish and rats. It could degrade protein substrates and lead to hemolysis on the blood agar plate. It could obviously accelerate growth of M3 as M3 cultured in 2216E liquid medium. A virulent protease secreted by M3 was purified following the procedures of ultrafiltration, gel filtration on sephadex G-100 column, ion-exchange chromatography on DEAE-cellulose A52 column. Specific antisera against M3 cell and the protease were prepared by immunization to rabbits. Slide agglutination assay, indirect fluorescent antibody technology, enzyme-linked immunosorbent assay (ELISA), and PCR were used to detect M3 qualitatively and quantitatively. It was proved that PCR was the most sensitive and specific method by comparison. Result showed that M3 had invaded and diffused into the viscera tissues through the blood circulating system 12 hours after muscular injection. M3 was first detected in the blood 2 hours after infection. Four hours after infection, M3 was detected in liver, kidney and spleen. M3 wasn't detected in gill and intestine until 12 hours after infection. Indirect ELISA and Western blot and dot-ELISA were performed to detect the virulent protease secreted by V. anguillarum M3. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in dot-ELISA test, while 7.8 ng in indirect ELISA and 6.25 ng in Western blot respectively. Protease could be detected 2 hours after incubation of M3 in 2216E liquid medium but enzyme activity was very low at the moment. From 6~12 hours, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. It was 5~6 hours after infection the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either from medium or from blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards. The whole cell inactivated vaccine preparations were made of formalin-killed V. anguillarum strain M3 and left-eyed flounders were vaccinated muscularly. Specific antibody production, bactericidal and SOD activity of serum, phagocytic activity of blood cell were measured. Results indicated that the vaccinated group possessed higher immune activity than the control group. It's interesting that the red cell of flounders showed distinct phagocytosis as well as the phagocytes. The challenges were performed artificially by muscular injection to flounders with V. anguillarum and vaccinated group presented better protection effect. Mutation of V. anguillarum M3 was induced according to insertional mutagenesis of angR gene. The conserved region of angR gene was cloned and inserted into suicide vector pNQ705 and transformed into the conjugation donor E.coli S17-1. Insertional mutation in V. anguillarum chromosomal angR gene was completed via mating the recombinant into M3 strain. The mutated strain was screened by antibiotic and selective medium TCBS. PCR and sequence were used to validate the correct mutation. The virulence of mutant dropped at least 10-fold by challenging goldfish and the activity of sequestering iron also decreased.